Minh Dao Nguyen

Alexis Weinreb

Laure Granger

Maëlle Jospin

Jean-Louis Bessereau
email: jean-louis.bessereau@univ-lyon1.fr

Keywords: C.elegans, muscle activity, axonal plasticity, neuron morphology, axon morphology, axon growth, calcium signalling, voltage-gated calcium channel i

During neural development, activity of the post-synaptic targets can affect the morphology of its axons in certain cases. Using Caenorhabditis elegans as a model system, we studied the activity-dependent morphological changes of one type of motor neurons in the worm's head, called the SAB neurons. They are cholinergic motor neurons that innervate head muscle in C. elegans. SAB neurons are highly sensitive during development, a decrease in muscle activity during a critical period of development leads to an overgrowth of SAB axons, either a failure of termination (looping) or a branching event in the middle (sprouting). With a gene candidate approach, we found several mutations of genes coded for voltage-gated calcium channel (VGCCs) that almost completely suppressed the overgrowth phenotype, for example mutations in egl-19 (coded for the α1 subunit, the main pore of the L-type channel) and unc-36 (coded for the α2δ auxiliary subunit). We propose a tentative model for the SAB axonal overgrowth: during development, as the muscle is deactivated, this is communicated to the SAB neurons with a yet unknown mechanism; then the neurons over-activate and the VGCCs are opened, increasing the intracellular calcium concentration, which leads to the overgrowth of SAB axons. Evidences for this model are three-pronged: First, SAB axons do not show histamine-induced overgrowth defects if the activity in the neurons is inhibited, meaning SAB activity is necessary for overgrowth. Second, SAB hyperactivity is sufficient to induce overgrowth as extra cations influx into the neurons produces overgrowth. Third, we have evidences showing that the calcium level in L1 larvae, the critical period of SAB overgrowth, is higher in histamine-treated animals than control, indicating a higher level of activity.

Résumé en francais:

Pendant le développement des certains neurons, activité postsynaptique est capable de changer la morphologie de leur axons. En utilisant le C. elegans comme l'animal modèle, nous avons étudié la changement morphologique d'un type de motor neurons dans la tête du vers, appelés les neurones SAB, en fonction de l'activity musculaire. Les neurones SAB sont cholinergiques et ils innervent les muscles de la tête du C. elegans. Ces neurones sont hautement sensitifs au cours du développement, une diminution de l'activité musculaire pendant une période critique entraîne une surcroissance (looping) ou un branchement ectopique (sprouting) de ses axons. Avec l'approche gène candidat, nous avons trouvé que la surcroissance axonale est quasiment éliminée dans des mutants des gènes codés pour les canaux calciques dépendent du voltage (CCDV), par exemple la sous-unité α1 egl-19 qui constitue le pore principal du canal de type L, ou la sous-unité auxiliaire α2δ unc-36. Nous proposons un modèle provisoire de la surcroissance des axons SAB: pendant developpement, lorsque le muscle est désactivé, cela est signalé aux neurones SAB avec un mécanisme inconnu; puis, les neurones suractivent et les CCDV s'ouvrent, augmentant la concentration de calcium intracellulaire, ce qui conduit à la surcroissance des axones SAB. Les preuves de ce modèle sont comme suite: premièrement, les axones SAB ne présentent pas de défauts de prolifération induits par l'histamine si l'activité des neurones est inhibée, ce qui signifie que l'activité SAB est nécessaire à la prolifération. Deuxièmement, l'hyperactivité des neurones SAB est suffisante pour induire une surcroissance car un afflux supplémentaire de cations dans les neurones produit une prolifération. Troisièmement, nous observons que le niveau de calcium dans les larves L1, la période critique de prolifération SAB, est plus élevé chez les animaux traités à l'histamine que chez les témoins, indiquant un niveau d'activité plus élevé.

Mot-clés:

C.elegans, activité musculaire, plasticité axonale, morphologie neuronale, morphologie axonale, croissance axonale, signalisation calcique, canaux calcique dépendent du voltage

Goal and strategies

"Form follows function". This maxim was stated by early 20 th century architect Louis Sullivan as "the pervading law of all things organic and inorganic". However, it is yet unknown whether this principle fully applies to the brain, as the relationship between the neuron's shape and its function is not clearly understood. There are examples of the activity of a neuron's post-synaptic target affecting the morphology of its axon, notable one being the motor neuron sprouting when its muscle target is inactivated. The study of these phenomenons is challenging in mammalian models due to the complexity of both the mechanisms and the model. Instead, the nematode Caenorhabditis elegans, with its simple nervous system and powerful genetic tools, is a suitable model to study how neurons and their post-synaptic target's activity are related.

Previous works from Zhao and Nonet have demonstrated the plasticity of SAB neurons in C. elegans [Zhao and Nonet, 2000]. These three motor neurons innervate the worm's head muscle. Evidence showed that a reduction of activity in muscle resulted in an overgrowth of SAB axons. During his PhD in our laboratory, Alexis Weinreb, has developed axonal fluorescent markers and a tool to manipulate muscle activity in order to study this relationship between SAB axonal morphology and muscular activity. He was able to reproduce the phenomenon of SAB axonal overgrowth, and to determined the critical period where the overgrowth happened.

With the gene candidate approach, we identified an essential role of voltage-gated calcium channels in provoking SAB axonal overgrowth when muscle activity is reduced. We demonstrated that these VGCCs function cell-autonomously in the neuron to induce overgrowth using tissue-specific single-copy expression of VGCC in a mutant background. This suggests involvement of neural activity in SAB axon plasticity, in addition to muscle activity. Our hypothetical model of the mechanism underlying SAB axon plasticity is that reduced muscle activity leads to hyperactivity in SAB neurons, which causes axonal

Caenorhabditis elegans as a model organism

General description

Caenorhabditis elegans is a species of non-parasitic roundworms that feeds on microbes, mainly bacteria. It has become an important model organism for many fields in biological research, such as neuroscience, aging and cell biology. In laboratory, the animal is grown on agar plates or liquid cultures with E. coli as food sources. C. elegans are small (about 1 mm in length at adulthood) and transparent, thanks to the clear cuticle layer surrounds the animal (Figure 1.1). This makes it easy to observe its internal organs and even its individual cells with appropriate microscope techniques.

The collagenous cuticle is secreted by the hypodermis, which is made up of one main body syncytium and several smaller syncytiums/mononucleate cell in the head and the tail. This epithelial system surrounds the pseudocoelomic cavity, which encloses the intestine and the two gonad arms. The body wall muscles of the worm are contained inside the hypodermis, separated by a thin basal lamina.

C. elegans feeds by pumping in the bacteria and grinding it with its pharynx. The ground food is transfer to the intestine, which is one long tube across the worm's length, connecting the pharynx and the anus. The excretion is then expelled to the outside by a rectal valve located between the guts and the rectum/anus. For reproduction, C. elegans are most commonly self-fertilizing hermaphrodite (XX sex chromosomes), which allows for homozygous worms to produce genetically identical progeny. Image from WormAtlas [START_REF] Altun | WormAtas Hermaphrodite Handbook -Introduction[END_REF] The worm has two bilaterally symmetric, U-shaped gonad arms connecting to a central uterus through the spermatheca. Oocytes form in the gonad arm and move towards the uterus as they mature. The oocytes are then fertilized by the sperm in spermatheca to become eggs. The eggs are stored in the uterus and laid outside through the vulva, which protrudes out of the ventral side in the middle of the worm. One adult hermaphrodite 1.1. C. elegans neuromuscular system worm alone can produce around 300 offsprings. The less common males (X0 sex chromosome) can spontaneously appear by non-disjunction of the X chromosomes in the hermaphrodite germ line (0.1%) [START_REF] Corsi | A Transparent Window into Biology: A Primer on Caenorhabditis elegans[END_REF]. Male mating facilitates transferring alleles between strains and isolating alleles from a particular strain.

Despite its relatively limited number of organs, C. elegans has a decently large number of behaviors, such as locomotion, feeding, defecation, foraging, egg laying, sensory responses to stimuli, male mating, learning and memory, etc... [de Bono, 2003, Liu and[START_REF] Liu | Sensory regulation of male mating behavior in caenorhabditis elegans[END_REF].

Genetics

The genome of C. elegans is quite compact. Aside from the sex chromosome X, C. elegans has another 5 pairs of autosomes, numbered from I to V in Roman numeral. C. elegans was the first multicellular eukaryotic genome sequenced to completion [C. elegans Sequencing Consortium, 1998], and even resequenced to fill the gaps [START_REF] Yoshimura | Recompleting the Caenorhabditis elegans genome[END_REF]. The genome sequence is 97Mb in total, with each chromosome pair roughly similar in size and in DNA content. C. elegans genome is surprisingly similar to that of humans, with 36% of the worm's protein-coding genes has a human match [START_REF] Shaye | OrthoList: A Compendium of C. elegans Genes with Human Orthologs[END_REF]. Furthermore, C. elegans is a promising model to study human disease in vivo, as it has been estimated that 42% of the human disease genes have an ortholog in the genome of C. elegans [START_REF] Baumeister | The worm in us -Caenorhabditis elegans as a model of human disease[END_REF][START_REF] Culetto | A role for Caenorhabditis elegans in understanding the function and interactions of human disease genes[END_REF][START_REF] Poulin | Genome-wide RNAi screens in Caenorhabditis elegans: impact on cancer research[END_REF].

Transgenesis and mutation engineering

One appeal of the C. elegans model is the powerful genetic tools that have been developed on it. Generating random mutations is relatively simple by exposing the worms to mutagens such as ethyl methane sulfonate (EMS) and ionizing radiation. Mutants can then be screened for a phenotype of interest (or the lack thereof), a much more laborious step. Mutations then can be isolated by outcrossing and identified by sequencing, giving insights into the function of the mutated gene.

It is also possible to generate genetically modified animals by injecting DNA directly into the worm's gonad. The resulting transgene is an extrachromosomal array, which consists of multiple copies of the injected DNA sequences [START_REF] Mello | Efficient gene transfer in C.elegans: extrachromosomal maintenance and integration of transforming sequences[END_REF]]. These arrays have very complex structure. The injected worms suffer from genetic mosaicism: not all descendants will inherit the transgene and not all cells of a transgenic worms will carry the transgene. It is common practice then to inject a marker (e.g. resistance to an antibiotic, fluorescence marker) along with the desired DNA to effectively screen the transgenic animals. It is also possible to integrate the multicopy extrachromosomal array Chapter 1. Introduction into the animal genome, by breaking it with UV or radiation then relying on the worm's DNA reparation.

A method to create integrated single-copy transgene was developed in C. elegans, called the miniMos [Frøkjaer-Jensen et al., 2014], named after the Mos1 transposon in Drosophila mauritania. In principle, the injection mix contains: a plasmid containing the DNA sequence to be inserted, flanked between the first and last 300 base pairs of the Mos1 transposon, another plasmid containing the transposase, and co-injection markers. The transposase can cut out the artificial transposon (desired transgene in between two ends of Mos1 transposon) from the resulting extrachromosomal array and paste the insert into the animal's genome. This method provides a way to obtain heritable transgenic strains with a constant level of expression.

To make changes in the C. elegans genome itself, the CRISPR/Cas9 system has been adapted for usage in the model, generating knock-in and knock-out strains [Chen et al., 2013a[START_REF] Dickinson | Engineering the Caenorhabditis elegans genome using Cas9-triggered homologous recombination[END_REF]. Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins were discovered to be a defense system for bacteria and archaea against invading viral DNA and plasmids [START_REF] Horvath | CRISPR/Cas, the Immune System of Bacteria and Archaea[END_REF]: specific DNA sequences (called spacer) of the invader are recognized with CRISPR RNAs and the Cas9 protein can cleave the DNA at a specific location. In the C. elegans model, single-guide RNA (sgRNA) can be engineered to direct the Cas9 protein to a spacer at a specific locus in the genome, creating double-strand breaks in that gene. The DNA repair mechanisms of the worm can either repair these breaks by nonhomologous end joining (NHEJ), generating small deletions and insertions in the region, resulting in gene knockout or, when provided a transgene template flanked by homologous sequences, the genome can incorporate the transgene by homologous recombination and resulting in knock-in of the transgene.

More recently, a method has been developed in order to obtain an integrated multiple copies transgene, named Modular safe-harbor Transgene Insertion (MosTI) [El Mouridi et al., 2022]. Making use of CRISPR/Cas9, this method requires the mutant strain unc-119(ed3) (which is almost completely paralyzed) and the injection of the following elements: a plasmid containing the desired DNA sequence, the Cas9 protein or a plasmid expressing the Cas9 protein (either constitutively or under control of a heat-shock promoter), a sgRNA that recognizes a spacer in the unc-119 locus, a plasmid containing unc-119 array fragment as well as the aforementioned spacer. After injection, the worms have an extrachromosomal array containing a great number of copies of the desired transgene, as well as multiple spacers. Cas9 causes double strands break in both the unc-119 locus, and the extrachromosomal array into multiple fragments (each containing multiple 1.1. C. elegans neuromuscular system copies of the desired transgene). Then, the worm's genome will repair itself by NHEJ using the array fragment of unc-119, repairing the unc-119 mutation and integrating multiple copies of the desired DNA in the process [START_REF] Yoshina | Locus-specific inte-BIBLIOGRAPHY gration of extrachromosomal transgenes in C. elegans with the CRISPR/Cas9 system[END_REF]. Worms having incorporated the multiple copies of transgene are easily recognizable and selected since they are able to move normally thanks to the repaired unc-119 locus. Array integration with MosTI is decently fast (2-3 weeks) and it is useful for situations where the transgene expression in single-copy insertion is not strong enough.

Development

The life cycle of C. elegans is relatively short: around 3 days in optimal conditions [START_REF] Byerly | The life cycle of the nematode Caenorhabditis elegans[END_REF]. The worms go through an embryonic stage, then four larval stages (L1-L4) and adulthood (Figure 1.2). [START_REF] Altun | WormAtas Hermaphrodite Handbook -Introduction[END_REF].

The speed of development of C. elegans doubled as the surrounding temperature increase from 15°C to 25°C [START_REF] Corsi | A Transparent Window into Biology: A Primer on Caenorhabditis elegans[END_REF]. Temperature over 25°C is hostile to the worm, Chapter 1. Introduction as the heat shock can kill the worm or render it sterile. It is common in laboratory condition to keep the worms at 15°C for maintaining strains or leave worms at 25°C to obtain descendants quickly.

At 20°C, adult hermaphrodite produces eggs which develop in utero for 2.5 hours after fertilization and then ex utero for another 14 hours. After this embryonic stage, the egg hatches into a larva at L1 stage, which lasts for about 16 hours. Each larval stage finishes with a molt, synthesizing a new cuticle and shedding the old one. During the molt, the animal becomes lethargic for 2 hours, stopping all movements and feeding. The other larval stages (L2, L3 and L4) last around 12 hours each. After L4, the larva becomes a young adult worm. It takes approximately 12 hours more for the worm to be capable of laying eggs and start propagating a new generation. Adult worms will continue to live for about another 2-3 weeks.

When there is no food left and/or the animals are too crowded, L2 larvae develop into an alternative L3 stage called "dauer"-meaning "lasting" in German [START_REF] Golden | The Caenorhabditis elegans dauer larva: Developmental effects of pheromone, food, and temperature[END_REF]. Dauer larvae develop special cuticle which is more resistant to environmental stresses and chemical agent. They can survive for months without eating, as the dauer cuticle completely covers the larva, including the mouth. When dauer larvae are transferred onto plates with bacteria, they molt and continue their development into L4 larvae.

The nervous system

Morphology of a neuron

The nervous system is a complex part of an animal that processes sensory information to make decisions by transmitting signals to and from different parts of the body. The cellular unit of the nervous system is the neuron, a highly specialized, electrically excitable cell. The structure of a typical neuron consists of the cell body (the "soma"), dendrites and an axon (Figure 1.3). Dendrites receive information, be it sensory input from the environment or inputs from other neurons. The soma processes the information and it is also where most of the protein synthesis happens. The axon transmits the information to different neurons or an effector, i.e. muscles.

Neurons can be classified according to their function:

• Sensory neurons: they receive environmental stimuli and send information to other neurons

• Motor neurons: they receive innervation from other neurons and innervate the muscle.

1.1. C. elegans neuromuscular system [START_REF] Blausen | Medical gallery of Blausen Medical[END_REF] • Interneurons: they receive innervation from other neurons and send information to other neurons. Further distinctions can be made in this class:

-Modulatory neurons that regulate other neurons via metabotropic action.

-Associative neurons that link sensory and motor neuron.

The structure that connects a neuron with its target is called a synapse. Synapses can form between the nerve terminal of the axon and the innervation targets, or synapses can form all along the axon. The latter is called synapses en passant, meaning they are formed in passing as the axon projection makes contact with the target. In vertebrates, synapses are primarily at the nerve terminal. For C. elegans, the majority of synapses is en passant.

Anatomy

For vertebrates, the nervous system can be separated into two parts, the central nervous system (CNS) and the peripheral nervous system (PNS). The CNS is the brain and the spinal cord, where information are centralized into a collective of numerous neurons.

The PNS is the nerve cells connecting to other non-neuronal cells, in order to transmit information from environmental stimuli and act out commands (from the brain or from automatic signals).

Chapter 1. Introduction

In C. elegans, the structure of the nervous system has been described in detail with reconstructions of series of images taken with electronic microscope [White et al., 1986]. The worm has in total 302 neurons, grouped into 118 classes by their morphologies and connectivities (Figure 1.4A). There is no clearly defined CNS in C. elegans, the quasiequivalence of a CNS is the nerve ring (Figure 1.4B), a large group of neurons located close to each other in the pharynx of the worm. The connectome, a map of how each neuron wires with each other, of the C. elegans hermaphrodite has been established the first time in totality in the 1980s [White et al., 1986]. An important grouping of neurons in the tail ganglia of male C. elegans, which governs mating behaviors, has later been described [START_REF] Jarrell | The Connectome of a Decision-Making Neural Network[END_REF]. Then, the connectome of the whole worm for both hermaphrodite and male was established [Cook et al., 2019]. The pharyngeal nervous system which is mostly independent from any other somatic nervous systems was re-evaluated by [START_REF] Cook | The connectome of the <span style="font-variant:small-caps;"> Caenorhabditis elegans </span> pharynx[END_REF]. The nerve ring as well as the ventral ganglion has also been characterized more in detail [Witvliet et al., 2021]. In the ventral line of the worm, multiple motor neurons cell bodies are located. They project their axons along both the ventral line and the dorsal line, forming nerve cords (Figure 1.4B). These nerve cords form synapse with the muscles, enabling movements. These motor neurons are separated into 8 classes: VA, VB, VD, VC innervating the ventral muscles, and DA, DB, DD, AS innervating the dorsal muscles [White et al., 1986]. Type A motor neurons like VA and DA send their axons anteriorly and are responsible 1.1. C. elegans neuromuscular system for the worm's backward movements, whereas type B motor neurons (VB and DB) send their axons posteriorly and enable forward movements [START_REF] Zhen | C. elegans locomotion: small circuits, complex functions[END_REF]. Type D motor neurons (VD and DD) are inhibitory neurons. VD neurons innervate ventral muscles but they receive innervation from DA and DB. When DA (or DB) send excitatory signal to activate dorsal muscle, it also activates VD which will inhibit ventral muscle. It is also true for the other side, DD is innervated by VA and VB. When VA/VB activate ventral muscle, DD is also activated to inhibit dorsal muscle.

Excitability

As with other cells, neurons have a negative membrane potential. The membrane potential depends on the intrinsic electrical properties of the motor neuron, relying on the quantity and the nature of leak channels present at the plasma membrane, and the inputs received from the upstream neurons. If the membrane potential decreases, then the neurons are even more negatively charged, or hyperpolarized. If the membrane potential increases, then the neurons are less negatively charged, or depolarized. In vertebrates, depolarization activates voltage-gated sodium and potassium channels and is then propagated as action potentials along the plasma membrane. In C. elegans motor neurons, depolarization propagation involves only passive electrical properties and no action potential has been recorded in motor neurons [START_REF] Goodman | Active Currents Regulate Sensitivity and Dynamic Range in C. elegans Neurons[END_REF][START_REF] Liu | Graded synaptic transmission at the Caenorhabditis elegans neuromuscular junction[END_REF].

As the depolarization reaches the nerve terminal, it triggers the opening of calcium channel and calcium ions rush in, prompt the vesicles containing neurotransmitters at the nerve terminal to release its content into synaptic cleft. This conversion from an electrical signal (depolarization) to a chemical signal (neurotransmitters release) happens in a specialized compartment called the presynaptic active zone. Neurotransmitters can have a direct effect on ion channels in neuron (ionotropic signaling) or it can trigger downstream signaling mechanisms that indirectly affect ion channels (metabotropic signaling).

Depolarization can also propagate between pre-and postsynaptic elements directly without the help of neurotransmitters. This requires a special structure between cells called the gap junction. This structure consists of channels connecting the cytoplasm of the two cells, allowing the ions to directly flow from one to the other and pass on the action potential or depolarization. Gap junctions are present between motor neurons and interneurons, between muscle cells but not between motor neuron and muscle cells [START_REF] Wen | Proprioceptive Coupling within Motor Neurons Drives C. elegans Forward Locomotion[END_REF].

The activity level of neurons can be experimentally manipulated by the use of exogenous ion channels. For example, the channelrhodopsin-2 (ChR2) is a light-gated cation channel first identified in the green algae Chlamydomonas reinhardtii [START_REF] Nagel | Channelrhodopsin-2, a directly light-gated cationselective membrane channel[END_REF]. This Chapter 1. Introduction channel opens when exposed to light, letting in various cations e.g. Na + , Ca 2+ and depolarizing the cells. ChR2 can be expressed in neurons of C. elegans to enable neural activity by stimulating the neurons with light [START_REF] Leifer | BIBLIOGRAPHY Optogenetic manipulation of neural activity in freely moving Caenorhabditis elegans[END_REF][START_REF] Stirman | Real-time multimodal optical control of neurons and muscles in freely behaving Caenorhabditis elegans[END_REF].

It is noted that ChR2 requires a chromophore i.e. all-trans retinal (ATR), which is not naturally present in C. elegans. In order to make use of this optogenetic approach, worms must be raised in presence of ATR, normally added to their food [START_REF] Nagel | Light Activation of Channelrhodopsin-2 in Excitable Cells of Caenorhabditis elegans Triggers Rapid Behavioral Responses[END_REF].

Another important genetic tool to manipulate activity in excitable cells of C. elegans is the histamine-gated channels. Histamine is a neurotransmitter in the nervous system of vertebrates and insects, but not C. elegans [START_REF] Chase | Efficient genome editing in Caenorhabditis elegans by CRISPR-targeted homologous recombination[END_REF]. Two ionotropic histamine receptors were identified in Drosophila melanogaster, histamine-gated chloride channel (HisCl), HisCl1 and HisCl2 [START_REF] Gengs | The Target of Drosophila Photoreceptor Synaptic Transmission Is a Histamine-gated Chloride Channel Encoded byort (hclA)[END_REF][START_REF] Gisselmann | Two cDNAs coding for histamine-gated ion channels in D. melanogaster[END_REF][START_REF] Zheng | Identification of Two Novel Drosophila melanogaster BIBLIOGRAPHY Histamine-gated Chloride Channel Subunits Expressed in the Eye[END_REF]. These channels are permeable to chloride ions in presence of histamine, thus hyperpolarizing and silencing the activity in the cells. Expression of HisCl1 channel in C. elegans neurons inhibits their activity in response to exogenously applied histamine [Pokala et al., 2014]. This is a robust approach to silence cell's activity in C. elegans, as the response to histamine is dose-dependent and reversible, and adding histamine to the food has no apparent effects on the worm's behaviors. A genetically modified version of HisCl1 that is permeable to cations was constructed, called HisCationic [Alkema, not yet published]. HisCat channel provides a way of using histamine to induce cell's activity, in addition to existing tools i.e. HisCl1 channel that silences activity.

Neurotransmission

The common neurotransmitters are listed in Table 1.1. The main excitatory neurotransmitter in vertebrates is Glutamate (Glu). It is also a neurotransmitter in C. elegans for sensory neurons and interneurons, but not motor neurons [START_REF] Brockie | Ionotropic Glutamate Receptors in Caenorhabditis elegans[END_REF]. Glutamate can have an ionotropic action on sodium, potassium and calcium channel, as well as a metabotropic action.

For vertebrates, acetylcholine (ACh) is the excitatory neurotransmitter in the neuromuscular junction (NMJ), the synapse between muscle and motor neurons. Same for C. elegans, except for a pharyngeal motor neuron, RIM [START_REF] Pereira | A cellular and regulatory map of the cholinergic nervous system of C. elegans[END_REF]. Otherwise, acetylcholine is also excitatory in some neuronal synapses. The acetylcholine receptor (AChR) are pentameric ion channel, meaning they are formed by five subunits, either of the same type (homomeric) or of five different types (heteromeric). The binding of acetylcholine on its receptor opens the channel, causing depolarizing influx of cations that results in an excitatory signal.

γ-Aminobutyric acid or GABA is the main inhibitory neurotransmitter in the vertebrate

Neurotransmitter Effect Chemical formula

Glutamate Excitatory

Acetylcholine Excitatory GABA Inhibitory Glycine Inhibitory nervous system. It is also the only inhibitory neurotransmitter that has been identified in C. elegans. GABA reduces the excitability of postsynaptic elements by opening ion channels that let in anion e.g. chloride ion or let out cation e.g. potassium ion, hyperpolarizing its synaptic target. Glycine is also another important inhibitory neurotransmitter in vertebrate, but no role has been identified in C. elegans neurotransmission. Another important type of neurotransmitter is neuropeptide. They are made up of small chains of amino acid (aa) that are mainly synthesized and released by neurons. The C. elegans genome has over 100 genes coding for more than 250 different peptides. There are three main types of neuropeptides in the nematode [START_REF] Li | Neuropeptides. WormBook[END_REF]:

• The insulin-like peptides. They are structurally similar to insulin or insulin-like growth factors in human. They mostly act on the insulin-like receptor tyrosine kinase DAF-2. There are 40 genes coding for the insulin-like peptides: daf-28 and the other 39 are prefixed by ins-and numbered from 1 to 39. daf-28 promotes reproductive growth and overexpression of some insulin-like peptides causes larval arrest.

• The FMRFamide (Phe-Met-Arg-Phe-NH2)-related peptides. These peptides all have in common the C-terminal sequence X 1 X 2 R(Arg)-F(Phe) where X 1 is an aromatic amino acid (Phe, Trp, Tyr) and X 2 is an amino acid with a hydrophobic side chain (Gly, Ala, Val, Leu, Ile, Pro, Phe, Met and Trp) [START_REF] Peymen | The FMRFamide-Like Peptide Family in Nematodes[END_REF]. There • The neuropeptide-like protein that does not belong to any of the previous group. There are 42 genes coding for these peptides, prefixed by nlp-and numbered from 1 to 42.

Neuropeptides are synthesized in form of longer chain peptide precursors called prepropeptides which are inactive. One prepropeptide can contain multiple copies of the same neuropeptide or multiple different neuropeptides or both. To get to an active neuropeptide, the prepropeptide goes through several cleavage and maturation processes (Figure 1.5). One important step is the cleavage of the long propeptide chain by a proprotein convertase into individual peptides. In C. elegans, there are 4 genes coding for proprotein convertase: egl-3, aex-5, bli-4 and kpc-1. EGL-3, ortholog of the proprotein convertase 2 (PC2) in human, seems to be responsible for processing the majority of neuropeptides [Husson et al., 2006]. egl-3 mutant worms have many defected phenotypes, notably slow locomotion and eggs retention. The proprotein convertases generally cleave the propeptides at sites with basic residues, either monobasic (lysine-K and arginine-R) or dibasic (KR, RR, RK and KK). The resulting peptides are only active after the cleavage of these basic amino acids by a carboxypeptidase. Three genes in the C. elegans genome encode carboxypeptidases, egl-21, cpd-1 and cpd-2. EGL-21 is the ortholog of the carboxypeptidase E, and it is required for the maturing process of around 80% of flp and nlp neuropeptide [Husson et al., 2007].

The last step of the neuropeptide processing pathway is the modification at the N or Cterminus, notable example being the amidation of the C-terminus glycine residue. These modifications protect the neuropeptides from degradation and even confer biological activity to the peptides [START_REF] Li | Neuropeptides. WormBook[END_REF].

SAB neurons

The object of this study revolves around the SAB neurons and its morphologies. The SAB is a class of 3 motor neurons in the head (Figure 1.4B and 1.6): SABVL (SAB Ventral Left), SABVR (SAB Ventral Right) and SABD (SAB Dorsal). The cell bodies of the SAB are located in the retrovesicular ganglion, close to the terminal bulb of the pharynx, on the ventral side. They project their axons anteriorly towards the tips of the nose, running along the muscle and forming en passant synapses. The SABVL neuron send its axon ventrally to the left, and the SABVR neuron send its axon ventrally to the right. The SABD neuron send its axon dorsally and to the right. Upon reaching the dorsal side, the SABD axon splits into two, one branch continues anteriorly on the right, the other crosses over to the left side and then continues anteriorly.

SABs are cholinergic motor neurons [Rivard et al., 2010]. As a group, the SAB motor neurons receive synaptic input from multiple interneurons (AVA, AVD, AVE, AVL, AVJ, DVA). The AVA and AVD are two bilaterally symmetric interneuron pairs with large diameter axons running along the entire length of the ventral nerve cord, providing input to the ventral cord motor neurons. The two AVE interneurons have similar synaptic target as AVD but its pair of axons only extend to about half of the ventral cord, from the nerve ring to the vulva. Together, AVA, AVD and AVE command backward locomotion in the worm. Together, AVA, AVD and AVE innervate SAB and other type A motor neurons in order to drive backward locomotion. The SAB ventrals receive inputs from dorsal motor neurons (DA) and the SABD receives inputs from ventral motor neurons (VA, VB).

SAB motor neurons have been visualized by using the promoter of the homeodomain protein UNC-4 coupled to a fluorophore [Zhao and Nonet, 2000]. Indeed, this protein has been shown to be expressed in type A motor neurons, including SAB [START_REF] Miller | Expression of the unc-4 homeoprotein in Caenorhabditis elegans motor neurons specifies presynaptic input[END_REF]. The identification of the neurons was done based on their location. More recently, Image from [START_REF] Reilly | Unique homeobox codes delineate all the neuron classes of C. elegans[END_REF]. D. Neural connections of SAB motor neurons. Image from [START_REF] Bhatla | WormWeb.org -C. elegans Interactive Neural Network -by nikhil bhatla[END_REF] the expression pattern of the unc-4 promoter has been re-characterized using the Neu-roPAL system [START_REF] Reilly | Unique homeobox codes delineate all the neuron classes of C. elegans[END_REF]. NeuroPAL stands for Neuronal Polychromatic Atlas of Landmarks [Yemini et al., 2021]. NeuroPAL transgene contains a combination of 41 selectively overlapping neuron-specific reporters, each displays a subset of four distinctly colored fluorophores, excluding GFP. Insertion of the NeuroPAL transgene in C. elegans genome results in a strain for which each neuron is being identifiable by its unique combi-1.1. C. elegans neuromuscular system nation of color and position. Crossing the NeuroPAL strain with a strain expressing GFP under unc-4 promoter has allowed to unambiguously identified all the neurons expressing unc-4. It has been shown that the unc-4 promoter drives expression in other neurons than SAB, namely I5, AVF, VA, DA, AS, VD.

The muscle system

Muscle makes movement possible in animals. In human, there are skeletal striated muscles that are controlled by the somatic nervous system, smooth muscles in hollow organs like the stomach, intestines and bladder that contracted involuntarily, and striated cardiac muscle of the heart that contract automatically. Smooth and cardiac muscles are under the control of autonomic nervous system.

Anatomy

A muscle cell is an electrically excitable cell. There are three different types of muscle cells: the long skeletal muscle with many nuclei (also known as muscle fiber) that allows for controlled movements, the cardiac muscle cell with a single nucleus joining together by intercalated discs to form the muscle of the heart, and the smooth muscle cell also with a single nucleus that control involuntary movements.

The striated muscles possess a repeating functional unit called sarcomeres. These sarcomeres show up as a series of parallel bands in microscopic image of striated muscle tissues, hence the name "striated". A sarcomere made of actin (the thin filament) and myosin (the thick filament) that can slide on each other in presence of calcium, which happens following calcium channel activation, such as ryanodine receptor (RyR) and voltage-gated calcium channels (VGCCs). A motor unit is made up of a motor neuron and all the muscle fibers that are innervated by that neuron, including the NMJs formed between the axon and the fibers. The larger a motor unit is, the more connections there are between the motor neurons and its postsynaptic muscle targets. A motor end plate of a muscle fiber is where the end point of a nerve terminal touches that fiber.

Striated muscle contraction is regulated by tropomyosin and the troponin complex, which consists of three proteins: troponin I (Inhibitory subunit binding to actin), troponin C (Calcium binding subunit), and troponin T (Tropomyosin binding subunit) [START_REF] Mukund | Skeletal muscle: A review of molecular structure and function, in health and disease[END_REF]. At low intracellular calcium level i.e. resting, tropomyosin/troponin cover the active sites on actin and prevents the actin-myosin interaction. When muscle cells are activated and the calcium level increases, troponin C binds to Ca 2+ , changing the conformation of tropomyosin/troponin and exposing the myosin binding sites on the Chapter 1. Introduction actin filaments. A multitude of troponin isoforms exists in mammals as well as in C.

elegans, which allows for a diversity of troponin expression and function between tissues [START_REF] Cao | Invertebrate troponin: Insights into the evolution and regulation of striated muscle contraction[END_REF][START_REF] Jin | Isoform Diversity, Regulation, and Functional Adaptation of Troponin and Calponin[END_REF][START_REF] Kagawa | Elegans Model for Studying Tropomyosin and Troponin Regulations of Muscle Contraction and Animal Behavior[END_REF].

Striated muscles made up the body wall of C. elegans (Figure 1.1 which allows it to move by curving its body. There are 95 body wall muscle cells organized into four quadrants, positioned around the worm's body. The two dorsal quadrants flank the dorsal nerve cord, and on the other side are the two ventral quadrants flanking the ventral cord. Each quadrant has two longitudinal rows of diamond-shaped mononuclear muscle cells which make contacts with its motor neurons by forming specialized membrane extensions called muscle arms [START_REF] Dixon | Muscle arm development in Caenorhabditis elegans[END_REF]. In addition to the 95 striated body wall muscle cells, the hermaphrodite worm also has smooth muscle cells: 20 in the pharynx, 4 in the anus, 8 vulva muscles, 8 in the uterus and the contractile sheath of the gonad. The SAB motor neurons are among the neurons that innervate the head muscle (Figure 1.8). The nose muscles (1 and 2) receive innervations from RMD, RME, SMB and SMD.

The muscle cells from 3 to 6 are innervated by the nerve ring, as well as the ventral nerve cord [START_REF] Gray | A circuit for navigation in Caenorhabditis elegans[END_REF]. The SAB neurons run along the head muscle and form en passant synapse directly with cells number 1, 3, 4 and 6.

The striated head muscles of C. elegans are slightly different from the rest of the body wall muscles. The first difference is in the type of movements. In liquid medium, the worm swims by bending their whole body back and forth. On solid medium, ventral and dorsal muscles of the worm coordinate their contraction to crawl in a two-dimensional sinusoidal wave pattern. But, the head muscles of the worm often move the nose circularly in three dimensions to explore their environment [START_REF] Croll | Behavioural Analysis of Nematode Movement[END_REF]. Furthermore, the worm's head moves in a random and irregular manner, while the body's movements are periodic and regular [START_REF] Ouellette | A Gate-and-Switch Model for Head Orientation Behaviors in Caenorhabditis elegans[END_REF].

Another difference between head and body wall muscles lies in their innervation. The body wall muscles are innervated mainly by ventral and dorsal nerve cords. Meanwhile, head muscles are innervated by neurons in the nerve rings as well as the nerve cords. Head muscles also seem to be regulated by a different mechanisms than body wall muscles, as expression of the tyramine-gated chloride channel LGC-55 specifically in the neck (muscle 3) induces a backward movement as an escape response [START_REF] Pirri | A Tyramine-Gated Chloride Channel Coordinates Distinct Motor Programs of a Caenorhabditis elegans Escape Response[END_REF].

In addition, the sarcomeric composition seems to differ between head and body muscles. Among the four genes coding for troponin I, the gene tni-3 is preferentially expressed in the head muscle [START_REF] Ruksana | Tissue expression of four troponin I genes and their molecular interactions with two troponin C isoforms in Caenorhabditis elegans[END_REF]. The promoter sequence of tni-3 drives strong expression in the head [Takashima et al., 2012]. Additionally, tropomyosin is encoded by the gene lev-11. One tropomyosin isoform, lev-11o, is predominantly expressed in the nose, while the other 4 isoforms are expressed in all muscle cells except the nose.

Excitability

Striated muscle cells of C. elegans have a relatively depolarized resting membrane potential, around -25 mV, as most invertebrates [START_REF] Gao | Action potentials drive body wall muscle contractions in Caenorhabditis elegans[END_REF], Jospin et al., 2002, Chapter 1. Introduction Liu et al., 2011]. This potential is set by the intrinsic properties of the muscle cells, but also by the innervation from motor neurons. In C. elegans, muscle cells received a double innervation through cholinergic and GABAergic motor neurons. Three types of ionotropic receptors are present at the NMJ of this animal: two acetylcholine receptors (AChR) and one GABA receptor [Richmond and Jorgensen, 1999]. Two types of acetylcholine receptors can be functionaly distinguished in regard to their agonist sensitivity: levamisole-sensitive acetylcholine receptor (L-AChR) and nicotine-sensitive acetylcholine receptor (N-AChR). L-AChR consists of 5 different subunits, coded by the following genes: unc-29, unc-38, unc-63, lev-1 and lev-8 [Lewis et al., 1980], of which unc-38, unc-63 and unc-29 are necessary for the formation of L-AChR in the muscle [Rayes et al., 2007, Richmond andJorgensen, 1999]. Levamisole is an agonist of L-AChR, while nicotine has no effect on L-AChR [START_REF] Boulin | Eight genes are required for functional reconstitution of the Caenorhabditis elegans levamisole-sensitive acetylcholine receptor[END_REF]. N-AChR is a homomeric channel, composed of only the ACR-16 subunit [START_REF] Francis | The Ror Receptor Tyrosine Kinase CAM-1 Is Required for ACR-16-Mediated Synaptic Transmission at the C. elegans Neuromuscular Junction[END_REF][START_REF] Rand | Genetic analysis of the cha-1-unc-17 gene complex in Caenorhabditis[END_REF]. Nicotine activates N-AChR but levamisole partially blocks N-AChR. GABA receptor in C. elegans is encoded by the gene unc-49 which has three isoforms (UNC-49A, UNC-49B and UNC-49C), each constitutes a subunit of the receptor.

As with motor neurons, muscle excitability can be experimentally manipulated by the use of exogenous channels gated by light or exogenous neurotransmitter.

Axonal growth and guidance

Axons grow to great lengths in search of the neuron's synaptic target, some axons extend to be as long as 1 m and more [START_REF] Debanne | Axon Physiology[END_REF]. Developing axon have an actinsupported extension at the growing tip called the growth cone. This structure is motile and highly dynamic, containing two main regions, peripheral (P-) and central (C-) regions.

Each region has a unique actin cytoskeleton structure (Figure 1.9b). The P-region is often seen as the form of a webbed hand with its fingerlike filopodia constantly extending and retracting to probe its surrounding environment and the lamellipodia-the webbing in the middle-consisting of a network of short and branched actin filaments. The C-region is behind the P-region, connected to the axonal shaft. Aside from the cellular organelles, this region also contains a dense microtubule array to support the growth cone movement.

A transitional zone (T-zone) exists between the two main regions, regulating both actin and microtubules [START_REF] Omotade | Actin-based growth cone motility and guidance[END_REF].

In order for axons to reach the synaptic target, the growth cone interacts with the local extracellular environment, resulting in directed outgrowth in response to certain features of the environment such as the guidance cues. The growth cone moves forward on a road 1.2. Axonal growth and guidance Image adapted from [START_REF] Omotade | Actin-based growth cone motility and guidance[END_REF].

made up of adhesive molecules on a neighboring cell surface such as transmembrane cell adhesion molecules (CAMs), or assembled into a dense extracellular matrix [START_REF] Lowery | The trip of the tip: understanding the growth cone machinery[END_REF]. In addition, various proteins are involved in different intracellular and extracellular processes that control axon navigation (Figure 1.10, [START_REF] Chisholm | The Genetics of Axon Guidance and Axon Regeneration in Caenorhabditis elegans[END_REF]).

The process of directed axonal outgrowth requires continuous remodeling of the actin cytoskeleton, which occurs through cyclical polymerization and depolarization of the actin filaments. There are over 100 of accessory proteins known to regulate the process: controlling the pool of actin monomers, polymerization, cross-linking, length of actin filaments and the disassembly of filament networks [START_REF] Pollard | Actin, a Central Player in Cell Shape and Movement[END_REF]. The first step of actin remodeling is the trafficking of surface receptor and its signaling components to the cell surface. Then, effectors are recruited to the site of the receptor, facilitating the remodeling of the cytoskeleton. This includes altering the cell's structural scaffold and adhesive properties; assembling and disassembling of actin filaments. One molecule or one pathway can be involved in multiple of the aforementioned roles.

Important signaling pathways involved in the this process, with their C. elegans homologs, are the Ena/VASP proteins (UNC-34 in C. elegans) controlling directed outgrowth in responses to extracellular cues; the GTPases and their regulators, such as Rac (CED-10), RhoG (MIG-2), Trio (UNC-73) and Dock180 (CED-5) [START_REF] Chisholm | The Genetics of Axon Guidance and Axon Regeneration in Caenorhabditis elegans[END_REF], having multiple roles e.g. regulating cytoskeleton dynamics, trafficking. The notable example of a guidance cue was discovered in the C. elegans model [START_REF] Hedgecock | The unc-5, unc-6, and unc-40 genes guide circumferential migrations of pioneer axons and mesodermal cells on the epidermis in C. elegans[END_REF]: the secreted extracellular UNC-6 (netrin) molecule and its receptors, UNC-5 (UNC5) and UNC-40 (DCC), which are highly conserved in invertebrates and vertebrates [START_REF] Chisholm | Conservation and divergence of axon guidance mechanisms[END_REF]. UNC-6 was first found in the nematode, then netrins were later identified in vertebrates as a homolog to UNC-6, both are structurally similar to laminins, a family of extracellular matrix glycoprotein that is the main components of the basal lamina-one of the layers of the basement membrane [START_REF] Kennedy | Netrins are diffusible chemotropic factors for commissural axons in the embryonic spinal cord[END_REF], Serafini et al., 1994]. UNC-6/netrin is expressed along the ventral midline [START_REF] Asakura | UNC-6 expression by the vulval precursor cells of Caenorhabditis elegans is required for the complex axon guidance of the HSN neurons[END_REF][START_REF] Wadsworth | Neuroglia and Pioneer Neurons Express UNC-6 to Provide Global and Local Netrin Cues for Guiding Migrations in C. elegans[END_REF]. Deleted in Colorectal Cancer (DCC) and UNC5 are the two netrin receptors in vertebrates, homologous to UNC-40 and UNC-5, respectively.

It has been shown that UNC-6 controls both ventral migrations (towards UNC-6) and dorsal migrations (away from UNC-6), as unc-6 mutants have guidance defects in both directions [START_REF] Hedgecock | The unc-5, unc-6, and unc-40 genes guide circumferential migrations of pioneer axons and mesodermal cells on the epidermis in C. elegans[END_REF]. Initially, it was hypothesized that UNC-5 mediates dorsal growth away from UNC-6 [Leung-Hagesteijn et al., 1992] and UNC-40 mediates ventral growth towards UNC-6 [START_REF] Chan | UNC-40, a C. elegans Homolog of DCC (Deleted in Colorectal Cancer), Is Required in Motile Cells Responding to UNC-6 Netrin Cues[END_REF] (the attraction-repulsion model). Later studies have demonstrated that the roles of UNC-40 and UNC-5 in ventral and dorsal 1.2. Axonal growth and guidance growth are in truth more complex: UNC-40 can act in dorsal growth, likely as a heterodimer with UNC-5 [START_REF] Hong | A Ligand-Gated Association between Cytoplasmic Domains of UNC5 and DCC Family Receptors Converts Netrin-Induced Growth Cone Attraction to Repulsion[END_REF][START_REF] Macneil | UNC-129 regulates the balance between UNC-40 dependent and independent UNC-5 signaling pathways[END_REF][START_REF] Norris | UNC-6/netrin and its receptors UNC-5 and UNC-40/DCC modulate growth cone protrusion in vivo in C. elegans[END_REF][START_REF] Norris | The UNC-6/Netrin receptors UNC-40/DCC and UNC-5 inhibit growth cone filopodial protrusion via UNC-73/Trio, Rac-like GTPases and UNC-33/CRMP[END_REF]; UNC-5 might have an effect on the localization of UNC-40, driving UNC-40 more ventrally towards UNC-6 [Levy-Strumpf and Culotti, 2014[START_REF] Limerick | A Statistically-Oriented Asymmetric Localization (SOAL) Model for Neuronal Outgrowth Patterning by Caenorhabditis elegans UNC-5 (UNC5) and UNC-40 (DCC) Netrin Receptors[END_REF][START_REF] Yang | Extracellular Matrix Regulates UNC-6 (Netrin) Axon Guidance by Controlling the Direction of Intracellular UNC-40 (DCC) Outgrowth Activity[END_REF]. Another molecule that regulates the responses to UNC-6 is UNC-129, a transforming growth factor beta. UNC-129 can bind to UNC-5, blocking its activity in order to enhance UNC-5+UNC-40 signaling at low concentration of UNC-6 [MacNeil et al., 2009].

The mechanism behind Netrin's action in the growth cone is yet unclear. Multiple models of Netrin and its receptors have been explored, such as the Statistically-Oriented Asymmetric Localization model (SOAL) [START_REF] Limerick | A Statistically-Oriented Asymmetric Localization (SOAL) Model for Neuronal Outgrowth Patterning by Caenorhabditis elegans UNC-5 (UNC5) and UNC-40 (DCC) Netrin Receptors[END_REF], which relies on the following concepts:

1. The netrin receptors i.e. UNC-40 on the surface of the membrane change position.

It is known that partial distribution of receptors can influence the movement of neurons in response to extracellular ligands [START_REF] Nguyen | The influence of receptor positioning on chemotactic information[END_REF][START_REF] Nguyen | How receptor diffusion influences gradient sensing[END_REF]. Then it could be the case that the differences in surface localization of UNC-40 can orient the movement of the growth cone, presumably towards the direction where UNC-40 is clustered. There are evidence of UNC-40 undergoes dramatic changes in localization, for example during the development of HSN axons where in the beginning UNC-40 is asymmetrically localized to the ventral surface of the cell body, where it is closest to a ventral source of netrin. After 1 hour, UNC-40 is strongly and uniformly localized more to the posterior side. Then at the L3 stage, UNC-40 is localized much more anteriorly. This suggests a dynamic pattern of UNC-40 localization at the developing leading edge of HSN axons [START_REF] Kulkarni | Experimental evidence for UNC-6 (netrin) axon guidance by stochastic fluctuations of intracellular UNC-40 (DCC) outgrowth activity[END_REF].

2. The localization of UNC-40 and the outgrowth activity that it mediates are stochastically oriented. evidence shown that if there is no UNC-6, UNC-40 can asymmetrically localized to a random surface (ventral, dorsal, anterior or posterior) instead of just ventral when there is UNC-6 [START_REF] Xu | The Roles of Multiple UNC-40 (DCC) Receptor-Mediated Signals in Determining Neuronal Asymmetry Induced by the UNC-6 (Netrin) Ligand[END_REF]. It was also discovered that the activity of UNC-40 is to cause another UNC-40 receptor to localize, either to the site of UNC-6 interaction, or to a different site [START_REF] Limerick | A Statistically-Oriented Asymmetric Localization (SOAL) Model for Neuronal Outgrowth Patterning by Caenorhabditis elegans UNC-5 (UNC5) and UNC-40 (DCC) Netrin Receptors[END_REF]. Thus, UNC-40 activity is both a positive feedback loop that recruits more UNC-40, or a negative loop that pushes UNC-40 away from the site. One type of loop informs the other, changing over time as the axon develops. Extracellular cues (either molecules present at the surfaces of surrounding cells and extracellular matrix, or cues interacting physically with receptors in the growth cone) other than UNC-6 can also affects UNC-40 asymmetric localization, in turn influencing the probability Chapter 1. Introduction of UNC-40 distribution and outgrowth. The direction that the axon will develop towards is a probability calculated from the combination of all the probabilities of what UNC-40 feedback loop will happens and where it will happens.

3. Neuronal membrane outgrowth is a mass transport phenomenon, which is driven by the receptors. The interaction of UNC-40 with netrin leads to cytoskeletal changes, creating force and membrane movements. This force that pushes outgrowth in the direction of UNC-40 + UNC-6 is called advection. Membrane mass transport can also occurs through random movements and it is called diffusion. The direction of outgrowth is a stochastic process, a product from both advection and diffusion movement which evolve over time.

This model makes several predictions on how axon extension is guided, that are different from the classic attraction/repulsion model. First, forward extension of the axon towards a cue that promotes outgrowth i.e. UNC-6 can be inhibited over time. The leading edge of an outgrowth has a strong directional bias at first towards UNC-6, then as the outgrowth reaches the UNC-6 sources, more UNC-6 molecules surround the growth cones interacting with the UNC-40 at the flank, increasing the probabilities of outgrowth towards the sides and decreasing the probability of forward movement. Second, extension could move towards the source of a cue that inhibits outgrowth, if only for a brief time. If the leading edge has a strong directional bias at first towards that direction by the combination of all the extracellular cues including the inhibitory cue, it continues to move towards that direction, interacting with more and more inhibitory cue until the inhibitory effect becomes too much for the initial bias. In general, movement towards a cue has an end result where the probabilities of outgrowth in different directions are equal (Figure 1.11). Third, multiple extensions from the same neuron could move in the same direction without following a prepatterned extracellular pathways, just by autonomously forming the same asymmetric localization of UNC-40. Fourth, concentration gradient of a guidance cue is not necessary to direct movement. A directional bias can be maintained along a track of uniformly distributed cue, as the probabilities of movement in each direction would not change and growth cone can still initiate movement. Last, extracellular cues cannot affect the direction of outgrowth directly. Cues can only influence UNC-40's localization and that might affect the direction. This model was able to explain the phenotype of unc-5 mutants, and determine that UNC-5 activity increases the positive feedback loop of UNC-40 + UNC-6. Without UNC-40, UNC-5 interacts with UNC-6 to inhibit outgrowth.

Another model that complements the SOAL model is polarity/protrusion . It is known that UNC-5 is required for an asymmetric accumulation of F-actin in the growth cone of VD neurons [START_REF] Norris | UNC-6/netrin and its receptors UNC-5 and UNC-40/DCC modulate growth cone protrusion in vivo in C. elegans[END_REF]. UNC-5 inhibits protrusion, since unc-5

1.2. Axonal growth and guidance as it develops toward a ventral source of a cue (blue). (B) SOAL process illustrated for the three positions shown in (A). Shown are scenarios for movement toward the blue cue [A] that promotes outgrowth (blue +) or that inhibits outgrowth (blue -). The cues act to increase (promote) and decrease (inhibit) the probability of outgrowth from a surface. At positions 1 and 2, cues along the anterior-posterior axis (orange -and red -) inhibit outgrowth in the anterior or posterior directions. At position 3, the cue from the ventral source predominates. Image adapted from [START_REF] Limerick | A Statistically-Oriented Asymmetric Localization (SOAL) Model for Neuronal Outgrowth Patterning by Caenorhabditis elegans UNC-5 (UNC5) and UNC-40 (DCC) Netrin Receptors[END_REF].

mutants have increased VD growth cone area and longer filopodial protrusions. It is suggested that UNC-6/netrin functions with UNC-5 to polarize the growth cone, creating an asymmetric accumulation of F-actin on the dorsal side, then limiting ventral and lateral protrusion of the growth cone by restricting the entry of microtubule [START_REF] Gujar | Control of Growth Cone Polarity, Microtubule Accumulation, and Protrusion by UNC-6/Netrin and Its Receptors in Caenorhabditis elegans[END_REF]. UNC-6 also acts with UNC-40 to stimulate the dorsal protrusion along the polarity of actin accumulation. This model supports UNC-40 interaction with UNC-6 as the main force of outgrowth, and that UNC-5 functions to polarize and focus the site of UNC-40-mediated protrusion.

Chapter 1. Introduction

Another molecule that affects dorso-ventral navigation of axons is the secreted protein SLT-1 (Slit), first discovered in Drosophila [START_REF] Seeger | Mutations affecting growth cone guidance in drosophila: Genes necessary for guidance toward or away from the midline[END_REF]. Slit is expressed primarily on the dorsal side and directs navigation towards the ventral midline [START_REF] Hao | C. elegans Slit Acts in Midline, Dorsal-Ventral, and Anterior-Posterior Guidance via the SAX-3/Robo Receptor[END_REF], playing the role of a chemorepellent guidance cue. This is accomplished by Slit interacting with the Roundabout (Robo) receptor expressed in the developing axons [START_REF] Brose | Slit Proteins Bind Robo Receptors and Have an Evolutionarily Conserved Role in Repulsive Axon Guidance[END_REF]. The homolog of Slit in C. elegans is SLT-1 which has two receptors, the major one is the Robo homolog, SAX-3. SAX-3 also has a function independent from SLT-1 by binding to the other SLT-1 coreceptor EVA-1 [START_REF] Fujisawa | The Slit Receptor EVA-1 Coactivates a SAX-3/Robo-Mediated Guidance Signal in C. elegans[END_REF]. Without SLT-1 or EVA-1, SAX-3 inhibits UNC-6 signaling to enhance ventral navigation defects [START_REF] Fujisawa | The Slit Receptor EVA-1 Coactivates a SAX-3/Robo-Mediated Guidance Signal in C. elegans[END_REF].

Semaphorins play a role in axonal guidance for insects and vertebrates. The C. elegans genome has one secreted semaphorin (mab-20 ) and two transmembrane semaphorins (smp-1 and smp-2 ). mab-20 affects the navigation of SMD and SDQ neurons [START_REF] Wang | The C. elegans L1CAM homologue LAD-2 functions as a coreceptor in MAB-20/Sema2-mediated axon guidance[END_REF], but otherwise there is no major role of semaphorins as a guidance cue in C. elegans.

The Wnt family was first discovered in Drosophila, containing 19 secreted glycoproteins [START_REF] Xu | Hippocampal Wnt3a is Necessary and Sufficient for Contextual Fear Memory Acquisition and Consolidation[END_REF] that functions in various vital cellular processes, such as metabolisms [START_REF] Karner | Wnt signaling and cellular metabolism in osteoblasts[END_REF], cell polarity [START_REF] Yang | Wnt-Frizzled/Planar Cell Polarity Signaling: Cellular Orientation by Facing the Wind (Wnt)[END_REF], synaptic plasticity [START_REF] Narvaes | Role of Wnt signaling in synaptic plasticity and memory[END_REF]. There are three known Wnt pathways: the canonical Wnt/βcatenin pathway (canonical since it is the first pathway to be discovered), and the two non-canonical pathway Wnt/Ca 2+ and Wnt/JNK/planar cell polarity(PCP). The Wnt pathway has an important role in neural cell migration along the anterior-posterior axis, but they do not seem to be a major guidance cue in C. elegans. Wnt mutants disrupt the initiation and the termination of outgrowth in few neurons, and even fewer neurons have axon navigation defects [START_REF] Chisholm | The Genetics of Axon Guidance and Axon Regeneration in Caenorhabditis elegans[END_REF].

The PHR system

A single highly conserved family of proteins called PAM-Highwire-RPM-1 (PHR) proteins function in all the key steps of axonal development: outgrowth, guidance, synapse formation and terminating growth [START_REF] Grill | The PHR proteins: intracellular signaling hubs in neuronal development and axon degeneration[END_REF]. The PHR stands for the human Protein Associated with Myc (PAM), Drosophila Highwire and C. elegans Regulator of Presynaptic Morphology 1 (RPM-1). PHR are large proteins, from C. elegans' RPM-1 (3766 amino acids) to Drosophila's Highwire (5233 amino acids), containing several protein interaction domains [START_REF] Baker | Defining Minimal Binding Regions in Regulator of Presynaptic Morphology 1 (RPM-1) Using Caenorhabditis elegans Neurons Reveals Differential Signaling Complexes[END_REF][START_REF] Grill | The PHR proteins: intracellular signaling hubs in neuronal development and axon degeneration[END_REF][START_REF] Sampathkumar | Structures of PHR Domains from Mus musculus Phr1 (Mycbp2) Explain the Loss-of-Function Mutation (Gly1092→Glu) of the C. elegans Ortholog RPM-1[END_REF]. One domain in particular is a RING-H2 domain of E3 ubiquitin ligase which can target a protein, attach a chain of ubiquitin on a lysine residue to mark the protein for degration by the proteasome.

Axonal growth and guidance

In Drosophila, Highwire mutants have a remarkable increase in motor axon branch length [START_REF] Wan | Highwire Regulates Synaptic Growth in Drosophila[END_REF] and overgrown sensory axon terminal [START_REF] Kim | Dscam Expression Levels Determine Presynaptic Arbor Sizes in Drosophila Sensory Neurons[END_REF]. In C. elegans, rpm-1 mutant worms have severe defects in axon termination in many different types of neurons, meaning the growth cone does not stop at the stereotypical termination point but continue to overgrowth. These neurons include:

• mechanosensory neurons (ALM and PLM) [Grill et al., 2007, Schaefer et al., 2000] • GABAergic motor neurons (DD and VD) [START_REF] Opperman | RPM-1 is localized to distinct subcellular compartments and regulates axon length in GABAergic motor neurons[END_REF] • pharyngeal neurons (M1) [START_REF] Refai | Extension of the Caenorhabditis elegans Pharyngeal M1 Neuron Axon Is Regulated by Multiple Mechanisms[END_REF] RPM-1 was initially found to be a part of an Skp-1 -Cullin -F-box (SCF) complex (stands for Skp-1 -Cullin -F-box) with the F-box protein FSN-1/Fbxo-45 [Liao et al., 2004]. The FSN-1 complex (containing SKP, CUL and FSN-1) binds to RPM-1 on the conserved FSN-1 binding domain (FBD1), forming the SCF complex. This complex functions as an E3 ubiquitin ligase. FSN-1 acts as the target recognition module, and RPM-1 connects FSN-1 with the E2 ubiquitin-conjugating enzyme via the RING finger domain. In ASI sensory neuron, FSN-1 acts independently of RPM-1 to regulate the secretion of the insulin-like peptides INS-4 and INS-6 (Figure 1.12). These peptides act on muscle DAF-2 insulin receptors to activate the canonical insulin age-1 /PI3Kpdk-1 /PDPK1daf-16 /FOXO pathway, which could then regulate the synaptic growth of GABAergic neurons through an unidentified retrograde signal. Interestingly, the regulation of INS-4 and INS-6 by FSN-1 would go through the regulation of the proprotein convertase EGL-3/PC2, which cleaves the propeptides containing INS-4 and INS-6, thus is responsible for the maturation of these two neuropeptides. FSN-1 promotes ubiquitination and degradation of EGL-3/PC2 to maintain its constant level: in fsn-1 mutants the amount of EGL-3 protein is doubled (in whole worm extracts). As a consequence, in fsn-1 mutants, the biosynthesis of INS-4 and INS-6 is hyper-activated which leads to overactivation of the muscle insulin pathway so that GABAergic synapses do not develop correctly [Hung et al., 2013].

Normally, ins-6 is expressed in the ASI neuron, but in food-deprived worms it is expressed in ASJ [START_REF] Cornils | Specific insulinlike peptides encode sensory information to regulate distinct developmental processes[END_REF]. ins-4 is expressed in ASI, ASJ and GABAergic motor neurons. Neuropeptides and in particular insulin-like peptides have already been implicated in the mechanisms of developmental choice and the control of neuronal connectivity [START_REF] Bargmann | Beyond the connectome: How neuromodulators shape neural circuits[END_REF][START_REF] Cornils | Specific insulinlike peptides encode sensory information to regulate distinct developmental processes[END_REF]. This differential expression of ins-4 and ins-6 might controls a choice of developmental program depending on the food available, as ASJ controls entry into dauer stage.

Another target of RPM-1 and the SCF complex is a MAP kinase kinase kinase (MAP3K) Cross-talk between these two signaling events by a yet unknown retrograde signal regulates synapse development along the axons. Image from [Hung et al., 2013].

called the Dual Leukine zipper-bearing Kinase 1 (DLK-1). DLK-1 is essential for axon outgrowth and regeneration: dlk-1 loss-of-function mutation suppress defective axon termination in rpm-1 mutants [Grill et al., 2007, Nakata et al., 2005]. In C. elegans, there are two isoforms of DLK-1, a long functional isoform DLK-1L, and a short inhibitory isoform DLK-1S that can bind to a dephosphorylated DLK-1L resulting in an inactive dimer [START_REF] Yan | Regulation of DLK-1 Kinase Activity by Calcium-Mediated Dissociation from an Inhibitory Isoform[END_REF]. Later studies found that the protein phosphatase PPM-2 inhibits DLK-1L by specifically dephosphorylating a single serine residue [START_REF] Baker | RPM-1 Uses Both Ubiquitin Ligase and Phosphatase-Based Mechanisms to Regulate DLK-1 during Neuronal Development[END_REF]. Inhibition by PPM-2 and by DLK-1S are similar in that it involved dephosphorylating DLK-1L, but it is not clear if PPM-2 dephosphorylation is directly required for DLK-1S inhibition. On the other hand, a high concentration of calcium can trigger the dissociation of the inactive complex DLK-1L/DLK-1S, thus activating the DLK-1 function [START_REF] Yan | Regulation of DLK-1 Kinase Activity by Calcium-Mediated Dissociation from an Inhibitory Isoform[END_REF]. The multitude of inhibitory pathways for DLK-1L indicates a fine level of control over the DLK-1 pathway in order to precisely terminate axonal growth.

Recently, the cyclin-dependent kinase Cdk5, another kinase that plays an important role in neural development, was discovered to be a target for inhibition by the RPM-1/FSN-1 ubiquitin ligase complex [START_REF] Desbois | Ubiquitin ligase activity inhibits Cdk5 to control axon termination[END_REF]. Cdk-5 is involved in neurite outgrowth, 1.2. Axonal growth and guidance synapse formation, among other roles in the nervous system [START_REF] Su | Cyclin-Dependent Kinases in Brain Development and Disease[END_REF]. The C.

elegans homolog, CDK-5, was found to interact with the RPM-1/FSN-1 complex by an in vivo proteomic approach: using a CRISPR-engineered RPM-1 that mutated the RING domain to shut down its ubiquitin ligase function but still retains the FSN-1 binding domain for target recognition. This "dead" RPM-1 is then expressed in rpm-1 mutant worms to trap potential targets without degrading it. The protein trapped in the dead RPM-1/FSN-1 is then purified by using the G::Streptavidin binding peptide affinity tag in the engineered RPM-1. Examination of axon termination in ALM mechanosensory neuron shows that cdk-5 mutant partially suppresses the axonal termination defects in rpm-1 mutant background and completely suppresses termination defects in fsn-1 mutant background.

Finally, the RPM-1 protein physically interacts with ANC-1, ortholog of nesprin-1, to regulate the axon termination of ALM and PLM sensory neurons, as well as the number of GABAergic NMJ [START_REF] Tulgren | The Nesprin Family Member ANC-1 Regulates Synapse Formation and Axon Termination by Functioning in a Pathway with RPM-1 and β-Catenin[END_REF]. Nesprin is a family of nuclear envelope pro-Chapter 1. Introduction teins (nesprins1-4 + KASH5), defined by their Klarsicht/ANC-1/Syne homology domain (KASH) [START_REF] Lombardi | The Interaction between Nesprins and Sun Proteins at the Nuclear Envelope Is Critical for Force Transmission between the Nucleus and Cytoskeleton[END_REF]. This domain allows the protein to attach to the nuclear lumina, forming the mechanical link between the cytoskeleton and the nucleus. Its 'giant' isoforms, nesprin-1 and nesprin-2, are enormous in size, approximately 1MDa and 800kDa respectively [START_REF] Rajgor | Nesprins: from the nuclear envelope and beyond[END_REF].

To regulate axon termination of sensory neuron in C. elegans, ANC-1 is anchored to the nucleus by UNC-84/SUN with the KASH domain and functions with RPM-1 to regulate EMR-1/Emerine. The canonical Wnt/β-catenin pathway pathway comes into play here to determine axon termination. In ALM neurons, EMR-1 facilitates the export of bar- A previous study had shown that, in cell culture, α-catenin and Nesprin-2 physically interact with each other and colocalize at the nuclear envelope or plasma membrane depending on the conditions. They also demonstrated that β-catenin, Emerin and Nesprin-2 interact physically, and that Nesprin-2 can control the activity of β-catenin [START_REF] Neumann | Nesprin-2 Interacts with α-Catenin and Regulates Wnt Signaling at the Nuclear Envelope[END_REF]. Another study suggests the existence of finer interactions between the different isoforms of Nesprin-2 and β-catenin in different cell types [START_REF] Zhang | Syne-1 and Syne-2 play crucial roles in myonuclear anchorage and motor neuron innervation[END_REF]. A more recent study has demonstrated that the reduction of β-catenin activity is associated with faulty regulation of the expression level of Emerin and Nesprin-2. Emerin, an inner nuclear membrane protein, was shown to promote export of β-catenin and Nesprin-2, an outer nuclear membrane protein, promotes nuclear import [START_REF] Sola-Carvajal | Accumulation of Progerin Affects the Symmetry of Cell Division and Is Associated with Impaired Wnt Signaling and the Mislocalization of Nuclear Envelope Proteins[END_REF].

Axon regeneration

The capacity of neural regeneration is widespread in the animal kingdom, such as zebrafish, axolotl, and of course, the nematode C. elegans. The C. elegans axons displayed regenerative growth after laser surgery (axotomy) (Figure 1.15, [START_REF] Yanik | Functional regeneration after laser axotomy[END_REF]), which opens the door for C. elegans to be a model organism to study axon regeneration. Further studies of the model have been able to differentiate regeneration from developmental outgrowth. The process of forming growth cones from severed axon stumps might be fundamentally similar to developing axons, but additional pathways are required from the neurons to detect the injury, response to injury, synthesize and transport building material to the growing ends (often over longer distances than the axons of developping 1.2. Axonal growth and guidance neurons) [START_REF] He | Intrinsic Control of Axon Regeneration[END_REF].

Immediately after axon injury, a rapid rise in intracellular calcium level is detected, starting at the injury site and spreading along the axon to the soma within seconds [START_REF] Ghosh-Roy | Calcium and Cyclic AMP Promote Axonal Regeneration in Caenorhabditis elegans and Require DLK-1 Kinase[END_REF][START_REF] Sun | Neuronal Regeneration in C. elegans Requires Subcellular Calcium Release by Ryanodine Receptor Channels and Can Be Enhanced by Optogenetic Stimulation[END_REF]. The amplitude of this calcium transient depends on the voltage-gated calcium channels and also calcium-induced Ca 2+ release from internal stores, and it is positively correlated to the extent of axon regrowth. Two independent genetic screens for injury-response and regeneration defects were carried out in two types of axons, the mechanosensory neuron PLM axon [START_REF] Chen | Axon Regeneration Pathways Identified by Systematic Genetic Screening in C. elegans[END_REF] and the GABAergic motor neuron axon [START_REF] Nix | Axon Regeneration Genes Identified by RNAi Screening in C. elegans[END_REF]. These studies discovered more than 100 genes that influence regeneration, including the DLK-1 kinase which regulates gene expression and [START_REF] Wu | Caenorhabditis elegans neuronal regeneration is influenced by life stage, ephrin signaling, and synaptic branching[END_REF]. Image from [START_REF] Chisholm | The Genetics of Axon Guidance and Axon Regeneration in Caenorhabditis elegans[END_REF].

microtubule cytoskeleton, promoting axonal regrowth.

Activity-dependant axonal outgrowth

Motor nerve sprouting in vertebrates

It was discovered in the 1940s and 1950s that partial denervated muscles can almost completely restored by motor nerves outgrowth [Brown et al., 1981[START_REF] Edds | Collateral Nerve Regeneration[END_REF]. Cutting part of the motor neurons innervating a muscle leads to excessive sprouting (Figure 1.16A) of the remaining motor neurons, which then take over the denervated muscle fiber and restore normal muscle activity [Diamond et al., 1976, Tam and[START_REF] Tam | Mechanisms controlling axonal sprouting at the neuromuscular junction[END_REF]. Drugs that suppress muscle activity also produce motor nerve sprouting. A notable example is the treatment of muscle with tetrodotoxin (TTX), an antagonist of the voltage-gated sodium channel, which prevents the propagation of action potential (Figure 1.16B, [Brown and Ironton, 1977]). Same goes for treatment with botulinum toxin which prevents the release of acetylcholine by degrading the acetylcholine exocytosis machinery, thus paralyses the muscle: intramuscular injection of a nonlethal dose induces motor neuron sprouting after 2 weeks [START_REF] Duchen | THE EFFECTS OF BOTULINUM TOXIN ON THE PATTERN OF INNERVATION OF SKELETAL MUSCLE IN THE MOUSE[END_REF]. Early study of the effect of treatment with α-bungarotoxin (BTX), an AChR antagonist, did not show any extra nerve sprouting [START_REF] Pestronk | Motor Nerve Sprouting and Acetylcholine Receptors[END_REF], but this result was contested by later studies (Figure 1.16E) [Holland and Brown, 1980[START_REF] Pestronk | Motor nerve terminal outgrowth and acetylcholine receptors: inhibition of terminal outgrowth by alpha-bungarotoxin and antiacetylcholine receptor antibody[END_REF], Wines and Letinsky, 1.2. Axonal growth and guidance 1991]. Image from [Brown and Ironton, 1977]. C, D. Mouse soleus muscle stained with zinc oxide and osmium tetroxide after control treatment (C) and after a week of daily BTX injection. E. Proportion of endplates showing terminal sprouting in control condition (top) or after BTX treatment (bottom). Images adapted from [Holland and Brown, 1980].

Interestingly, contrary evidence were found that treatments with BTX do not induce Chapter 1. Introduction motor nerve sprouting, but in fact BTX treatment even suppresses the nerve sprouting in partially denervated muscle compared to just partially denervated muscle alone [Connold and[START_REF] Connold | The effect of muscle activity on motor unit size in partially denervated rat soleus muscles[END_REF][START_REF] Connold | Temporary loss of activity prevents the increase of motor unit size in partially denervated rat soleus muscles[END_REF]. The rat soleus muscles (the wide flat muscle found on the posterior leg) are partially denervated by removing one side of the L5 ventral ramus (an anterior division of the spinal nerve, containing major neural input to the soleus muscle. After 2-to 10-months of recovery, with just the sectioned L5 ventral ramus and no treatment, the mean motor unit size almost doubled compared to sectioned L5 with BTX treatment (Figure 1.17A). The lack of motor unit expansion in paralyzed muscle is likely to be caused by the inability to form sprouts. L5+NaCl muscles have 24.3 ± 4 sprouting compared to 5 ± 2 sprouting of L5+BTX muscles [START_REF] Connold | Temporary loss of activity prevents the increase of motor unit size in partially denervated rat soleus muscles[END_REF]. This result is inconsistent with previous studies showing that BTX does induce motor neuron sprouting [Holland and Brown, 1980[START_REF] Pestronk | Motor nerve terminal outgrowth and acetylcholine receptors: inhibition of terminal outgrowth by alpha-bungarotoxin and antiacetylcholine receptor antibody[END_REF][START_REF] Wines | Inactivity-induced motor nerve terminal sprouting in amphibian skeletal muscles chronically blocked by α-bungarotoxin[END_REF]. But, it is similar to the first study on the effect on BTX and motor neuron, in the sense that BTX did not effectively induce nerve sprouting [START_REF] Pestronk | Motor Nerve Sprouting and Acetylcholine Receptors[END_REF]. One possible reason for this inconsistent result is that BTX could have had an effect on presynaptic motor neurons [START_REF] Connold | Temporary loss of activity prevents the increase of motor unit size in partially denervated rat soleus muscles[END_REF].

BTX is not the only drug that produces this kind of contrary evidence. TTX blockade of neuromuscular activity during the sprouting phase significantly reduces sprouting in extensively denervated muscle (Figure 1.17B) [Tam et al., 2002]. The L4 or L5 spinal root avulsion results in partial denervation of rat hindlimb muscles: extensive denervation (over 80% loss of innervation) in tibialis anterior (TA, muscle in the anterior compartment of the leg, along the surface of the tibia) and medical gastrocnemius (MG, muscles in the back part of the leg, posterior to the soleus muscle), moderate denervation (around 50% loss) in soleus (SOL) and plantaris (PL, superficial muscle of the superficial posterior compartment of the leg). After 1 month, extensive denervated muscles with normal caged activity have enlarged motor units and around 25 sprouts per 100 innervated endplates.

On the other hand, blocking neuromuscular activity of extensive denervated muscle with TTX almost completely prevents the enlargement of motor units as well as the motor nerve sprouting: 5 sprouts per 100 endplates, the same as control condition with no operation done on the motor nerve [Tam et al., 2002]. The suggested mechanism for TTX's sprouting suppression effect is that TTX would reduce calcium influx into the nerve terminal by blocking nerve conduction of action potentials [Tam et al., 2002]. Calcium being a critical part of neurite outgrowth (this will be further elaborated in section Calcium's role in axon outgrowth), TTX blockade of electrical activity may be enough to reduce the outgrowth of sprouts from deactivated motor neurons. One could think that BTX might also have the same effect on the motor neurons, which is electrically silencing the neurons, resulting in a suppression of sprouting. There are sufficient evidence pointing to a role of muscle activity in the induction of nerve sprouting. Denervated or pharmalogically deactivated muscle might produce a growth factor to promote sprouting, or a retrograde signal reporting inactivity to the nerve and that signal triggers sprouting. This hypothesis is further reinforced by studies showing that stimulating activity in inactive muscle can prevent terminal nerve sprouting [START_REF] Brown | Nodal and terminal sprouting BIBLIOGRAPHY from motor nerves in fast and slow muscles of the mouse[END_REF]. After 4 to 6 days of direct electrical stimulation of muscle paralysed by botulinum toxin, terminal sprouting is essentially only 6% of nerve terminal have sprouts, compared to 53% in control condition. For soleus muscles 4-18 days after denervation, no endplate was innervated by terminal sprouts in electrically stimulated muscles, compared to 8.4 ± 5.8% of endplates was innervated by sprouts in control. These results indicate that the electrical activity of the muscle suppresses terminal sprouting in motor nerves. Interestingly, terminal sprouts were observed in non-denervated muscles but physically immobilized, either by a plaster cast [START_REF] Pachter | Neuromuscular plasticity following limb immobilization[END_REF] or by pinning the joint at a right angle [START_REF] Fahim | Remodelling of the neuromuscular junction after subtotal disuse[END_REF]. Assuming there is still electrical activity in both muscle and motor neurons as there was no treatment or operation applied (except to restrain the muscle), the presence of sprouts may suggest a retrograde signal reporting muscle disuse to the motor neuron. A noteworthy point is that there is a difference between terminal sprouting and nodal sprouting (see Figure 1.16A for visual comparison) in the amount of sprouts or what muscle produces what sprouts, suggesting a different cause for each type of sprouting [Brown et al., 1981].

The precise mechanism of motor nerve sprouting has not yet been explained, and several causal agents have been proposed. One of the early theories is a soluble growth factor which might be excreted by either the inactive muscle or a type of glial cell in the PNS called Schwann cells. There are two types of Schwann cells, one that is responsible for forming a myelin sheath insulating the axons, the other is located at the NMJ and is non-myelinating. The latter type has been shown to be able to sense neurotransmission at the NMJ, and to induce and guide sprouting in denervated or paralyzed muscle [START_REF] Gordon | Peripheral Nerve Regeneration and Muscle Reinnervation[END_REF]Borschel, 2017, Son et al., 1996]. When the terminal Schwann cells detect no neuromuscular activity, it upregulates its expression of glial fibrillary acidic protein and extends its processes across innervated and denervated muscle endplates, guiding axon sprouts to the denervated NMJs [START_REF] Gordon | Peripheral Nerve Regeneration and Muscle Reinnervation[END_REF].

A 56 kDa protein antigen secreted by the muscle was found to have a functional role in nerve sprouting, as monoclonal antibody against this antigen suppresses the terminal sprouting in paralyzed muscle by 50% [START_REF] Gurney | Suppression of sprouting at the neuromuscular junction by immune sera[END_REF], Gurney et al., 1986a]. This antigen was named neuroleukin, for being a neurotrophic factor and a lymphokine product [Gurney et al., 1986b]. Later studies have identified this antigen to be identical to glucose-6-phosphate isomerase (GPI), an enzyme that catalyzes the reversible transformation between glucose-6-phosphate and fructose-6-phosphate [START_REF] Chaput | The neurotrophic factor neuroleukin is 90% homologous with phosphohexose isomerase[END_REF][START_REF] Faik | Mouse glucose-6-phosphate isomerase and neuroleukin have identical 3' sequences[END_REF]. Monoclonal antibodies against neuroleukin does not inhibit the enzymatic activity of GPI, and rabbit antiserum partially inhibit GPI (Gurney's replies to [START_REF] Faik | Mouse glucose-6-phosphate isomerase and neuroleukin have identical 3' sequences[END_REF]). Neuroleukin and its potential receptor are shown to be expressed in the developing and adult rat brain [START_REF] Leclerc | Expression of the AMF/neuroleukin receptor in developing and adult brain cerebellum[END_REF][START_REF] Repiso | Expression Patterns in Mouse Embryos of Neuroleukin/Glucose-6-Phosphate Isomerase and Autocrine Motility Factor Receptor[END_REF]. After this point, the relation between neuroleukin/GPI and nerve sprouting is not explored and studied further.

Seven neurotrophic factors are shown to be secreted at a higher level in the NMJ than in muscle cells with no contact with neurons: neurotrophic factors Brain-Derived Neurotrophic Factor (BDNF), glial-cell-line-derived neurotrophic factor (GDNF), insulin-like growth factor-binding protein-3 (IGFBP-3), insulin-like growth factor (IGF)-1, neurotrophin (NT): NT-3 and NT-4, and vascular endothelial growth factor (VEGF) [START_REF] Saini | Cross-talk between motor neurons and myotubes via endogenously secreted neural and muscular growth factors[END_REF]. This demonstrates a cross-talk between muscle and motor neuron to promote regeneration. Among the seven, BDNF, NT-3 and NT-4 are expressed by muscle [Griesbeck et al., 1.2. Axonal growth and guidance 1995[START_REF] Ip | The expression profiles of neurotrophins and their receptors in rat and chicken tissues during development[END_REF][START_REF] Koliatsos | Neurotrophin 4/5 is a trophic factor for mammalian facial motor neurons[END_REF], 1993[START_REF] Maisonpierre | NT-3, BDNF, and NGF in the developing rat nervous system: Parallel as well as reciprocal patterns of expression[END_REF][START_REF] Timmusk | Widespread and Developmentally Regulated Expression of Neurotrophin-4 mRNA in Rat Brain and Peripheral Tissues[END_REF].

After the muscle is denervated, the expression of NT-3 stays the same, but NT-4's expression decreased [START_REF] Funakoshi | Muscle-Derived Neurotrophin-4 as an Activity-Dependent Trophic Signal for Adult Motor Neurons[END_REF]. In fact, the production of NT-4 is dependent on muscle activity, it also increased in response to electrical stimulation of the muscle [START_REF] Funakoshi | Muscle-Derived Neurotrophin-4 as an Activity-Dependent Trophic Signal for Adult Motor Neurons[END_REF]. Intramuscular injection of NT-4 promotes sprouting of intact motor nerves [START_REF] Funakoshi | Muscle-Derived Neurotrophin-4 as an Activity-Dependent Trophic Signal for Adult Motor Neurons[END_REF].

The expression of BDNF seems to be a debated topic. Early study of BDNF suggests that its expression seems to decreased slowly after denervation, which can be due to the effect of denervation on the Schwann cells [START_REF] Griesbeck | Expression of neurotrophins in skeletal muscle: Quantitative comparison and significance for motoneuron survival and maintenance of function[END_REF]. However, one other study found a strong diminution of BDNF and NT-3 expression in response to denervation [START_REF] Loeb | Neuregulin Expression at Neuromuscular Synapses Is Modulated by Synaptic Activity and Neurotrophic Factors[END_REF]. Later study shows that BDNF is upregulated in denervated rat facial muscle, correlating with regeneration of axons [START_REF] Grosheva | Early and continued manual stimulation is required for long-term recovery after facial nerve injury[END_REF]. BDNF seems to enhance the survival of innervating motor neurons [Oppenheim et al., 1992, Zhang and[START_REF] Zhang | Localized Synaptic Potentiation by BDNF Requires Local Protein Synthesis in the Developing Axon[END_REF], reinnervate denervated muscle and regulate postsynaptic AChR at the NMJ [START_REF] Bendella | Putative roles of soluble trophic factors in facial nerve regeneration, target BIBLIOGRAPHY reinnervation, and recovery of vibrissal whisking[END_REF].

There are 2 IGF coding genes in mammals, IGF-1 and IGF-2. An increase in the transcription of IGF-1 and IGF-2 has been identified following muscle denervation [START_REF] Caroni | Nerve sprouting in innervated adult skeletal muscle induced by exposure to elevated levels of insulin-like growth factors[END_REF]. Exogenous addition of a protein capable of binding IGF suppresses the sprouting induced by botulinum toxin [START_REF] Caroni | Role of muscle insulinlike growth factors in nerve sprouting: suppression of terminal sprouting in paralyzed muscle by IGF-binding protein 4[END_REF], suggesting a functional role of IGF in motor nerve sprouting. IGF-2 was proposed to be the important factor between the two IGFs in motor nerve sprouting [START_REF] Ishii | Role of insulin-like growth factors in peripheral nerve regeneration[END_REF], while IGF-1 seems to have a general neuroregenerative effect [START_REF] Bianchi | Neurotrophic and Neuroregenerative Effects of GH/IGF1[END_REF]. Application of IGF-1 seems efficacious in recovery of temporary denervated facial muscle (crush injury) [START_REF] Bayrak | The Effect of Insulin Like Growth Factor-1 on Recovery of Facial Nerve Crush Injury[END_REF].

Neuregulin (NRG) is another neurotrophic factor that was recently found to play a role in the muscle reaction to nerve injury. The expression of neuregulin's receptors, ErbB2 and ErbB3, is upregulated in denervated muscle over time, then downregulated after the muscle is reinnervated and its function recovered [START_REF] Morano | Modulation of the Neuregulin 1/ErbB system after skeletal muscle denervation and reinnervation[END_REF]. NRG-1 overexpression in muscle accelerates the sprouting in denervating muscles [START_REF] Mancuso | Neuregulin-1 promotes functional improvement by enhancing collateral sprouting in SOD1G93A ALS mice and after partial muscle denervation[END_REF].

As direct electrical stimulation of the paralysed muscle suppresses terminal sprouting [START_REF] Brown | Nodal and terminal sprouting BIBLIOGRAPHY from motor nerves in fast and slow muscles of the mouse[END_REF], electrical stimulation of the nerve accelerates the motor axon regeneration [START_REF] Ahlborn | One hour electrical stimulation accelerates functional recovery after femoral nerve repair[END_REF][START_REF] Al-Majed | Brief Electrical Stimulation Promotes the Speed and Accuracy of Motor Axonal Regeneration[END_REF][START_REF] Gordon | Brief post-surgical electrical stimulation accelerates axon regeneration and muscle reinnervation without affecting the functional measures in carpal tunnel syndrome patients[END_REF][START_REF] Nix | Electrical stimulation of regenerating nerve and its effect on motor recovery[END_REF][START_REF] Pockett | Acceleration of peripheral nerve regeneration after crush injury in rat[END_REF]. The expression of several neurotrophic factors is upregulated in the Schwann cells when the injured nerve is directly stimulated [Huang et al., Chapter 1. Introduction 2009[START_REF] Koppes | Electrical Stimulation of Schwann Cells Promotes Sustained Increases in Neurite Outgrowth[END_REF][START_REF] Wenjin | Electrical Stimulation Promotes BDNF Expression in Spinal Cord Neurons Through Ca2+-and Erk-Dependent Signaling Pathways[END_REF], notably the nerve growth factor (NGF) and BDNF, by activating the calcium-dependent signaling pathway.

Activity-dependent morphology of motor neuron in C. elegans

In C. elegans, two classes of motor neurons show a morphology regulated by postsynaptic muscle activity. The first case is the SAB motor neurons. SAB neurons send projections into the worm's nose, which directly innervate the muscles of the nose (see Section SAB neurons). The synapses formed are cholinergic, but contain only L-AChR receptors, and no N-AChR. SABs are type A motor neurons, i.e. their axons grow in an anterograde direction.

The seminal study of Zhao and Nonet (2000) revealed that in mutants with reduced cholinergic transmission, such as cha-1 (choline acetyltransferase), unc-29 (a component of L-AChR), unc-18 (syntaxin-binding protein), SAB axons show morphological defects, for example sprouting to form ectopic branches, or failure to terminate growth, as in the axon does not stop at the tip of the nose but it overextends and continues in another direction (looping) [Zhao and Nonet, 2000]. Disruption of GABAergic, glutamatergic, or monoaminergic signaling has no effect, but direct reduction of muscle activity by ectopic expression of the potassium channel EGL-36 induces defects in SAB axons. unc-54 (coded for heavy-chain myosin) mutant worms which are completely paralyzed, do not show overgrowth. Finally, the use of a temperature-sensitive allele shows that this defect induction only takes place in the early larval stages (L1 and L2), and that a reduction in cholinergic activity for 8 hours is sufficient to effectively induce defects [Zhao and Nonet, 2000]. This therefore suggests that, in a critical period during the early larval stages, in the event of a defect in the activity of the muscles, these can send a retrograde signal to the SAB neurons and induce overgrowth defects.

Alexis Weinreb, a previous alumni of our team, has reproduced the activity-dependent overgrowth of SAB motor neuron, by using a robust method to inhibit the muscle activity: the HisCl1 ion channel. This ionotropic receptor of histamine is present in Drosophila melanogaster, but there is no histaminergic neurotransmission in C. elegans. The HisCl1 channel is permeable to chloride ion and structurally similar to GABA receptors. Expression of HisCl1 channel in C. elegans allows the formation of a functional ion channel. In presence of histamine, electrically excitable cell expressing HisCl1 is hyperpolarized and deactivated by the influx of chloride ion [Pokala et al., 2014]. The expression of HisCl1 channel in the worm's muscle by the myo-3 promoter can effectively paralyze the worm at a histamine concentration of 1 mM [START_REF] Weinreb | Impact de l'activité postsynaptique sur le développement et le maintien de la jonction neuromusculaire de C. elegans[END_REF]. With this tool, it was demonstrated that reduced muscle activity leads to overgrowth of SAB axon. 82 ± 7.2 % of histamine-1.2. Axonal growth and guidance induced paralyzed worms show SAB axonal defects (Figure 1.18). Alexis Weinreb has also determined that L1 is the critical period of developing SAB overgrowth, and that local inhibition of the head muscle is sufficient to induce SAB axonal defects. Two genes involved in neuropeptide processing was found to have a role in the formation of the SAB overgrowth. His results are further discussed in a later chapter. The second case is the DVB neuron, which locates at the tail of the worm and projects its axons anteriorly along the ventral cord in both sexes, male and hermaphrodite. DVB, along with AVL neuron, innervate enteric muscles to control the defecation cycle. These are two excitatory GABAergic neurons as their postsynaptic target expresses the EXP-1 channel, a cation channel opened by GABA [START_REF] Beg | EXP-1 is an excitatory GABA-gated cation channel[END_REF]. In mutants of the homeobox gene lim-6, the axons of these GABAergic neurons terminate early on, before reaching their synaptic targets. These morphological defects of the AVL and DVB neurons seem to prevent the contraction of the worm's enteric muscle during defecation [START_REF] Hobert | The Caenorhabditis elegans lim-6 LIM homeobox gene regulates neurite outgrowth and function of particular GABAergic neurons[END_REF]. The DVB neuron of these worms exhibits visually similar overgrowth defects to SAB's, the sprouting happens in or near the cell body [START_REF] Hobert | The Caenorhabditis elegans lim-6 LIM homeobox gene regulates neurite outgrowth and function of particular GABAergic neurons[END_REF]. Furthermore, the DVB axon also shows sprouting defects close to the cell body in unc-13 mutants where neuromuscular activity is reduced, and unc-25 mutants, where GABA biosynthesis is disrupted. The induction of sprouting is due to a decrease in the activity of the enteric muscles since they also appear in mutants of the transcription factor hlh-8 /Twist necessary for development normal enteric muscle, as well as in the constitutively active potassium channel mutant egl-2, which inhibits enteric muscle [START_REF] Loria | Two Neuronal, Nuclear-Localized RNA Binding Proteins Involved in Synaptic Transmission[END_REF].

A study of DVB neuron in male C. elegans has discovered a case of activity-dependent and Chapter 1. Introduction sexually dimorphic axonal morphology [START_REF] Hart | Neurexin controls plasticity of a mature, sexually dimorphic neuron[END_REF]. DVB neuron has another function in male worm in addition to innvervating enteric muscle. It controls the neurons and muscles of the spicules which are used during mating. The activation of DVB causes an immediate protraction of the spicule. Repeated activation of the spicule protraction circuit leads to a significant increase in DVB neurites. Vice versa, by exposing males to exogenous GABA, the activity of the spicule as well as the DVB neurites are reduced. The growth of these neurites depends on the expression of neuroligin and neurexin. In hermaphrodites, neurexin or neuroligin mutation or overexpression has no effect on DVB morphology [START_REF] Hart | Neurexin controls plasticity of a mature, sexually dimorphic neuron[END_REF].

Activity-dependent synaptic plasticity in Drosophila melanogaster

The neuromuscular junction of Drosophila is a powerful model for the study of synaptic plasticity since the 1990. Many molecules, proteins and pathways implicated in Drosophila's NMJ plasticity have been shown to have similar functions at other synapse and in higher organism. One of the first studies about activity and NMJ morphology examined the mutants of the potassium channels Sh (Shaker), eag (ether a go-go) and Hk (Hyperkinetik) [START_REF] Budnik | Morphological plasticity of motor axons in Drosophila mutants with altered excitability[END_REF]. These mutants are hyperexcitable and there are numerous extra branches at the NMJ compared to wild type (Figure 1.19A, B). Conversely, the gene mle regulates a sodium channel para: mle mutants do not generate action potential and double mle plus hyperexcitable mutants negate the hyperexcitable phenotype as well as prevent all excess sprouting at the nerve terminal.

A cell adhesion molecule at the NMJ, Fasciclin II (Fas II), has been determined as a necessary component for long-term synaptic structural plasticity. Fas II mutants displayed pre-synaptic sprouting phenotype similar to Shaker and eag double mutant as well as the dunce (Cyclic adenosine monophosphate (cAMP) phosphodiesterase II) mutants [START_REF] Byers | Defect in cyclic AMP phosphodiesterase due to the dunce mutation of learning in Drosophila melanogaster[END_REF]. It was discovered that the double hyperexcitable mutants and dunce mutants downregulate the level of Fas II in the NMJ, and FasII functions downstream of the cAMP pathway with the cAMP response element-binding protein (CREB) to control presynaptic strength and sprouting (Figure 1.20, [START_REF] Schuster | Genetic Dissection of Structural and Functional Components of Synaptic Plasticity. I. Fasciclin II Controls Synaptic Stabilization and Growth[END_REF]). cAMP pathway activation depends pre-synaptic calcium accumulation, which activates the calcium/calmodulindependent adenylate cyclase rutabaga. rutabaga and Dunce regulate the level of cAMP, which controls the activity of cAMP-dependent protein kinase (PKA), and protein phosphatase 1 (PP1).

Later studies discovered that during development, motor neurons make connections with numerous muscle fibers. Then there is a process of synaptic refinement to remove a significant amount of connections, until there is only 1 or 2 muscle fibers innervated per motor neurons. This refinement process requires a calcium signaling pathway in neurons, as muscle activity has no effect on the sprouting [START_REF] Vonhoff | In Vivo Calcium Signaling during Synaptic Refinement at the Drosophila Neuromuscular Junction[END_REF]. The pathway involved is the calmodulin-dependent protein kinase II (CaMKII) which is triggered by an accumulation of pre-synaptic calcium. CaMKII regulates the Clustering of Discs large (DLG), which controls synaptic molecules such as Fas II. The exact mechanism of retraction and refinement is not yet understood. What is known now is that the cAMP pathway function with CaMKII to regulate chemorepulsion mediated by Semaphorin-2a (Sema2a) during activity-dependent synaptic refinement, in order to reduce aberrant neuromuscular connections (Figure 1.20, [START_REF] Bai | Activity-Dependent Synaptic Plasticity in Drosophila melanogaster[END_REF]).

Axonal growth and guidance

Activity-dependent plasticity in the visual system

The mammalian visual system, known as the retinotectal system in other vertebrates, is very studied in the tadpoles of African clawed frog (Xenopus laevis) and larval zebrafish (Danio rerio), due to the reliance on vision for their survival. These organisms already make extensive use of their visual system for movement during their developmental stage. The studies of the development of the visual system in these model organisms have given a lot of insight into how sensory neurons read out and response to activity [Kutsarova et al., 2017].

The retinal ganglion cell (RGC) receive visual information from the photoreceptors and from amacrine and bipolar cells. Then, RGCs send their axons towards the brain, forming optic nerves. The majority of optic nerves went to the thalamus where the sensory inputs are processed, the rest went to the midbrain. In other non-mammal vertebrates, the retinal optic nerves connect with the tectum (the dorsal side of the midbrain), forming retinotectal projections. The ensemble of retinotectal projections constitutes a retinotopic map that shows the spatial organization of retinal neurons.

The process of refinement-removing excess connections-of the retinotectal projections is highly dependent on activity. For example, treatment by TTX, blocking the action potential of the RGC prevents the refinement process, causing an increase in the number of axon branches in Xenopus [START_REF] Cohen-Cory | BDNF Modulates, But Does Not Mediate, Activity-Dependent Branching and Remodeling of Optic Axon Arbors In Vivo[END_REF]] and an enlargement of the multiunit receptive field size in goldfish [START_REF] Schmidt | Activity sharpens the map during the regeneration of the retinotectal projection in goldfish[END_REF]. Similarly, by blocking the NMDA receptor (an excitatory glutamate ion channel) in the retina of tadpoles [START_REF] Cline | NMDA receptor antagonists disrupt the retinotectal topographic map[END_REF]] and rats [START_REF] Simon | Nmethyl-D-aspartate receptor antagonists disrupt the formation of a mammalian neural map[END_REF], many axon terminals are mistargeted and the normal refinement of termination zone of RGC axons is disrupted.

Interestingly, constantly stimulating the retina of goldfish during development with stroboscopic light also prevents the refinement process, axons are long with many scattering branches and the receptive field is abnormally large [START_REF] Schmidt | Stroboscopic illumination and dark rearing block the sharpening of the regenerated retinotectal map in goldfish[END_REF]. This suggests that activity is instructive to the refinement process and not permissive. However, it was found in rat model that asynchronous stimulation of the eyes substantially improved the segregation of synapses into the correct eye-specific region [START_REF] Zhang | Visual map development depends on the temporal pattern of binocular activity in mice[END_REF].

Additionally, it was found that electrical activity strongly enhance axon growth of RGCs in response to neurotropic factors [Goldberg et al., 2002]. Without activity, RGC axons do not response to peptide neurotropic factors and there are little growth. But if the cells were electrically active, even short bursts once every minute can drastically increase axon growth in presence of neurotrophins. Although, electrical activity alone has no effect.

Chapter 1. Introduction

Activity-dependent plasticity of the corpus callosum in mammals

The corpus callosum (CC) is the major axonal tract of the mammalian brain, located beneath the cerebral cortex, visibly connecting the two hemispheres (Figure 1.21, [START_REF] Fenlon | Contralateral targeting of the corpus callosum in normal and pathological brain function[END_REF]). The callosal connections mediate communications between the two hemispheres. The development of CC begins at embryonic stages, when the hemispheres are fused together creating a route in the middle, and continues into an extended period after birth. The neurons of the cingulate cortex (the part located above this midline route) are the first one to elongate their axons along this path, paving the way for the bulk of the other cortical neurons at the outer layers to project their axons across the callosal tract to the opposite hemisphere. These neurons are called callosal projecting neurons (CPNs), and their projection is made contralateral, from one hemisphere to the other. The developmental axon projections are subject to refinement and this process is informed by activity [START_REF] De León Reyes | Development and plasticity of the corpus callosum[END_REF]. Calcium imaging have discovered distinct pattern of neuronal activity in the neonatal mouse cortex during the development period of CPNs [START_REF] Khazipov | Early patterns of electrical activity in the developing cerebral cortex of humans and rodents[END_REF][START_REF] Siegel | Peripheral and Central Inputs Shape Network Dynamics in the Developing Visual Cortex In Vivo[END_REF][START_REF] Yang | Three Patterns of Oscillatory Activity Differentially Synchronize Developing Neocortical Networks In Vivo[END_REF]. The refinement of CPNs depends on activity at three different levels:

1. alteration in the sensory inputs from the periphery 2. alteration in the thalamic inputs (which processes the sensory inputs then relay it to the cerebral cortex)

3. alteration in the synaptic transmission of pre-and postsynaptic CPN 1.2. Axonal growth and guidance

Manipulating any, or all, three of these levels yields effects only during a specific temporal window known as "critical periods" [START_REF] De León Reyes | Development and plasticity of the corpus callosum[END_REF].

There does not seem to be an established general rules in the relationship between sensory activity and the refinement or the developmental decision of CPNs. For example, in cats or rats, removing one eye increased the CPNs in a specific area which is normally not innervated by callosal axons, but removing both eyes leads to a reduction of CPNs in an typically callosal-rich area (between the two hemispheres). Removing whiskers from one side of the rat leads to a decrease of CPNs in both hemispheres, but cauterizing whiskers from both sides resulted in an increase of CPNs.

The thalamic inputs seem to determine three aspects of callosal assembly: the refinement rates and CPN number, the distribution of CPN, and the postsynaptic targets. Ablation of some thalamic axons doubled the number of CPNs in all cortical layers, resulting in hyperconnectivity. Ablation of the ventral posterior nucleus reduces refinement and causes CPNs to develop in non-callosal region.

Overexpression of a potassium channel (Kir2.1) in a CPN inhibits the presynaptic activity of the neuron by hyperpolarization, consequently decreases contralateral innervation [START_REF] Mizuno | Evidence for Activity-Dependent Cortical Wiring: Formation of Interhemispheric Connections in Neonatal Mouse Visual Cortex Requires Projection Neuron Activity[END_REF] and disturbs the callosal axon growth at 9 days after the mice is born [Mizuno et al., 2010]. Silencing the postsynaptic activity by overexpressing the same channel disturbed axon growth at a later stages, 9 to 15 days post-natal. Inhibiting both pre-and postsynaptic activity results in impairment of callosal axon projections. This suggests that the activity of neurons is essential for callosal axon growth. Furthermore, the combination of Kir2.1 expression and the blockage of synaptic transmission in the presynaptic CPN resulted in a more severe reduction in the contralateral projections than just the potassium channel expression [START_REF] Mizuno | Evidence for Activity-Dependent Cortical Wiring: Formation of Interhemispheric Connections in Neonatal Mouse Visual Cortex Requires Projection Neuron Activity[END_REF]. More recent studies show that presynaptic mitochondrial entry in some callosal axons affects axon termination and branching [START_REF] Courchet | Terminal Axon Branching Is Regulated by the LKB1-NUAK1 Kinase Pathway via Presynaptic Mitochondrial Capture[END_REF][START_REF] Lewis | MFF-dependent mitochondrial fission regulates presynaptic release and axon branching by limiting axonal mitochondria size[END_REF], indicating the importance of synaptic vesicles release for axonal connectivity.

Recent studies have demonstrated that neural activity is required for callosal axon development during day 6 to day 15, and the critical period of development was found to be before day 9 [Tezuka et al., 2022]. By expressing the Kir2.1 in a Tet-off system, it is possible to control the temporal expression of this channel and turn off the expression when needed by doxycycline treatment, allowing in turn a temporal control of the presynaptic activity of the CPN. A doxycycline treatment on mice expressing the potassium channel will restore the spontaneous activity to the neurons, and a restoration by day 6 rescued the stalled axon growth phenotype. The activity of the neurons were monitored Chapter 1. Introduction by calcium imaging, and high activity during day 6 to day 15 were shown to play a role in forming the specific projection pattern of callosal axons.

Calcium signaling

Calcium is a universal second messenger in eukaryotic cells. The cytoplasmic concentration of calcium is normally kept very low, around 100 nM. This is 10000 times lower than typical extracellular calcium concentration [START_REF] Clapham | Calcium Signaling[END_REF]. Calcium is so important that the calcium homeostatic system was developed very early on in the evolutionary history of multicellular life to maintain this calcium gradient across plasma membranes [START_REF] Case | Evolution of calcium homeostasis: From birth of the first cell to an omnipresent signalling system[END_REF]. Even the most primitive bacteria e.g. Synechococcus sp. has a form of calcium ion pump that is structurally similar to eukaryotic calcium pumps (plasma membrane Ca 2+ ATPase (PMCA) or sarco/endoplasmic reticulum Ca 2+ -ATPase (SERCA) type) and ion exchangers (Ca 2+ /H + and Na + /Ca 2+ exchangers). Later eukaryotic lifeforms evolved organelles storing calcium and calcium-binding proteins, further improving the control of calcium level in addition to other biological effects.

Calcium signaling happens when there is an increase of intracellular calcium level, either by release of Ca 2+ ions from intracellular stores, or by Ca 2+ influx through plasma membrane ion channels, or both. The most common signaling pathway that increase intracellular calcium concentration is the phospholipase C (PLC) pathway, which is activated by many cell surface receptors such as G protein-coupled receptors and receptor tyrosine kinases. PLC cleaves phosphatidylinositol 4,5-bisphosphate (PIP2) into diacyl glycerol (DAG) and inositol 1,4,5-trisphosphate (IP3), two important secondary messengers. IP3 is diffused to the ER and acts on IP3 receptor, releasing Ca 2+ from the endoplasmic reticulum into the cytoplasm. If the calcium store in the ER is depleted, activation of store-operated calcium channels (SOCs) will replenish the calcium storage by Ca 2+ influx (called store-operated calcium entry (SOCE)) from outside the cell. SOCs are composed of stromal interaction molecules (STIM1/2 proteins) which monitor Ca 2+ level in the ER, and Orai1/2/3 proteins which form the Ca 2+ release-activated calcium channel (CRAC) in the membrane. Upon depletion of ER calcium, STIM binds to Orai, opening the calcium channel for Ca 2+ entry [START_REF] Zhang | Store-Operated Calcium Channels in Physiological and Pathological States of the Nervous System[END_REF].

There are many calcium-binding proteins functioning downstream of calcium influx, mediating a myriad of events for numerous biological functions. A notable example is the regulatory protein calmodulin. Calmodulin has two globular domains, a N-domain and a C-domain, each containing a pair of calcium-binding EF motif, for a total of 4 calcium binding sites for each molecule of calmodulin [START_REF] Chin | Calmodulin: a prototypical calcium sensor[END_REF]. Calcium- For neurons, intracellular calcium regulates many essential functions such as the release of neurotransmitters, excitability and cell viability [START_REF] Berridge | Calcium signalling: dynamics, homeostasis and remodelling[END_REF], as well as nearly every process in the neural development: neural induction [START_REF] Moreau | The choice between epidermal and neural fate: a matter of calcium[END_REF], proliferation [START_REF] Somasundaram | Store-operated CRAC channels regulate gene expression and proliferation in neural progenitor cells[END_REF], migration [START_REF] Zheng | Calcium signaling in neuronal motility[END_REF] and differentiation [START_REF] Rosenberg | Calcium Signaling in Neuronal Development[END_REF].

Voltage-gated calcium channels

Type and composition

The voltage-gated calcium channels (Ca V ), or VGCCs, are a group of conserved ion channel found in the membrane, responsible for the intracellular calcium transient that initiates many physiological events. Normally, the concentration of intracellular calcium is kept extremely low (10 -7 M) and its variations can trigger muscle contraction, release of neurotransmitters, gene regulation. These physiological responses rely on the multiprotein complex structure of the channels as well as the regulatory proteins that coupled the signal of calcium influx with the downstream effectors. This type of ion channel is called 'voltage-gated' due to their mode of function, as they response to a change in the cell's membrane potential. If the membrane potential depolarizes past a certain threshold, then the VGCCs are triggered and become permeable to calcium ion.

The VGCCs have one main subunit, α1, that forms the actual pore that ions pass through.

There are 3 main groups of VGCC, separated based on their homology (the percentage identity between their protein sequences) and their properties (Figure 1.23a, [START_REF] Dolphin | Presynaptic calcium channels: specialized control of synaptic neurotransmitter release[END_REF]). First group is the 'L-type' VGCCs (Ca V 1) named after their 'long' opening in presence of dihydropyridine agonists which can bind to and block the channel, prevent the influx of calcium ion [START_REF] Hess | Different modes of Ca channel gating behaviour favoured by dihydropyridine Ca agonists and antagonists[END_REF]. Second group is the 'non-L type' VGCCs (Ca V 2) which differentiate themselves by their responses to various specific toxins: 'P/Q type' (Ca V 2.1) is blocked by ω-agatoxin IVA, 'N-type' is blocked by ω-conotoxin GVIA [START_REF] Starr | Primary structure of a calcium channel that is highly expressed in the rat cerebellum[END_REF], and 'R-type' is blocked by SNX-482 [START_REF] Mori | Primary structure and functional expression from complementary DNA of a brain calcium channel[END_REF]. Ca V 1 and Ca V 2 are both composed of multiple subunits: a main pore α1 and the auxiliary subunits α2, β, and sometimes γ. The third group is the 'T-type' VGCCs (Ca V 3) named after the short duration of their current (Figure 1.23b). Ca V 3 channels are activated at a much lower membrane potential compared to Ca V 1 and Ca V 2, and also contain only the main pore α1 and no auxiliary subunits [START_REF] Soong | Structure and Functional Expression of a Member of the Low Voltage-Activated Calcium Channel Family[END_REF].

Initially when the VGCCs were first characterized, they were separated into low-voltageactivated and high-voltage-activated channels [START_REF] Bean | Two kinds of calcium channels in canine atrial cells. Differences in kinetics, selectivity, and pharmacology[END_REF][START_REF] Carbone | A low voltage-activated, fully inactivating Ca channel in vertebrate sensory neurones[END_REF][START_REF] Hagiwara | Voltage clamp analysis of two inward current mechanisms in the egg cell membrane of a starfish[END_REF]. Later studies found that, rather than a clear division into low and high, the activation voltage ranges of VGCCs resemble a continuum of activation threshold for the different channels, which can be further modified by the auxiliary subunits and alternative splicing (Figure 1.23b, [START_REF] Dolphin | Calcium channel auxiliary α2δ and β subunits: trafficking and one step beyond[END_REF]Lee, 2020, Liao et al., 2009]).

The human genetic nomenclature for VGCC starts with CACN (for CAlcium ChaNnel), followed by their subunit type (A1 for α1, A2 for α2, B for β, D for δ, G for γ) and finish with a letter (for α1 subunit) or a number to specify the subtype. The main subunit α1 of the VGCC comprise of 4 homologous domain (I to IV), each with 6 transmembrane α-helical segments (S1 to S6), for a total of 24 segments (Figure 1.23c). The S5 and S6 segments form the channel pore, a pathway for ions to pass through, as well as a selective filter to restrict the passage of calcium ion thanks to a key amino acid residue (usually glutamate or aspartate). The S4 segment is a voltage sensor, containing motif of positively charged residues. When the membrane is sufficiently depolarized and the intramembrane potential became more positive, the positively charged S4 segment moves towards the cell surface, resulting in a conformational change and the opening of the channel. The segment S1 and S2 are linked together by a short loop. This S1-S2 linker, the intracellular loops and the C-terminal of VGCC are the preferred sites for protein interaction. The opening of the VGCC causes an incoming calcium current that is attenuated in time due to two parameters: the voltage dependent inactivation (VDI) and the calcium dependent inactivation (CDI). This prevents unwanted prolonged depolarization, finely control the activity of the cells and avoid excessive ion influx. VDI is the process that happens in many voltage-gated channel, where the channel is closed shortly after being opened by the depolarizing membrane potential. The channels recover from the inactivation and open again when the membrane potential returns to its negative resting potential. Some VGCCs can enter into an inactivated state by the binding of calcium coming through the channel with the specific regulatory protein calmodulin (CaM). Calcium-bound calmodulin goes through a conformational change and reinforces its bonding with the C-terminal part of the VGCC, promoting inactivation of the channel.

The auxiliary subunit β regulates the VGCC levels at the cell surface and their voltage dependent activation and inactivation [START_REF] Buraei | The beta Subunit of Voltage-Gated Ca 2+ Channels[END_REF]. β subunits bind to the I-II intracellular loop of Ca V 1 and Ca V 2 to exercise their function.

The two polypeptides α2 and δ are encoded into a preprotein by the same gene, then that preprotein is proteolytically processed into α2 and δ, and they make up the α2δ subunit [START_REF] De Jongh | Subunits of purified calcium channels. Alpha 2 and delta are encoded by the same gene[END_REF][START_REF] Jay | Structural characterization of the dihydropyridine-sensitive calcium channel alpha 2-subunit and the associated delta peptides[END_REF]. There are four types of α2δ subunits: CACNA2D1, CACNA2D2, CACNA2D3 and CACNA2D4. The α2 subunit is an extracellular glycoprotein linked by a disulfide bond with the δ subunit, which is itself bound on the membrane with a GPI anchor (Figure 1.23c). The α2δ interacts with the first extracellular loop of domain I of the α1 subunit via its Von Willebrand factor-A domain (Figure 1.23c, [START_REF] Cantí | The metal-ion-dependent adhesion site in the Von Willebrand factor-A domain of α 2 δ subunits is key to trafficking voltage-gated Ca 2 + channels[END_REF]). This VWA domain has a metal-ion-dependent adhesion site (MIDAS) that binds to a divalent metal (usually magnesium), and mutation in this MIDAS disabled the effect of α2δ subunit on the VGCC. α2δ subunits regulate the activation and inactivation of VGCC (to a variable degree depending on the subtype) and increase the current density (the amount of ions pass through the channel) [START_REF] Dolphin | Calcium channel auxiliary α2δ and β subunits: trafficking and one step beyond[END_REF][START_REF] Zamponi | The Physiology, Pathology, and Pharmacology of Voltage-Gated Calcium Channels and Their Future Therapeutic Potential[END_REF]. α2δ subunits, with the presence of β subunits, also increase the cell surface expression of VGCC.

Only one of subunit γ (γ1) is detected with Ca V 1 at the muscle, and none of the γ subunits are present with Ca V 2 subtypes in the brain [START_REF] Müller | Quantitative proteomics of the Cav2 channel nano-environments in the mammalian brain[END_REF]. γ subunits do not seem to play a major role in VGCC, as various γ subunits such as γ2 (stargazin), γ3, γ4 and γ8 were identified as members of the transmembrane AMPA regulatory proteins family (TARP) [START_REF] Tomita | Functional studies and distribution define a family of transmembrane AMPA receptor regulatory proteins[END_REF].

Calcium signaling

VGCC in human

There are 4 'L-type' α1 subunit in vertebrate, Ca

V 1.1, Ca V 1.2, Ca V 1.3, and Ca V 1.4 (Fig- ure 1.23a
). These channels function quite similarly, but they are expressed in different parts of the body with different biophysic properties. Ca V 1.1 is the only α1 subunit expressed in the skeletal muscle fibre, working directly with the ryanodine receptor (RyR) to induce muscle contraction through the process of excitation-contraction coupling. Ca V 1.4 is expressed in the photoreceptor neurons of the retina, playing an important role in our ability to see, as mutations in Ca V 1.4 decrease visual acuity and cause night blindness [START_REF] Boycott | A summary of 20 CACNA1F mutations identified in 36 families with incomplete X-linked congenital stationary night blindness, and characterization of splice variants[END_REF]. Ca V 1.2 and Ca V 1.3 are expressed in a variety of tissues, not just in one tissue like Ca V 1.1 and Ca1.4, such as the neurons, cardiac muscle cells, sinoatrial node, ... They are implicated in neuronal, cardiovascular and endocrine functions. However, Ca V 1.2 and Ca V 1.3 do have differences in their functions and their expression. In the heart, Ca V 1.2 is largely in the heart muscle to control muscle contraction and Ca V 1.3 is in the sinoatrial node to play the pacemaker role. Ca V 1.2 represents 90% of calcium channel in the brain and the rest is Ca V 1.3. These channels are located in the soma and the dendrites of neurons, regulating excitability and participating the calcium signaling pathway that results in gene regulation. Mutations in Ca V 1.2 that removed the VDI of the channel cause a form of autism known as Timothy syndrome, which is characterized by physical deformations as well as neurological and developmental defects [START_REF] Splawski | Severe arrhythmia disorder caused by cardiac L-type calcium channel mutations[END_REF]. There is evidence that mutations in other Ca V are also associated with autism [START_REF] Yuen | Whole genome sequencing resource identifies 18 new candidate genes for autism spectrum disorder[END_REF].

The 3 Ca V 2 α1 subunits (Figure 1.23a) are located at the nerve terminal. They are primarily involved in synaptic function, they trigger the release of neurotransmitters as the membrane is depolarized. The closer the channels are to the exocytosis machinery the more neurotransmitters are released

The Ca V 3 family consists of 3 members (Figure 1.23a), defining the T-type VGCC. Their threshold of activation and inactivation are more negative than the other two family. These proteins contribute to the pacemaker activity, hormone secretion and they regulate neuron excitability [START_REF] Zamponi | The Physiology, Pathology, and Pharmacology of Voltage-Gated Calcium Channels and Their Future Therapeutic Potential[END_REF].

VGCC in C. elegans

In C. elegans, there are 9 genes coding for VGCC, listed in Table 1.2. Three genes are considered homologs to human α1 subunits by comparing percentage of identity between the protein sequences: egl-19 belongs with the Ca V 1 family (59% identity) [Lee et al., 1997], unc-2 is a Ca V 2 (60% identity) [Schafer and Kenyon, 1995] and cca-1 resembles a Ca V 3 (40% identity) [Steger et al., 2005]. EGL-19 is expressed in both the muscle system Chapter 1. Introduction (body wall muscle, pharyngeal muscle, anal depressor muscle) and the nervous system (head ganglion, motor neurons) [START_REF] Arellano-Carbajal | Macoilin, a Conserved Nervous System-Specific ER Membrane Protein That Regulates Neuronal Excitability[END_REF], Jospin et al., 2002[START_REF] Kim | The Dystrophin Complex Controls BK Channel Localization and Muscle Activity in Caenorhabditis elegans[END_REF], Lee et al., 1997]. UNC-2 is expressed exclusively in the nervous system [Mathews et al., 2003]. The two α2δ subunit UNC-36 and TAG-180 as well as the two β subunits CCB-1 and CCB-2 were identified by genomic analysis [START_REF] Bargmann | Neurobiology of the Caenorhabditis elegans Genome[END_REF]. UNC-36 is expressed in neurons and muscles [Frøkjaer-Jensen et al., 2006].

The body wall muscle is controlled by EGL-19, since specific inhibition of EGL-19 by nemadipine (a dihydropyridine agonist that blocks Ca V 1) shuts down activity in the muscle [Lainé et al., 2011]. EGL-19 is also the only functional α1 subunit in the muscle, as mutations in unc-2 and cca-1 do not change the current of calcium compared to wild type. Null mutations in egl-19 are embryonic lethal, thus the viable hypomorph mutants are more often studied. tag-180, ccb-2 mutants also do not affect the calcium current, but unc-36 mutants have slower activation kinetic, lower maximum conductance, faster to deactivate. Furthermore, muscle cells without ccb-1 do not produce voltage-dependent calcium courant. The auxiliary subunits present in the muscle are then the α2δ subunit UNC-36 and the β subunits CCB-1.

The Ca V 2 UNC-2 localizes to presynaptic active zone, mediating the release of both acetylcholine and GABA [Mathews et al., 2003]. UNC-2 calcium current also drives the oscillation in motor neurons [START_REF] Gao | Excitatory motor neurons are local oscillators for backward locomotion[END_REF]. unc-2 loss-of-function mutants are slow and uncoordinated, as well as reduced intrinsic neuronal calcium oscillations in motorneurons.

In contrast, gain-of-function mutations in unc-2 increase calcium currents and lead to hyperactive motor behaviors [START_REF] Huang | Gain-of-function mutations in the UNC-2/CaV2α channel lead to excitation-dominant synaptic transmission in Caenorhabditis elegans[END_REF].

The Ca V 3 CCA-1 and Ca V 1 EGL-19 control the excitability of cells in the pharyngeal muscle. CCA-1 initiates the depolarization with its lower activation threshold, then EGL-19 takes over and extending the depolarizing current. The pharynx's pumping frequency is reduced by 20% in cca-1 mutants compared to wild type [Steger et al., 2005]. egl-19 loss-of-function mutants show slow kinetics of the terminal bulb action potential during depolarization phase, while egl-19 gain-of-function mutants increase the duration of the action potential [Lee et al., 1997].

Calcium's role in axon outgrowth

The complex relationship of calcium and neurite growth

As for the role of calcium in axonal outgrowth, early studies about the relationship between calcium and growth cone were contentious. Addition of inorganic calcium channel blockers to the growth medium was reported to prevent neurite outgrowth [Nishi and 1.3. Calcium signaling Berg , 1981[START_REF] Suarez-Isla | Blockers of calcium permeability inhibit neurite extension and formation of neuromuscular synapses in cell culture[END_REF]. However, other studies suggested that outgrowth could happen in presence of calcium channel blockers [START_REF] Letourneau | MIGRATORY CELL LOCOMOTION VERSUS NERVE AXON ELONGATION[END_REF], as well as in the absence of extracellular calcium [START_REF] Bixby | Early differentiation of vertebrate spinal neurons in the absence of voltage-dependent Ca2+ and Na+ influx[END_REF]. Raising the level of intracellular calcium increases neurite outgrowth in some case [Anglister et al., 1982, Nishi and[START_REF] Nishi | Effects of high K+ concentrations on the growth and development of ciliary ganglion neurons in cell culture[END_REF] but inhibits outgrowth in others [START_REF] Haydon | Serotonin Selectively Inhibits Growth Cone Motility and Synaptogenesis of Specific Identified Neurons[END_REF][START_REF] Robson | L-type calcium channels in the regulation of neurite outgrowth from rat dorsal root ganglion neurons in culture[END_REF][START_REF] Suarez-Isla | Blockers of calcium permeability inhibit neurite extension and formation of neuromuscular synapses in cell culture[END_REF]. Then, with the development of the first calcium indicator fura-2 [START_REF] Grynkiewicz | A new generation of Ca2+ indicators with greatly improved fluorescence properties[END_REF], a lot of work was done to examine what actually happen to the intracellular calcium level during all the different treatments e.g. inorganic calcium channel blockers [START_REF] Mccaig | Studies on the mechanism of embryonic frog nerve orientation in a small applied electric field[END_REF], ionophore [START_REF] Mills | Neuron-specific and state-specific differences in calcium homeostasis regulate the generation and degeneration of neuronal architecture[END_REF], calcium-free medium [START_REF] Campenot | Growth of sympathetic nerve fibers in culture does not require extracellular calcium[END_REF]] ... The results showed that changes in the intracellular calcium level of each treatment were as intended but short-lived (less than an hour), suggesting a very efficient system maintaining calcium homeostasis. Calcium concentrations in the growth cone are regulated either by calcium influx through the VGCCs or by release of calcium from intracellular stores (either by the mechanism of Calcium-induced Calcium Release (CICR) [START_REF] Bedlack | Localized membrane depolarizations and localized calcium influx during electric field-guided neurite growth[END_REF][START_REF] Holliday | Calcium-induced release of calcium regulates differentiation of cultured spinal neurons[END_REF] or store-operated calcium entry [START_REF] Shim | A critical role for STIM1 in filopodial calcium entry and axon guidance[END_REF]).

Shortly after, the relationship between specific intracellular calcium levels and growth cone activity was established [START_REF] Lankford | Roles of actin filaments and three secondmessenger systems in short-term regulation of chick dorsal root ganglion neurite outgrowth[END_REF]. Increase in calcium level (by addition of an ionophore) or decreases in calcium levels (by removing extracellular calcium) both prevent neurite outgrowth, and both effects are reversible. It was then suggested that axonal outgrowth occurs in a determined range of intracellular calcium [Kater and Mills, 1991]. Within that range there is an optimal level of calcium that promotes outgrowth: if the cell calcium level is suboptimal but permissible, increasing calcium level to an optimal level would lead to more outgrowth. On the other hand, if the calcium level is at optimal, increasing it further above the permissible range would stop any neurite outgrowth. This optimal level of calcium is dubbed the "set-point".

Eventually, later work has demonstrated that calcium can regulate growth cone in ways Chapter 1. Introduction much more complex than simple shift of intracellular calcium level over the entire growth cone [START_REF] Gomez | Regulation of growth cone behavior by calcium: new dynamics to earlier perspectives[END_REF]. For example, depolarizing treatments that cause a rise in intracellular calcium level such as high KCl pulses, neurotransmitters or electric field, applied broadly to the growth cone usually result in loss of filopodia and occasional growth cone collapse [START_REF] Fields | Accommodation of mouse DRG growth cones to electrically induced collapse: Kinetic analysis of calcium transients and set-point theory[END_REF][START_REF] Neely | Role of substrate and calcium in neurite retraction of leech neurons following depolarization[END_REF][START_REF] Rehder | Regulation of neuronal growth cone filopodia by intracellular calcium[END_REF]. However, those treatments applied locally to the growth cone do not cause collapse but steer it in a certain direction instead. Local stimulation of calcium influx with electric field [START_REF] Bedlack | Localized membrane depolarizations and localized calcium influx during electric field-guided neurite growth[END_REF], calcium ionophore [START_REF] Gundersen | Characterization of the turning response of dorsal root neurites toward nerve growth factor[END_REF] or neurotransmitters [START_REF] Zheng | Essential role of filopodia in chemotropic turning of nerve growth cone induced by a glutamate gradient[END_REF][START_REF] Zheng | Turning of nerve growth cones induced by neurotransmitters[END_REF] can guide axon growth in the direction of the localized elevation of intracellular calcium level.

It was established then that calcium is an essential component of axon guidance pathway. A calmodulin-dependent protein kinase II (CaMKII)-calcineurin (CaN) switch often initiates either an attractive or repulsive response to elevations of intracellular calcium [START_REF] Sutherland | The interdependent roles of Ca 2+ and cAMP in axon guidance: The Interdependent Roles of Ca 2+ and cAMP in Axon Guidance[END_REF]. This turning response necessitates an asymmetric distribution of signaling molecules between the intracellular and the extracellular side of the growth cone. It was reported that an asymmetric calcium elevation is sufficient to cause growth cone turning (Figure 1.24): large intracellular calcium increases with normal extracellular calcium level causes attraction response [START_REF] Zheng | Turning of nerve growth cones induced by localized increases in intracellular calcium ions[END_REF]. Vice versa, small intracellular increases causes repulsion response [START_REF] Wen | A CaMKII/Calcineurin Switch Controls the Direction of Ca2+-Dependent Growth Cone Guidance[END_REF].

More evidence of calcium signaling positively affecting the growth rate of growth cone were found by [START_REF] Arie | Developmental changes in the regulation of calcium-dependent neurite outgrowth[END_REF]. Inhibition of the calcium channel IP3 receptors diminishes the neurite growth during early development but not later stages. Conversely, inhibition of RyR inhibits the neurite outgrowth at later stage, but not in earlier one. Furthermore, higher frequencies of spontaneous local calcium transients correlate with more rapidly growing processes, at the expense of processes in other place of the growth cone. Vice versa, reducing frequencies of calcium transients reduces the incidence of outgrowth [START_REF] Hutchins | Differential Outgrowth of Axons and their Branches Is Regulated by Localized Calcium Transients[END_REF].

Calcium is thought to be involved in growth cone motility through two possible pathway. The first is through the brain-derived neurotrophic factor (BDNF). BDNF is found to induce axonal growth by triggering a cytoplasmic calcium signaling pathway that results in a clustering of the membrane receptor called β-1 integrin onto the growth cone. This cluster of β-1 integrin increases the adhesion of the growth cone to attractive chemotropic cues e.g. netrin and promotes axon outgrowth. By blocking the entry of extracellular calcium into the cytoplasm, both the clustering of β-1 integrin and the axon outgrowth are prevented, suggesting that calcium influx is necessary for BDNF-induced axon outgrowth [START_REF] Carlstrom | Bidirectional remodeling of β1-integrin adhesions during chemotropic regulation of nerve growth[END_REF]. The second mechanism involving calcium which modulates axonal growth is the eukaryotic elongation factor-2 (eEF2), a crucial transcription factor regulated by calcium and calmodulin [START_REF] Kaul | Eukaryotic elongation factor-2 (eEF2): its regulation and peptide chain elongation[END_REF]. eEF2 in its dephosphorylated form promotes mRNA translation, but phosphorylated eEF2 inhibits translation [START_REF] Iketani | Regulation of neurite outgrowth mediated by localized phosphorylation of protein translational factor eEF2 in growth cones[END_REF]. It has been found that particular calcium elevations in the growth cone lead to phosphorylation of eEF2 by eEF2 kinase [START_REF] Iizuka | Calcium-induced synergistic inhibition of a translational factor eEF2 in nerve growth cones[END_REF][START_REF] Iketani | Regulation of neurite outgrowth mediated by localized phosphorylation of protein translational factor eEF2 in growth cones[END_REF], meaning an inhibition of mRNA translation and a reduction of axonal growth. Calcium elevations also decrease the total amount of eEF2 in the growth cone. Conversely, BDNF is known to increase the level of active, dephosphorylated eEF2 [START_REF] Takei | Brain-derived Neurotrophic Factor Enhances the Basal Rate of Protein Synthesis by Increasing Active Eukaryotic Elongation Factor 2 Levels and Promoting Translation Elongation in Cortical Neurons[END_REF]. This suggests that the nature of the calcium elevations is important to understand eEF2 phosphorylations, as influx of extracellular calcium functions with BDNF to promote axonal growth.

A recent study has found that a mechanosensitive ion channel, DmPiezo, functions to restrict axon regrowth in Drosophila [START_REF] Song | The Mechanosensitive Ion Channel Piezo Inhibits Axon Regeneration[END_REF]. This ion channel is permeable to calcium and it transforms mechanical stimulation into electrical signal by influx of cations [START_REF] Coste | Piezo1 and Piezo2 Are Essential Components of Distinct Mechanically Activated Cation Channels[END_REF]. Knock-out of the DmPiezo channel is sufficient to promote axon regeneration, and overexpression of the DmPiezo channel reduces the percentage of regenerated axons. Furthermore, it was demonstrated that, after axon injury, Piezo inhibits axon regeneration by a calcium-dependent CaMKII, which activates a nitric oxide synthase (NOS) and the downstream cGMP kinase. This seems to indicate that calcium entry from different sources such as VGCC, RyR, IP3R or CICR, can have varying effects on axon growth.

Role of VGCC in neurite growth

As mentioned in the Axon regeneration and The PHR system section, L-type VGCC is responsible for the immediate response to axon injury. A rapid rise in intracellular calcium was observed by calcium imaging, starting at the injury site then quickly spread to the soma. This rise in calcium is due to the opening of L-type VGCC. Then, intracellular calcium can trigger the DLK-1 pathway by dissociating the inhibitory dimer DLK-1L/DLK-1S [START_REF] Yan | Regulation of DLK-1 Kinase Activity by Calcium-Mediated Dissociation from an Inhibitory Isoform[END_REF], promoting axon regrowth [START_REF] Ghosh-Roy | Calcium and Cyclic AMP Promote Axonal Regeneration in Caenorhabditis elegans and Require DLK-1 Kinase[END_REF].

For many sensory neurons in vertebrates, VGCC seems to play an inhibitory role in axon regrowth. Several studies have shown that electrical activity is a negative signal for axon regrowth in both immature and adult sensory neurons of the dorsal root ganglia (DRG) [Enes et al., 2010, Robson and[START_REF] Robson | L-type calcium channels in the regulation of neurite outgrowth from rat dorsal root ganglion neurons in culture[END_REF]]. L-type VGCC is implicated as the mediator of axon growth inhibition, activated by the electrical activity. The short term effect of L-type VGCC seems to be changing the cytoskeletal structure of the growth cone, and the long term effect could be at the transcriptional level, promoting either intrinsic growth inhibitory factors, or suppressors of growth-promoting proteins [START_REF] Enes | Electrical Activity Suppresses Axon Growth through Cav1.2 Channels in Adult Primary Sensory Neurons[END_REF]. The subunit α2δ which regulates the activity of α1 subunits was also found to suppress axon regeneration of DRG neurons [START_REF] Tedeschi | The Calcium Channel Subunit Alpha2delta2 Suppresses Axon Regeneration in the Adult CNS[END_REF]. Overexpression of CACNA2D2 in dissociated adult DRG neurons impaired axon elongation: there was a drastic reduction in axon length as well as increased branching compared to control condition. In control animal, peripheral DRG axons regenerated 4 days after injury, while overexpressing CACNA2D2 in peripheral DRG axons prevented regeneration over long distances. Furthermore, cultured Cacna2d2 mutant cells displayed increased axon length and decreased branching. Downregulated Cacna2d2 expression also promoted axon growth. It was also shown that CACNA2D2 inhibits axon growth by stimulating calcium influx through N-and P/Q-type VGCC. In C. elegans, a new regeneration pathway, independent from the DLK-1 pathway, was found in the sensory neuron ASJ [START_REF] Chung | Novel DLK-independent neuronal regeneration in Caenorhabditis elegans shares links with activity-dependent ectopic outgrowth[END_REF]. This regeneration pathway is triggered by reduction of sensory activity i.e. severing sensory dendrites, or by reduction of calcium activity. Blocking EGL-19 (Ca V 1.2 homolog) with nemadipine-A and loss-of-function egl-19 mutants both triggered 1.3. Calcium signaling regeneration on their own, as well as causing ectopic outgrowths. Loss-of-function mutants of unc-43 /CaMKII also displayed regenerative effect, suggesting that CaMKII functions to mediate the inhibitory effect of calcium influx on axon growth.

Outside of sensory neurons, L-type VGCC is reported to play a crucial role in neuronal development [START_REF] Wong | Activity-dependent regulation of dendritic growth and patterning[END_REF], for example by promoting neutrophin-induced dendritic growth in cortical neurons [START_REF] Mcallister | Neurotrophin regulation of cortical dendritic growth requires activity[END_REF]. Presynaptic and postsynaptic neuronal activities are found to be essential during axon development of callosal projection neurons [Mizuno et al., 2010, Tezuka et al., 2022] For motor neurons and retinal ganglion cells (mentioned in section Activity-dependent plasticity in the visual system), activity seems to enhance axon growth [Goldberg et al., 2002, Singh andMiller, 2005]. Electrical activity potentiates axon growth in sympathetic motor neurons in two ways: the activation of CaMKII-MAPKK pathway by the calcium influx in the axon, and the neutrophin-mediated axon growth [Singh and Miller, 2005]. In PC12 cells cultured in low serum medium, calcium influx through VGCC induces neurite growth by activating the protein tyrosine kinases, including the proto-oncogene tyrosine-protein kinase Src. This in turn forms an Shc/Grb2 complex, activates Ras and MAPK, subsequently induces the nerve growth factor regulating gene NGFI-A [START_REF] Rusanescu | Calcium influx induces neurite growth through a Src-Ras signaling cassette[END_REF].

In cortical neurons, it is reported that calcium influx via VGCC induced dendritic growth by activating the CaM kinase IV [START_REF] Redmond | Calcium Regulation of Dendritic BIBLIOGRAPHY Growth via CaM Kinase IV and CREB-Mediated Transcription[END_REF]. A constitutionally active form of CaMKIV also induced dendritic growth. The mechanism seems to be that CaMKIV activates the transcription factor cAMP response element-binding (CREB), as dominant negative form of CREB negates dendritic growth induced by both calcium influx and CaMKIV.

Recent studies have shown more evidence of L-type VGCC's role in neurite extension and morphology. It is reported that the developing mouse excitatory cortical neurons produce Spontaneous Regenerative Calcium Transients (SRCaTs) [Kamijo et al., 2018]. These calcium transients originated from L-type VGCC since they are strongly inhibited by hyperpolarization, blocked by a dihydropyridine derivative and are not prevented by inhibiting action potential (by using TTX that blocks sodium channels). Furthermore, expression of a dihydropyridine-resistant L-type VGCC succeed in restoring these calcium transients. The SRCaTs seem to promote neurite growth, as knock-out of the L-type VGCC produces shorter neurites than the control. In addition, gain-of-function mutation in L-type VGCC, the one that causes Timothy syndrome in human, causes an increase in SRCaTs, resulted in aberrant migration of the cortical neurons. The same gain-of-function mutation causing Timothy syndrome in C. elegans' L-type VGCC, egl-19, produces an axon termination failure phenotype in the touch receptor neurons ALM and PLM [Buddell Chapter 1. Introduction et al., 2019, Buddell andQuinn, 2021]: the axons of these 2 neurons do not stop growing at the designated target, they overextended a significant amount. It was also discovered that VGCCs negatively regulate axon termination, as loss-of-function or null mutations of VGCC genes such as the α1 subunits egl-19 and unc-2, along with the α2δ subunit unc-36, all suppressed the axon termination defect of fsn-1 mutants, but not rpm-1. Thus, VGCCs function specifically to counteract the FSN-1 pathway, presumably promoting axon growth.

Calcium imaging

Given the importance of calcium as a second messenger, monitoring calcium level is a useful way to gain information about the activities of a particular system. fura-2, a ratiometric dye, was the first widely used calcium indicator [START_REF] Grynkiewicz | A new generation of Ca2+ indicators with greatly improved fluorescence properties[END_REF], opening new doors for research in calcium in homeostasis. As of now, the synthetic dyes were replaced by the genetically encoded calcium indicator (GECI) which can be engineered to be expressed in a tissue of choice and to be used in C. elegans.

A popular GECI family is the GCaMP, first developed by [START_REF] Nakai | A high signal-to-noise Ca2+ probe composed of a single green fluorescent protein[END_REF]. In principle, it is a permuted Green Fluorescent Protein (GFP), engineered to contain a calmodulin (CaM) domain at the C-terminus and a calmodulin-binding peptide RS20 at the N-terminus [START_REF] Lukas | Calmodulin binding domains: characterization of a phosphorylation and calmodulin binding site from myosin light chain kinase[END_REF]. At resting state, GCaMP is exposed to water and has little fluorescence. In presence of calcium ion, the CaM binds to the calcium ion and go through a conformational change, thus binding tightly to the RS20 domain. This prevents water from reaching the chromophore which quickly deprotonates and fluoresces brightly in its anionic form. The GCaMP family has been through multiple engineering improvements, the version used in this study is GCaMP6s [START_REF] Chen | Ultrasensitive fluorescent proteins for imaging neuronal activity[END_REF]].

An approach to make use of GECI is pseudo-ratiometric imaging. Unlike the ratiometric dye which fluoresces in two wavelengths and the intensity of both colors shifts with the concentration of calcium, this approach uses a fusion of the GECI with a calcium-insensitive fluorophore. The fluorescence of the GECI and the calcium-insensitive fluorophore both depends on the expression level of the protein, but the former depends on the intracellular calcium level as well. The ratio of the GECI against the other fluorophore gives a measure of the calcium level while eliminating most of the variation due to protein expression level.

Chapter 2

Results

Regulation of axonal morphology in SAB neurons

Work-in-progress article below.

List of figures presented in the article: [START_REF] Lowrie | Dependence of postnatal motoneurones on their targets: review and hypothesis[END_REF]Vrbová, 1992, Navarrete and[START_REF] Navarrete | Activity-dependent interactions between motoneurones and muscles: Their role in the development of the motor unit[END_REF] and following muscle denervation: undamaged motor nerves extend their axons towards the denervated fibers, forming neuromuscular junctions (NMJ) and [START_REF] Brown | A central role for denervated tissues in causing nerve sprouting[END_REF], Brown and Ironton, 1977, Holland and Brown, 1980[START_REF] Pamphlett | Early terminal and nodal sprouting of motor axons after botulinum toxin[END_REF]]. The hypothesized model is that inactive muscles release a retrograde factor that acts on the motor axon to regulate axonal morphology [Brown et al., 1981].

February 18, 2023 1/32

Despite the identification of several important factors, the molecular mechanism underlying this retrograde communication remains unclear [START_REF] English | Cytokines, growth factors and sprouting at the neuromuscular junction[END_REF].

The Caenorhabditis elegans organism model is a suitable model system to study axon development and shaping. The nematode's nervous system is simple, containing only 302 neurons, whose connectome has been extensively studied [Cook et al., 2019, White et al., 1986, Witvliet et al., 2021]. Its genome is completely sequenced and easy to manipulate with up-to-date genome engineering techniques, such as CRISPR/Cas9 and the use of Mos1 transposons for single-copy transgene insertions [START_REF] Chen | Targeted genome engineering in Caenorhabditis elegans[END_REF][START_REF] Farboud | Targeted genome editing in Caenorhabditis elegans using CRISPR/Cas9: CRISPR/Cas9 genome editing in C. elegans[END_REF][START_REF] Nance | The Caenorhabditis elegans Transgenic Toolbox[END_REF]. 36% of predicted C. elegans genes have a human match [C. elegans Sequencing Consortium, 1998[START_REF] Rubin | Comparative genomics of the eukaryotes[END_REF], and about 42% of the human genes involved in disease have an ortholog in C. elegans [START_REF] Markaki | Modeling human diseases in Caenorhabditis elegans[END_REF]. Activity-dependent plasticity has been early described in C. elegans amphid sensory neurons [START_REF] Peckol | Sensory activity affects sensory axon development in C. elegans[END_REF] and SAB neurons [Zhao and Nonet, 2000]. SAB are cholinergic motor neurons, whose cell bodies are located in the head of the worm, on the ventral side, in the retrovesicular ganglion (Figure 1A). SABVL and SABVR (SAB ventral left and ventral right) send axons anteriorly, whereas SABD (SAB dorsal) sends an axon dorsally at first, then branches to give two neurites growing anteriorly. The four SAB projections run toward the tip of the nose along the muscle cells, forming en passant neuromuscular synapses on head muscle cells. When synaptic transmission or muscle activity is impaired, SAB exhibit axonal overgrowth [Zhao and Nonet, 2000]. Axonal overgrowth has been well studied in the touch receptor neurons AVM and PVM: axonal termination defects were observed in several mutants, including rpm-1 and fsn-1 [START_REF] Borgen | RPM-1 regulates axon termination by affecting growth cone collapse and microtubule stability[END_REF], Grill et al., 2007, Schaefer et al., 2000]. rpm-1 encodes a ubiquitin ligase that belongs to the well-conserved PHR (Highwire in Drosophila, Phr-1 in mouse and Pam/MYCB2 in human) protein family. RPM-1 forms a multi-subunit complex with the F-box protein FSN-1 [Liao et al., 2004] that promotes axon termination [Nakata et al., 2005, Schaefer et al., 2000, Zhen et al., 2000]. In addition, RPM-1 also acts on axon termination through a FSN-1 independent pathway, involving the guanine nucleotide exchange factor GLO-4 [Grill et al., 2007]. PLM axonal overgrowth phenotype observed in fsn-1 can be suppressed by mutations in genes encoding voltage-gated calcium subunits [Buddell et al., 2019, Buddell andQuinn, 2021].

Calcium signaling plays a major role in axon development. It is thought that a certain level of intracellular calcium is required for axon growth, and there is an optimal level of calcium, known as the "set-point", that maximizes outgrowth [Kater and Mills, 1991].

Evidence indicate that global calcium activity in the growth cone seems to have a negative regulating effect on axon growth [START_REF] Gomez | In vivo regulation of axon extension and pathfinding by growth-cone calcium transients[END_REF][START_REF] Jacques-Fricke | Ca2+ Influx through Mechanosensitive Channels Inhibits Neurite Outgrowth in Opposition to Other Influx Pathways and Release from Intracellular Stores[END_REF][START_REF] Tang | Spontaneous Calcium Transients in Developing Cortical Neurons Regulate Axon Outgrowth[END_REF], while calcium transients localized to a specific area of the growth cone promote outgrowth in the direction of attractive guidance cues [Tang andKalil, 2005, Zheng, 2000]. Voltage-gated calcium channels (VGCCs) are a source of calcium influx from the extracellular compartment and several studies have suggested February 18, 2023 2/32 that they play a crucial role in neuronal development [Singh andMiller, 2005, Wong andGhosh, 2002]. In C. elegans, there are 3 genes coding for α1 subunits that constitute the main pore of VGCC: egl-19 -homolog of human (Ca V 1.2) CACNA1C [Lee et al., 1997], unc-2 -homolog of human (Ca V 2.2) CACNA1B [Schafer and Kenyon, 1995],

and cca-1 -homolog of human (Ca V 3.3) CACNA1I [Steger et al., 2005]. EGL-19 and UNC-2 are regulated by the auxiliary subunit α2δ UNC-36 [Lainé et al., 2011, Saheki and Bargmann, 2009, Tong et al., 2017]. Evidence show that gain-of-function mutations of the egl-19 gene produce an axon termination failure phenotype in the touch receptor neurons ALM and PLM [Buddell et al., 2019, Buddell andQuinn, 2021].

Here we demonstrated that decrease of muscle activity during a critical developmental phase can induce overgrowth of SAB axons, leading to ectopic supplemental synapses.

We showed that local inhibition of the head muscles is sufficient to impact SAB morphological development. Furthermore, we identified the neuronal VGCCs as one important component underlying the mechanism of SAB muscle-activity dependent overgrowth. Finally, we established that activity in SAB neurons is essential to develop axon overgrowth.

Materials and methods

Plasmids

The plasmids used in this work are listed in Table 1. All plasmids were built using Gibson assembly [Gibson, 2011] The strains used in this work are listed in Table 3.

Table 1. List of plasmids

All strains were maintained at 20°C on nematode growth medium (NGM) using

Escherichia coli OP50 as food source [Brenner, 1974].

Microscopy and SAB scoring

To decrease the activity of muscle, we expressed the histamine-gated chloride channel (HisCl1) [Pokala et al., 2014] tagged with BFP in the muscle cells (krSi16

[Pmyo-3::HisCl1::SL2::tagBFP]).

The following protocol is used to score SAB axonal defects: For each genotype, one control plate and one Histamine-treated plate are prepared with 20 gravid worms each.

The gravid worms were to lay eggs for 2 to 5 hours (in order to obtain a synchronized population), then they are removed from the plates. The control plate is treated with 300µl of water and the treatment plate is treated with 300µl of Histamine (Sigma Aldrich H7250, stock concentration of 1M in water, treatment concentration of 1/30 M) to a final concentration of 1mM. After three days, worms were mounted on dry pads February 18, 2023 4/32 Reporter for SAB neurons [Nonet, 1999] krSi16 [Pmyo-3::HisCl1::SL2::tagBFP] miniMos single-copy insertion of pAW24 Alexis Weinreb krSi24 miniMos single-copy insertion of pAW28 Alexis Weinreb krSi66 krSi-alleles were constructed by miniMos insertions [Frøkjaer-Jensen et al., 2014]. krIs-alleles are integration of extrachromosomal array using the modular safe-harbor transgene insertion (MosTI) method [El Mouridi et al., 2022], except for krIs80 which was integrated by irradiation [Mariol et al., 2013].

(2% agarose in water, dry overnight) and immobilized with 4% polyethylene beads :mNeonGreen], are then observed on a Zeiss Axioskop compound microscope.

For each experiment, at least 3 replicates were performed. The n indicated for each figure corresponds to the number of individual worms per condition.

To examine the impact of histamine-induced muscle inhibition on the synaptic receptor's density and count varicosities along the SAB axons, we used the EN3939 strain carrying the unc-29(kr208::tagRFP-T) allele which is a reporter of levamisole-sensitive acetylcholine receptor (L-AChR). The protocol is the same as one described in previous paragraph but imaged on Andor Spinning Disk confocal microscope. Raw fluorescent intensity of the unc-29(kr208::tagRFP-T) reporter for both conditions is normalized to the control condition to obtain the relative fluorescence of L-AChR.

To induce SAB overgrowth with the histamine-gated cation channel (HisCat) in SAB neurons, we used the EN8873 and EN8874 strains carrying the Punc-4::HisCat::SL2::tagBFP transgene. The protocol is as follows: One control plate and one Histamine-treated plate are prepared with 20 gravid worms of each. The gravid February 18, 2023 6/32 worms were to lay eggs for 2 to 5 hours then they are removed from the plates. The control plate is treated with 300µl of water and the treatment plate is treated with 300µl of histamine (treatment concentration of 1M) to a final concentration of 30mM.

SAB axons are then observed with the aforementioned protocol.

Calcium imaging

To monitor activity in SAB neurons, we used a ratiometric calcium imaging system [START_REF] Zheng | Calcium imaging of multiple neurons in freely behaving C. elegans[END_REF]. EN8871 and EN8872 strains were made to carry Punc-4(short)::NLS(SV40)::GCaMP6s::mCherry::NLS(EGL13): expressing a calcium indicator, GCaMP6s [START_REF] Chen | Ultrasensitive fluorescent proteins for imaging neuronal activity[END_REF], fused with a stable fluorophore mCherry as reference, specifically in the nucleus of SAB cell bodies. SAB cell bodies' positions are identified using NeuroPAL [Yemini et al., 2021]. The protocol is as follows: One control plate and one Histamine-treated plate are prepared with 20 gravid worms of each. The gravid worms were to lay eggs for 2 hours then they are removed from the plates. The control plate is treated with 300µl of water and the treatment plate is treated with 300µl of histamine (treatment concentration of 1/30 M) to a final concentration of 1mM.

After 16 hours, L1 worms in control condition are mounted on dry pads with only M9.

L1 worms in histamine-treated condition are mounted on dry pads with histamine (treatment concentration of 1/30 M). Mounted worms are then filmed using the dual channel microscope Nikon Eclipse Ti2 for 1 minute at 2 frame per second, then analyzed with a macro made by fellow team member Franklin Florin. The ratio of GCaMP6s/mCherry is weighted by the local contribution of the calcium indicator GCaMP fluorescence to the total fluorescence of the channel. This is done to better highlight calcium increase events. SAB activity is measured by the weighted ratio of indicator/reference of each worm in the region-of-interest (containing SAB cell bodies) over 120 frames, then normalized to the weighted ratio of control worms.

Statistics

All statistical analysis were done in R. The penetrance of SAB defect is compared using the Cochran-Mantel-Haenszel (CMH) test [Cochran, 1954, Mantel andHaenszel, 1959].

This modified χ 2 test takes into account the date when the experiments are performed when testing whether or not a genotype/treatment affects the amount of worms carrying SAB defects. As every experiment usually has 3 to 4 replicates, each replicate is performed in a different day and so the data is stratified by date/replicate. A series of 2 x 2 contingency tables is constructed by taking the a binary outcome of observed worms (normal SAB axons or defects SAB axons), as well as a binary predictor (either the genotype or the histamine treatment), one

A i B i N 1i Normal C i D i N 2i Column total M 1i M 2i T i 0.001).
The p-values of the statistical tests performed are listed in Supplementary Information S3.

Results

Inhibition of muscle electrical activity induces axonal overgrowth in SAB motor neuron

The SAB axons have been reported to display morphological changes, such as looping and sprouting (Figure 1B), when synaptic activity of SAB neurons is disturbed [Zhao and Nonet, 2000]. As muscle activity is decreased upon perturbation of synaptic activity, we tested whether the decrease in muscle activity alone also led to SAB overgrowth. To visualize SAB axons, we expressed the green fluophore mNeonGreen in SAB neurons using the promoter Punc-4. To decrease activity in the muscle, we expressed a single-copy of the histamine-gated chloride channel (HisCl1) in the muscle cells [Pokala et al., 2014]. Exposure of transgenic worms to histamine activates the HisCl1, then chloride ions flow in through the ion channel, hyperpolarizing the muscle cells and inhibiting electrical activity. This resulted in near complete paralysis of the transgenic worms upon histamine treatment, while no gross effect was visible on control wild-type animals.

Without histamine treatment, only 8 ± 6% of animals expressing HisCl1 exhibited either sprouting or looping, which was similar to the fraction observed in control worms (SAB overgrowth: 8 ± 6%). Quantification of SAB axonal defects showed a clear overgrowth phenotype with histamine for animals expressing HisCl1 in muscle : 82 ± 7% of the animals exhibited SAB axon sprouting or looping. In the absence of HisCl1, histamine did not induce any significant defect, validating the specificity of our assay (Figure 1C). Onward, we will refer to the animals expressing mNeonGreen in SAB and HisCl1 in muscle as the WT* genotype. WT* served as control for histamine effect and it is statistical significant in all cases.

SAB overgrowth could be induced either by the decrease of muscle activity, or indirectly by the complete paralysis and lack of movement of the worms. To discriminate between these two possibilities, we examined SAB axons in worms that carry a mutation in the gene encoding a Myosin Heavy Chain, unc-54. These mutants retain wild-type levels of electrical activity in the muscle but are paralysed due to the absence of a functional contractile apparatus [START_REF] Dibb | Sequence analysis of mutations that affect the synthesis, assembly and enzymatic activity of the unc-54 myosin heavy chain of Caenorhabditis elegans[END_REF]. We did not observe an increase of SAB axon overgrowth (13 ± 3% in unc-54 mutants, 7 ± 2% in WT* worms, 77 ± 3% in WT* February 18, 2023 8/32 worms on histamine) (Figure 1D). This indicates that the lack of electrical activity of 201 the muscle, rather than its contractile activity, gives rise to SAB overgrowth.

Inhibition of muscle electrical activity does not impact the number of SAB synapses

Since SAB motor neurons are cholinergic [Rivard et al., 2010], we monitored the expression of the two muscle acetylcholine receptors, the levamisole-sensitive receptors (L-AChR) and the nicotine-sensitive receptors (N-AChR) [Richmond and Jorgensen, 1999]. Synaptic expression was not observed for N-AChR (Supplementary Information S1), only for L-AChR. When the ectopic axonal branches grew in contact with muscle membrane, we could often observe the accumulation of postsynaptic L-AChRs in proximity to the ectopic varicosities (Figure 2A). This indicates that the ectopic branches can form functional synapses. To determine if there is a difference in synaptic density between normal and paralyzed worm, we measured the number of varicosities in WT* worms with histamine. For dorsal SAB axons of histamine-treated worms, we observed a significant increase in numbers of varicosities (13 ± 2 varicosities) compared to control worms (10 ± 2 varicosities). The same effect were measured in ventral axons, histamine-treated worms have 15 ± 2 varicosities, compared to 11 ± 2 in control worms. The increase of the amount of varicosities in SAB axons of histamine-treated worms is presumably due to the additional synapses formed at the overgrown axons.

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Inhibition of head muscle activity is sufficient to induce SAB axon overgrowth

We showed that SAB overgrowth was induced by inhibiting the electrical activity of the entire body wall muscle system. However the SAB axons only innervate the first muscle cells in the head of the worm (Figure 3A). SAB overgrowth could be mediated by a local signal; in that case inhibition of the head muscles would be sufficient for its induction. Alternatively, induction of SAB overgrowth could require inhibition of a high fraction of the muscle cells and be mediated by a long-range signal. To discriminate between these two hypotheses, we drove the expression of HisCl1 specifically in the head muscles by using a promoter sequence upstream of the gene tni-3 /troponin I [Takashima et al., 2012]. We observed a strong expression of HisCl1 in the head muscles innervated by the SAB axons (Figure 3A), as well as expression in the vulva and the stomatointestinal muscle (not shown). Upon addition of histamine, we could efficiently inhibit the head muscles of the worms, as was made evident by paralysis restricted to the head and neck region (Supplementary Movie S5). This reliably induced SAB overgrowth (50 ± 3% of the worms displayed SAB overgrowth on histamine, Figure 3B).

We concluded from this data that SAB overgrowth is mediated by a signaling pathway spatially restricted to the head region.

Axonal overgrowth is dependent of muscle activity during L1 stage

To determine the time window during which decreased muscle activity induces SAB overgrowth, we exposed worms to histamine only during part of the larval development (Figure 3C,D). While there was no overgrowth in worms not exposed to histamine (4 ± 2% of worms) and overgrowth in worms exposed during the whole larval development (80 ± 4%), exposure restricted to the L1 larval stage, but not during subsequent larval development, induced overgrowth (71 ± 5% and 17 ± 4% respectively). The SAB axons are thus sensitive to muscle activity only during a critical period included in the L1 stage. This is partially consistent with results previously reported with a temperature-sensitive mutant [Zhao and Nonet, 2000]. We next tested whether the critical period spans the entire L1 stage. Exposure to histamine only at the beginning, middle, or end of the L1 stage was sufficient to induce SAB overgrowth (34 ± 5%, 31 ± 5% and 16 ± 4% of the worms respectively, Figure 3C,D). Together, these results show that the SAB axons are sensitive to muscle activity throughout the L1 larval stage and only during this stage.

February 18, 2023 11/32 (scd-2 ), two in neuropeptide processing (egl-3 and egl-21 ), one in miRNA processing (alg-2 ) and one in signal transduction (plx-2 ).

The only gene candidate which displayed significant reductions in the penetrance of SAB overgrowth was egl-3 (Figure 4A). Mutant of egl-3 /PC2, a proprotein convertase required for the processing of almost all C. elegans neuropeptides [Husson et al., 2006],

suppressed the overgrowth of SAB axons to 35 ± 3%. It is noted that mutant of egl-21 /CpE, a carboxypeptidase implicated in the maturation of about half of C.

elegans neuropeptides [Husson et al., 2007], reduced the overgrowth rate to 67 ± 3% (Figure 4A), but this reduction is not statistically significant.

After having identified egl-3 as a factor potentially involved SAB overgrowth, we aimed to narrow down the location where egl-3 functions to induce axonal overgrowth: EGL-3 was specifically expressed in several tissues in egl-3(gk238) mutant background (Figure 4B, Supplementary Information S2). As a positive control, we expressed EGL-3 in the whole worm and this ubiquitous expression did rescue the overgrowth phenotype as 89 ± 6% of rescued worms showed overgrowth compared to 85 ± 5% in WT* worms.

However, EGL-3 expression in neither muscle nor neurons could restore the overgrowth phenotype, with only 48 ± 13% and 51 ± 6% of worms displaying overgrowth, respectively. Strain containing both EGL-3 expressing transgenes also did not rescue SAB overgrowth either (48 ± 15% of worms displaying overgrowth defect).

Furthermore, expression of EGL-3 in the intestine as well as in all three tissues (muscle, neuron and intestine) did not reproduce the rescue effect of ubiquitous EGL-3 expression (Supplementary Information S2).

Neuronal voltage-gated calcium channels are involved in SAB axonal overgrowth

L-type voltage-gated calcium channels (VGCCs) have been reported to negatively regulate axon termination in the mechanoreceptors PLM and ALM [Buddell et al., 2019, Buddell andQuinn, 2021]. We thus tested whether SAB axonal overgrowth induced by reduced muscle activity was modified in VGCC mutants. We quantified SAB axonal overgrowth in mutants carrying strong loss-of-function mutations in genes encoding the three pore-forming VGCC subunits, EGL-19 (L-type) [Lee et al., 1997], UNC-2 (P/Q type) [Schafer and Kenyon, 1995] and CCA-1 (T-type) [Steger et al., 2005], as well as the auxiliary alpha2/delta UNC-36 subunit that is common to P/Q and L-type VGCC [Frøkjaer-Jensen et al., 2006, Lainé et al., 2011, Saheki and Bargmann, 2009, Tong et al., 2017].

Hypomorph egl-19 mutations almost completely suppressed the SAB axonal overgrowth.

Only 15 ± 6% of egl-19(n582) and only 26 ± 6% of egl-19(ad1006) mutant worms exhibited SAB overgrowth compared to 84 ± 3% in WT* (Figure 5A). Overgrowth induced by reduced activity in muscle was also significantly suppressed in unc-2 and unc-36 null mutants, with SAB defects observed in 82 ± 8% of WT*, 36 ± 5% of unc-2

February 18, 2023 13/32 and 20 ± 9% of unc-36 (Figure 5A). No suppression of overgrowth was observed in cca-1 mutants (93 ± 6%).

To determine in which tissues the VGCCs were required to induce SAB overgrowth in paralysed worms, we rescued UNC-36 in either muscle or neurons of unc-36 null mutant background. Expression of UNC-36 in SAB neurons using the Punc-4 promoter completely rescues the overgrowth phenotype, from 16 ± 6% of worms exhibiting overgrowth defect in null unc-36 mutants to 83 ± 5% with UNC-36 neuronal expression (Figure 5B). On the other hand, muscle expression of UNC-36 with the Pmyo-3

February 18, 2023 14/32 promoter did not seems to have an effect, with only 21 ± 8% worms carrying defects (Figure 5B). These reslts showed that VGCCs functions cell-autonomously in SAB neurons to induce axonal overgrowth when muscle activity is reduced. and 33 ± 13% of transgenic worms with overgrowth, compared to 5 ± 3% in wild-type animals) (Figure 6A). It has to be noticed that even without HisCat activation through histamine, transgenic worms exhibited more frequent axonal defects than wild type ( 23± 17% and 20 ± 9% of transgenic worms with overgrowth, compared to 4 ± 3% in wild type), suggesting that the transgene might be active without histamine treatment.

A second prediction arising from our model is that SAB neurons should be hyperactive during histamine-induced paralysis. To test this prediction, we monitored intracellular calcium concentration in SAB neurons by pseudo-ratiometric calcium imaging of WT* worms during L1, in two conditions: control versus histamine-treatment (Figure 6B,C).

We generated two independent lines from WT* worms expressing the calcium indicator GCaMP6s, in a lite-1(ce314) mutant background to mitigate the animal's response to blue light [Edwards et al., 2008]. For these two independent lines, we observed a higher calcium level in SAB cell bodies of paralyzed worms compared to worms in control condition. The amplitude of the increase was different between the two lines. For the first line, the average calcium signal in histamine-treated L1 larvae is 146% that of control larvae, with a small-to-medium effect size of d = 0.495. On the other hand, the average calcium signal in histamine-treated L1 larvae of the second line is only 116% that of control, and the effect size is small (d = 0.23).

The final prediction coming from our model is that decreasing SAB activity in the context of muscle shutdown should suppress SAB overgrowth. So we expressed HisCl1 channel in the SAB neurons of worms already expressing this channel in muscle. The transgene by itself did not seem to cause any overgrowth defect (Figure 6D). When excitability was decreased only in muscle, SAB motor neurons exhibited a clear phenotype, as previously described on our study (80± 9% of animals with axonal overgrowth). When excitability was decreased only in SAB neurons, these neurons exhibited no overgrowth phenotype (5 ± 5%). When excitability was decreased only in muscle, SAB motor neurons exhibited a clear phenotype, as previously described on our

February 18, 2023 16/32 study (80 ± 9% of animals with axonal overgrowth). A strong reduction of SAB overgrowth was observed when excitability was reduced in both muscle and SAB motor neurons (23 ± 11%).

Discussion

SAB axonal morphology depends on muscle activity

Our study showed that decreasing muscle activity led to an overgrowth phenotype of SAB motor neurons. To manipulate muscle activity, we have built a strain expressing a integrated monocopy of a transgene encoding the hyperpolarizing HisCl1 channels from Drosophila melanogaster in C. elegans muscle cells. Since histamine is not a physiological neurotransmitter used in C. elegans, exposure of animals to histamine specifically induces the opening of HisCl1 channels and thus a hyperpolarization of muscle cells [Pokala et al., 2014]. In their pioneer work, Zhao and Nonet have also established that SAB axonal overgrowth is induced by decreased muscle activity [Zhao and Nonet, 2000]. Their conclusions were based on two experiments. First, they expressed in muscle a transgene encoding a gain-of-function potassium channels, EGL-36, as a non-integrated multicopy array. Second, they rescued only in muscle the expression of one subunit of the L-AChR, again using a non-integrated multiple array.

The tool we developed offers two main advantages: it is not massively overexpressed and it allows a temporal control of muscle hyperpolarization.

The temporal control of muscle activity allowed us to inhibit muscle activity during well-defined periods of post-embryonic development. Our results showed that the SAB axonal morphology were sensitive to postsynaptic activity throughout the L1 larval stage and only during this stage. This is partialy consistent with previous results pointing to the L1 and L2 stages as a critical period [Zhao and Nonet, 2000]. The difference regarding the L2 stage was probably due to a slower response of the temperature-sensitive allele that was used. Other systems also display a critical period during development where the morphology of neurons can be determined. For example, it is the case in vertebrate visual system, where postsynaptic activity influences the connectivity and branching of presynaptic neurons [Kutsarova et al., 2017]. In C.

elegans, the L1 stage is one of particular interest, as the nervous system of C. elegans undergoes a major rewiring event [White et al., 1986].

Two pieces of evidence favor the fact that SAB overgrowth induced by decreased-muscle activity potentially leads to the addition of new synapses. First, there is an increase of the number of SAB varicosities, where synapses are thought to be localized. Second, postsynaptic receptors are enriched at these structures. Testing whether the synapses are functional will require the development of molecular tools, such as calcium indicators tethered at the postsynaptic sites. In any case, the number of additional synapses is limited and we could not observe any phenotype correlated with the presence of ectopic synapses.

February 18, 2023 18/32

Neuronal voltage-gated calcium channels are required for SAB overgrowth Axonal SAB overgrowth induced by reduced-muscle activity is suppressed in animals carrying mutations in egl-19 , unc-2 and unc-36 encoding VGCC subunits [Lee et al., 1997, Saheki and Bargmann, 2009, Schafer and Kenyon, 1995]. These subunits are present in both muscle cells (EGL-19 and UNC-36) and neurons (EGL-19, UNC-36 and UNC-2) [Frøkjaer-Jensen et al., 2006, Jospin et al., 2002, Mathews et al., 2003]. UNC-36 is an auxiliary subunit common to both L-type (containing the pore-forming EGL-19 subunit) and P/Q-type (containing the pore-forming UNC-2) VGCC. Tissue-specific rescue experiments showed that the VGCCs function cell-autonomously in the motor neurons to induce overgrowth. UNC-36 expression in motor neurons driven by unc-4

promoter successfully restored the overgrowth phenotype, while UNC-36 expression in muscles driven by myo-3 promoter did not have any effect on the percentage of worms showing axonal defects. In other contexts, VGCCs have been shown to be involved in axon development [Mizuno et al., 2010, Tezuka et al., 2022], for review [Arjun McKinney et al., 2022]. Calcium activity in rat's callosal projection neurons during a critical period, from 6 to 15 days post-natal is essential to axon development.

However, this result was here surprising at first since dysfunction of neuronal VGCC should decrease SAB excitability. If motor neurons are less active, one could expect less active muscle cells and thus overgrowth, while VGCC mutations lead to suppression of the induced-overgrowth. A similar paradoxical situation has been described in rat NMJ:

tetrodotoxin blockade of neuromuscular activity significantly reduces the NMJ sprouting observed in extensively denervated muscle [Tam et al., 2002]. Moreover, a similar situation has already been described in C. elegans: mutants for the FSN-1/F-box protein have impaired neurotransmission at NMJ [Hung et al., 2013], yet overgrowth of the PLM touch receptor neuron observed in fsn-1 mutant is suppressed by mutations in VGCC [Buddell et al., 2019].

Muscle inactivity induces hyperactivity in SAB neurons, resulted in axonal overgrowth

Our hypothetical model of the histamine-induced SAB axonal overgrowth proposes that a decrease in muscle activity could induce hyperactivity of SAB that leads to overgrowth. Decrease of VGCCs in neurons would suppress SAB hyperactivity and in turn suppress the axonal overgrowth. Several lines of evidence support our model.

Firstly, SAB motor neuron activity is necessary to induce a great majority of axonal overgrowth in response to decreased muscle activity. Indeed, the animals display overwhelmingly less axonal overgrowth when both muscle and motor neuron are rendered less active.

Secondly, SAB hyperactivity alone is sufficient to induce axonal overgrowth: increasing February 18, 2023 19/32 motor neuron activity by expressing and activating an exogenous cationic channel led to axonal overgrowth, albeit at a weaker penetrance than when muscle activity is decreased.

Finally, we have evidence that the level of calcium in SAB motor neurons of L1 worms with decreased muscle activity is higher than that of neurons in control condition.

Calcium levels were measured in animals with a lite-1(ce314) mutant background to mitigate its response to blue light [Edwards et al., 2008], thus decreasing the influence of phototoxicity on the results. This suggests that SAB neurons may be more active when the muscle is paralysed by hyperpolarisation of muscle cells.

Neural activity has been shown to enhance axon growth in motor neurons and retinal ganglion cells. Retinal axons do not extend in presence of neutrophic factors, but short electrical bursts once every minute can drastically increase axon growth [Goldberg et al., 2002]. Furthermore, electrical activity potentiates axon growth in sympathetic motor neurons in two ways: the activation of CaMKII-MAPKK pathway by the calcium influx in the axon, and the neutrophin-mediated axon growth [Singh and Miller, 2005] [Doi and Iwasaki, 2002] proposed that the retrograde signal was a neuropeptide, of unknown nature, secreted by muscle cells. More recently, a tripartite structure has been described: muscle contraction would be detected by the interneuron DVA, through the stretch-activated channels TRP-4, that would in turn secrete the NLP-12 neuropeptide, that will modulate motor neuron activity by acting on its receptor CKR-2 [Hu et al., 2011[Hu et al., , 2015]]. We found that mutations in egl-3 partially suppress histamine-induced SAB overgrowth. This could indicate that functional neuropeptides could act as signaling molecules to induce SAB overgrowth. Conversely, neuropeptides could act upstream of muscle activity, for example by decreasing SAB activity. We did not further investigate EGL-3 role in SAB axonal overgrowth, because overgrowth phenotype suppression by egl-3 was partial, no other gene encoding proteins involved in neuropeptides processing was identified, and we did not manage to determine in which tissue EGL-3 was required for SAB axonal overgrowth. The evidence presented here does not allow distinguishing between these two hypotheses.

February 18, 2023 20/32

Besides neuropeptides, Tong et al. [Tong et al., 2017] have proposed that muscle cells could secrete a soluble version of neurexin NRX-1 that would bind to the VGCC UNC-36 subunit and thus modulate presynaptic activity. We have no evidence of such mechanism in the case of SAB overgrowth induced by muscle activity since decreasing nrx-1 expression did not suppress SAB overgrowth. To go further, tissue-specific transcriptomic could be a powerful approach to identify the neuronal and muscle players involved in SAB axonal plasticity.

Functional test of EGL-3 tissue-specific expression

As mentioned in the previous section, EGL-3 expression in various tissue types did not successfully rescue the histamine-induced SAB overgrowth phenotype. We decided to do a functional test of these tissue-specific expressions of EGL-3 to see if they are capable of restoring different phenotypes related to the egl-3(gk238) deletion mutant background, namely eggs retention and locomotion [START_REF] Kass | The EGL-3 Proprotein Convertase Regulates Mechanosensory Responses of Caenorhabditis elegans[END_REF].

Firstly, we quantified the number of eggs laid by egl-3(gk238) mutant worms expressing EGL-3 in different tissues, after 24 hours starting from late L4 larvae. The results are represented on Figure 2.10. N2 and WT* were positive controls for the egg-laying phenotype. N2 and WT* adult worms were capable of laying substantially more eggs than egl-3(gk238) mutant worms, at 34 ± 2 and 31 ± 4 comparing to 7 ± 2. Neither muscle expression nor neuronal expression of EGL-3, as well as both expressions combined succeed at restoring the egglaying capacity, with only 9 ± 2, 10 ± 2 and 12 ± 2 eggs laid per worms in 24 hours, respectively. Ubiquitous expression of EGL-3 did rescue the phenotype as each worm laid 24 ± 1 eggs. Interestingly, intestinal expression of EGL-3 seemed to even further decrease the number of eggs laid to 2 ± 1, and the effect is statistically significant. The same effect is reproduced in strain with all 3 transgenes expressing EGL-3 in muscle, neuron and intestine (average 1 ± 0.5 eggs laid per worm).

The second phenotype tested is locomotion. We quantified the number of full body bends Same as with egg-laying, N2 and WT* are used as positive control. N2 and WT* adult worms are more mobile in liquid medium than egl-3(gk238) mutant worms, completing 1.1 ± 0.3 and 1.2 ± 0.3 full comparing to 7 ± 2. Neither muscle expression nor neuronal expression of EGL-3, as well as both expressions combined succeed at restoring the egglaying capacity, with only 9 ± 2, 10 ± 2 and 12 ± 2 eggs laid per worms in 24 hours, respectively. Ubiquitous expression of EGL-3 did rescue the phenotype as each worm laid 24 ± 1 eggs. Interestingly, intestinal expression of EGL-3 seems to even further decrease the number of eggs laid to 2 ± 1, and the effect is statistically significant. The same effect is reproduced in strain with all 3 transgenes expressing EGL-3 in muscle, neuron and intestine (average 1 ± 0.5 eggs laid per worm).

SAB axonal defects scoring of worms with VGCCs gain-of-function mutations

We also scored SAB axonal overgrowth in VGCC gain-of-functions mutants, namely egl-19(n2368)(G365R) [START_REF] Lainé | Hyperactivation of L-type voltage-gated Ca2+ channels in C. elegans striated muscle can result from point mutations in the IS6 or the IIIS4 segment of the α1 subunit[END_REF] and unc-2(G1132R) [START_REF] Huang | Gain-of-function mutations in the UNC-2/CaV2α channel lead to excitation-dominant synaptic transmission in Caenorhabditis elegans[END_REF] Results of SAB scoring with unc-2(G1132R) mutant backgrounds are represented on Figure 2.13. There are 3 independent lines of unc-2(G1132R) mutant background, engineered by CRISPR. In control condition, gain-of-function of UNC-2 did not produce a significant increase in the proportion of worms showing SAB defects, 3 ± 3% for WT* compared to 6 ± 4% for unc-2 gain-of-function. With histamine treatment, only one line showed a significant increase in percentage of worms with defect, from 78% to 96%. The other two lines did not increase the percentage of worms with defect. Chapter 3

Discussion

Previous works by Alexis Weinreb have established that a decrease in muscle activity, by activation of HisCl1 channel, during a critical period of development leads to axonal overgrowth defects in SAB neurons [START_REF] Weinreb | Impact de l'activité postsynaptique sur le développement et le maintien de la jonction neuromusculaire de C. elegans[END_REF]. Preliminary results also showed that inhibition of activity only in head muscle was sufficient to induce axonal defects. We have reproduced and confirmed these results at the beginning of this project. The experiments done in this work built upon the gene candidate approach to study the mechanism behind the histamine-induced SAB axonal overgrowth. With that approach, the gene egl-3 coding for proprotein convertase PC2 was found to be involved, as egl-3 null mutant suppresses SAB overgrowth. We attempted to determine in which tissue EGL-3 functions to induce overgrowth. Curiously, only the ubiquitous expression of EGL-3 was successful in restoring the level of worms displaying overgrowth. In addition, the VGCCs were identified as an important component behind SAB axonal plasticity. We established that activity in SAB neurons is essential to develop axon overgrowth. Furthermore, we showed that SAB hyperactivity is sufficient to induce axonal overgrowth. Finally, by calcium imaging, we compared the calcium level in SAB neurons of L1 larvae under control and histaminetreated conditions, and we found a slight increase in average calcium level. Together, our results suggest that SAB axonal overgrowth is a consequence of hyperactivity in SAB neurons.

VGCCs are essential in SAB axonal plasticity

Axonal SAB overgrowth induced by reduced-muscle activity is suppressed in animals carrying mutations in egl-19 , unc-2 and unc-36 encoding VGCC subunits [Lee et al., 1997, Saheki and Bargmann, 2009, Schafer and Kenyon, 1995]. These subunits are present in both muscle cells (EGL-19 and UNC-36) and neurons (EGL-19, UNC-36 and UNC-2) [Frøkjaer-Jensen et al., 2006, Jospin et al., 2002, Mathews et al., 2003]. UNC-36 is an auxiliary subunit common to both L-type (containing the pore-forming EGL-19 subunit) and P/Q-type (containing the pore-forming UNC-2) VGCC. Tissue-specific rescue experiments showed that the VGCCs function cell-autonomously in the motor neurons to induce overgrowth. UNC-36 expression in motor neurons driven by unc-4 promoter successfully restored the overgrowth phenotype, while UNC-36 expression in muscles driven by myo-3 promoter did not have any effect on the percentage of worms showing axonal defects. In other contexts, VGCCs have been shown to be involved in axon development [Mizuno et al., 2010, Tezuka et al., 2022], for review [Arjun McKinney et al., 2022]. Calcium activity in rat's callosal projection neurons during a critical period, from 6 to 15 days post-natal is essential to axon development.

However, this result was here surprising at first since dysfunction of neuronal VGCC should decrease SAB excitability. If motor neurons are less active, one could expect less active muscle cells and thus overgrowth, while VGCC mutations lead to suppression of the induced-overgrowth. A similar paradoxical situation has been described in rat NMJ: tetrodotoxin blockade of neuromuscular activity significantly reduces the NMJ sprouting observed in extensively denervated muscle [Tam et al., 2002]. Moreover, a similar situation has already been described in C. elegans: mutants for the FSN-1/F-box protein have impaired neurotransmission at NMJ [Hung et al., 2013], yet overgrowth of the PLM touch receptor neuron observed in fsn-1 mutant is suppressed by mutations in VGCC [Buddell et al., 2019].

Experiment silencing the activity of both muscle and motor neurons by HisCl1 channel activation demonstrate that VGCCs are essential in SAB axonal plasticity. Indeed, the animals do not display a significant amount of axonal overgrowth when both muscle and motor neuron are rendered less active.

Interestingly, activation of HisCl1 channel expressed by unc-4 promoter to silence SAB neural activity alone does not induce SAB overgrowth. This result seems inconsistent with previous works that found SAB overgrowth in mutations in synaptic transmission gene unc-13 [Weinreb, 2018, Zhao andNonet, 2000]. It is possible that the inhibition of activity only in motor neurons is not sufficient to generate overgrowth, while mutations in unc-13 affect a wider range of neurons that could play a role in producing overgrowth.

It is noted that silencing of SAB neural activity alone does not produce the same severe paralysis effect as unc-13 mutants, as worms with just SAB silenced are still able to move.

3.2. Muscle inactivity induces hyperactivity in SAB neurons, resulted in axonal overgrowth

Muscle inactivity induces hyperactivity in SAB neurons, resulted in axonal overgrowth

Our hypothetical model of the histamine-induced SAB axonal overgrowth proposes that a decrease in muscle activity could induce hyperactivity of SAB that leads to overgrowth. Decrease of VGCCs in neurons would suppress SAB hyperactivity and in turn suppress the axonal overgrowth. Several lines of evidence support our model.

Firstly, SAB motor neuron activity is necessary to induce axonal overgrowth in response to decreased muscle activity. Indeed, the animals do not display a significant amount of axonal overgrowth when both muscle and motor neuron are rendered less active. Secondly, SAB hyperactivity alone is sufficient to induce axonal overgrowth: increasing motor neuron activity by expressing and activating an exogenous cationic channel led to axonal overgrowth, albeit at a weaker penetrance than when muscle activity is decreased. Finally, we have evidence that the level of calcium in SAB motor neurons of L1 worms with decreased muscle activity is higher than that of neurons in control condition. Calcium levels were measured in animals with a lite-1(ce314) mutant background to mitigate its response to blue light [Edwards et al., 2008], thus decreasing the influence of phototoxicity on the results. This suggests that SAB neurons may be more active when the muscle is paralysed by hyperpolarisation of muscle cells.

Neural activity has been shown to enhance axon growth in motor neurons and retinal ganglion cells. Retinal axons do not extend in presence of neutrophic factors, but short electrical bursts once every minute can drastically increase axon growth [Goldberg et al., 2002]. Furthermore, electrical activity potentiates axon growth in sympathetic motor neurons in two ways: the activation of CaMKII-MAPKK pathway by the calcium influx in the axon, and the neutrophin-mediated axon growth [Singh and Miller, 2005] It has been shown that gain-of-function mutations of UNC-2 increase the intracellular calcium current [START_REF] Huang | Gain-of-function mutations in the UNC-2/CaV2α channel lead to excitation-dominant synaptic transmission in Caenorhabditis elegans[END_REF]. Since UNC-2 is only expressed in the nervous system, this mutation will only affect neural activity and not muscle. However, after scoring of 3 different unc-2 gain-of-function mutant strains in control, no significant amount of SAB overgrowth was observed. This result seems to contradict our hypothetical model that SAB hyperactivity induces overgrowth. We can imagine that the intracellular calcium level in unc-2 gain-of-function mutant worms might not reach the optimal range for axon growth [Kater and Mills, 1991]. It would be interesting to measure the calcium level in L1 larvae of unc-2 gain-of-function mutant strains and compare it to WT* L1 larvae in histamine-treated condition and larvae expressing the exogenous cationic HisCat channel.

Neuropeptides as retrograde signal in SAB axonal overgrowth?

What is still missing from our model is how muscle cells can induce hyperactivity in the SAB motor neurons. Our model implies that a retrograde signal is sent by muscle cells either directly or indirectly to SAB motor neurons. Retrograde signals have been already described in C. elegans neuromuscular junction. The first study, by [Doi and Iwasaki, 2002] proposed that the retrograde signal was a neuropeptide, of unknown nature, secreted by muscle cells. More recently, a tripartite structure has been described: muscle contraction would be detected by the interneuron DVA, through the stretch-activated channels TRP-4, that would in turn secrete the NLP-12 neuropeptide, that will modulate motor neuron activity by acting on its receptor CKR-2 [Hu et al., 2011[Hu et al., , 2015]]. We found that mutations in egl-3 partially suppress histamine-induced SAB overgrowth. This could indicate that functional neuropeptides could act as signaling molecules to induce SAB overgrowth. Conversely, neuropeptides could act upstream of muscle activity, for example by decreasing SAB activity. The evidence presented here does not allow distinguishing between these two hypotheses. Investigation into EGL-3 role in SAB axonal overgrowth proved to be difficult. Overgrowth phenotype suppression by egl-3 was partial, no other gene encoding proteins involved in neuropeptides processing was identified, and we did not manage to determine in which tissue EGL-3 was required for SAB axonal overgrowth. Phenotypes other than SAB overgrowth such as egg laying and thrash in liquid medium were also evaluated in EGL-3 rescue strains. Results indicate that the tissue-specific rescue effects of these two phenotypes were all partial, insignificant, even. This leads to suspicions of technical problem with our tissue-specific expression of EGL-3.

Manipulating expression in SAB neurons

Besides neuropeptides, Tong et al. [Tong et al., 2017] have proposed that muscle cells could secrete a soluble version of neurexin NRX-1 that would bind to the VGCC UNC-36 subunit and thus modulate presynaptic activity. Previous RNAi experiment by Alexis Weinreb on nrx-1 showed a possible overgrowth suppression effect, but it was not statistically significative and not easy to reproduced [START_REF] Weinreb | Impact de l'activité postsynaptique sur le développement et le maintien de la jonction neuromusculaire de C. elegans[END_REF].

To go further, tissue-specific transcriptomic could be a powerful approach to identify the neuronal and muscle players involved in SAB axonal plasticity. Recently, a study comparing between the transcriptome of adult skeletal muscle with motor nerves crushed and control found 7133 differentially expressed genes (DEG), 3656 upregulated and 3477 downregulated [START_REF] Staunton | Skeletal muscle transcriptomics identifies common pathways in nerve crush injury and ageing[END_REF]. This sizeable amount of DEG in denervated muscle indicates a wide-ranging reconfiguration of the muscle, such as loss of presynaptic NMJ and early phases of nerve regeneration. The most upregulated DEG included nervecrushed muscle neuronal acetylcholine receptor subunit alpha-9 (CHRNA9 ), cholinergic receptor nicotinic gamma (CHRNG) and notably growth differentiation factor 5 (GDF5 ) which stimulates axon growth and branching, as well as increasing the number of cells in cultures of rat midbrain neurons [START_REF] O'keeffe | Effects of growth/differentiation factor 5 on the survival and morphology of embryonic rat midbrain dopaminergic neurones in vitro[END_REF]. It is feasible to perform an analysis of the transcriptome of C. elegans head muscle, in control and in histaminetreated condition. As local inhibition of activity in the head is sufficient to induce SAB overgrowth, transcriptome profile of the head muscle during histamine-induced paralysis may provide insight into the nature of the retrograde signal. At the time of writing, we have done cell-sorting experiments to select for head muscle cells and sequenced the extracted RNA. We obtained the raw data, awaiting further bioinformation analysis.

Manipulating expression in SAB neurons

The promoter of the unc-4 gene is often used in order to drive genetic expressions in the SAB neurons. The expression pattern of the unc-4 promoter has been characterized byReilly et al. [2020]. It has been shown that the unc-4 promoter drives expressions in other neurons than SAB, namely I5, AVF, VA, DA, AS, VD. The expression map is established by using a fluorescent reporter of unc-4 e.g. unc-4::gfp in the multicolorlandmark neuron identification strain NeuroPAL [Yemini et al., 2021].

In order to better identify SAB cell bodies for calcium imaging, we used a truncated version of unc-4 promoter that still shows strong expressions in SAB but weak expression in DA motor neurons. This reduces the number of neurons expressing calcium indicators, making it easier to identify our target. The truncated version, designated unc -4 p romC, is 1048 bp long. Only the first conserved region, a part of the second conserved region and the zone in-between are covered by this shortened version. In the first region, there is a 8 pb deletion to remove a COE motif, which binds the Collier/Olf/Ebf transcription family in C. elegans [START_REF] Kratsios | Transcriptional Coordination of Synaptogenesis and Neurotransmitter Signaling[END_REF]. The removal of COE motif is likely to decrease the expression in certain neurons.

It is mentioned in SAB neurons section that the unc-4 promoter drives expressions in the SAB, I5, AVF, VA, DA, AS, VD [START_REF] Reilly | Unique homeobox codes delineate all the neuron classes of C. elegans[END_REF]. As we are only interested in the expression of a transgene in SAB neurons when using the unc-4 promoter, it is important to determine if the transgene's expression in other neurons can have undesired effects related to SAB's function. The I5 neuron is a glutamatergic neuron that regulates the muscle relaxation of the pharynx [START_REF] Avery | Motor neuron M3 controls pharyngeal muscle relaxation timing in Caenorhabditis elegans[END_REF]. As the pharyngeal nervous system is well isolated, genetic expression in I5 should not affect head muscles or SAB. AVF is a polymodal neurons as it has the functions of both motorneurons and interneurons. Its axons pass around the nerve ring and along the ventral cord, forming few synapses [White et al., 1986]. AVF forms NMJs with muscle cells numbered 11, 12 and 14 [START_REF] Varshney | Structural Properties of the Caenorhabditis elegans Neuronal Network[END_REF], a non-negligible distance away from SAB's synaptic targets. All in all, transgene expression in AVF probably does not disturb the SAB system, saved for some unknown indirect effect. VA, DA, AS and VD are all motor neurons. It is noted that VA and DA also innervate SAB directly. Minh-Dao Nguyen krSi-alleles were constructed by miniMos insertions [Frøkjaer-Jensen et al., 2014]. krIsalleles are integration of extrachromosomal array using the modular safe-harbor transgene insertion (MosTI) method [El Mouridi et al., 2022], except for krIs80 which was integrated by irradiation [Mariol et al., 2013].

Name

To generate single-copy insertion of a plasmid of interest, we used the miniMos insertion method [Frøkjaer-Jensen et al., 2014]. N2 animals were injected with 10-15 ng/µl the plasmid of interest containing the promoter and the open reading frame, 50 ng/µl pCFJ601 (Mos1 transposase), 10 ng/µl pMA122 (negative selective marker Phsp16.2::peel-1 ), and 2.5 ng/µl pAV41 (Pmyo-2::BFP). Neomycin (G418) was added to plates 24 h after injection at 0.75 µg/µl final concentration. Plates were kept at 25°C until starvation. Starved plates were heat shocked for 2 hours at 34°C. Selected lines were then bred to homozygosity.

To generate multi-copies integration of a plasmid of interest, we used the modular safeharbor transgene insertion (MosTI) method [El Mouridi et al., 2022]. unc-119(ed3) mutant animals were injected with 10-25 ng/µl the plasmid of interest, 25 ng/µl pMDJ231 (Phsp-16.41::Cas9::gpd2::tagRFP-T, Cas9 protein under heat shock promoter), 5 ng/µl pSEM319 (ce-unc-119 MosTI array fragment), and 15 ng/µl pSEM319 (single-guide RNA to MosTI landing site). Plates were kept in 15°C for 24 hours, after which neomycin (G418) was added to plates at 0.75 µg/µl final concentration. Plates were kept at 25°C until the worms were close to starvation. Almost starved plates were incubated for 20 hours at 30°C. Animals with integrated transgene are selected by lack of locomotion defects normally present in unc-119(ed3) mutant animals. Selected lines were then bred to homozygosity.

The strains used in this work are listed in Table 4.3.

All strains were maintained at 20°C on nematode growth medium (NGM) using Escherichia coli OP50 as food source [Brenner, 1974].

Microscopy and SAB scoring

To decrease the activity of muscle, we expressed the histamine-gated chloride channel (HisCl1) [Pokala et al., 2014] tagged with BFP in the muscle cells (krSi16 [Pmyo-3::HisCl1::SL2::tagBFP]).

The following protocol is used to score SAB axonal defects: For each genotype, one control plate and one Histamine-treated plate are prepared with 20 gravid worms each. The gravid worms were to lay eggs for 2 to 5 hours (in order to obtain a synchronized population), then they are removed from the plates. The control plate is treated with 300µl of water and the treatment plate is treated with 300µl of Histamine (Sigma Aldrich H7250, stock concentration of 1M in water, treatment concentration of 1/30 M) to a final concentration of 1mM. After three days, worms were mounted on dry pads (2% agarose in water, dry overnight) and immobilized with 4% polyethylene beads (Polysciences Poly-bead®polystyrene 0.1 µm). The SAB axons, reported by krSi24 :mNeonGreen], are then observed on a Zeiss Axioskop compound microscope. For each experiment, at least 3 replicates were performed. The n indicated for each figure corresponds to the number of individual worms per condition.

To examine the impact of histamine-induced muscle inhibition on the synaptic receptor's density and count varicosities along the SAB axons, we used the EN3939 strain carrying the unc-29(kr208::tagRFP-T) allele which is a reporter of levamisole-sensitive acetylcholine receptor (L-AChR). The protocol is the same as one described in previous paragraph but imaged on Andor Spinning Disk confocal microscope. Raw fluorescent intensity of the unc-29(kr208::tagRFP-T) reporter for both conditions is normalized to the control condition to obtain the relative fluorescence of L-AChR.

To induce SAB overgrowth with the histamine-gated cation channel (HisCat) in SAB neurons, we used the EN8873 and EN8874 strains carrying the Punc-4::HisCat::SL2::tagBFP transgene. The protocol is as follows: One control plate and one Histamine-treated plate are prepared with 20 gravid worms of each. The gravid worms were to lay eggs for 2 to 5 hours then they are removed from the plates. The control plate is treated with 300µl of water and the treatment plate is treated with 300µl of histamine (treatment concentration of 1M) to a final concentration of 30mM. SAB axons are then observed with the aforementioned protocol.

Calcium imaging

To monitor activity in SAB neurons, we used a ratiometric calcium imaging system [START_REF] Zheng | Calcium imaging of multiple neurons in freely behaving C. elegans[END_REF]. EN8871 and EN8872 strains were made to carry Punc-4(short)::NLS(SV40)::

GCaMP6s::mCherry::NLS(EGL13): expressing a calcium indicator, GCaMP6s [START_REF] Chen | Ultrasensitive fluorescent proteins for imaging neuronal activity[END_REF], fused with a stable fluorophore mCherry as reference, specifically in the nucleus of SAB cell bodies. SAB cell bodies' positions are identified using NeuroPAL [Yemini et al., 2021]. The protocol is as follows: One control plate and one Histaminetreated plate are prepared with 20 gravid worms of each. The gravid worms were to lay eggs for 2 hours then they are removed from the plates. The control plate is treated with 300µl of water and the treatment plate is treated with 300µl of histamine (treatment concentration of 1/30 M) to a final concentration of 1mM. After 16 hours, L1 worms in control condition are mounted on dry pads with only M9. L1 worms in histamine-treated condition are mounted on dry pads with histamine (treatment concentration of 1/30 M). Mounted worms are then filmed using the dual channel microscope Nikon Eclipse Ti2 for 1 minute at 2 frame per second, then analyzed with a macro made by fellow team member Franklin Florin. The ratio of GCaMP6s/mCherry is weighted by the local contribution of the calcium indicator GCaMP fluorescence to the total fluorescence of the channel. This is done to better highlight calcium increase events. SAB activity is measured by the weighted ratio of indicator/reference of each worm in the region-of-interest (containing SAB cell bodies) over 120 frames, then normalized to the weighted ratio of control worms.

Egg-laying and thrashing phenotype assays

egl-3 mutant worms have severe egg-laying defects, as well as locomotion defects slowing their crawl speed. Functional test of tissue-specific expressions of EGL-3 were done to see if they are capable of rescuing these two phenotypes.

To measure the number of eggs laid by worms with EGL-3 expression in different tissues, 10 late L4 larvae of each strain were sustained on separated plates for 24 hours at 20°C. During these times, the larvae will grow into adults capable of eggs-laying. We counted the number of eggs on each plate after the 24 hours period, then divided that number by 10 to get the average number of eggs laid per worm.

To assess the frequency of worms thrashing in water, we picked out young adult worms of each genotype and put them in a small well containing a room temperature liquid (M9 salts solution). The worms are filmed for 30 seconds on AZ100 macroscope (Nikon) equipped with a Flash 4.0 CMOS camera (Hamamatsu Photonics). The footage is then analyzed by a macro to count the number times each worm complete a full body bend during those 30 seconds. The frequency of body bends in liquid represents the locomotion capacity of the worms

Statistics

All statistical analysis were done in R. The penetrance of SAB defect is compared using the Cochran-Mantel-Haenszel (CMH) test [Cochran, 1954, Mantel andHaenszel, 1959]. This modified χ 2 test takes into account the date when the experiments are performed when testing whether or not a genotype/treatment affects the amount of worms carrying SAB defects. As every experiment usually has 3 to 4 replicates, each replicate is performed in a different day and so the data is stratified by date/replicate. A series of 2 x 2 contingency tables is constructed by taking the a binary outcome of observed worms (normal SAB axons or defects SAB axons), as well as a binary predictor (either the genotype or the histamine treatment), one

Figure 1 . 1 :

 11 Figure 1.1: General anatomy of C. elegans. A. Differential interference contrast (DIC) image of an adult hermaphrodite. Scale bar 0.1 mm. B. Schematic drawing of anatomical structures.Image from WormAtlas[START_REF] Altun | WormAtas Hermaphrodite Handbook -Introduction[END_REF]

Figure 1 . 2 :

 12 Figure 1.2: Life cycle of C. elegans at 22°C. The length of time the animal spends at a certain stage is noted in blue along the arrows. The length of the animal at each stage is marked next to the stage name in micrometers. Image from WormAtlas [Altun and Hall, 2006].

Figure 1 . 3 :

 13 Figure 1.3: Morphology of a neuron. Image adapted from WikiJournal of Medicine[START_REF] Blausen | Medical gallery of Blausen Medical[END_REF]

Figure 1 . 4 :

 14 Figure 1.4: C. elegans nervous system. A. Digital model of the C. elegans nervous system. Numerous neurons are located around the head and the pharynx, and multiple neurons are on the ventral line. Image from the Openworm project. B. Fluorescent marker of excitatory synapses, showing the nerve ring with the nerve cords, as well as the SAB axons at the nose. Image from Bérangère Pinan-Lucarré.

Chapter 1 .

 1 Introduction are 34 genes coding for over 70 different FMRFamide-like peptides, prefixed flp-and numbered from 1 to 34. Their receptors usually are G protein-coupled receptors, although few other receptors have been identified. Multiple flp-peptides can activate the same receptors, and one flp-peptide can act on multiple different receptors.

Figure 1 . 5 :

 15 Figure 1.5: Schematic representation of the neuropeptide processing pathway. A prepropeptide has its N-terminal signal peptide cleaved by a peptidase. Then the proprotein convertase cleaves the propeptide at dibasic (KR, RR, RK, KK) or monobasic (K, R) sites. After cleavage, the resulting intermediate peptides are treated by a specific carboxypeptidase to remove the Cterminal basic amino acids. Finally, if the C-terminal residue is a glycine, it is transformed into an amide. Mature neuropeptides are encircled in red. Image from[Husson et al., 2007]

Figure 1 . 6 :

 16 Figure 1.6: The SAB motor neurons. A. Schematic representation of the SAB neurons, showing the position of the SAB cell bodies and the axon projections. B. Cross sectional schema of the head, showing the four SAB axons in contact with the muscle. Image from Alexis Weinreb. C. Confocal image showing the expression pattern of the unc-4 gene, showing the SAB neurons.Image from[START_REF] Reilly | Unique homeobox codes delineate all the neuron classes of C. elegans[END_REF]. D. Neural connections of SAB motor neurons. Image from[START_REF] Bhatla | WormWeb.org -C. elegans Interactive Neural Network -by nikhil bhatla[END_REF]

Figure 1 .

 1 Figure 1.7: C. elegans muscle. Two muscle quadrants are visible (one marked by green brackets). Each quadrant consists of two rows of muscle cells. The individual cell has a diamond shape (one cell highlight in cyan). The arrows point to the smooth muscles in the pharynx, the intestine and the constricting anal muscle. The arrowhead indicates the vulva. Rhodamine phalloidin staining done by Charlotte Scholtes, scale bar 250 nm.

Figure 1 . 8 :

 18 Figure 1.8: Innervation of the C. elegans head muscle[START_REF] Gray | A circuit for navigation in Caenorhabditis elegans[END_REF]. The muscle cells are represented as colored rhombi, numbered from the most anterior to the most posterior. The SAB neurons (in blue) innervate cells numbered 1, 3, 4 and 6.

Figure 1 . 9 :

 19 Figure 1.9: Illustration of the actin-based growth cone. a. Growth cones protrude towards attractive cues or retract away from repulsive cues during guidance, according to the classical attraction-repulsion model. b. Representative fluorescent image of a rat growth cone showing the peripheral region rich in actin (blue) and the microtubule-concentrated central region (red).Image adapted from[START_REF] Omotade | Actin-based growth cone motility and guidance[END_REF].

Figure 1 . 10 :

 110 Figure 1.10: Important proteins controlling axonal navigation. Image from [Chisholm et al., 2016].

Figure 1 .

 1 Figure 1.11: SOAL model of outgrowth movement toward extracellular cues that promote or inhibit outgrowth activity. (A) Schematic diagram of the outgrowth of a neuron through an environment of multiple extracellular cues. The extracellular cues are represented as color gradients of blue, orange, and red. Model assumes the cues along the anterior-posterior axis (orange and red) create an equal probability for UNC-40 asymmetric localization and outgrowth in the anterior and posterior directions. The extension transverses three different positions (1-3)as it develops toward a ventral source of a cue (blue). (B) SOAL process illustrated for the three positions shown in (A). Shown are scenarios for movement toward the blue cue [A] that promotes outgrowth (blue +) or that inhibits outgrowth (blue -). The cues act to increase (promote) and decrease (inhibit) the probability of outgrowth from a surface. At positions 1 and 2, cues along the anterior-posterior axis (orange -and red -) inhibit outgrowth in the anterior or posterior directions. At position 3, the cue from the ventral source predominates. Image adapted from[START_REF] Limerick | A Statistically-Oriented Asymmetric Localization (SOAL) Model for Neuronal Outgrowth Patterning by Caenorhabditis elegans UNC-5 (UNC5) and UNC-40 (DCC) Netrin Receptors[END_REF].

Figure 1 . 12 :

 112 Figure1.12: The schematic presentation of a tentative model for FSN-1. In motor neuron, FSN-1 functions independently to regulate postsynaptic IGF signaling in sensory neuron. RPM-1 functions with FSN-1 to inhibit pre-synaptic Mitogen-activated protein kinase (MAPK) activity. Cross-talk between these two signaling events by a yet unknown retrograde signal regulates synapse development along the axons. Image from[Hung et al., 2013].

Figure 1 . 13 :

 113 Figure1.13: The schematic presentation of the negative regulatory network for DLK-1 pathway in the motor and mechanosensory neurons in C. elegans. The SCF complex ubiquitinates DLK-1L and marks it for degradation. RPM-1 binds to PPM-2 and PPM-2 directly dephosphorylates and inhibits DLK-1L. PPM-1, not bound to RPM-1, inhibits the DLK-1 pathway, most likely by dephosphorylating and inhibiting PMK-3 or MKK-4. Image from[START_REF] Grill | The PHR proteins: intracellular signaling hubs in neuronal development and axon degeneration[END_REF].

Figure 1 .

 1 Figure1.14: RPM-1 signaling through the ANC-1 pathway to regulate ALM and PLM axons termination. Both the canonical Wnt pathway and the RPM-1/ANC-1/UNC-84 complex cooperate to positively regulate the BAR-1 level. BAR-1 increases nuclear import and activates the transcription factor POP-1. Image from[START_REF] Tulgren | The Nesprin Family Member ANC-1 Regulates Synapse Formation and Axon Termination by Functioning in a Pathway with RPM-1 and β-Catenin[END_REF].

Figure 1 .

 1 Figure 1.15: Illustration of morphological stages in regrowth of neuron after laser injury, based on studies of mechanosensory neurons[START_REF] Wu | Caenorhabditis elegans neuronal regeneration is influenced by life stage, ephrin signaling, and synaptic branching[END_REF]. Image from[START_REF] Chisholm | The Genetics of Axon Guidance and Axon Regeneration in Caenorhabditis elegans[END_REF].

Figure 1 . 16 :

 116 Figure 1.16: Motor nerve sprouting. A. Schema of different types of motor nerve sprouting. a: axon, m: myelin, m.e-p: motor end-plate, m.f: muscle fiber, n.s: nodal sprouting, p.s: preterminal sprouting, u.s: ultraterminal sprouting. Image from [Barker and Ip, 1966]. B. Muscle end-plate of mouse stained with zinc oxide and osmium tetroxide after tetrodotoxin treatment. a. Control mouse, no paralysis. b. 3.5 days of paralysis. c. 5 days of paralysis.Image from[Brown and Ironton, 1977]. C, D. Mouse soleus muscle stained with zinc oxide and osmium tetroxide after control treatment (C) and after a week of daily BTX injection. E. Proportion of endplates showing terminal sprouting in control condition (top) or after BTX treatment (bottom). Images adapted from[Holland and Brown, 1980].

Figure 1 .

 1 Figure 1.17: Sprouting suppression effects of TTX and BTX on partially denervated muscle.A. Mean motor unit size of 2-to 10-months old control (dashed horizontal line) and treated soleus muscle, calculated by dividing the muscle activity by the number of motor neurons plus muscle fibers, normalized to the control. The treatment is indicated at the bottom of each bar, L5+: L5 ventral ramus (the anterior division of a spinal nerve, L5 innervates the mouse's soleus muscle) is severed. Images from[START_REF] Connold | Temporary loss of activity prevents the increase of motor unit size in partially denervated rat soleus muscles[END_REF]. B. Number of sprouts in plantaris and soleus muscles, in control condition (horizontal line, n = 38), moderately dernervated (PD < 75%, n = 7 in normal caged activity, n = 4 in TTX treatment) or extensively denervate (PD < 75%, n = 7 in normal caged activity, n = 5 in TTX treatment)[Tam et al., 2002].

Figure 1 .

 1 Figure 1.18: Plasticity of SAB morphology. A. Images of SAB axons using the Punc-4::mNeonGreen. Axon without overgrowth (top), axon with looping (middle), axon with sprouting (bottom). Scale bar 10 µm. Nose is left, pharyngeal bulbs are right. B.Proportions of worms displaying SAB overgrowth in wild-type or Pmyo-3::HisCl1 transgenic worms, with or without histamine. Bars represent proportion ± standard error, n > 30.

Figure 1 .

 1 Figure 1.19: Branching at the NMJ of 3rd stage Drosophila larvae with hyperexcitable mutations. A, B, C. Drawings of anti-HRP staining of axon terminals at the NMJ of wild-type (A), hyperexcitable mutants (B), and hyperexcitable mutants with a mutation suppressing action potential (C).D, E, F. Number of branches of each order of wild-type and hyperexcitable mutant (D), double mutants of hyperexcitable + suppressed action potential (E), and in mutants with suppressed action potential (F). Image adapted from[START_REF] Budnik | Morphological plasticity of motor axons in Drosophila mutants with altered excitability[END_REF].

Figure 1 .

 1 Figure 1.20: Activity-dependent synaptic plasticity at the Drosophila larval NMJ. Two calcium-dependent pathway are involved.The first is cAMP pathway regulated by calcium/calmodulin-dependent adenylate cyclase, rutabaga and Dunce. Cell adhesion molecule Fas II and cAMP response element-binding proteins (CREB) are downstream of the cAMP pathway and responsible for increasing sprouting and pre-synaptic transmitter release. PKA and PP1 are also regulated by increased cAMP levels, and they, in turn, regulate Ca 2+ /calmodulindependent protein kinase (CaMK)II, which is the second calcium-dependent pathway. The PKA-PP1-CaMKII interaction controls the withdrawal/retraction process through Plexin B receptor and Semaphorin-2a (Sema2a) chemorepulsion. The synaptic localization of Discs large (DLG) regulated by CaMKII controls the clustering of synaptic molecules, such as Fas II or Shaker. Image from[START_REF] Bai | Activity-Dependent Synaptic Plasticity in Drosophila melanogaster[END_REF].

Figure 1 .

 1 Figure 1.21: The general organization of the corpus callosum. (A-C) The structure and location of the human CC (hCC), shown in blue. (A) Dorsal view of the hCC. (B) Sagittal view of the hCC. (C) Coronal view of the hCC. Axons of cortical neurons from both hemispheres meet in the midline and cross to the opposite hemisphere in order to target specific contralateral regions. Most of these projections will connect homotopic regions within the brain, and fewer will connect heterotopic regions. Image from [De León Reyes et al., 2020].

  activate the Ca 2+ -calmodulin-dependent protein kinases (such as CaMKII) or Ca 2+ -calmodulin-dependent protein phosphatase calcineurin. Other notable examples include the nitric oxide synthase (NOS), protein kinase C (PKC), troponin C (Figure 1.22).

Figure 1 . 22 :

 122 Figure 1.22: Overview of calcium signaling pathway in human. Each rectangle represents a gene product, usually proteins. Lines with black arrowhead indicate molecular interaction or relation. Dashed lines with black arrowhead means indirect link or unknown reaction. Each circle represents a chemical compound, DNA or other molecules. Lines between 2 rectangles represent protein-protein interaction, arrows mean activation and T-shaped lines mean inhibition. Image from the Kyoto Encyclopedia of Genes and Genomes [KEGG, 2020].

Figure 1 .

 1 Figure 1.23: Voltage-gated calcium channel (VGCC, or Ca V ) nomenclature and properties. a. VGCC nomenclature, homology, human genetic nomenclature and protein classification, tissue distribution. b Normalized current-voltage relationships for VGCC currents recorded from tsA201 (transformed human kidney) cells. c VGCC subunit interactions. The α1 subunit contains four homologous domains (I-IV) each with six transmembrane segments (S1-S6), an S4 voltage sensor (yellow), containing a motif of positively charged amino acid residues, and a P loop between S5 and S6 (red segments), which form the pore domain, containing key acidic residues (cyan circles) involved in the selectivity filter. The α2δ subunit is shown with its glycosyl phosphatidylinositol (GPI) anchor linking it into the membrane and von Willebrand factor A (VWA) domain binding to the first extracellular loop of the α1 subunit. The two domains of the β-subunit (SRC homology 3 domain (SH3) and guanylate kinase-like domain (GK)) are also shown, with the GK interacting with the α1-interaction domain (AID) motif on the I-II linker intracellular loop. Image from [Dolphin and Lee, 2020].

Figure 1 . 24 :

 124 Figure 1.24: Asymmetric calcium signals drive lamellipodial and filopodial protrusion. The bundle of F-actin in the filopodia responds to extracellular guidance cues because of downstream effects of calcium. Image from [Gasperini et al., 2017].

Figure 2 . 1 :Figure 2 . 2 :Figure 2 . 4 :Figure 2 . 9 :

 21222429 Figure 2.1: Plasticity of the SAB system Figure 2.2: Effect of histamine-induced axonal overgrowth on SAB synapses Figure 2.3: Spatio-temporal determinism of SAB plasticity Figure 2.4: Overgrowth suppression effect of different mutant backgrounds Figure 2.5: Overgrowth suppression effect of VGCCs mutant backgrounds Figure 2.6: Effect of SAB neural activity on axonal overgrowth Figure 2.7: ACR-16 expression pattern in the head of the worm Figure 2.8: Attempts at phenotypic rescue by specific expression of EGL-3 Figure 2.9: Histograms of normalized weighted indicator/reference ratio

  Prgef-1::egl-3(cDNA)::SL2::wScarlet::miniMos1 Express EGL-3 in neurons pCR10 miniMos1::Pmyo-3::egl-3(cDNA)::SL2::wScarlet::miniMos1 Express EGL-3 in muscles pCR11 miniMos1::Peft-3::egl-3(cDNA)::SL2::wScarlet::miniMos1 Express EGL-3 ubiquitously pCR10 miniMos1::Pmyo-3::egl-3(cDNA)::SL2::wScarlet::miniMos1 Express EGL-3 in muscles pMDN2 miniMos1::Punc-4::GFPunc-36::miniMos1 Express UNC-36 in SAB neurons pMDN3 miniMos1::Pmyo-3::GFPunc-36::miniMos1 Express UNC-36 in muscles pMDN4 miniMos1::Pvha-6::egl-3(cDNA)::SL2::wScarlet::miniMos1 Express EGL-3 in the intestine pMDN7 miniMos1::Punc-4(short)::NLS(SV40)::GCaMP6s::mCherry:: NLS(EGL13)::miniMos1 Calcium indicator in SAB cell bodies pMDN9 miniMos1::Punc-4::HisCl1::SL2::TagBFP::miniMos1 Express HisCl1 channel in SAB neurons pMDN10 miniMos1::Punc-4::HisCat::SL2::TagBFP::miniMos1 Express HisCat channel in SAB neurons

  )::SL2::wScarlet::miniMos1] miniMos single-copy insertion of pMDN4 Minh-Dao Nguyen krSi193[Punc-4::GFPunc-36] miniMos single-copy insertion of pMDN2 Minh-Dao Nguyen krSi194[Pmyo-3::GFPunc-36] miniMos single-copy insertion of pMDN3 Minh-Dao Nguyen krSi257[Punc-4::HisCl1::SL2::tagBFP] miniMos single-copy insertion of pMDN9 Minh-Dao Nguyen krIs80 [Ptni-3::HisCl1::SL2::wScarlet] Multicopies integration of pAW34 by irradiation Alexis Weinreb krIs89[Punc-4::HisCat::SL2::tagBFP] MosTI Integration n°1 of extrachromosomal array formed by injecting pMDN10 Minh-Dao Nguyen krIs90[Punc-4::HisCat::SL2::tagBFP] MosTI Integration n°2 of extrachromosomal array formed by

Fig 1 .

 1 Fig 1. Plasticity of the SAB system.

Fig 2 .

 2 Fig 2. Effect of histamine-induced axonal overgrowth on SAB synapses

Fig 3 .

 3 Fig 3. Spatio-temporal determinism of SAB plasticity

Fig 4 .

 4 Fig 4. Overgrowth suppression effect of different mutant backgrounds

Fig 5 .

 5 Fig 5. Overgrowth suppression effect of VGCCs mutant backgrounds and phenotypic rescue by UNC-36 tissue-specific expression

Fig 6 .

 6 Fig 6. Effect of SAB neural activity on axonal overgrowth

Fig

  Fig S1. ACR-16 expression pattern in the head of the worm

Figure 2 . 10 :

 210 Figure 2.10: Number of eggs laid per worms after 24h of each EGL-3 rescue strain. 3 replicates were performed, N = 10 for all genotypes. The numbers of eggs laid of egl-3(gk238) mutant worms are used as reference for statistical comparison. (n.s.: not significant, *: p < 0.05, ***: p < 0.001)

Chapter 2 .

 2 Resultseach egl-3 rescue strain did during 30 seconds. The results are represented on Figure2.11.

Figure 2 .

 2 Figure 2.11: Number of body bends per second of young adult worms from each EGL-3 rescue strain. N is the number of worms observed. The body bend per second of egl-3(gk238) mutant worms are used as reference for statistical comparison. (n.s.: not significant, **: p < 0.01, ***: p < 0.001)

Figure 2 . 12 :

 212 Figure 2.12: Proportion of worms displaying SAB overgrowth with (+) or without (-) addition of histamine, in a wild-type or egl-19(n2368) gain-of-function mutant background. For each combination of genotype and condition, bars were ordered from left to right by date, so that bars of the same order are matched together, constituting a replicate. For example, the first bar from the left of each cohort form a replicate. The number of worms observed for each cohort is shown in between parenthesis on top of each bar (n.s.: not significant, ***: p < 0.001).

Figure 2 . 13 :

 213 Figure 2.13: Proportion of worms displaying SAB overgrowth with (+) or without (-) addition of histamine, in a wild-type or unc-2 gain-of-function mutant background. For each combination of genotype and condition, bars were ordered from left to right by date, so that bars of the same order are matched together, constituting a replicate. The number of worms observed for each cohort is shown in between parenthesis on top of each bar. Statistical tests are performed between cohorts of different genotype but in the same condition, to assess the effect of unc-2 gain-of-function mutant background (n.s.: not significant, ***: p < 0.001).

  ::SL2::wScarlet::miniMos1] miniMos single-copy insertion of pMDN4

Table 1 .1: Common neurotransmitters in both vertebrates and nematodes, their effects as well as their chemical formula.

 1

Table 1 .2: Voltage-gated calcium channels in C.elegans. Source: [Hobert, 2013]

 1

	Subunit Gene α1 egl-19 unc-2 cca-1 nca-1 nca-2 α2δ unc-36 tag-180 β ccb-1 ccb-2	Type L-type (ortholog of Ca V 1) P/Q type (ortholog of Ca V 2.1) T-type (ortholog of Ca V 3.3) sodium leak (ortholog of NALCN) neurons Expression neurons, muscles, hypodermis, pharynx neurons neurons, pharynx sodium leak (ortholog of NALCN) neurons neurons, muscles neurons pharynx, muscles neurons

Table 2 . 1 :

 21 List of plasmids used in this work

Table 2 . 2

 22

: List of alleles used in this work Table 2.3: List of C. elegans strains used in this work Table 2.4: Example of a 2x2 contingency table for the i replicate Table 2.5: p-value of statistical tests performed Axonal Plasticity In C.

elegans Motor Neurons SAB Depends On Both Pre-and Postsynaptic Activity Introduction

	During development, axonal morphology is influenced by multiple environmental factors,
	including the electrical activity of the postsynaptic targets. For example, motor axons
	are shaped by muscle activity during development

Generation of single-copy insertion and multi-copies integrated alleles

  The alleles used in this work are listed in Table2. Protein expressions are driven by

	myo-3 -(body-wall muscle), unc-4 -(motor neurons), tni-3 -(head muscle), rgef-1 -
	(pan-neuronal), vha-6 -(intestine), or eft-3 -(ubiquitous) promoters.
	To generate single-copy insertion of a plasmid of interest, we used the miniMos insertion
	method [Frøkjaer-Jensen et al., 2014]. N2 animals were injected with 10-15 ng/µl the
	plasmid of interest containing the promoter and the open reading frame, 50 ng/µl
	pCFJ601 (Mos1 transposase), 10 ng/µl pMA122 (negative selective marker
	Phsp16.2::peel-1 ), and 2.5 ng/µl pAV41 (Pmyo-2::BFP). Neomycin (G418) was added
	to plates 24 h after injection at 0.75 µg/µl final concentration. Plates were kept at 25°C
	until starvation. Starved plates were heat shocked for 2 hours at 34°C. Selected lines
	were then bred to homozygosity.

To generate multi-copies integration of a plasmid of interest, we used the modular safe-harbor transgene insertion (MosTI) method

[El Mouridi et al., 2022]. unc-119(ed3)

mutant animals were injected with 10-25 ng/µl the plasmid of interest, 25 ng/µl pMDJ231 (Phsp-16.41::Cas9::gpd2::tagRFP-T, Cas9 protein under heat shock promoter), 5 ng/µl pSEM319 (ce-unc-119 MosTI array fragment), and 15 ng/µl pSEM319 (single-guide RNA to MosTI landing site). Plates were kept in 15°C for 24 hours, after which neomycin (G418) was added to plates at 0.75 µg/µl final concentration. Plates were kept at 25°C until the worms were close to starvation.

Almost starved plates were incubated for 20 hours at 30°C. Animals with integrated transgene are selected by lack of locomotion defects normally present in unc-119(ed3) mutant animals. Selected lines were then bred to homozygosity.

Table 2 . List of alleles used in this work.

 2

	Name acr-16(kr463::degron::wScarlet) alg-2(ok304) egl-3(gk238) egl-19(n582) egl-21(n476) lite-1(ce314) plx-2(ev773) scd-2(ok565) unc-2(e55) unc-29(kr208::tagRFP-T) unc-36(e251) unc-54(e190) jsIs42 [Punc-4::snb-1::GFP]	Description Reporter for N-AChRs Early stop-codon substitution mutant of a protein in Argonaute family Deletion mutant of proprotein convertase PC2 Loss-of-function substitution mutant of L-type calcium voltage-gated channel subunit α1 Deletion mutant of carboxypeptidase CpE Loss-of-function substitution mutant of blue light photo-ceptor Deletion mutant of plexin Deletion mutant of Anaplastic lymphoma kinase Early stop-codon substitution mutant of non L-type calcium voltage-gated channel subunit α1 Reporter for L-AChRs Early stop-codon substitution mutant of calcium voltage-gated channel auxiliary subunit α2δ Deletion mutant of heavy chain myosin	Source Océane Romatif CGC CGC CGC CGC CGC CGC CGC CGC [Richard et al., 2013] CGC CGC

Table 3 . List of C. elegans strains used in this work.

 3

	Figure Name all EN3940 krSi24; krSi16 Genotype 1, 6 EN3831 krSi24 1 EN7133 unc-54(e190); krSi16; krSi24 2 EN2383 unc-29(kr208::tagRFP-T); jsIs42 2 EN3939 unc-29(kr208::tagRFP-T); krSi24; krSi16 3 EN7922 krIs80; krSi24 4 EN3951 alg-2(ok304); krSi16 ; krSi24 4, S2 EN3953 egl-3(gk238); krSi16 ; krSi24 4 EN3955 egl-21(n476); krSi16 ; krSi24 4 EN7195 scd-2(ok565); krSi16 ; krSi24 4 EN7197 plx-2(ev773); krSi16 ; krSi24 4 EN7402 egl-3(gk238); krSi16; krSi24; krSi66 4 EN7405 egl-3(gk238); krSi16; krSi24; krSi69 4, S2 EN7318 egl-3(gk238); krSi16; krSi24; krSi74 4, S2 EN8085 egl-3(gk238); krSi16; krSi24; krSi66; krSi69 5 EN8086 egl-19(n582); krSi16; krSi24 5 EN8087 unc-2(e55); krSi16; krSi24 5 EN8088 unc-36(e251); krSi16; krSi24 5 EN8423 krSi193; unc-36(e251); krSi16; krSi24 5 EN8424 krSi194; unc-36(e251); krSi16; krSi24 6 EN8787 krSi257; krSi24 6 EN8788 krSi257; krSi16; krSi24 6 EN8873 krIs89; krSi24 6 EN8874 krIs90; krSi24 6, S3 EN8871 krIs91; krSi16; lite-1(ce314) 6, S3 EN8872 krIs92; krSi16; lite-1(ce314) S1 EN7643 acr-16(kr463::degron::wScarlet; krSi81 S2 EN8422 egl-3(gk238); krSi16; krSi24; krSi191 S2 EN8609 egl-3(gk238); krSi16; krSi24; krSi66; krSi69; krSi191 Minh-Dao Nguyen Source Alexis Weinreb Alexis Weinreb Alexis Weinreb Alexis Weinreb Alexis Weinreb Alexis Weinreb Alexis Weinreb Alexis Weinreb Alexis Weinreb Alexis Weinreb Alexis Weinreb Alexis Weinreb Alexis Weinreb Alexis Weinreb Minh-Dao Nguyen Minh-Dao Nguyen Minh-Dao Nguyen Minh-Dao Nguyen Minh-Dao Nguyen Minh-Dao Nguyen Minh-Dao Nguyen Minh-Dao Nguyen Minh-Dao Nguyen Minh-Dao Nguyen Minh-Dao Nguyen Minh-Dao Nguyen Océane Romatif Minh-Dao Nguyen

Table 4 . Example of a 2x2 contingency table for the i replicate

 4

	table per replicate. (n.s.: not significant,

Decrease of muscle activity leads to motor neuron hyperactivity that induces axonal overgrowth.

	From our results, we hypothesized that muscle activity decrease could induce
	hyperactivity of SAB that would lead to overgrowth. Then, decrease of VGCC activity
	in neurons (through unc-36 mutation for example) would counteract this SAB
	hyperactivity induced by muscle shutdown and in turn would suppress axonal
	overgrowth.
	If this model is true, a first prediction is that increasing activity in SAB neurons should
	induce axonal overgrowth defect. We expressed in SAB neurons a modified version of
	HisCl1 channel, named HisCat, that let in cations (e.g. calcium ion) instead of chloride
	ions when the channel is exposed to histamine [Alkema, not yet published]. Upon
	histamine treatment, two independent lines of transgenic HisCat worms displayed SAB
	overgrowth with a significant higher penetrance than that of control animals (35 ± 11%
	February 18, 2023	15/32

  . More recently, a new type of calcium transients mediated by Ca V 1.2 (L-type VGCC, including EGL-19 in C. elegans and CACNA1C in human) was identified in cultured prenatal cortical mouse neurons, called Spontaneous Regenerative CAlcium Transients (SRCaTs) [Kamijo et al., 2018]. During development, Ca V 1.2 seems to open at resting potential and being necessary for the SRCaTs to develop. Axon length of cortical neurons from conditional Ca V 1.2 knockout mice exhibit reduced neurite length. This indicates that Ca V 1.2 functions to stimulate axon growth during development.

	What is still missing from our model is how muscle cells can induce hyperactivity in the
	SAB motor neurons. Our model implies that a retrograde signal is sent by muscle cells
	either directly or indirectly to SAB motor neurons. Retrograde signals have been
	already described in C. elegans neuromuscular junction. The first study, by

Table S1 . p-value of statistical tests performed

 S1

	Fig Population 1 1C Wild-type control 1C krSi16 control 1D krSi16 histamine-treated 2B dorsal axons control 2B ventral axons control 3B Ptni-3::HisCl control 3D H0 3D H0 3D H0 3D H0 3D H0 3D H0 3D H1 3D H1 3D Hinf 4A alg-2 control 4A egl-21 control 4A egl-3 control 4A mlk-1 control 4A plx-2 control 4A scd-2 control 4B egl-3 histamine-treated 4B egl-3 histamine-treated 4B WT* histamine-treated 4B egl-3 histamine-treated 4B egl-3 histamine-treated 4B WT* histamine-treated 5A egl-19 (n582) control 5A egl-19(ad1006) control 5A unc-2 control 5A unc-36 control 5A cca-1 control 5B unc-36 histamine-treated 5B unc-36 histamine-treated 5B WT* histamine-treated 6A HisCl in muscle + histamine 6A HisCl in SAB control 6B Wild-type control 6B HisCat #1 control 6B HisCat #2 control 6B Wild-type control 6B Wild-type control 6D GCaMP6s #2 control 6D GCaMP6s #5 control 8 egl-3 histamine-treated 8 egl-3 histamine-treated 8 EGL-3 ubiquitous rescue + histamine EGL-3 triple rescue + histamine Population 2 Wild-type histamine-treated krSi16 histamine-treated unc-54 control dorsal axons histamine-treated ventral axons histamine-treated Ptni-3::HisCl histamine-treated H1 H2 Hinf H1a H1b H1c Hinf H2 H2 alg-2 histamine-treated egl-21 histamine-treated egl-3 histamine-treated mlk-1 histamine-treated plx-2 histamine-treated scd-2 histamine-treated EGL-3 muscle rescue + histamine EGL-3 neuronal rescue + histamine EGL-3 neuronal rescue + histamine EGL-3 double rescue + histamine EGL-3 ubiquitous rescue + histamine CMH test Test CMH test CMH test CMH test Welch t-test 2.39E-02 p-value 6.66E-01 1.05E-06 2.20E-16 Welch t-test 1.70E-03 CMH test 2.26E-14 CMH test 2.20E-16 CMH test 9.72E-03 CMH test 2.20E-16 CMH test 1.28E-07 CMH test 4.23E-06 CMH test 1.46E-02 CMH test 2.19E-01 CMH test 2.20E-16 CMH test 2.20E-16 CMH test 4.31E-01 CMH test 7.24E-02 CMH test 2.20E-16 CMH test 1.62E-02 CMH test 4.09E-01 CMH test 8.95E-01 CMH test 7.93E-01 CMH test 3.53E-03 CMH test 8.91E-09 CMH test 1.65E-01 2.20E-16 EGL-3 ubiquitous rescue + histamine CMH test 3.94E-01 egl-19(n582) histamine-treated CMH test 2.20E-16 egl-19(ad1006) histamine-treated CMH test 2.34E-15 unc-2 histamine-treated CMH test 4.78E-16 unc-36 histamine-treated CMH test 2.20E-16 cca-1 histamine-treated CMH test 1.64E-01 UNC-36 muscle rescue + histamine CMH test UNC-36 neuronal rescue + histamine CMH test 2.20E-16 UNC-36 neuronal rescue + histamine CMH test 4.99E-01 HisCl in both + histamine CMH test 2.20E-16 HisCl in SAB + histamine CMH test 5.39E-01 Wild-type histamine-treated CMH test 7.84E-01 HisCat # 1 + histamine CMH test 1.88E-02 HisCat # 2 + histamine CMH test 1.67E-01 HisCat #1 control CMH test 2.86E-06 HisCat #2 control CMH test 4.02E-05 GCaMP6s #2 histamine-treated Welch t-test 2.20E-16 GCaMP6s #5 histamine-treated Welch t-test 2.20E-16 EGL-3 intestinal rescue + histamine CMH test 8.80E-04 EGL-3 triple rescue + histamine CMH test 6.38E-01 CMH test 1.48E-09 1.00E+00 n.s. Note n.s. *** *** * ** *** *** *** n.s. *** *** *** *** *** * * n.s. n.s. *** n.s. *** n.s. *** *** *** *** n.s. *** n.s. *** ** n.s. n.s. n.s. * *** n.s. n.s. *** *** n.s. * *** *** *** ** Fig S4.
	February 18, 2023	24/32

Movement of Ptni-3::HisCl1 worms on histamine

		2.2. Functional test of EGL-3 tissue-specific expression
	February 18, 2023	25/32

  . More recently, a new type of calcium transients mediated by Ca V 1.2 (L-type VGCC, including EGL-19 in C. elegans and CACNA1C in human) was identified in cultured prenatal cortical mouse neurons, called Spontaneous Regenerative CAlcium Transients (SRCaTs)[Kamijo et al., 2018]. During development, Ca V 1.2 seems to open at resting potential and being necessary for the SRCaTs to develop. Axon length of cortical neurons from conditional Ca V 1.2 knockout mice exhibit reduced neurite length.This indicates that Ca V 1.2 functions to stimulate axon growth during development.Gain-of-function mutations of EGL-19 seem to both induce SAB overgrowth in control condition and suppress overgrowth in histamine-treated condition. However, this result does not provide a good conclusion since both muscle and neurons are hyperactive in this strain. EGL-19 gain-of-function mutant worms are not completely paralysed in 1 mM of

	Chapter 3. Discussion
	histamine as other strains do.

Table 4 .2: List of alleles used in this work.

 4

  table per replicate. (n.s.: not significant, *: p < 0.05, **: p < 0.01, ***: p < 0.001) Mean numbers of varicosities in SAB axons are compared using Welch t-test. Mean normalized weighted ratios of calcium indicator/reference in SAB cell bodies are also compared using Welch t-test (n.s.: not significant, *: p < 0.05, **: p < 0.01, ***: p < 0.001).

Table 4 .3: List of C. elegans strains used in this work.

 4

	Figure Name	Genotype

Table 4 .4: Example of a 2x2 contingency table for the i replicate

 4

		Genotype 1	Genotype	2	Row total
		(or control)	(or histamine-
			treated)
	Defect	A i	B i		N 1i
	Normal	C i	D i		N 2i
	Column total M 1i	M 2i		T i

(Polysciences Polybead®polystyrene 0.1 µm). The SAB axons, reported by krSi24

[START_REF] Nonet | Visualization of synaptic specializations in live C. elegans with synaptic vesicle protein-GFP fusions[END_REF]

February 18, 2023 9/32

Remerciements

First and foremost, I would like to express my gratitude to members of my thesis jury: Bénédicte DURAND, Michel LABOUESSE, and Homaira NAWABI. Thank you for agree-

Acknowledgments

We thank Sonia El Mouridi and Christian Frøkjaer-Jensen for sharing the reagents for MosTI multicopy integration, as well as the scientific discussion. We thank Mark Alkema for sharing the HisCat plasmid. We thank Paschalis Kratsios for sharing his insights on promoters driving expressions in motor neurons. Some strains were provided by the CGC, which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440).

February 18, 2023 21/32

Supplementary Information

Chapter 4

Materials and methods

Plasmids

The plasmids used in this work are listed in Table 4.1.

All plasmids were built using Gibson assembly [Gibson, 2011]. All plasmids' open reading frame were confirmed by sequencing. Plasmids named pAW are built by Alexis Weinreb, plasmids named pMDN are built by Minh-Dao Nguyen. Punc-4(short) is a truncated Punc-4 promoter that still shows strong expression in SABs but weak expression in other motor neurons.

Generation of single-copy insertion and multi-copies integrated alleles

The alleles used in this work are listed in Table 4.2