Feng B Quan

Anne-Laure Gaillard

Faredin Alejevski

Guillaume Pézeron
email: guillaume.pezeron@mnhn.fr

Hervé Tostivint
email: htostivi@mnhn.fr

Urotensin II-related peptide (Urp) is expressed in motoneurons in zebrafish, but is dispensable for locomotion in larva

Keywords: Urotensin 2, URP, motoneurons, zebrafish

The urotensin 2 (uts2) gene family consists of four paralogs called uts2, uts2-related peptide (urp), urp1 and urp2. uts2 is known to exert a large array of biological effects, including osmoregulation, control of cardiovascular functions and regulation of endocrine activities.

Lately, urp1 and urp2 have been shown to regulate axial straightening during embryogenesis.

In contrast, much less is known about the roles of urp. The aim of the present study was to investigate the expression and the functions of urp by using the zebrafish as a model.

For this purpose, we determined the expression pattern of the urp gene. We found that urp is expressed in motoneurons of the brainstem and the spinal cord, as in tetrapods. This was confirmed with a new Tg(urp:gfp) fluorescent reporter line. We also generated a urp knockout mutant by using CRISPR/Cas9-mediated genome editing and analysed its locomotor activity in larvae. urp mutant did not exhibit any apparent defect of spontaneous swimming when compared to wild-type.

We also tested the idea that urp may represent an intermediary of urp1 and urp2 in their role on axial straightening. We found that the upward bending of the tail induced by the overexpression of urp2 in 24-hpf embryos was not altered in urp mutants. Our results indicate that urp does probably not act as a relay downstream of urp2.

In conclusion, the present study showed that zebrafish urp gene is primarily expressed in motoneurons but is apparently dispensable for locomotor activity in the early larval stages.

Introduction

Urotensin 2 (Uts2) is a cyclic neuropeptide that was first isolated from the urophysis of the teleost fish goby Gillichthys mirabilis [START_REF] Pearson | Urotensin II: a somatostatin-like peptide in the caudal neurosecretory system of fishes[END_REF] and subsequently identified in most vertebrate species [START_REF] Conlon | Liberation of urotensin II from the teleost urophysis: an historical overview[END_REF]. The primary structure of Uts2 has been strongly conserved during evolution, especially at the level of the cyclic region (CFWKYC) which is shared by all species investigated so far [START_REF] Conlon | Somatostatin-and urotensin II-related peptides: molecular diversity and evolutionary perspectives[END_REF][START_REF] Jarry | The vasoactive peptides urotensin II and urotensin II-related peptide regulate astrocyte activity through common and distinct mechanisms: involvement in cell proliferation[END_REF]. In teleosts, uts2 mRNA mainly occurs in neurosecretory neurons located in the caudal part of the spinal cord, called Dahlgren cells, that send their axons into the urophysis [START_REF] Ohsako | Cloning and sequence analysis of cDNAs encoding precursors of urotensin II-alpha and -gamma[END_REF][START_REF] Parmentier | Comparative distribution of the mRNAs encoding urotensin I and urotensin II in zebrafish[END_REF]. In contrast, in mammals, which are devoid of this caudal neurosecretory system, the uts2 gene is expressed primarily in motoneurons of the brainstem and spinal cord [START_REF] Coulouarn | Cloning, sequence analysis and tissue distribution of the mouse and rat urotensin II precursors[END_REF][START_REF] Coulouarn | Cloning of the cDNA encoding the urotensin II precursor in frog and human reveals intense expression of the urotensin II gene in motoneurons of the spinal cord[END_REF][START_REF] Dun | Urotensin IIimmunoreactivity in the brainstem and spinal cord of the rat[END_REF]. Uts2 has been shown to exert various effects both in mammals and in teleosts, including osmoregulation, control of cardiovascular functions and regulation of endocrine activities [START_REF] Vaudry | International Union of Basic and Clinical Pharmacology. XCII. Urotensin II, Urotensin II-Related Peptide, and their receptor: from structure to function[END_REF][START_REF] Vaudry | Urotensin II, from fish to human[END_REF]. Biological actions of Uts2 are mediated through activation of G-proteincoupled receptors called Uts2 receptor (Uts2r, aka Utr). In mammals, there is only one Uts2r [START_REF] Ames | Human urotensin-II is a potent vasoconstrictor and agonist for the orphan receptor GPR14[END_REF][START_REF] Liu | Identification of Urotensin II as the endogenous ligand for the orphan G-protein-coupled receptor GPR14[END_REF][START_REF] Mori | Urotensin II Is the endogenous ligand of a G-Protein-Coupled Orphan Receptor, SENR (GPR14)[END_REF][START_REF] Nothacker | Identification of the natural ligand of an orphan G-protein-coupled receptor involved in the regulation of vasoconstriction[END_REF], but the occurrence of up to five uts2r subtype genes (uts2r1-5) has been demonstrated in other vertebrates. Among these subtypes, uts2r1 is the counterpart of the mammalian uts2r [START_REF] Cui | Characterization of four urotensin II receptors (UTS2Rs) in chickens[END_REF][START_REF] Konno | Identification and signaling characterization of four urotensin II receptor subtypes in the western clawed frog, Xenopus tropicalis[END_REF][START_REF] Tostivint | Molecular evolution of GPCRS: somatostatin/urotensin II receptors[END_REF].

It is now well established that Uts2 belongs to a family of structurally related peptides that, in addition to Uts2, includes three Uts2-related peptides (Urps) called Urp, Urp1 and Urp2 [START_REF] Tostivint | Molecular evolution of GPCRS: somatostatin/urotensin II receptors[END_REF][START_REF] Tostivint | Impact of gene/genome duplications on the evolution of the urotensin II and somatostatin families[END_REF]. Synteny analysis suggested that the four genes of the uts2 family arose through the two whole-genome duplication events, called 2R, that took place early during vertebrate evolution [START_REF] Tostivint | Molecular evolution of GPCRS: somatostatin/urotensin II receptors[END_REF][START_REF] Tostivint | Impact of gene/genome duplications on the evolution of the urotensin II and somatostatin families[END_REF][START_REF] Parmentier | Occurrence of two distinct urotensin II-related peptides in zebrafish provides new insight into the evolutionary history of the urotensin II gene family[END_REF][START_REF] Quan | Characterization of the true ortholog of the urotensin II-related peptide (URP) gene in teleosts[END_REF]. Uts2, Urp, Urp1 and Urp2 exhibit the same cyclic hexapeptide core sequence while their N-and C-terminal regions are more variable.

The urp gene has been initially characterized in rat [START_REF] Sugo | Identification of urotensin II-related peptide as the urotensin II-immunoreactive molecule in the rat brain[END_REF] then in chicken [START_REF] Sugo | Identification of urotensin II-related peptide as the urotensin II-immunoreactive molecule in the rat brain[END_REF] and frog [START_REF] Konno | Urotensin II receptor (UTR) exists in hyaline chondrocytes: a study of peripheral distribution of UTR in the African clawed frog, Xenopus laevis[END_REF]. In teleosts, urp1 and urp2 were first reported as two co-orthologs of urp [START_REF] Nobata | Potent cardiovascular effects of homologous urotensin II (UII)-related peptide and UII in unanesthetized eels after peripheral and central injections[END_REF] but this view was invalidated when it appeared that both genes represent two distinct paralogs of the uts2 family [START_REF] Parmentier | Occurrence of two distinct urotensin II-related peptides in zebrafish provides new insight into the evolutionary history of the urotensin II gene family[END_REF]. Finally, the occurrence of the genuine urp was demonstrated soon after in zebrafish and medaka [START_REF] Quan | Characterization of the true ortholog of the urotensin II-related peptide (URP) gene in teleosts[END_REF]. In tetrapods, urp, like uts2, is primarily expressed in motoneurons of the brainstem and the spinal cord [START_REF] Konno | Urotensin II receptor (UTR) exists in hyaline chondrocytes: a study of peripheral distribution of UTR in the African clawed frog, Xenopus laevis[END_REF][START_REF] Bougerol | Generation of BAC transgenic tadpoles enabling live imaging of motoneurons by using the Urotensin II-related peptide (ust2b) gene as a driver[END_REF][START_REF] Dubessy | Characterization of urotensin II, distribution of urotensin II, urotensin II-related peptide and UT receptor mRNAs in mouse: evidence of urotensin II at the neuromuscular junction[END_REF][START_REF] Pelletier | Androgenic down-regulation of urotensin II precursor, urotensin II-related peptide precursor and androgen receptor mRNA in the mouse spinal cord[END_REF]. In the mouse spinal cord, urp and uts2 transcripts were seen to co-localize in most motoneurons [START_REF] Pelletier | Androgenic down-regulation of urotensin II precursor, urotensin II-related peptide precursor and androgen receptor mRNA in the mouse spinal cord[END_REF]. The urp1 and urp2 genes have been initially found only in teleost species [START_REF] Parmentier | Occurrence of two distinct urotensin II-related peptides in zebrafish provides new insight into the evolutionary history of the urotensin II gene family[END_REF][START_REF] Nobata | Potent cardiovascular effects of homologous urotensin II (UII)-related peptide and UII in unanesthetized eels after peripheral and central injections[END_REF] but the occurrence of either one of the two genes has subsequently been reported outside this taxon, notably in spotted gar, elephant shark, coelacanth [START_REF] Tostivint | Molecular evolution of GPCRS: somatostatin/urotensin II receptors[END_REF][START_REF] Tostivint | Impact of gene/genome duplications on the evolution of the urotensin II and somatostatin families[END_REF] and frog [START_REF] Alejevski | Conserved role of the urotensin II receptor 4 signaling pathway to control body straightness in a tetrapod[END_REF]. In zebrafish, urp1 and urp2 mRNAs have primarily been detected in the brainstem and the spinal cord [START_REF] Parmentier | Occurrence of two distinct urotensin II-related peptides in zebrafish provides new insight into the evolutionary history of the urotensin II gene family[END_REF][START_REF] Quan | Comparative distribution and in vitro activities of the Urotensin II-Related Peptides URP1 and URP2 in zebrafish: evidence for their colocalization in spinal Cerebrospinal Fluid-Contacting Neurons[END_REF]. In the spinal cord, urp1 and urp2-expressing cells have been demonstrated to correspond to cerebrospinal fluid-contacting neurons (CSF-cNs) [START_REF] Quan | Comparative distribution and in vitro activities of the Urotensin II-Related Peptides URP1 and URP2 in zebrafish: evidence for their colocalization in spinal Cerebrospinal Fluid-Contacting Neurons[END_REF].

Up to now, the functional properties of the three Urps have been poorly studied, in contrast to those of Uts2. In mammals, Urp is able to activate Uts2r1 with the same potency as Uts2 [START_REF] Sugo | Identification of urotensin II-related peptide as the urotensin II-immunoreactive molecule in the rat brain[END_REF], suggesting that the two peptides have some redundant functions [START_REF] Vaudry | International Union of Basic and Clinical Pharmacology. XCII. Urotensin II, Urotensin II-Related Peptide, and their receptor: from structure to function[END_REF][START_REF] Vaudry | Urotensin II, from fish to human[END_REF]. However, Uts2 and Urp have also been shown to exert distinct effects, for example on astrocyte activity [START_REF] Jarry | The vasoactive peptides urotensin II and urotensin II-related peptide regulate astrocyte activity through common and distinct mechanisms: involvement in cell proliferation[END_REF]. Despite these data, the genuine functions of Urp are still largely unknown. In zebrafish, in a recent study based on gain-and loss-of-function experiments, Urp1 and Urp2 have been suggested to regulate axial straightening during development [START_REF] Zhang | Cilia-driven cerebrospinal fluid flow directs expression of urotensin neuropeptides to straighten the vertebrate body axis[END_REF]. This action appeared to require one of the zebrafish Uts2 receptor subtypes, Uts2r3, specifically expressed in muscles [START_REF] Zhang | Cilia-driven cerebrospinal fluid flow directs expression of urotensin neuropeptides to straighten the vertebrate body axis[END_REF] The study by Zhang et al. [START_REF] Zhang | Cilia-driven cerebrospinal fluid flow directs expression of urotensin neuropeptides to straighten the vertebrate body axis[END_REF] illustrated that the zebrafish is a suitable model to investigate the roles of the peptides of the Uts2 family. The aim of the present study was to focus on zebrafish Urp [START_REF] Quan | Characterization of the true ortholog of the urotensin II-related peptide (URP) gene in teleosts[END_REF]. For this purpose, we determined the expression pattern of the urp gene and generated urp knockout mutant by using CRISPR/Cas9-mediated genome editing.

Materials and methods

Nomenclature

In mammals, the gene encoding Urp neuropeptide has been named Uts2b (e.g. for mouse: ENSMUSG00000056423 in Ensembl). In bony fish, the gene was named uts2d (ENSDARG00000098149 for zebrafish in Ensembl). To avoid confusion, we will refer to this gene as urp in our manuscript.

Ensembl gene ID of the Uts2 receptor genes in zebrafish are the following: uts2r1:

ENSDARG00000009624; uts2r2: ENSDARG00000078481; uts2r3: ENSDARG00000040816; uts2r4: ENSDARG00000096322; uts2r5: ENSDARG00000096870.

Animals

Zebrafish (Danio rerio) were bred and maintained according to standard procedures [START_REF] Westerfield | The zebrafish book. A guide for the laboratory use of zebrafish (Danio rerio)[END_REF].

Embryos were raised at 28.5°C in E3 embryo medium and staged according to Kimmel et al. [START_REF] Kimmel | Stages of embryonic development of the zebrafish[END_REF]. Wild-type animals were Tubingen and TL. Transgenic line Tg(mnx1:gfp) was previously described [START_REF] Flanagan-Steet | Neuromuscular synapses can form in vivo by incorporation of initially aneural postsynaptic specializations[END_REF].

All procedures were approved by the Institutional Ethics Committee Cuvier at the Muséum national d'Histoire naturelle (protocols # 68-020; APAFIS#6945). In accordance to these guidelines, all efforts were made to minimize the number of animals used and their suffering.

Real time quantitative RT-PCR.

Total RNA samples were purified from entire wild type embryos, larvae or juveniles collected at the appropriate stage, and from tissues including brain, spinal cord, eyes, skin, muscle, heart, spleen, gill, gas bladder, intestine, liver, kidney, ovary and testis dissected from adults.

All samples were lysed using a TissueLyser II system (Qiagen) with RNAble solution (Eurobio). Samples were treated with DNAse I (Roche) to remove potential contamination by genomic DNA and then purified with phenol/chloroform extraction. The integrity of RNA was then assayed by electrophoresis on a 1% agarose gel. cDNA samples were obtained from 2 µg of purified total RNA using Goscript reverse transcriptase (Promega) with random primers (Promega). Finally, specific cDNAs were quantified by real-time PCR using PowerUp SYBR Green (Applied Biosytem) and a QuantStudio 6 Flex instrument (Applied Biosytem). For all RT-qPCR, lsm12B and mob4 were used as housekeeping genes [START_REF] Hu | Identification of novel Reference genes suitable for qRT-PCR normalization with respect to the zebrafish developmental stage[END_REF].

Primer sequences are listed in Table S1. Prior to be used in qPCR, all primers pairs were assayed on 4 serial dilutions of corresponding cDNA. Linearity of CT variation on dilutions and single peak of melting curved were verified. Relative quantities were calculated using the 2e-∆CT formula.

In situ hybridization

To generate the urp probe, we used a previously described full length cDNA [START_REF] Quan | Characterization of the true ortholog of the urotensin II-related peptide (URP) gene in teleosts[END_REF] cloned into pGEMT-easy (Promega). The urp1 and gfp probes were previously described [START_REF] Quan | Comparative distribution and in vitro activities of the Urotensin II-Related Peptides URP1 and URP2 in zebrafish: evidence for their colocalization in spinal Cerebrospinal Fluid-Contacting Neurons[END_REF][START_REF] Labalette | Molecular dissection of segment formation in the developing hindbrain[END_REF]. Sense and antisense RNA probes were synthesized with SP6 or T7 transcriptase by using Digoxigenin (Dig)-and Fluorescein (Fluo)-RNA labelling mix (Roche). In situ hybridization (ISH) experiments were carried out following the protocols reported in previous studies [START_REF] Quan | Comparative distribution and in vitro activities of the Urotensin II-Related Peptides URP1 and URP2 in zebrafish: evidence for their colocalization in spinal Cerebrospinal Fluid-Contacting Neurons[END_REF].

In brief, embryos and dissected adult brains and spinal cords, were fixed overnight at 4°C in 4% PFA in PBS. The hybridization step was performed overnight at 65°C. Dig-/Fluo-labelled probes were detected with anti-Dig/Fluo antibodies conjugated to alkaline phosphatase (1/4000, Roche) followed by a chromogenic reaction using a solution of BM Purple (Roche) as substrate. For fluorescent ISH, labelled probes were detected using anti-Dig/Fluo antibodies conjugated to peroxidase (1/150, Roche) and revealed by Tyramide Signal Amplification using Tyramide-FITC (PerkinElmer) or -TAMRA (Invitrogen) as substrate. For double ISH, Fluo-and Dig-labelled probed were incubated together but revealed successively, in this order. Following staining, brains and spinal cords were included in 3% agarose and sectioned at 50 μm using a vibratome (VT1000S Leica).

Immunohistochemistry

For urp -ChAT double staining, ISH was performed before immunohistochemistry and revealed using FITC-or TAMRA-conjugated tyramide. After several washes in PBS, tissues were submitted to immunostainning as described in [START_REF] Bougerol | Generation of BAC transgenic tadpoles enabling live imaging of motoneurons by using the Urotensin II-related peptide (ust2b) gene as a driver[END_REF]. The primary antibodies used for IHC were goat anti-choline-acetyltransferase (ChAT; 1:100, Millipore)

while the secondary antibodies were donkey anti-rabbit or anti-goat IgGs coupled to Alexa

Fluor 488 and Alexa Fluor 546, respectively (1:500, Life Technologies).

To detect GFP, embryos were fixed for 2 hours at room temperature or overnight at 4°C in 4% PFA in PBS. Following several washes in PBS -0.01% Tween 20 (PBS-Tw), embryos were blocked for 1 hour at room temperature in blocking solution (1% DMSO, 1% Triton, 1%

BSA and 10% Normal Goat Serum in PBS) then incubated overnight at 4°C with primary antibody in blocking solution (rabbit anti:GFP, 1:500, Life Technologies). Samples were washed in PBS -0.5% Triton and incubated overnight at 4°C with secondary antibody in blocking solution (Alexa 488-conjugated goat anti-rabbit, 1:500, Life Technologies). Finally, embryos were washed in PBS -0.5% Triton and mount in 80% glycerol.

Image acquisition

For single labelling, embryos were imaged on an Olympus SZX12 stereomicroscope while brain and spinal cord sections were imaged on a Leica DM5500B microscope. For fluorescent double labelling, samples were imaged on an Olympus FV1000 confocal microscope (urpgfp / ChAT double staining) or on a Zeiss Axiozoom v16 (urp -urp1 double staining). Whole mount GFP stained embryos were imaged on a Zeiss Axiozoom v16. Images were then processed using ImageJ [START_REF] Schneider | NIH Image to ImageJ: 25 years of image analysis[END_REF].

Generation of transgenic lines

Transgene constructs were generated using plasmids from the tol2kit [START_REF] Kwan | The Tol2kit: a multisite gateway-based construction kit for Tol2 transposon transgenesis constructs[END_REF] and Gateway recombination system (Life technologies). For the urp:GFPcaax construct, a 2.6kb genomic DNA fragment, corresponding to the sequence immediately upstream of the ATG of urp, was amplified by PCR (Q5 polymerase, NEB) and cloned by BP recombination into pDONRP4-P1R to produce p5E-2.6kb-urp. The final transgene was then obtained by LR recombination with the entry plasmids p5E-2.6kbURP, pME-eGFPcaax and p3E-polyA into the pDestTol2CG2 destination vector. For the hsp:urp2 construct, the coding sequence from urp2 was amplified by PCR (Q5 polymerase, NEB) from cDNA and cloned into pME-MCS using BamHI -XbaI. Final transgenes were then obtained by recombination with p5E-hsp70l and p3E-polyA into the pDestTol2CG2 destination vector. For transgenesis, 2 nl of a solution with 30 ng/µl of plasmid and 25 ng/µl of tol2 transposase mRNA were injected in 1 or 2 cellstage embryos.

CRISPR/Cas9 RNA guide design and injection

Two small guide RNA (sequences in Table S1) were designed using CRISPOR (http://crispor.tefor.net/, [START_REF] Haeussler | Evaluation of off-target and on-target scoring algorithms and integration into the guide RNA selection tool CRISPOR[END_REF]), in order to frame the region encoding for the mature peptide, and produced in vitro as previously described [START_REF] Auer | CRISPR/Cas9-mediated conversion of eGFP-into Gal4-transgenic lines in zebrafish[END_REF]. sgRNA were individually mixed with Cas9 protein to form ribonucleoprotein complexes (160 ng/µl sgRNA and 0.8 µg/µl Cas9).

Embryos at 1-2 cell-stage were injected with 2 nl of a mix of the two sgRNA-Cas9 complexes and raised to adulthood. These founders were crossed against wildtype fish to screen for their ability to transmit a deleted allele by genotyping their progeny. F1 from two identified founders were raised to establish the two different mutant lines.

For genotyping, genomic DNA was extracted from either fin clips (adult) or whole embryos in 50 mM NaOH for 15 min at 95°C. After buffer neutralisation with 1/10 volume of Tris 10 mM pH 8, the DNA solution was directly used for PCR amplification (wildtype: 731 bp, mutants 556 bp and 565 bp. Primer sequences in Table S1).

Quantification of locomotor activity

Six days old larvae from crosses between urp -/-mutant females and heterozygous males, giving 50% of MZurp -/-and 50% of urp +/-, were individually placed in 48-well plates.

Spontaneous swimming was captured using a Noldus Daniovision system and the distance moved per minute was measured using the Ethovision software. To increase spontaneous swimming, after a habituation period of 15 minutes, larvae were submitted to a cycle of 15 minutes of dark followed by 15 minutes of light, repeated 2 times. Following records, larvae were euthanized and genotyped.

Statistics

Quantitative data were analysed using R software [START_REF] Core | R: A Language and Environment for Statistical Computing[END_REF] and the tidyverse package [START_REF] Wickham | Welcome to the tidyverse[END_REF]. Plots were produced using the ggplot2 packages [START_REF] Wickham | ggplot2: Elegant Graphics for Data Analysis[END_REF]. Values are presented as mean ± SEM or boxplot (median ± interquartile range). Welch two sample t-test and Fisher's exact test were performed as indicated in figure legends.

Results

The urp gene is expressed in the brainstem and the spinal cord both in embryos and adults

The distribution of zebrafish urp mRNA in various adult tissues was examined by RT-qPCR.

Figure 1 displays the relative expression of urp in various tissues. The urp transcript was mostly detected in the spinal cord with a much lower expression in the brain. To analyse the expression of urp in the zebrafish nervous system, we performed ISH with a Dig-labelled probe on adult tissue sections and on whole embryos from 10 hours post fertilization (hpf) to 3 days post fertilization (dpf). In the adult brain, urp-expressing cells were detected in two regions of the brainstem, namely the motor nucleus of the vagal nerve and in the intermediate reticular formation (Figure 2a). In the spinal cord, urp mRNA-containing cells were located in two parallel rows along its full length in a ventro-lateral position (Figure 2b-d).

The expression of urp during development was assessed using RT-qPCR. Figure 3 shows the relative expression of urp in whole animals from one-cell stage to juvenile stage (28 dpf). A very low expression was detected in one-cell stage embryos suggesting maternal expression.

During early embryonic development, urp expression remained low until mid somitogenesis (16 hpf) when it increased slightly. A peak of expression was observed at 24 hpf, at the end of somitogenesis. urp expression was decreased at 2 dpf and a second peak was observed at 3-4 dpf. After 4 dpf, urp expression gradually decreased until 28 dpf.

By ISH, the first urp-expressing cells were observed at 16 hpf (Figure 4a) in the anterior spinal cord. At the end of somitogenesis (24 hpf, Figure 4b-c), the urp transcript was distributed in two parallel rows, as in adult, both in the hindbrain and the rostral part of the spinal cord. Thereafter, urp staining gradually disappeared from the spinal cord and was only visible in the hindbrain at 2 dpf (Figure 4d-e).

Most cells containing urp mRNA express motoneuronal markers

In both embryos and adults, urp-expressing cells were primarily found in the ventro-lateral part of the spinal cord, a region that corresponds to the motoneuronal domain. To test the motoneuronal identity of these cells, their ability to express two motoneuronal markers, Mnx1

(also named Hb9), a transcription factor involved in motoneuron specification [START_REF] Arber | Requirement for the homeobox gene Hb9 in the consolidation of motor neuron identity[END_REF], and ChAT (Choline-AcetylTransferase), was analysed. The expression of mnx1 was assayed using a Tg(mnx1:gfp) transgenic line [START_REF] Flanagan-Steet | Neuromuscular synapses can form in vivo by incorporation of initially aneural postsynaptic specializations[END_REF] and ChAT expression was determined using an anti-ChAT antibody. In Tg(mnx1:gfp) 24-hpf embryos, due to the weak expression of urp in the spinal cord, double-stained urp -gfp cells could be viewed only in the hindbrain. As depicted in figure 5a-c, all urp mRNA containing cells appeared gfp positive, but conversely, only a part of the latter (about 20%) contained urp transcript. In the adult spinal cord (Figure 5d-g), all urp-expressing cells were stained by the anti-ChAT antibody, but numerous ChAT immunoreactive cells did not appear to contain the urp mRNA.

urp is co-expressed with urp1 in some cells of the adult hindbrain.

In a previous study [START_REF] Quan | Comparative distribution and in vitro activities of the Urotensin II-Related Peptides URP1 and URP2 in zebrafish: evidence for their colocalization in spinal Cerebrospinal Fluid-Contacting Neurons[END_REF], we have observed that the urp1 gene is expressed in two structures of the adult hindbrain, namely the motor nucleus of the vagal nerve and the reticular formation. Because both structures appeared to also contain urp-expressing cells, we asked whether urp and urp1 are expressed in the same cells. As depicted in figure 6, urp and urp1 mRNAs co-localized in the same cells of the motor nucleus of vagal nerve. In contrast, no colocalization was seen in the reticular formation (data not shown).

A transgenic reporter line suggests that urp is expressed in all motoneuron subtypes.

Our results suggested that urp is expressed in motoneurons in both embryos and adults. To determine whether urp expression is restricted to a subtype of motoneurons, we sought to visualise axonal projections of urp-expressing cells with a GFP reporter transgenic line. To do so, a genomic DNA fragment corresponding to the urp presumptive promotor, 2.6 kb immediately upstream of urp start codon, was cloned and placed upstream of a membrane bound GFP (GFPcaax) in a Tol2 plasmid (see methods). Consistent with urp expression profile, embryos injected with this plasmid exhibited mosaic GFP expression restricted to what appeared to be motoneurons (data not shown). Thus, these animals were raised to establish the Tg(urp:gfp) transgenic line. We assayed GFP expression in Tg(urp:gfp) using both ISH and immunofluorescence staining. At 1 dpf, GFP expression could not be detected.

At 2 dpf, gfp mRNA was detected in two rows of cells in the anterior half of the spinal cord (Figure 7a, a') and the GFP protein pattern was quite similar (Figure 7b,b').

In 3-dpf embryos, gfp mRNA could not be detected, however, likely due to protein stability and accumulation, GFP immunofluorescence staining was detected all along the spinal cord.

GFP positive cells were organised along two rows with projections towards ventral, medial and dorsal part of each somite (Figure 7c, c', c''), suggesting GFP expression in all three subtypes of primary motoneurons (RoP, CaP, MiP) [START_REF] Lewis | From cells to circuits: development of the zebrafish spinal cord[END_REF][START_REF] Myers | Development and axonal outgrowth of identified motoneurons in the zebrafish[END_REF]. Besides, a large number of cells was observed in each somite, suggesting that not only primary but also secondary motoneurons expressed GFP. Overall, this profile suggested that the Tg(urp:gfp) line globally recapitulates the endogenous urp expression pattern and that Urp neuropeptide is specifically expressed in motoneurons in the spinal cord.

urp is not required for correct development and locomotion.

To investigate the function of Urp, we used CRISPR/Cas9-mediated genome edition to produce an urp knockout mutant line. Since the function of urp is likely to reside within its last exon, the one encoding for the mature peptide, we aimed at deleting this entire region. We designed two sgRNA, one on each side of the target region (Figure 8a), that were injected simultaneously into 1-or 2-cell stage embryos. We recovered two alleles, which differed for only a few nucleotides, corresponding to a complete deletion of the targeted region and thus most likely to be null (Figure 8a). We then produced homozygous urp -/-mutants but no abnormal phenotype was observed at morphological or global behaviour levels at any stage.

Adult urp -/-mutant fish appeared normally viable and fertile. To test for a putative compensation effect from maternal mRNA contribution, we produced maternal and zygotic (MZ) mutants from homozygous mutant females but again these animals developed normally.

Since, urp appeared expressed in motoneurons, we reasoned that the loss of urp could result in alterations of locomotor ability. To test for this possibility, we analysed spontaneous swimming of 6-dpf larvae using an automated tracking system. Control (heterozygous siblings) and MZurp mutant larvae were submitted to two cycles of 15 min of dark and 15 min of light and the distanced moved per minute was measure for each larva. Overall, no significant difference was observed. In particular, the total distances moved during dark and light periods were not different (Figure 8b-c; cumulated distance during light on periods: 569 mm ± 349 (sd) and 581 mm ± 276 for controls and mutants respectively; light off periods: 971 mm ± 579 and 980 mm ± 489 for controls and mutants respectively).

Next, to test whether the absence of Urp could influence the expression of uts2r genes, we used RT-qPCR to measure their expression levels in 24-hpf embryos, when urp expression is the highest. While expression of uts2r5 could not be reproducibly detected in neither control nor mutant (not shown), the levels of expression of ust2r1, uts2r2 and uts2r4 were not altered in MZurp mutants. In contrast, a slight decrease in uts2r3 expression level was observed in MZurp mutant embryos (Figure 8d, RQ = 1.01 ± 0.07 (sem) and 0.74 ± 0.06 for controls and mutants respectively).

It has been previously reported that the absence of phenotype of a null mutant can be due to genetic compensation. In particular, mutant mRNA degradation can trigger transcriptional adaptation [START_REF] El-Brolosy | Genetic compensation triggered by mutant mRNA degradation[END_REF]. To test if such a mechanism could explain the lack of apparent phenotype in urp mutants, we used RT-qPCR to measure the expression level of all genes of the uts2 family in 24-hpf embryos. While our results clearly showed a reduction of urp mRNA in MZurp-/mutants, likely due to mRNA decay, we could not detect any increase in the expression of the four other genes (Figure 8e).

Urp is not involved in axis straightness.

Several recent studies have reported the involvement of urp1, urp2 and uts2r3 in correct axis straightness in zebrafish [START_REF] Zhang | Cilia-driven cerebrospinal fluid flow directs expression of urotensin neuropeptides to straighten the vertebrate body axis[END_REF][START_REF] Cantaut-Belarif | Adrenergic activation modulates the signal from the Reissner fiber to cerebrospinal fluid-contacting neurons during development[END_REF][START_REF] Lu | Reissner fibre-induced urotensin signalling from cerebrospinal fluid-contacting neurons prevents scoliosis of the vertebrate spine[END_REF]. In particular, the overexpression of the peptides in embryos results in an upward bending of the tail, likely due to localized muscle contraction [START_REF] Zhang | Cilia-driven cerebrospinal fluid flow directs expression of urotensin neuropeptides to straighten the vertebrate body axis[END_REF][START_REF] Lu | Reissner fibre-induced urotensin signalling from cerebrospinal fluid-contacting neurons prevents scoliosis of the vertebrate spine[END_REF]. The expression of urp in motoneurons prompted us to assay whether this peptide was required in this process. To test this hypothesis, we injected wildtype and MZurp mutant embryos with a transgenic construct driving urp2 expression under a heat-inducible promoter.

We then occasioned overexpression at 24 hpf with a heat-shock for 1 hr at 37°C. Consistent with what was previously seen in wildtype embryos [START_REF] Zhang | Cilia-driven cerebrospinal fluid flow directs expression of urotensin neuropeptides to straighten the vertebrate body axis[END_REF][START_REF] Lu | Reissner fibre-induced urotensin signalling from cerebrospinal fluid-contacting neurons prevents scoliosis of the vertebrate spine[END_REF], we found that overexpression of urp2 provoked an upward bending of the tail. The effect was visible immediately at the end of the heat-shock treatment and lasted for several hours (Figure 9a Consistent with the absence of embryonic phenotype in urp mutants, these results suggest that Urp is not involved in axis straightness.

Discussion

The aim of the present study was to determine the expression pattern of the urp gene in zebrafish and to investigate its functional properties by genome editing and transgenesis technologies.

We found here that urp is primarily expressed in motoneurons of the brainstem and the spinal cord, both in embryos and adults. In support of this view, urp-expressing cells were shown to contain ChAT and to express gfp in the Tg(mnx1:gfp) fish transgenic line. Expression of urp in motoneurons has been previously reported in several species including mouse [START_REF] Dubessy | Characterization of urotensin II, distribution of urotensin II, urotensin II-related peptide and UT receptor mRNAs in mouse: evidence of urotensin II at the neuromuscular junction[END_REF], chicken (HT, unpublished observations) and frog [START_REF] Bougerol | Generation of BAC transgenic tadpoles enabling live imaging of motoneurons by using the Urotensin II-related peptide (ust2b) gene as a driver[END_REF]. Our results thus suggest that urp expression in motoneurons appeared early during vertebrate evolution, before the fish-tetrapod split.

Our study revealed that urp expression could be detected by ISH in only a fraction of the motoneurons both in adults and in embryos. This could mean that urp is only expressed in a subpopulation of motoneurons. Yet, our transgenic reporter line Tg(urp:gfp) suggests otherwise. In this line, gfp mRNA expression was also found restricted to a few cells in the anterior half of the spinal cord, very much like urp. Nevertheless, in 3-dpf embryos immunofluorescence staining revealed GFP expression all along the spinal cord, in three types of projections (towards ventral, medial and dorsal part of each somites), and a large number of cells in each segment suggesting expression in both primary and secondary motoneurons.

Thus, this observation suggests that urp is actually expressed in all motoneurons, although, in most cells, at a level too low to be detected by ISH.

The conserved expression of Urp in motoneurons in vertebrates suggests an important role for this peptide in these cells. In mammals however, the functional significance of Urp in motoneurons is still a matter of speculation, mainly due to the fact that its biological actions are very difficult to distinguish from those of Uts2. Two main reasons may be given for this fact: i) Urp and Uts2 expression patterns widely overlap in the central nervous system [START_REF] Dubessy | Characterization of urotensin II, distribution of urotensin II, urotensin II-related peptide and UT receptor mRNAs in mouse: evidence of urotensin II at the neuromuscular junction[END_REF]; in particular, the vast majority of the motoneurons express both genes [START_REF] Pelletier | Androgenic down-regulation of urotensin II precursor, urotensin II-related peptide precursor and androgen receptor mRNA in the mouse spinal cord[END_REF]. ii) Urp and Uts2 likely act through the same receptor since mammals possess only one Uts2 receptor, namely Uts2r1 [START_REF] Tostivint | Molecular evolution of GPCRS: somatostatin/urotensin II receptors[END_REF]. Despite these reservations, several lines of evidence support the idea that Urp and/or Uts2 may be involved in the control of the locomotor activity in mammals.

Intracerebroventricular (icv) injection of Uts2 in mouse and rat induces a dose-dependent increase of ambulatory movements [START_REF] Gartlon | Central effects of urotensin-II following ICV administration in rats[END_REF][START_REF] Do-Rego | Behavioral effects of urotensin-II centrally administered in mice[END_REF][START_REF] Rego | Behavioral actions of urotensin-II[END_REF][START_REF] Clark | Urotensin II acts as a modulator of mesopontine cholinergic neurons[END_REF]. In rat, Uts2 excites mesopontine cholinergic neurons [START_REF] Clark | Urotensin II acts as a modulator of mesopontine cholinergic neurons[END_REF]. In mouse cervical spinal synaptosomes, Uts2 induces a concentration-dependent stimulation of acetylcholine release [START_REF] Bruzzone | Urotensin II receptor and acetylcholine release from mouse cervical spinal cord nerve terminals[END_REF]. Urp and/or Uts2 could also indirectly affect the locomotor activity since Uts2 has been proposed to play a role in motoneuron differentiation, survival, and/or programmed cell death [START_REF] Coulouarn | Specific expression of the urotensin II gene in sacral motoneurons of developing rat spinal cord[END_REF].

Also in trout, icv injection of Uts2 causes a long-lasting increase in motor activity [START_REF] Vanegas | Divergent cardio-ventilatory and locomotor effects of centrally and peripherally administered urotensin II and urotensin II-related peptides in trout[END_REF].

However, in fish, unlike mammals, Uts2 is not expressed in motoneurons but only in a small population of neurosecretory neurons (Dahlgren cells) located at the caudal extremity of the spinal cord [START_REF] Ohsako | Cloning and sequence analysis of cDNAs encoding precursors of urotensin II-alpha and -gamma[END_REF][START_REF] Parmentier | Comparative distribution of the mRNAs encoding urotensin I and urotensin II in zebrafish[END_REF][START_REF] Lu | Molecular characterization and expression of Urotensin II and its receptor in the flounder (Platichthys flesus): A hormone system supporting body fluid homeostasis in euryhaline fish[END_REF]. Thus, in fish, Urp is apparently the only member of the Uts2 family to be expressed in motoneurons and is therefore in the better position to exert the actions on locomotion previously assigned to Uts2.

In this respect, zebrafish appears as a good model to investigate the functions of the Urp released by motoneurons. For this purpose, we generated an urp mutant zebrafish using the CRISPR/Cas9 system. The two alleles recovered were likely to be null since the region encoding the mature cyclic peptide was deleted in both. Besides, RT-qPCR revealed that urp mRNA was largely decreased in urp mutants, suggesting mRNA decay. Since motoneurons play a central role in muscle activity and since one Uts2 receptor, Uts2r3, was recently reported to be expressed in muscle, at least in embryos [START_REF] Zhang | Cilia-driven cerebrospinal fluid flow directs expression of urotensin neuropeptides to straighten the vertebrate body axis[END_REF], we analysed the locomotor activity of urp mutants.

To examine the impact of the urp knockout on the locomotor activity, we analysed spontaneous swimming of 6-dpf larvae. However, no significant difference was observed in distance travelled during recorded time between mutants and wild-type fish. This absence of a mutant phenotype was unlikely due to transcriptional adaption since we could not detect any upregulation of the other genes of the Uts2 family. Our assay was based on an automated tracking system which is relatively easy to use and was therefore perfectly suitable for a preliminary study. However, this assay could have failed to reveal subtle locomotor defects.

During the last years, powerful tools have been developed based on high frequency camera that are capable of tracking simultaneously hundreds of fish and monitoring many fine swimming kinematic parameters [START_REF] Bishop | Three-dimensional motion tracking reveals a diving component to visual and auditory escape swims in zebrafish larvae[END_REF][START_REF] Johnson | Probabilistic models of larval zebrafish behavior eeveal structure on many scales[END_REF][START_REF] Mirat | ZebraZoom: an automated program for high-throughput behavioral analysis and categorization[END_REF][START_REF] Quan | Somatostatin 1.1 contributes to the innate exploration of zebrafish larva[END_REF]. A reanalysis of our urp mutant by using such tools will be essential for the future. Taken together, our results do not necessarily rule out a role of Urp in the locomotor activity in zebrafish. Nevertheless, if such a role exists, it is likely either modest or redundant with that of other factors. The only thing we can assume is that these factors do not belong to the Uts2 family.

Recently, two other peptides of the Uts2 family, namely Urp1 and Urp2, where proposed to regulate body straightness in zebrafish, together with the receptor Uts2r3. [START_REF] Zhang | Cilia-driven cerebrospinal fluid flow directs expression of urotensin neuropeptides to straighten the vertebrate body axis[END_REF] reported that knockdown of urp1 and urp2, as well as that of uts2r3 led to similar embryonic phenotypes. Moreover, they showed that, in embryos, the overexpression of peptides of the Uts2 family can induce an upward bending of the tail and that these peptides act through Uts2r3 since they had no effect in a uts2r3 null background. We previously reported that in the spinal cord, urp1 and urp2 are specifically expressed in CSF-cNs [START_REF] Quan | Comparative distribution and in vitro activities of the Urotensin II-Related Peptides URP1 and URP2 in zebrafish: evidence for their colocalization in spinal Cerebrospinal Fluid-Contacting Neurons[END_REF]. While these cells do not contact muscles, they are known to form synaptic contacts with some motoneurons [START_REF] Hubbard | Intraspinal sensory neurons provide powerful inhibition to motor circuits ensuring postural control during locomotion[END_REF]. Besides, our results revealed a reduction of uts2r3 expression in embryos deprived of Urp, suggesting that Urp could exert some function trough Uts2r3. It was thus conceivable that Urp, being expressed in motoneurons, represented an intermediary between Urp1 and Urp2 and Uts2r3. However, our results indicated that it is not likely the case since the effect of urp2 overexpression was not affected in the urp null mutant.

In conclusion, the present study showed that zebrafish urp gene is primarily expressed in motoneurons, as in tetrapods. However, in the face of that, urp is apparently not required for zebrafish locomotor activity in the early larval stages. Further investigations will be needed to test its possible role in locomotion by using more accurate tools. However, in the absence of any functional evidence for an implication of Urp in locomotion, the idea that it could be involved in totally different functions must not be ruled out.

Figure legends

Supplementary data

  , b. n= 33/38). Strikingly, the effect of urp2 overexpression was also observed in MZurp mutants (Figure 9c, d. n= 22/26).

Fig. 1 .Fig. 2 .Fig. 3 .

 123 Fig. 1. Tissue distribution of urp mRNA in adult zebrafish assessed by RT-qPCR. Means from four to six independent experiments are presented. Error-bars are s.e.m.

Fig. 4 .

 4 Fig. 4. Expression profile of urp during zebrafish embryogenesis. Localization of urp mRNA revealed by in ISH in zebrafish embryos. During somitogenesis, urp is detected in two rows of cells in the anterior half of the forming spinal cord (a-c). At 24 hpf, urp is also detected in a small cluster of cells in the hindbrain (b). At 34 hpf, urp expression in the spinal cord is progressively reduced to the most anterior part of the spinal cord. The expression in the hindbrain is increased (d). From 48 hpf, urp is only detected in the hindbrain. a, b, d and e, lateral view; c, dorsal view. h, hindbrain; sc, spinal cord.

Fig. 5 .Fig. 6 .

 56 Fig. 5. urp is expressed in motoneurons both in embryos and adults. (a-c) Localization of urp and gfp mRNAs revealed by double fluorescence whole-mount ISH on Tg(mnx1:gfp) zebrafish embryos at 24 hpf (dorsal views). gfp is in green (a) and urp in red (b). A merge with DAPI in blue is presented in (c). Double-labelled cells are indicated by arrows. Scale bar: 15 μm. (d-f) Localization of urp mRNA revealed by fluorescent ISH together with a fluorescent immunostaining for ChAT on adult zebrafish spinal cord (sagittal section). ChAT

Fig. 7 .

 7 Fig. 7. Expression profile of Tg(urp:gfp). (a, a') Localization of gfp mRNA revealed by ISH in 2-dpf embryos. (b-c'') Localization of GFP protein revealed by immunofluorescent staining in 2-dpf embryos (b, b') and 3-dpf embryos (c-c''). Note the two rows of gfpexpressing cells in the spinal cord (blue and white arrowhead) and the three types of projection (ventral, dorsal and medial, arrows in c''). GFP expression in the heart (asterisks) is due to the presence of transgene marker (clmc:gfp) in the transgene. a, b, c and c'', lateral view; a', b' and c', dorsal view. Scale bar: 100 μm.

Fig. 8 .

 8 Fig. 8. MZurp mutant exhibit normal locomotor ability in spontaneous swimming. (a) The wildtype genomic sequence of the urp locus, around the fifth exon encoding the core of the mature peptide, is aligned with the two mutant alleles obtained in this study. The corresponding wildtype peptide sequence is indicated above with the mature peptide sequence highlighted in orange. On the wildtype sequence, exons are in bold, coding sequence in upper case and the position of the two guide RNA is indicated in green (+ PAM in yellow). The two mutant alleles correspond to a deletion of the sequence between the two guides with a small insertion in the second allele (in red). A motility assay was performed on MZurp mutants (n = 51 mutants and 45 sibling controls). (b) Average distance covered per minutes during the motility assay. Dark periods (i.e. light off) are indicated by grey area. (c) Cumulated distance covered during light on and light off (dark) periods (30 min total per period; Welch Two Sample t-test, p = 0.80 and p = 0.91 and for light on and light off (dark) periods respectively). (d, e) Expression levels of genes encoding for four Uts2 receptors (d) and the five peptides of the Uts2 family (e) assayed by RT-qPCR in wildtype or MZurp mutant 24-hpf embryos. Data were collected from five independent clutches and represented as mean ± SEM. Welch two sample t-test, p-value are indicated on each panel.

Fig. 9 .

 9 Fig. 9. urp is not involved in axis straightness in zebrafish embryos. (a, b) In wildtype embryos, overexpressing urp2 resulted in upward bending of the tail (n = 33/38). (c, d) The effect of urp2 overexpression was not altered in MZurp mutants (n = 22/26. Fisher's exact test, p-value = 1).

Figure 6

 6 Figure 2

Table S1 .

 S1 Sequences of the oligonucleotides used for PCR amplifications and genome editing

	sgRNA (CRISPR)
	sgRNA#1	AAAACTGGTGTCAAACGCAATGG
	sgRNA#2	TCAAGTGAAGTGCGAGGAGAAGG
	cloning of CDS
	Oligoname urp2_CDS_F	sequence ggatccGTATCTGTAGAATCTGCTTTGCTGC
	genotyping urp2_CDS_R	tctagaGGCAGAGGGTCAGTCGTGTTAT
	URP_genotyp_F	GACTGGAGTTGGCCAGGAAA
	URP_genotyp_R Cloning of urp promotor	ACACTAGAGGGAGACAACAGC
	URP_prom_F	ggggacaactttgtatagaaaagttgATATTTGATGGCATACATTGTTCCT
	Real-time PCR URP_prom_R	ggggactgcttttttgtacaaacttgTGCACTTGTAAAATGTTGGGTTTCTC
	urp_qPCR_F	GCGGAAAAATGTCATGCCTCTTC
	urp_qPCR_R	TTGAGCTCCTTTTGAAGCTCCTG
	uts2a_qPCR_F	CACTGCTCAACAGAGACAGTATCA
	uts2a_qPCR_R	CCAAAAGACCACTGGGAGGAAC
	uts2b_qPCR_F	TACCCGTCTCTCATCAGTGGAG
	uts2b_qPCR_R	TTTTCCAGCAAGGCCTCTTTTAC
	urp1_qPCR_F	ACATTCTGGCTGTGGTTTGTTC
	urp1_qPCR_R	CTCTTTTGCACCTCTCTGAAGC
	urp2_qPCR_F	ACCAGAGGAAACAGCAATGGAC
	urp2_qPCR_R	TGAGGTTTCCATCCGTCACTAC
	uts2r1_qPCR_F	CTCCTTTCTCTTTGGCACAAGC
	uts2r1_qPCR_R	GGAGATCCAGTAGGTTCTTGCC
	uts2r2_qPCR_F	AGTCTGCGTCATCCAGTAATCAG
	uts2r2_qPCR_R	AAACCCCAATCTTACCTTCGCA
	uts2r3_qPCR_F	CTTTGCCATCGCTCTCAGTTTG
	uts2r3_qPCR_R	CAGGCAGGAACACACAACTTTC
	uts2r4_qPCR_F	TGCCTTCATCTTGACTCTTCCC
	uts2r4_qPCR_R	GTCCACGTTGGGAAACAAATCC
	uts2r5_qPCR_F	TTTCTCGTGTGGCTCTTCTCTC
	uts2r5_qPCR_R	GCCATTGTGCACGTCTATCTTG
	mob4_qPCR_F	AAGAGTGCCCTGCCATTGATTA
	mob4_qPCR_R	AGTTTGGCCACAGATGATTCCT
	lsm12b_qPCR_F	GAGACTCCTCCTCCTCTAGCAT
	lsm12b_qPCR_R	GATTGCATAGGCTTGGGACAAC
		26

Acknowledgements

We thank Jean-Paul Chaumeil, Philippe Durand and Céline Maurice (PhyMA MNHN) for zebrafish care; Anne De Cian and Dr Jean-Paul Concordet (String MNHN) for preparing sgRNA and providing with Cas9 protein; Mathieu Carrara (PhyMA MNHN) designing and validating qPCR oligos for lsm12 and mob4; Dr Caroline Parmentier (Sorbonne Université, Paris) for help with sections; Sonya Galant (Université Paris Saclay) for help in double ISH; Prof. Patrick Charnay (IBENS, Paris) for providing us with the pBS-gfp plasmid; Dr Edor Kabashi (Institut Imagine, Paris) for sharing the Tg(mnx1:gfp) fish transgenic line. Dr Lydia Djenoune and the team of the "Plateforme d'Imagerie Cellulaire Pitié Salpêtrière" for help in confocal imaging. Cyril Willig and the "Centre de Microscopie de fluorescence et d'IMagerie numérique" for help with in axiozoom imaging.

This work was supported by CNRS, MNHN (ATM "Formes possibles, formes réalisées" 2015 and ATM "blanche" 2017 to G.P. and H.T.). F.A. was the recipient of a fellowship from the Alliance Sorbonne Université (programme Emergence).

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