Stephanie Bertrand
email: stephanie.bertrand@obs-banyuls.fr

Hector Escriva
email: hescriva@obs-banyuls.fr

Daniel Aldea

Anthony Leon

Yann Le Petillon

Guillaume Luxardi

Pierluigi Scerbo

Marie Cibois

Lucie Subirana

Manuel Irimia

Laurent Kodjabachian

Expression of Fox genes in the cephalochordate Branchiostoma lanceolatum

Keywords: Fox genes, amphioxus, Evo-Devo, chordates, embryonic development

Forkhead box (Fox) genes code for transcription factors that play important roles in different biological processes. They are found in a wide variety of organisms and appeared in unicellular eukaryotes. In metazoans, the gene family includes many members that can be subdivided into 24 classes. Cephalochordates are key organisms to understand the functional evolution of gene families in the chordate lineage due to their phylogenetic position as an early divergent chordate, their simple anatomy and genome structure. In the genome of the cephalochordate amphioxus Branchiostoma floridae, 32 Fox genes were identified, with at least one member for each of the classes that were present in the ancestor of bilaterians. In this work we describe the expression pattern of 13 of these genes during the embryonic development of the Mediterranean amphioxus, Branchiostoma lanceolatum. We found that FoxK and FoxM genes present an ubiquitous expression while all the others show specific expression patterns restricted to diverse embryonic territories. Many of these expression patterns are conserved with vertebrates, suggesting that the main functions of Fox genes in chordates were present in their common ancestor.

Preface

"Thus, from the war of nature, from famine and death, the most exalted object which we are capable of conceiving, namely, the production of the higher animals, directly follows.

There is grandeur in this view of life, with its several powers, having been originally breathed into a few forms or into one; and that, whilst this planet has gone cycling on according to the fixed law of gravity, from so simple a beginning endless forms most beautiful and most wonderful have been, and are being, evolved." (Charles Darwin, The origin of species, 1859) My thesis work belongs to the field of Evolutionary Developmental Biology (Evo-Devo), therefore, I will try to explain the main concept of this area. Evo-devo is the discipline that contains and combines two important research lines in biology: Developmental Biology, which studies the development of organisms, from the time of fertilization, through embryogenesis, growth, reproduction, until further death; and Evolutionary biology, which seeks to explain the evolution of organisms and their diversification from ancestral forms to modern species. The result of linking these two areas creates a "new discipline" that aims to identify the developmental mechanisms leading to the acquisition of new morphological structures and molecular signatures throughout evolution, and to unravel therefore potential evolutionary scenarios that explain how these events took place.

Since the publication of "The origin of species" by Charles Darwin in 1859, embryology has been considered to provide the best evidence for evolution. This assumption was closely followed by Ernst Haeckel in 1866, with his book "General morphology of organisms", where he postulated that the development of an embryo would trace the evolution of the species (recapitulation theory, biogenetic law or simply called "ontogeny recapitulates phylogeny").

From then, many zoologists assumed that embryos provided a way to study evolution, giving origin and popularity to a discipline called evolutionary embryology in 1870's. This comparative new area was later overpassed by the rediscovery of the Mendelian laws in 1900, and the further use of Drosophila as a model organism. However, it was not until 1977 with Stephen J. Gould's book "Ontogeny and Phylogeny" that researchers revisited one punctual concept: heterochrony (alteration in the timing of the developmental process leading to changes in morphology), that was first conceived by Haeckel, and elaborated by Gavin de Beer in 1930. This reborn concept and the discovery of the Hox genes in 1978 led to the first major impetus for evo-devo [1,2,3].

Such discovery was later supported by the studies carried out by Walter Gehring and collaborators in 1984, when they started to use the "Homeobox" (Hox) in homology searches in several phyla because of their conservation. Nevertheless, the first use of the term "evolutionary developmental biology" dates from 1983 by the zoologist Peter Calow, of the University of Sheffield in England, and it officially emerged in 1999 after obtaining its own division in the Society for Integrative and Comparative Biology; and since then, it has gained enormous importance in recent years.

The beginning of the new genomic era and the emergence of cutting edge molecular genetics tools have revolutionized evo-devo over the past two decades. This has allowed researchers to reveal unknown mechanisms of development in the emergence of novel structures during evolution, but yet, much is still to be known, and integrated, in order to expand our understanding of gene regulation. In this context, the main objective of my PhD work has been to understand the evolution of the gene regulatory network underlying the process of neural induction in chordates by using the cephalochordate amphioxus as a model system.

Introduction Chapter Metazoans and the evolution of the nervous system

During the last decades, the next generation sequencing (NGS) techniques, also known as highthroughput sequencing, have strikingly increased the quantity of genomic data for several organisms. These new technologies have led to expand our knowledge of genomic and transcriptomic regulation of the major biological processes in several phyla, moreover it has led to improve cutting-edge genetics tools, and to refine phylogenetic analyses by generating enough data to construct more reliable and robust topologies that sustain the origin of modern animal lineages from single common ancestors, and decipher how these organisms have diverged over the last millions years.

It is believed that first animals evolved over at least 600 million years ago, and most importantly, that the last common ancestor (LCA) of animals was multicellular. This multicellularity state might have first appeared as a simple clonal organization of unicellular organisms. But these first organisms with multicellular complexity should have gone through drastic changes in their genetic composition before the emergence of specific cell types in order to adapt to their environment [4]. This adaptation led to the emergence of sensory cells, which allowed organisms to explore their habitats. In this context, it is undeniable that not only the acquisition of sensory cells, but the formation of extremely complex nervous systems is a major success of evolution.

However, it brings the question: when did the first neurons and neural circuits appear and how did they organize in such different forms in almost all eumetazoans? In order to answer this, several studies have generated new data that has helped to understand the evolutionary history of nervous system. In fact, we can track back the origin of the nervous system before the split between cnidarians and bilaterians, and claim that the genetic toolkit of neural development would have been formed before these two phyla appeared. However, little is known about the emergence and evolution of neural cells in non-bilaterian animals [5].

The acquisition of neural cells was a crucial event in animal evolution, and it could have had its origin with the appearance of first sensory cells and sensory systems coupled to effectors that helped animals to find food, mate or avoid unsuitable habitats. Nowadays, the simplest animal sensory cells can be found in poriferan larvae, which are not only sensitive to light but to other stimuli. Sponges lack neural system and, in many terms, constitute a primitive group when compared to eumetazoans. Nevertheless, they count on other sensory systems as for the case of A. queenslandica larvae, that present a specific type of epithelial cell, the flask cell, which is able to convert exogenous stimuli into internal developmental signals that will induce settlement and metamorphosis [6]. Besides this, they also exhibit expression of several pro-neural markers like AmqbHLH and genes of the Notch-Delta pathway. These characteristics are similar to what is also observed in larval sensory neurons in bilaterians and cnidarians [6,7,8].

In contrast to poriferans, cnidarians have been widely considered the earliest metazoans to have evolved a nervous system. They have a diffused nervous system consisting on nerve nets that are mainly spread in epithelium. However, some of them, mostly medusozoans, have evolved sensory systems showing neural regionalization. Besides, the genetic repertoire controlling several neurodevelopmental mechanisms in cnidarians exhibit striking similarities to the bilaterians, and therefore it is assumed that this repertoire was already present in the common eumetazoan ancestor [9]. Furthermore, some authors have proposed that the neural rings in medusozoans constitute the cnidarian Central Nervous System (CNS). This idea supports the hypothesis that the emergence of a rudimentary centralized nervous system appeared before the split with bilaterians, contrasting with the idea that both systems converged independently [10].

Similar to cnidarians, ctenophores also have an epithelial nerve net, and for more than a century both groups were united as jelly-like diploblastic organisms in a clade named Coelenterata.

However, these two lineages are very dissimilar at the genomic level, and their morphologic resemblance is probably due to convergent evolution. Nowadays, the phylogenetic position of ctenophores is on debate, from a sister group to bilaterians to the most basally divergent metazoans. In fact, recent publications support the position of ctenophores as sister group of all animals. This revelation points 2 possibilities: The nervous system existed in the LCA of metazoans and was lost in poriferans and placozoans, or ctenophores and the ancestor of cnidarians and bilaterians acquired their nervous system independently [8,11,12].

The possibility of ctenophores having a convergent nervous system is not parsimonious with the examination of the genomes of representative species from the phyla of Porifera, ctenophore and Placozoa. In fact, all key molecular components of the nervous system are conserved in the genomes of these animals, like voltage-gated ion channels and specialized SNARE proteins that mediate synaptic vesicle fusion. Even the simplified animal Trichoplax, the only member of the expression of marker genes [17]. However, other close groups to chordates, as echinoderms and hemichordates, show deep differences in comparison to chordates, making difficult to predict the structure of the nervous system of their common ancestor at the base of deuterostomes [18]. Indeed, the adult echinoderm nervous systems is morphologically distant from the one of chordates. Their neurons are dispersed forming a nerve net that is organized in nodes and interconnected to other adjacent nodes and radial nerves. Besides, these nerve cords do not express Hox genes during development, which is assumed to be a conserved trait in chordates [19,20].

In the case of hemichordates, even if they share morphological affinities with chordates, there is no robust evidence to confirm if one of their nerve cords (dorsal or ventral) is homologous to the dorsal chordate nerve cord, or if both evolved independently [21,22]. All this information draws several evolutionary landscapes, not only for the origin of neural cells, but also for the origin of centralized nervous systems in bilaterians. Throughout evolution, species diversified and their nervous systems diverged from their common ancestors, still, most of their genetic toolkit devoted to the first steps of neural cells formation is well conserved among them. For these reasons, comparative studies have been launched during the last years to identify the conserved key players triggering neural fate in many animals. However, the main research focus was retained on vertebrate nervous system formation due to the lack of genomics information in other species until the last decades. In vertebrates, the CNS starts to form very early in embryonic development via a neural induction process that takes place in the ectoderm. The neuroectodermal plate then folds into a closed tube during neurulation. As development continues, the neural tube will regionalize and give shape to a CNS.

Chapter 2. Neural Induction in vertebrates

The term "induction" refers to the interaction at close range between two or more cells, or tissues, of different history and properties. One of these group of cells (inducer) produces a signal that changes the cellular behavior of the adjacent group of cells (recipient), thereby causing them to change their fate. This particular case of specification happens during embryonic development, where signals emanate from a source tissue and instruct cell type fate acquisition in a neighboring competent tissue [23].

When the recipient acquires neural fate, then the event is called neural induction. This is the first step in the formation of the CNS in vertebrates, and it is one of the first events of cell fate induction during embryonic development. It consists on the transformation of pluripotent ectodermal cells into neuroectodermal cells (neuroectoderm) that will form the neural plate and then the neural tube.

Neural induction, the organizer and the default model

In 1907, Warren Lewis decided to graft the dorsal blastoporal lip from a gastrulating embryo of Rana palustris into a host, in order to determine "what organ-forming stuffs are present in the lip of the blastomere, but more specially to determine the extent of independent self-differentiation possessed by this structure". He observed the formation of a second axis that presented a neural tube and axial and paraxial mesoderm (notochord and somites), but attributed this structure to the graft itself [24]. It was not until 1924 that Hilde Mangold and Hans Spemann excised small tissues (dorsal blastoporal lip) from a salamander gastrula and grafted them into the ventral region of host embryos. Surprisingly, a secondary body axis formed at the transplant site, and it included a neural tube, notochord and somites, as the experiment of Warren. By using grafts and hosts from differently pigmented species so that the donor cells could be identified, they demonstrated that the notochord from the second axis originated from the graft itself, while the somites and neural tube originated from the host tissue (Figure 3) [25].

They reasoned that the transplanted cells from the graft induced adjacent host cells to become the Activin receptor induces neural fate commitment in amphibian ectoderm [44,45]. These experiments suggested that these molecules might not be directly inducing neural fate but act as inhibitors of TGFβ/BMP signals. Besides, the presence of these inhibitory mechanisms in different species highlights their conservation between different phyla over evolution [46].

Since the Bone morphogenetic proteins (BMP) signaling promotes epidermal differentiation in ectoderm, its inhibition constitutes a key process for neural differentiation. However, BMPinhibition itself cannot explain the events taking place in the initiation of the nervous system formation [33,46,[START_REF] Hawley | Disruption of BMP Signals in Embryonic Xenopus Ectoderm Leads to Direct Neural Induction[END_REF][START_REF] Stern | Neural Induction: Old Problem, New Findings, yet More Questions[END_REF]. Additional works in more organisms have revealed the presence of other secreted proteins such as fibroblast growth factors (FGFs), wingless/int-1 class proteins (WNTs) and Nodal, that are also responsible for the neural fate commitment [40,46,[START_REF] Stern | Neural Induction: Old Problem, New Findings, yet More Questions[END_REF][START_REF] Stern | Neural Induction: 10 Years on since the 'default Model[END_REF][START_REF] Wilson | Neural Induction: Toward a Unifying Mechanism[END_REF].

Signaling pathways involved in neural induction

For several decades, many laboratories have experimented with several molecules to understand the molecular regulation of neural induction. It was first observed that mis-expression of a dominant-negative Activin receptor (later discovered to inhibit the signaling by several TGF-β related factors) in Xenopus embryos blocked mesoderm formation, but also unexpectedly generated ectopic neural tissue. After this, experiments with Noggin, Follistatin and Chordinwhich are expressed in the organizer-confirmed that they are binding partners of bone morphogenetic proteins that antagonize BMP signaling [START_REF] Stern | Neural Induction: Old Problem, New Findings, yet More Questions[END_REF]. Later on, Bmp-4 was described as a potent epidermal inducer and neural inhibitor, and its expression pattern was mainly observed throughout the ectoderm during gastrulation until the formation of the presumptive neural plate.

It was also shown that other members of the BMP family, as Bmp-2 and Bmp-7, were good epidermal inducers [START_REF] Wilson | Induction of Epidermis and Inhibition of Neural Fate by Bmp-4[END_REF][START_REF] Fainsod | On the Function of BMP-4 in Patterning the Marginal Zone of the Xenopus Embryo[END_REF][START_REF] Suzuki | Regulation of Epidermal Induction by BMP2 and BMP7 Signaling[END_REF]. But the first evidences of the presence of other signaling pathways involved in the neural induction process were observed when Chordin and Noggin were overexpressed after microinjection of a dominant-negative receptor of FGFs [START_REF] Launay | A Truncated FGF Receptor Blocks Neural Induction by Endogenous Xenopus Inducers[END_REF][START_REF] Celli | Soluble Dominant-Negative Receptor Uncovers Essential Roles for Fibroblast Growth Factors in Multi-Organ Induction and Patterning[END_REF][START_REF] Sasai | Endoderm Induction by the Organizer-Secreted Factors Chordin and Noggin in Xenopus Animal Caps[END_REF]. The description of FGF as a potential signal for neural induction, as well as studies of other signals that were identified as directly or indirectly involved in the process of neural induction, changed the simple vision of the default model and brought to discussion a more complex developmental mechanism.

BMP Signaling pathway

Bone morphogenetic proteins (BMPs) are secreted growth factors with several biological functions during development. They are dimeric proteins with a single interchain disulfide bond, and form the largest subgroup of the transforming growth factor-beta (TGF-β) superfamily. This superfamily consists of more than 30 ligands, identified in mammals, and they all possess seven highly conserved cysteine residues in their carboxy-terminal region [START_REF] Kingsley | The Mouse Short Ear Skeletal Morphogenesis locus Is Associated with Defects in a Bone Morphogenetic Member of the TGFβ Superfamily[END_REF][START_REF] Eimon | In Xenopus Embryos, BMP Heterodimers Are Not Required for Mesoderm Induction, but BMP Activity Is Necessary for Dorsal/Ventral Patterning[END_REF][START_REF] Mueller | Promiscuity and Specificity in BMP Receptor Activation[END_REF]. The BMP subfamily comprises more than 15 ligands in mammals, and can be devided into the BMP-2/-4, the BMP-5/-6/-7/-8, the GDF-5/-6/-7, the GDF-8/-11, the BMP-9(GDF-2)/BMP-10, the GDF-1/-3 and the GDF-10/BMP-3 subgroups (Figure 5b) [START_REF] Mueller | Promiscuity and Specificity in BMP Receptor Activation[END_REF]. They were first isolated for their capability to induce formation of ectopic bone, cartilage condensation, chondrocyte maturation and interdigital cell death. In fact, Wozney et al. identified 3 different mammalian BMP genes (BMP-1, BMP-2A, and BMP-3) based on their ability to induce formation of ectopic bone and cartilage in rats [START_REF] Wozney | Novel Regulators of Bone Formation: Molecular Clones and Activities[END_REF]. Moreover, it was shown that the mammalian BMP-2 and BMP-4 are more closely related to the Drosophila Decapentaplegic (dpp) gene than to other mammalian BMPs. Similarly, BMP-5, BMP-6 and BMP-7 are also closer to Drosophila 60A gene than to either dpp or other BMP genes. These observations suggested the homology of Drosophila dpp and 60A to vertebrate BMP genes, their conservation across phyla, and that these two BMP classes were potentially present in the common ancestor of bilaterians. Further work confirmed the involvement of BMPs in more developmental processes as cell proliferation, cell differentiation, apoptosis and other cell-to-cell interactions during morphogenesis [START_REF] Kingsley | The Mouse Short Ear Skeletal Morphogenesis locus Is Associated with Defects in a Bone Morphogenetic Member of the TGFβ Superfamily[END_REF][START_REF] Miyazono | Bone Morphogenetic Protein Receptors and Signal Transduction[END_REF][START_REF] Wozney | Novel Regulators of Bone Formation: Molecular Clones and Activities[END_REF][START_REF] Wharton | Drosophila 60A Gene, Another Transforming Growth Factor Beta Family Member, Is Closely Related to Human Bone Morphogenetic Proteins[END_REF][START_REF] Blanco Calvo | Biology of BMP Signalling and Cancer[END_REF].

The action of BMP ligands is mediated by two types of transmembrane serine/threonine kinase receptors. These specific receptor subunits that bind to BMPs include type-I receptors (BMPR-I) and type-II (BMPR-II). Both types of receptors are needed to form a functional complex to initiate a linear signaling pathway from the type-II to the type-I receptor after ligand binding.

There are two types of BMP signaling: The Smad-dependent signaling, which constitutes the main intracellular signaling, and the Smad-independent signaling, which includes the ERK, p38/MAPK and JNK pathway. In the Smad-dependent signaling, activation of the BMPR-I by the BMPR-II results in phosphorylation of a R-Smad (Smad1, Smad5 and Smad8). After phosphorylation, these Smads form a complex with the Co-Smad (common Smad, Smad4, As stated before, BMP signaling is actively involved in ectodermal cell fate, by promoting epidermal specification at the ventral side of the embryo. In fact, Bmp-4 in Xenopus is widely expressed in the entire ectoderm during gastrula stage, and its expression later disappear from the future neural plate when the organizer appears, supporting the anti-neural function of BMP signals [START_REF] Stern | Neural Induction: Old Problem, New Findings, yet More Questions[END_REF][START_REF] Stern | Neural Induction: 10 Years on since the 'default Model[END_REF]. It was later confirmed that Bmp-4 inhibits neuralization and induces epidermis in dissociated ectodermal cells [START_REF] Wilson | Neural Induction: Toward a Unifying Mechanism[END_REF][START_REF] Wilson | Induction of Epidermis and Inhibition of Neural Fate by Bmp-4[END_REF]. Similar experiments in zebrafish underlined the role of BMP signals in directing the fates of cells positioned on the ventral side of the gastrula ectoderm irrespective of whether or not these cells are destined to contribute to neural or non-neural structures. Moreover, it was shown in rats that autocrine production of BMPs induce dormancy of neural stem cells, and its antagonist, Noggin, can induce dormant cells to re-enter the cell cycle and reacquire neurogenic potential [START_REF] Kudoh | Combinatorial Fgf and Bmp Signalling Patterns the Gastrula Ectoderm into Prospective Neural and Epidermal Domains[END_REF][START_REF] Sun | Interplay between FGF2 and BMP Controls the Self-Renewal, Dormancy and Differentiation of Rat Neural Stem Cells[END_REF].

These results confirm the function of BMPs as epidermis inducer, and its depletion is a substantial and required step for the formation of neural cells. Considering this, inhibition of this signaling pathway can take place at different levels. In the cytosol, inhibitory Smad proteins (I-Smad), Smad6 and Smad7, can repress the action of BMP by inhibiting the receptor-mediated phosphorylation of Smad1, Smad5 or Smad8 or by competing with Smad4 for the binding to Smad1, Smad5 and Smad8. Besides them, the ubiquitin ligases SMURF1 and 2 (Smad ubiquitination regulatory factors) are able to target Smad1 and 2 and the TGF-β receptors for degradation by the proteasome. On the other side, inside the nucleus, some transcriptional repressor complexes that are recruited by the SKI oncoprotein can also block BMP signaling by directly interacting with and repressing the activity of Smad complexes. Extracellularly, inhibition of BMP signals is modulated by various secreted proteins that bind and inhibit BMP ligands, such as Noggin, Follistatin, Chordin, Xnr3 (Xenopus Nodal-related 3) (which are found to be expressed in the organizer region in several animals), DAN/Cerberus protein family and TSG (twisted gastrulation) (Figure 6) [35,46,[START_REF] Bragdon | Bone Morphogenetic Proteins: A Critical Review[END_REF][START_REF] Gazzerro | Bone Morphogenetic Proteins and Their Antagonists[END_REF][START_REF] Wang | Ski Represses Bone Morphogenic Protein Signaling in Xenopus and Mammalian Cells[END_REF][START_REF] Harland | Neural Induction[END_REF][START_REF] Chang | Twisted Gastrulation Can Function as a BMP Antagonist[END_REF][START_REF] Hansen | Direct Neural Induction and Selective Inhibition of Mesoderm and Epidermis Inducers by Xnr3[END_REF][START_REF] Balemans | Extracellular Regulation of BMP Signaling in Vertebrates: A Cocktail of Modulators[END_REF].

Chordin is not sufficient to impose neural character on naïve ectodermal cells. However, it is able to stabilize the expression of Sox3 if cells are exposed to a graft of Hensen's node for five hours, supporting the idea that BMP inhibition is not sufficient for neural induction and the ectoderm must be exposed to signals from the organizer before it can respond to BMP antagonists [START_REF] Streit | Neural Induction: A Bird's Eye View[END_REF][START_REF] Faure | Endogenous Patterns of BMP Signaling during Early Chick Development[END_REF].

Altogether, these experiments show that inhibition of BMP antagonists, or activation of BMP signals, induce a ventralization of the embryo and a loss of neural markers in different vertebrate models. Therefore, BMP inhibition can be considered necessary for the formation of neural tissue, during neural induction in Xenopus or during the formation of the neural plate in chick. On the other hand, BMP inhibition has not been proven to induce neural fate itself, in consequence, other signals are necessary to trigger this process and eventually give rise to all the nervous tissue of the embryo.

It is therefore undeniable that the study of differentiation of neural cells during embryogenesis is an exciting and complex field, but it also appears that the signaling factors and transcriptional regulators involved in this process are way more complicated than previously thought. The disproval of the default model, and the expression of different putative inductive candidates in neural tissues, puts in discussion which could be the instructive signal orchestrating neural fate acquisition. As an alternative, FGF signaling was observed to play a key role in neural induction, since FGF induces expression of Sox2 and Sox3 in Xenopus after injection of Smad5-somitabun (Smad5-sbn), an anti-morphic form of Smad5. Further experiments in chick have proven that blocking FGF signaling can lead to a loss of neural markers like Sox3, and experiments in the tunicate Ciona intestinalis showed that FGF appears to be an important neural inducing signal, supporting the idea that both FGF signaling and inhibition of BMP signals are essential for neural induction [START_REF] Bertrand | Neural Tissue in Ascidian Embryos Is Induced by FGF9/16/20, Acting via a Combination of Maternal GATA and Ets Transcription Factors[END_REF][START_REF] Streit | Initiation of Neural Induction by FGF Signalling before Gastrulation[END_REF][START_REF] Marchal | BMP Inhibition Initiates Neural Induction via FGF Signaling and Zic Genes[END_REF].

FGF signaling pathway

The Fibroblast Growth Factor (FGF) family is a group of proteins (secreted FGFs) that bind to receptor tyrosine kinases (RTKs) and of intracellular non-signaling proteins (iFGFs). Members of the FGF family are related by core sequence conservation (120-130 amino acids) and are found in diverse metazoan species [START_REF] Ornitz | The Fibroblast Growth Factor Signaling Pathway[END_REF][START_REF] Bertrand | FGF Signaling Emerged Concomitantly with the Origin of Eumetazoans[END_REF]. In vertebrates, there are 4 FGF receptors (FGFRs) and between 22 to 27 fibroblast growth factors, which are grouped into 8 subfamilies based on differences in sequence homology and phylogeny. This number of ligands and receptors is the consequence of vertebrate-specific whole genome duplications, whereas in cephalochordate there are only eight FGFs and one FGFR. The secreted FGFs are expressed in nearly all tissues and play essential roles in the earliest stages of embryonic development, during organogenesis, and in the adult, where they function as homeostatic factors that are important for tissue maintenance, repair, regeneration, and metabolism. At the cellular level, secreted FGFs regulate fundamental cellular processes as regulation of proliferation, survival, migration, differentiation, and metabolism [START_REF] Ornitz | The Fibroblast Growth Factor Signaling Pathway[END_REF][START_REF] Bertrand | FGF Signaling Emerged Concomitantly with the Origin of Eumetazoans[END_REF][START_REF] Aldea | Amphioxus Illuminates the Origin of the Vertebrates' Head[END_REF][START_REF] Oulion | Evolution of the FGF Gene Family[END_REF].

FGFs bind to a dimeric form of their receptor (FGFR) that comprise 3 immunoglobulin domains (IgI, IgII and IgIII). IgII contains a heparin binding domain (HBD) with high affinity to FGF ligands linked to a heparin sulphate proteoglycan (HSPG). The ligand-receptor interaction results in the transphosphorylation and activation of the intracellular tyrosine kinase domain, which finally activate several intracellular cascades as Ras/MAPK, PI3K/Akt, Jak/Stat and PLC [START_REF] Thisse | Functions and Regulations of Fibroblast Growth Factor Signaling during Embryonic Development[END_REF][START_REF] Lanner | The Role of FGF/Erk Signaling in Pluripotent Cells[END_REF] (Figure 9). The regulation of this pathway is mediated by FGF inhibitors such as Sprouty, which is a intracellular negative regulator of RTKs including FGFR; SEF (similar expression to Fgf), which is a transmembrane protein that functions as an antagonist of FGF signaling through the Ras-MAPK pathway; Dusp6 (Dual-specificity phosphatase 6), which encodes an ERK-specific MAPK phosphatase (MKP3); CBL, an E3 ubiquitin ligase, which forms a ternary complex with phosphorylated receptor FRS2α (FGF receptor substrate 2α) and GRB2 (Growth factor receptorbound protein 2), which results in the ubiquitination and degradation of FGFR [START_REF] Ornitz | The Fibroblast Growth Factor Signaling Pathway[END_REF][START_REF] Thisse | Functions and Regulations of Fibroblast Growth Factor Signaling during Embryonic Development[END_REF][START_REF] Lanner | The Role of FGF/Erk Signaling in Pluripotent Cells[END_REF].

As a matter of fact, FGF and BMP signals play opposing roles in the specification of neural and epidermal cells respectively, thus resulting in exclusive domains of FGF and BMP activities.

Although, results from several vertebrate models as mouse, chick, zebrafish and Xenopus have uniformly shown that BMP inhibition is necessary for the formation of neural cells, not all of them have supported the requirement of FGFs as instructive signal in neural fate commitment. In mouse, FGF inhibition does not block the expression of neural markers, whereas in zebrafish and ectodermal explants of chick, FGF acts as a signal controlling differentiation in specific neural territories, as the posterior neural tissue. Therefore, it is believed that FGFs are probably not direct neural inducers in vertebrates [102,103].

Wnt Signaling pathway

The wingless related (Wnt) pathway comprises secreted proteins (Wnt ligands) that bind to specific transmembrane receptors and trigger either β-catenin dependent or β-catenin independent signaling cascades, resulting in the transcription of target genes or other cellular responses. Wnts were suggested as potential inducers of neural fate after observing that microinjection of Wnt8 or β-catenin, triggered expression of the neural marker NCAM and blocked the expression of Bmp-4 in ectodermal explants of Xenopus [104]. Further work in the same model also showed that inhibition of Wnt pathway by using a negative dominant of β-catenin, inhibits the expression of the neural markers Sox2 and Sox3 in vivo [105]. In chick, Wnt activity seems to be involved in determining the neural plate border by mediating the formation of border cells that will later give origin to placodes and neural crest [103]. In contrast to these observations, there is also evidence suggesting that later in development, Wnt signaling may suppress the generation of neural cells.

In fact, if Wnts are overexpressed in Xenopus embryos at blastula stages, the resulting embryos are 'over-ventralized' and the generation of neural tissue is inhibited [START_REF] Wilson | Neural Induction: Toward a Unifying Mechanism[END_REF]. In addition to this, over-expression of Wnt3a in chick ectoderm explants also induces a loss of expression of neural markers, and over-expression of different inhibitors of Wnt signaling induces neural markers expression in animal cap cells [26]. Taken together, these results suggest that inhibition of Wnt signals might be necessary during neural induction, and its activation is only necessary for the formation of specific cell types, as neural crest cells.

Sonic Hedgehog signaling pathway

Sonic hedgehog (Shh) is a vertebrate homologue of the secreted protein encoded by the Drosophila gene hedgehog, and is expressed in the notochord and floor plate in early development. In zebrafish, Shh activity specifies placodal cells of adenohypophyseal character from the Pitx3 expressing progenitor cells at gastrula stage. Its over-expression blocks lens formation in zebrafish, and induces ectopic expression of pituitary genes in chick [106].

Furthermore, Shh was observed to initiate the formation of motor neurons from cells that are located in the ventral region of the neural tube in chick [107]. In contrast to several studies suggesting that neuroectoderm is established normally in absence of Sonic Hedgehog signaling, Maye et al demonstrated that mutants in the signaling molecules of this pathway in mouse embryonic stem cells (ESCs) could lead to defect in the generation of neuroectoderm, which is not capable of producing appropriate neuronal and glial progenitors derived from ESCs [108].

Taken together, these experiments suggest a role of Shh in the formation of competent neuroectoderm, and as inducer of some specific neuronal cell types.

Notch Signaling pathway

The Notch pathway functions as a core signaling system during embryonic development and is also required for the regulation of tissue homeostasis and stem cell maintenance in the adult. Its activation relies on the interaction of Notch transmembrane receptors that are expressed on the surface of one cell with membrane-bound ligands that are expressed on the surface of other neighboring cells. Binding of Notch DSL ligands (Delta, Serrate, Lag-2) results in the cleavage of the Notch protein, which leads to the release of the intracellular domain into the cytoplasm of the receiving cell, while the extracellular domain is endocytosed by the sending cell. As a result, the intracellular domain translocates into the nucleus to modulate Notch target gene transcription, whereas the endocytosed extracellular domain undergoes lysosomal degradation [109,110]. This signaling pathway has been reported to promote expansion of neural progenitor cells during embryonic stem cell differentiation. In fact, its pharmacological inhibition, or genetic ablation of RBPJk (Recombining binding protein suppressor of Hairless, transcriptional effector of the Notch pathway) significantly reduces the proportion of ESCs that enter the neural lineage. However,

Chapter 3: Nodal signaling pathway in vertebrates

The Nodal signaling pathway is involved in patterning and cell differentiation processes that take place during the pre-gastrulation and gastrulation stages of chordate development. In particular, it is essential for the specification of the primary body axes (anterior-posterior, dorso-ventral and left-right) as well as for mesoderm and endoderm formation [113].

The ligands of the Nodal pathway are members of the transforming growth factor-beta (TGF-β) superfamily which bind to type-I and type-II serine threonine kinase receptors. The type-I receptors recognized by Nodal are ACVR1B and ACVR1C (also called ALK4 and ALK7). The type-II receptors are thought to be ACVR2A and ACVR2B (also called ACTRII and ACTRIIB).

Besides them, there are some co-receptors of the EGF-CFC family, which are small cysteine-rich extracellular proteins that are attached to the plasma membrane through a glycosylphosphatidylinositol (GPI) linkage. These co-receptors are TDGF1 or CFC1 (previously known as Cripto and Cryptic, respectively), and are essential for Nodal signaling. Activation of type-I receptor by type-II receptor results in the phosphorylation of cytoplasmic Smad2/Smad3, leading to their interaction with co-Smad (Smad4) forming a complex that will translocate into the nucleus and regulate the transcription of target genes (Figure 10) [113,114]

Importance of Nodal signaling for development in vertebrates

Nodal signaling plays important roles in vertebrate developmental processes. As stated before, most of these roles have been reported during mesoderm formation, and axial patterning events along the anteroposterior, dorsoventral and left-right axes. In fact, Nodal ligands have the properties associated with a morphogen: a signal that acts over a distance to elicit dose-dependent In this context, gene expression, fate map and functional analyses in zebrafish have shown that Nodal signaling acts before gastrulation to specify the progenitors for mesoderm and endoderm.

In the absence of Nodal signaling, progenitors acquire inappropriate fates relative to their position. This was confirmed after studying the function of the Nodal-related ligands Cyclops (Cyc) and Squint (Sqt) (teleosts have three Nodal-related genes called Cyclops, Squint and Southpaw), which are required for germ-layer formation in zebrafish. In fact, dorsal marginal cells in the zebrafish blastula normally give rise to notochord and prechordal plate. But in the absence of Nodal signaling in Cyclops:Squint double mutants or maternal-zygotic One-eyed pinhead (the gene coding for tdgf1, a Nodal co-receptor) mutants, blastula cells give rise to hindbrain and midbrain. On the ventral side, cells that should give rise to blood or pro-nephros, give rise exclusively to tail cells. These fate changes are already reflected at the blastula stage by the abnormal expression of specific gene markers. For example, Floating head, a gene expressed in notochord progenitors, and Goosecoid, a gene expressed in prechordal plate progenitors, are not expressed in Nodal signaling mutants [118,119,120,121,122]. This Nodal signaling ablation can also make organ asymmetry to be randomized or isomeric. For example, in the zebrafish diencephalon, the parapineal organ is normally located on the left. In the absence of Nodal signaling, the parapineal is still located asymmetrically, but in approximately half of the mutants it is located on the right [123]. Additional to this function, Nodal gene was also found to be expressed asymmetrically on the left side in the lateral plate mesoderm during somitogenesis in zebrafish, chick, mouse and Xenopus. Moreover, several components of the Nodal signaling pathway have been found to be implicated in left-right asymmetry in such animals [118,124,125].

In fact, it was shown that left-right axis defects can be caused by hypomorphic Nodal mutations in mouse, whereas in zebrafish depletion of the Nodal protein Southpaw induces left-right abnormalities [126,127].

Recent studies have also suggested that Nodal signaling is required for the maintenance of undifferentiated human and mouse embryonic stem (ES) cells. Indeed, all key components of the Nodal pathway are highly expressed in both undifferentiated mouse and human ES cells and Nodal signaling is required in vivo to maintain epiblast pluripotency and prevent precocious neural differentiation [113]. Regarding neural tissue formation, it seems that the development of the nervous system requires a blockage of the Nodal signaling pathway in the presumptive neuroectoderm. Nonetheless, Nodal signaling is later required for the specification of ventral cell types in the zebrafish nervous system. Mutations in the Nodal ligand gene cyclops and partial loss of one-eyed pinhead lead to the absence of medial floor plate and ventral forebrain, and result in cyclopic embryos. Furthermore, chimera experiments have shown that some of these defects are not caused indirectly by defects in the development of the underlying axial mesoderm or endoderm, but by a direct requirement for Nodal signaling in neural cells [119].

Nodal signaling in non-vertebrates

The members of Nodal pathway, such as the ligand Nodal, have been reported across bilaterians.

The role of Nodal during axes patterning is conserved in non-vertebrate chordates, as amphioxus.

For example, the Nodal/Vg1 signaling pathway specifies dorsal identity, and opposition between Nodal/Vg1 and BMP signaling is fundamental to both D/V and A/P patterning in this model [128,129]. On the other hand, Nodal also plays an essential role in the establishment of the D/V axis in echinoderms, but not in endoderm or mesoderm induction. In hemichordates, inhibition of Nodal function disrupts dorsoventral fates and blocks formation of the larval mesoderm, indicating that Nodal is required for mesodermal and ventral fate in deuterostomes [130]. The Activin/Nodal signaling has also been shown to control both internal organ asymmetry and asymmetric external development in most deuterostomes. For example, Nodal was reported to be crucial to establish the positioning of the mouth in relation to the rest of the larval axis in amphioxus. Indeed, after Nodal pathway inhibition, the larvae exhibit a loss of the mouth opening and preoral pit, as well as a bilaterally symmetrical phenotype with duplication of the right-sided structures and suppression of left-sided morphology [131]. In echinoderms, Nodal pathway acts downstream on the right side of the embryo to maintain Nodal, Pitx2 and Lefty expression, and its inhibition after gastrulation causes formation of an ectopic rudiment on the right side [132].

Nodal signals not only activate asymmetric Pitx expression in deuterostomes, but also in snails, supporting the hypothesis of the existence of a Nodal-Pitx cascade in the last common ancestor of deuterostomes, spiralians, ecdysozoans and chaetognaths [133]. In this context, the Nodal signaling role in the establishment of asymmetry and axes patterning during embryonic development seems to be well conserved across several phyla. Strikingly, a recently found Nodalrelated gene (Ndr) in Hydra underlined the possibility that this pathway emerged before the emergence of bilaterians. Ndr is essential for setting up an axial asymmetry along the main body axis, and it is also required for bud induction and Pitx expression [134].

Regarding neural differentiation, Nodal signaling was also found to be playing key roles in nonvertebrates. In ascidians, Nodal regulates the expression of BMPs and Chordin in the lateral neural plate, thus regulating the specification of epidermal sensory neurons alongside BMP signaling. On the other hand, ablation of FGF and Nodal together leads to arrest neural differentiation and proper neural tube morphogenesis by undergoing sequential cell division of only neural progenitors (but not differentiated neural cells) [135,136]. In the case of echinoderms, ectopic activation of Nodal can generate siamese larvae with two skeletons, two oral lobes, two mouths, two dorsal sides and two ciliary bands, suggesting a whole duplication of the D/V axis. This mirror duplication contained neural territories that were induced and patterned, either directly or indirectly, by Nodal ectopic activation [137].

Recent work from our lab has also put in discussion the role of the Nodal pathway during neural differentiation and the formation of the CNS in amphioxus. Indeed, we showed that Nodal is the instructive signal triggering neural fate from the ectoderm, and that the signal originates from the organizer [102]. This study proposes Nodal as a conserved neural inductive signal in chordates.

In order to discuss this topic in Chapter 5, I will present the model in which this study was undertaken, which is also the model of my thesis project. the split of cephalochordates from the rest of chordates [143]. The genome of three species has been sequenced: The Caribbean species Branchiostoma floridae, the Asian species B. belcheri and the Mediterranean species B. lanceolatum. From their analyses, it was confirmed that cephalochordates did not go through the two round of whole genome duplications (2R) that occurred in vertebrates. Besides, their gene synteny is highly conserved with vertebrates [142].

Phylogenetic position and genomic organization.

The sub-phylum Cephalochordata belongs to the phylum Chordata, which also includes the subphyla Urochordata (Tunicata) and Vertebrata (Craniata), and comprises a small number of species that are similar in appearance and lifestyle, in contrast to tunicates and vertebrates that show a large range of habitats [144,145]. Cephalochordates were traditionally grouped with the vertebrates in the so-called "Euchordata" or "Notochordata". This classification was based on common developmental and morphological features as a similar vascular system, segmented paraxial mesoderm, a prominent notochord, homologue structures to the vertebrate liver, pituitary and thyroid gland (hepatic caecum, Hatschek's pit and endostyle, respectively) [146,147].

However, phylogenetic analyses with large sets of nuclear genes has allowed to relocate cephalochordates at the base of chordate phylum. This classification groups together vertebrates and tunicates, as sister groups, into a clade named Olfactores [148]. Even though tunicate genomes exhibit a fast evolutionary rate, they have retained enough phylogenetic signals for their position to be recovered through a maximum likelihood construction (Figure 13). This topology not only indicates that tunicates evolve rapidly, but also became simplified during evolution. In fact, they have lost several common characteristics with our chordate ancestor, like some key developmental genes, that have made them acquire divergent bodyplans. On the other hand, they present a type of migratory neural crest-like cells, which is a typical characteristic of vertebrates and which is absent in amphioxus. This morphologic novelty sustains their phylogenetic position as the sister group of vertebrates, despite their high degree of divergence [144,149]. three to six months per year depending on the species. The spawning season for B. belcheri and B. lanceolatum takes place during two or three months in spring-summer. A. lucayanum is an exception since it is able to spawn during two different periods of the year, during the summer and the autumn. Right before spawning, amphioxus swims up to the water column and release their gametes and come back into the sand afterwards. Different intervals of spawning have been observed in each species. For instance, the species B. belcheri spawn during a short period of days, whereas B. floridae is able to spawn in a synchronic way every two weeks. On the other hand, A. lucayanum is apparently influenced by the lunar cycle, thus most of the population tends to spawn the day after the full moon [START_REF] Aldea | Amphioxus Illuminates the Origin of the Vertebrates' Head[END_REF]157].

The species that was used in this project as a model is the Mediterranean species Branchiostoma lanceolatum, therefore the embryonic development described here correspond to this species at 19 °C, as previously published by Fuentes and collaborators (Figure 17) [159,160].

From fertilization until gastrulae stage, the development of amphioxus is similar to other nonvertebrate deuterostomes, which is not the case during neurulae stage with the formation of vertebrate-like structures as the notochord, the somites and the neural tube [143,161,162,163].

Amphioxus body plan axis appears to be already established from early development. In fact, during oogenesis in B. floridae, some organelles, mRNAs and molecules are not distributed uniformly. In the animal pole, there are presence of nucleus, striated fiber bundles, Nodal and TCF mRNAs, whereas in the vegetal pole there is presence of endoplasmic reticulum that is associated with ribosomes and mRNA of germ cell markers as Vasa and Nanos, which are inherited asymmetrically by a single blastomere during cleavage (Figure 18 a,b). Thus, the germ line of amphioxus seems to be already determined in the unfertilized egg, through the asymmetrical localization of germ cell markers [164,165,166]. other body structures. Thus the mouth is placed on the left side of the pharynx and the gill slits on the right ventro-lateral side. Moreover, the left-side somites are positioned more rostrally (halfsomite) than the right-side somites. In most species this asymmetry largely disappears during metamorphosis, which involves massive tissue re-organization, as it is the case for the mouth, which gets positioned rostrally by the end of metamorphosis. The post-metamorphic juveniles gradually grow into adults with estimated life spans ranging from 2 to 8 years depending on the species [START_REF] Aldea | Amphioxus Illuminates the Origin of the Vertebrates' Head[END_REF]166].

Chapter 5: Neural Induction in amphioxus and central nervous system development

Anatomical overview

The early formation of the CNS in cephalochordates and vertebrates depends on ectodermal dorsal patterning signals during early embryogenesis. The first step in this process is neural induction, which was proposed to take place at gastrula stage in both subphyla. In amphioxus, the neural induction process starts when the dorsal region of the ectoderm secretes proteins that antagonize pro-epidermal signals (BMPs) that are present in the ventral region of the embryo, leading to specify the neuroectoderm that will later become the neural plate. This pro-neural territory was identified as the homologous of the well-known Spemann organizer, which was previously thought to be exclusive in vertebrates. In fact, grafting experiments from Tung in 1961 suggested the inductive capacity of the dorsal blastoporal lip of amphioxus embryo to generate a secondary body axis containing dorsal structures, as a notochord and a neural tube. After this, Le Petillon and collaborators reproduced these experiments, confirming the inductive capacity of this structure in amphioxus (Figure 19) [102,167,168]. Once the organizer has defined the prospective neural plate at late gastrula, its border region detaches from the edges, forming neural ridges, and moves over it by lamellipodia. The neural plate sinks below the surface while the ridges slide across it to fuse in the dorsal midline. Once the neural plate is folded into a neural tube underneath the dorsal ectoderm, it detaches from it. The neural tube does not close completely, leaving an opening at the anterior end named neuropore. This process is different in vertebrates, where the neural plate border region remains attached to the neural plate while it folds until the neural tube is formed. Then the neural tube detaches from the ectoderm and the border regions will go through epithelial-to-mesenchymal transition and become a migratory cell population, known as neural crest cells. These cells form defining features of vertebrates including craniofacial skeleton, peripheral nervous system, and pigment cells (Figure 20) [17,18,169]. Lack of definitive neural crest in amphioxus, and their presence in ascidians, supports the hypothesis that this structure emerged as a novelty in the Olfactores clade.

In amphioxus, FGFs present dynamic embryonic expression patterns in the developing embryo, while their only receptor is ubiquitously expressed during all developmental stages. These expression patterns have been mainly associated to important processes as mesoderm and neural tissue formation. However, when FGF signal is blocked during early development, it leads to a loss of anterior somites whereas neural induction still occurs [START_REF] Aldea | Amphioxus Illuminates the Origin of the Vertebrates' Head[END_REF]. Similar to vertebrate models, the amphioxus ectodermal explants provide a sharper appreciation of the processes taking place during neural differentiation by excluding the full mesendoderm input. Such tissue can be obtained through microsurgery at gastrula stage (gastrula explant), or grown from the animal blastomeres isolated at 8 cell stage (blastomere explant). By using this technique, Le Petillon et al. showed that FGF activation is capable to promote neural fate, but its inhibition does not impede the formation of neural tissue in both ectodermal explants and treated embryos. In consequence, FGF is a bona fide neural inducer in cephalochordate embryos and explants, as it is also described in vertebrates. However, it is not playing an instructive role in amphioxus neural fate commitment [102,168,173].

In this context, Le Petillon also demonstrated that another signaling pathway is important during neural induction in amphioxus, the Nodal signaling pathway, which was previously described to confer anterior-dorsal identity during early development by opposing BMP signaling. In fact, Nodal is mainly expressed in the animal half during early cleavage stages, and later on becomes more restricted to the future dorsal ectoderm and mesoderm during gastrula stage [129]. It has been shown that ectopic activation of this pathway using recombinant Activin treatment induces embryo dorsalization, whereas its pharmacological inhibition induces ventralization, overexpression of BMPs and consequent loss of neural structures in amphioxus. This study used the pharmacological inhibitor SB505124, which blocks Alk4, Alk5 and Alk7 TGF-β receptors, in order to inhibit the Nodal pathway. Such treatment caused the loss of expression of dorsal markers SoxB1a and Chordin at the gastrula stage, and loss of neural tube marker Wnt3 during neurula stage [102,168]. Activin-treated embryos were not only dorsalized but completely neuralized, as confirmed by the expression of the pan-neural markers Neurogenin and Hu/Elav, and the complete loss of expression of the epidermal marker K1 (Figure 24) [102,168].

In order to observe if FGF signaling was involved in Nodal-mediated neuralization, FGFR

Abstract

Neural induction is the process through which pluripotent ectodermal cells are committed to a neural fate. It is known, in vertebrates, that the dorsal organizer produces BMP antagonists, and other signals that induce neural fate acquisition. However, not much is known about neural induction in other chordates. Our team has previously shown that the cephalochordate Branchiostoma lanceolatum (amphioxus) presents a functional organizer, and that the acquisition of epidermal fate relies on BMP activation. However, deprivation of BMP signals leads to an undifferentiated state of the ectoderm, indicating that BMP inhibition is not sufficient for neural induction. Moreover, inhibition of FGF signaling does not block neural induction, in the contrary to what is observed in several vertebrate lineages, and ascidians, suggesting that FGF is not the key signal to induce neural fate in amphioxus. Remarkably, activation of the Nodal pathway triggers neural induction and represent an instructive signal of this process in amphioxus. The objective of my project was therefore to determine the gene regulatory network (GRN) downstream of the Nodal signaling pathway during neural induction in embryos of the european amphioxus Branchiostoma lanceolatum.

The main approach of this work was to undertake comparative RNA-seq and ATAC-seq (Assay for Transposase-Accessible Chromatin) experiments on different samples. I compared data obtained at different time points between control embryos and embryos in which the entire ectoderm is neuralized thanks to the activation of the Nodal pathway (through treatment with the Activin recombinant protein). Moreover, I also generated transcriptomic and epigenomic data for ectodermal explants that we obtained by microdissecting the amphioxus embryos at the eight-cell stage. These explants, which correspond to the 4 smaller cells, naturally give epidermis when cultured whereas most of the cells differenciate into neural cells following Nodal pathway activation. From the ATAC-seq data obtained in collaboration with the Gomez-Skarmeta team in Sevilla, I identified a total of 679 putative early enhancers specific to treated explants and could define in these sequences the putative transcription factor binding sites. From these data and the RNA-seq data, I could propose a GRN for neural induction. To test this hypothetical GRN, I first focused on putative enhancers possessing Smad binding sites. Indeed, Smad2/3 is the transcription factor effector of the Nodal signaling pathway and such enhancers might therefore be present in the vicinity of Nodal direct target genes. A set of 8 transcription factors were identified as presumptive direct target genes and in order to determine how such targets could be orchestrating neural induction, I cloned their respective regulatory sequences into a GFP reporter vector with an amphioxus beta Actin minimal promoter. The reporter constructs were injected in unfertilized eggs that were then fixed at different developmental stages. The GFP expression patterns observed were not exclusive to pro-neuronal, or pro-ectodermal territories for any of them; suggesting that such regulatory elements could be pleiotropic, and could be driving a general gene expression when activated individually. Therefore, Nodal could be activating a large combination of transcription factors at a defined spatio-temporal situation in order to determine a specific neural pattern, and the regulation of neural induction would not be linear, which is consistent with the shape of the GRN that I propose. In order to functionally test the role of the transcription factors activated downstream of Nodal, a constitutive repressor approach was undertaken. Indeed, knock-down techniques (morpholinos, siRNA, etc...) are not efficient in amphioxus and the implementation of the CRISPR/Cas9 system is still in progress. Three transcription factors (Neurogenin, Pou3f and SoxB1a) were chosen as candidates and chimera constructions were realized by fusing their sequence to the Engrailed repressor domain sequence.

Injection of their respective mRNA into unfertilized eggs reduced the neural territory but did not deplete neural gene expression, which argues for a non-linear regulation downstream of Nodal. In addition to these experiments, we have identified Klf1/2/4 as a potential negative regulator of the whole GRN, and tested its putative role on neural fate acquisition. After injecting Klf1/2/4 mRNA into unfertilized eggs, there is a total depletion of neuroectodermal and neural markers expression in embryos. Together, the results here obtained show that the neural induction process is not controlled by a master gene but by a combination of transcription factors that are activated by Nodal at a specific time point. Moreover, the negative control executed by Klf1/2/4 suggests an interplay between pluripotency markers and Nodal targets during early neuroectoderm differentiation. My work illustrates a scenario where Nodal is antagonizing the expression of Klf1/2/4 in specific pro-neural territories in the ectoderm, as well as activating a large number of target genes during the neural induction process. This scenario offers new perspectives to understand how neural fate is acquired, and pro-neural cells differentiate from ectodermal cells. This chapter aims to discuss the results from the experiments that were carried out during my thesis project in order to understand the regulation of the neural induction process after Activin/Nodal activation. The first part is mainly focused on the identification of novel neural markers, and their expression patterns, during the neural induction and neural differentiation processes. These markers have allowed to confirm early neural patterns or differentiated neuronal structures (as observed with the synaptic vesicle marker Synaptotagmin).

The next part aims to determine the timing window at which Nodal signaling triggers neural induction. The third part will dig into the data obtained through RNA-seq data analysis from whole embryos and/or explants under Dorsomorphin (BMP-inhibitor) and Activin (Nodalactivator) treatments at different developmental time points in order to identify potential candidate genes involved in the neural induction process.

The following part discusses the results obtained through ATAC-seq from whole embryos and/or explants after Nodal activation at two developmental time points, in order to identify putative regulatory regions interacting with key transcription factors during the neural induction process.

In this part, a detailed workflow is presented with the steps that have been followed since the identification of the over-represented peaks specific to Activin-treated explants until the generation of a gene regulatory network (GRN) based on such ATAC-seq data.

Finally, the fifth part aims to validate this GRN by functional analysis of putative enhancers.

Here we used both enhancers possessing Smad binding sites present in putative Nodal direct targets, and enhancers of the locus containing the neural gene Neurogenin. In addition to this, we have used injections of mRNA of transcription factors or of their constitutive repressor chimeras to test the function of nodes of the GRN.

Characterization of neural territories by novel neural markers

In order to better understand the neural differentiation process in amphioxus, we studied though in situ hybridization (ISH) the expression of a set of classic neural markers that are expressed during neural differentiation in vertebrates: SoxB1b,SoxB2,Pou3f (Brn 1/2/4

SoxB1b

SoxB1b is one of the amphioxus-specific SoxB1 paralogous genes (SoxB1a, SoxB1b and SoxB1c).

All these paralogs have been described to have similar expression patterns in B. floridae, which is consistent with an ancient role of SoxB1 in neuroectoderm specification [174]. In fact, SoxB1a and SoxB1b present a high degree of sequence similarity, they both share the same locus, and their expression patterns are virtually equals; in comparison to SoxB1c, whose expression was described to start later and to remain at larval stages. In B. lanceolatum, the expression does not differ from the one observed in B. floridae. SoxB1b is expressed in the whole ectoderm in gastrulae, then the expression becomes restricted to the presumptive neuroectoderm at the end of gastrulation and continues to be detected alongside the neural plate at neurula stage. Once neurulation is over, SoxB1b expression gets restricted to the CNS and gradually disappears until the larva stage (Figure 26). This gene constitutes a good pan-ectodermal marker at early stages and pan-neural marker during neurulation, as well as the known SoxB1a, which has been widely used in amphioxus in previous experiments.

SoxB2

SoxB2 is also expressed in the presumptive neuroectoderm during gastrulation. The expression continues to be detected in the neural plate, and gets restricted to a posterior region of CNS at the end of neurulation. SoxB2 exhibits a quite similar pattern of expression to SoxB1a and SoxB1b, in both amphioxus species during the formation of the neural plate and tube [174]. However, only in B. lanceolatum it becomes restricted to the posterior region of the CNS in later stages (Fig 26).

Pou3f (Brn1/2/4)

Pou3f transcription factor is a well known neural gene in several vertebrate models. In B. floridae, its expression has been described to start during gastrulation, in the dorsal ectoderm at the mid-gastrula stage and becomes later restricted to the anterioir region of the neural plate [175]. In B. lanceolatum, the expression was observed in early neurula, and continues throughout the whole neural tube in later stages until larva (Figure 26). There is no evidence of an early expression, though its strong expression in larva suggest that this gene might be involved in the formation of some differentiated neurons.

FoxB

FoxB expression was first detected dorsally, both in the ectoderm and in the mesendoderm, as a weak signal in mid gastrula stage embryos. At early neurula stage, a signal could be observed in the neural plate on both sides of the midline, as well as in two patches in the posterior paraxial mesoderm. During the late neurula stage, expression was detected in the most posterior paraxial mesoderm that gives rise to the newly formed somites and in the neural tube posterior to the cerebral vesicle. Then, FoxB expression in the mesoderm faded away in late neurulae and get later restricted to the cerebral vesicle and to some neurons along the neural tube in larvae (Figure 26). This expression pattern was included in the publication of Aldea et al, 2016 [176].

FoxG

FoxG expression was first observed at the neurula stage in the anterior region of the first somites.

At the late neurula stage, FoxG was expressed in the anterior ventral region of the three most anterior somites. Later on, in late neurula before the mouth opens, a neural expression appeared in some individual neurons within the neural tube, while the expression observed in the first somites disappeared. This expression persisted in the larva stage embryos in which FoxG was also detected in some neurons of the cerebral vesicle (Figure 26). This expression pattern was included in the publication of Aldea et al, 2016 [176].

Collier/olfactory/early B-cell factor (Coe)

Coe gene has been associated to the formation of olfactory placode neurons and peripheral nervous system development in vertebrates. In B. floridae, it is first detected at early neurula stage throughout the neural plate. In later stages, the expression pattern was identified in isolated ectodermal cells that seem to correspond to subsets of sensory neurons [177]. In B. lanceolatum, the expression was detected at gastrula stage (earlier than in B. floridae) and continues to be expressed in the whole neural plate during neurulation. In 36hpf embryos, before going into the larval stage, the expression pattern gets restricted to patches alongside the CNS, similarly to what is observed in B. floridae (Figure 26). Therefore, Coe expression in later stages might correspond to such isolated subsets of ectodermal neural cells.

Huntingtin (Htt)

Htt is associated to the neurodegenerative disease Huntington, and it was described as a gene potentially involved in the development of the nervous system in vertebrates. It has been, however, only detected at late stages in B. floridae, starting from neurula stage and restricted to small neural compartments. In larval stages, the expression pattern is mainly detected in the anterior region of the neural tube [178]. In B. lanceolatum, we could not detect clear expression patterns at any stage of development (Figure 26).

Single minded (Sim)

Sim codes for a basic helix-loop-helix (bHLH) transcription factor that is required for the specification of the ventral midline in Drosophila, and it has been associated to neurogenesis [179]. In B. floridae, the expression was first observed at early neurula stage in the dorsal mesoderm, and later on in the forming cerebral vesicle. Besides, it presents expression in six cell clusters during late neurula stage [180]. In B. lanceolatum, the expression pattern is virtually similar to the expression in B. floridae until late neurula. Nonetheless, in larva, the expression seems to be less restricted. In fact, the expression is not only maintained in the cerebral vesicle but also in other non-neural regions alongside the larva, such as the pharynx (Figure 26).

Neural cell adhesion molecule (Ncam)

Ncam codes for a cell adhesion protein that is involved in the development and regeneration of the nervous system [181], and it is usually used as a paneural marker in several vertebrate models. However, its expression pattern has not been previously described in amphioxus. Here, Ncam is expressed maternally in egg and early stages of development. In fact, its expression during the first stages of development is ubiquitous, but becomes restricted to the neural plate at early neurula stage. At the end of neurulation there is no trace of expression of Ncam in the neural tube, but restricted patches in the posterior and anterior mesoderm. This temporary neural expression of Ncam suggest that this protein could be necessary for the first steps of the formation of the neural plate (Figure 26).

Synaptotagmin (Syt)

Syt codes for a synaptic vesicle protein involved in neurotransmitter release. All three paralogs of this gene in rat are distinctly expressed in different neural subsets of differentiated neurons. In amphioxus there is no previous description of its expression pattern [182]. Nonetheless, we could identify one gene coding for Synaptotagmin (Syt), and another with similar structure that we identified as Syt-like. The expression of Syt starts at late neurula and is limited to the CNS at later stages (Figure 26). Its protein is known to be found when axons and synaptic vesicles are formed, supporting this gene as a good neuronal marker in amphioxus. In this project, Syt was mainly used to mark the CNS in late/post neurula stages.

In order to asses the specific time of action of Nodal signaling during neural induction, it was necessary to use different pharmacological treatments in early embryos in order to activate and/or block the whole signaling pathway. The results are included in the publication of Le Petillon et al, 2017 (Supplementary data).

We made continuous treatments with recombinant human Activin protein* 50ng/ml, or with the pharmacological Nodal inhibitor SB505124* 50µM in amphioxus embryos starting at 3, 4, 5 and 7hpf at 19°C. Expression of the neural marker Neurogenin shows that early treatments with Activin at 3 and 4hpf were able to dorsalize the embryos and expand the neural territory at neurula stage (N2). However later treatments at 5 and 7 hpf led to a Neurogenin expression pattern similar to control embryos. Interestingly, treatment at 4 hpf led to a loss of anterior neural identity when compared to embryos treated at 3 hpf. In contrast, inhibition of the Nodal pathway via the application of SB505124 at any time between 3 to 5 hpf impeded the formation of Neurogenin positive neural tissue. On the other hand, treatment started at 7 hpf did not deplete the formation of neural cells at N2 stage (Figure 28a).

We also undertook discontinuous treatments in embryos at 3hpf with Activin 50ng/ml or SB505124 50µM for 1 and 3 hours, then the molecules were washed and the embryos left to develop until the N2 stage. In fact, 1 hour Activin treatment does not affect the formation of neural tissue (here observed by the normal expression of Neurogenin and normal morphology).

However, a treatment of at least 3 hours is able to dorsalize the embryo and expand the neural territory. On the other hand, embryos treated with SB505124 for 1 or 3 hours did not show any neural marker expression indicating a complete abrogation of neural induction (Figure 28b).

Generation of transcriptomic libraries

In order to define the genes that are regulated during neural induction triggered by Nodal in amphioxus, and to define the effect of BMP inhibition, a comparative transcriptomic approach was used. The results here presented support the specificity of the treatments and provide a source of data corresponding to target genes activated during the neural induction process. All experiments were done at least in duplicate, and raw data were analysed using the DESeq2 package with specific parameters**.

First, in order to determine if Nodal signal induces neural fate acquisition independently of BMP signal inhibition, a comparative transcriptomic analysis was performed. We exposed embryos to Dorsomorphin* 35µM (which is known to exclusively block BMP-mediated SMAD1/5/8 phosphorylation though the inhibition of type I receptors ALK2, ALK3 and AKL6) starting from 3hpf, in order to inhibit the differentiation of ectodermal cells. These embryos were collected together with controls at two developmental time points Gastrula G4 (11hpf) and Neurula N2 (19hpf) for RNA extraction and sequencing. In a similar way, we exposed embryos to the recombinant human protein Activin* 50ng/ml starting from 3hpf, in order to activate the Nodal signaling pathway. In fact, it has been demonstrated in previous publications that the Activin ligand is able to effectively bind type I ALK4/7 and type II TGF-b receptors leading to SMAD2/3 phosphorylation. These treated embryos were also collected together with controls at the same developmental time points that were previously considered for Dorsomorphin treatment (G4 and N2 stages). RNA extraction and sequencing was performed subsequently (Figure 30).

Comparative RNA-seq data analyses from embryos after BMP inhibition and Nodal activation

The comparative analysis carried out in Dorsomorphin-treated embryos at G4 stage (11hpf) show that 491 transcripts (from a total of 7149 transcripts) are over-expressed compared to controls (log2FC>2, padj<0,1), whereas 525 transcripts are underexpressed (log2FC<-2). We performed the same approach with N2 stage (19hpf) embryos and observed a total of 6156 transcripts (padj<0,1) with a differential expression level between treated and control embryos. From all these transcripts, 760 are over-expressed (log2FC>2) and 1409 are underexpressed (log2FC<-2).

For embryos treated with Activin, and stopped at G4 stage, the DESeq2 analysis (-2>log2FC>2; Padj <0,1) recovered 95 significantly up-regulated transcripts and 422 down-regulated transcripts in treated embryos vs control. In the oldest embryos, at N2 stage, the DESeq2 analysis (-2>log2FC>2; Padj <0,1) recovered 26 up-regulated and 60 down-regulated transcripts in Activintreated embryos vs control.

We have merged the data obtained from the analyses done in embryos treated to Activin and Dorsomorphin at G4 and N2, in order to have a comparative view of the effects of both Nodal and BMP signaling pathways. The results concerning the most interesting genes are presented as a heatmap in Figure 33. The data obtained first show that BMP and Nodal pathways are opoosite signals. Indeed, when Nodal pathway is activated, Nodal target expression is induced whereas BMP targets expression is down-regulated. When BMP pathway is inhibited, Nodal targets are overexpressed whereas BMP target gene expression is down-regulated. Moreover, our data show that both signals have different action on ectodermal cell fate. In fact, classical neural markers are positively regulated after Activin treatment, whereas their expression seem to decrease after Dorsomorphin treatment, supporting the proposition that BMP inhibition is not sufficient for neural induction to take place.

Comparative RNA-seq data analyses from 5hpf embryos treated with Activin and

Puromycin

Once we confirmed that Nodal signal is early required during the formation of neural tissue (Chapter 3.2), and that most of the up-regulated Nodal targets at 11hpf and 19hpf correspond to indirect targets involved in late neural development processes, we decided to generate transcriptomic data for Activin-treated embryos at an earlier developmental stage.

In order to determine the immediate downstream targets of the Nodal signaling pathway, we carried out Activin treatment in embryos at 3hpf until 5hpf, with a control experiment. In addition to this, we decided to perform Puromycin treatment in embryos at 2,5hpf and Puromycin/Activin treatments in embryos at 2,5/3hpf until 5hpf in order to block translation during early embryogenesis, and expose embryos to Activin as a unique signal. We finally proceeded to obtain the corresponding RNA libraries followed by sequencing (RNA-seq) for each treatment and control (Figure 31).

Analysis of transcriptomes from Activin-treated 5hpf embryos

The results corresponding to the embryos treated at 3hpf and processed at 5hpf showed only few genes that are sensitive to Nodal signal activation under low stringency parameters (log2FC >1,0; Padj <0,1) (Table1). Remarkably, the most up-regulated transcript (according to its Log2FC and Basemean value), corresponds to Nodal direct target Lefty, which is known to be mainly involved in mesoderm formation. Moreover, the following up-regulated transcripts correspond to Nodal ligand Vg1 and to the Nodal pathway target Pitx (isoform 1 and 2) which is also proposed to be involved in mesoderm formation during early embryogenesis. Besides, the neural marker gene Neurogenin was also found to be sensitive to Activin treatment at 5hpf. In addition to these results, the gene coding for the esterase PCED1 (PC-Esterase Domain Containing 1), and a non-characterized transcript (here named BL01555 according to its annotation number) were found to be over-expressed in treated embryos. Two more unknown transcripts were found to be over-expressed (BL01930 and BL11242), but both of them correspond to the same genomic locus (which contains 4 amphioxus-specific tandem paralogs).

For this reason, most of the annotations in this locus overlap such transcripts and characterization was not possible. From now, all the consequent transcripts referred to this particular overexpressed locus will be referred only as "BL11242*".

Analysis of transcriptomes from Puromycin-treated 5hpf embryos

In contrast to the comparative analysis of the data obtained from embryos treated with only Activin and their control, this approach aimed to find direct targets of Nodal. In fact, Puromycin is a pharmacological antibiotic that causes premature chain termination during translation, therefore blocking the synthesis of proteins. This treatment was based on exposing embryos at 2,5hpf to Puromycin for 30 minutes followed by wash -or Activin treatment-at 3hpf, in order to define Nodal pathway direct targets under Activin as a unique signal. All treatments were stopped at morula stage (5hpf) and isolated for RNA-seq (Figure 31). Since our objective was to define up-regulated targets of the Nodal signaling pathway, we only extracted the data corresponding to up-regulated transcripts from each analysis.

First, we compared the results obtained from the Puromycin/Activin vs Puromycin treatments, but we did not obtain any statistically supported result through DESeq2 analysis. For this reason, we decided to manually compare the results obtained from the comparative analysis of transcriptomes from Puromycin/Activin-treated embryos vs control, with the results obtained from the comparative analysis of transcriptomes from Puromycin-treated embryos vs control.

The Puromycin/Activin vs control analysis (Padj<0,1, -2>Log2FC>2) did not recover any overrepresented transcript. For this reason, we adjusted the DESeq2 cut-off parameters in order to identify targets that are slightly sensitive to the treatments (Padj<0,1, Log2FC>0,75). Similarly, the Puromycin-treated embryos vs control analysis did not show any up-regulated transcript. Still, we identified 36 transcripts that are down-regulated from a total of 444 significantly regulated transcripts (padj<0,1; -2>Log2FC>2). This supports that Puromycin is a treatment able to reduce general gene expression, and that blocking protein synthesis does not lead to any gene expression activation through the absence of translation of putative transcriptional inhibitors.

In this context, the cut-off for both analyses were changed (Padj<0,1, Log2FC>0,75), to less stringent parameters. As a result, we found 37 overexpressed transcripts from the analysis of Puromycin/Activin vs control, and 25 overexpressed transcripts from the analysis of Puromycin vs control. We then compared both data sets and identified a total of 18 overexpressed transcripts that are only present in the first treatment, and should correspond to direct targets of Nodal (Table2).

These results show targets that are sensitive to Activin treatment at 5hpf, after translation inhibition through Puromycin treatment. Such results are different from the data obtained in the analysis of Activin vs control at 5hpf. Here, we observe the up-regulation of several genes associated to structural functions and cell adhesion (as Cadherin-CDH13 and Sidekick-Sdk1/2).

In fact, we did not observe up-regulation of classic neural gene markers in this analysis.

However, some up-regulated targets correspond interesting genes as Auts2/Auts-like (Autismsusceptibility candidate), which has been described to be involved in neuronal migration in In conclusion, the analyses undertaken in 5hpf Activin-treated embryos confirm the up-regulation of known Nodal signaling downstream targets for mesoderm formation as Pitx and Lefty. On the other hand, the only neural gene over-expressed is Neurogenin, supporting that neural induction is an early process, where Neurogenin up-regulation seems to be one of the first steps. Additional candidates obtained after inhibition of translation (Puromycin treatment) did not elucidate the possibility of an early activation of neural markers leading to neural fate acquisition.

Comparative RNA-seq data analyses from ectodermal explants treated with Activin

Several up-regulated target genes that have been found in embryo analyses correspond to well known mesodermal genes. In order to avoid this mesodermal input, we have decided to reproduce micro-dissection techniques described by Le Petillon, and to generate transcriptomic data from dissected ectodermal explant treated with Activin from 3hpf until 11hpf and 19hpf (Figure 32).

Ectodermal explants at 11hpf were collected for RNAseq, and data was analysed with DESeq2 (2<log2FC<-2,0; Padj <0,1) using the amphioxus annotation, as previously done for whole embryos. We have found 3158 up-regulated transcripts and 4223 down-regulated transcripts in treated ectodermal explants at 11hpf vs untreated explants. On the other hand, analysis of RNAseq data from ectodermal explants at 19hpf under the same parameters-recovered 4041 upregulated transcripts and 5757 down-regulated transcripts in treated ectodermal explants vs untreated explants. The RNA-seq data from ectodermal explants show that there is a higher number of genes that are sensitive to Activin treatment in ectoderm compared to whole embryos.

Therefore, we decided to focus on the results obtained from these experiments, specially at 11hpf.

In order to determine potential target candidates, we did a more stringent analysis (log2FC>4,5; Padj <0,1, Basemean>100) using our reference transcriptome. This allowed us to identify Nodal targets previously identified at 5hpf, like: Lefty, Pitx, Vg1, Auts2 and Neurogenin (Table3). The over-expression of Lefty and Pitx in embryos highlighted the early role of Nodal signaling for the formation of mesoderm during gastrulation, and its role in the control of left-right asymmetry in later developmental stages. However, their up-regulation in ectodermal tissue suggests that Nodal signaling equally activates ectodermal and mesodermal targets in ectodermal explants. This

shows that Nodal is a massive gene activator for several developmental processes.

Interestingly, the over-expression of Neurogenin after Activin treatment, at 5hpf and 11hpf, confirms again that neural induction is a very early process. Taken together our results provide a wide outlook of Nodal as a general activator of several genes in embryos and ectodermal explants, but it does not provide a definitive clue about the direct targets that are controlled by this signaling pathway, or how the transcription of each target is regulated. In order to address this question, we adopted an additional approach through the Assay for Transposase-

Regulation of the neural induction process through genomic regulatory regions

The Assay for Transposase-Accessible Chromatin with high throughput sequencing (ATAC-seq) Considering this, we followed the workflow presented in Figure34. In collaboration with the team of Jose-Luis Gomez-Skarmeta at the CABD in Sevilla -Spain, we identified a total of 679 overrepresented peaks, and 2235 down-represented peaks in Activin-treated explants when compared to the control. Such peaks represent open chromatin regions containing putative regulatory sequences (from now on, named "enhancers") that were located throughout the whole genome.

After this, these results were processed following two approaches: a TRANS regulatory approach by determining the genomic position of putative active "super-enhancers" regions and affiliate them to their closest gene, and a CIS regulatory approach by predicting the TF binding sites that are present in each of the enhancer, and generating a list of candidate TFs that are involved in the neural induction process. Some of the TFs found with super-enhancers are orthologous to factors that have been well described as being involved in the formation of neural structures in vertebrates, as it is the case of Neurogenin, SoxB2, Vent, FoxAB, Pax3/7, Nkx6.1, Otx, Sim, whereas others as Zic, Snail, Hey1

Vg1 5 Iqub 4 BL02475 3
FoxAB 4 Tmem59 3 BL11242* 3
Hey1 4 Twsg1 3 BL19334 3
Otx 4 Cnr3-l 3 BL83847 3
Twist 4 Fhod1 3 BL20607 3
Zic 4 Gcc2 3 BL24287 3
Znf503 4 Hs3st 3
Znf888 4 KcnQ3 3
FoxB 3 Lrig1 3
Gli1 3 Lrp4 3
Gsc 3 Ndufa1 3
Insm1 3 Pcdhb 3
or Insm1 are orthologous of genes that play several roles in other developmental processes. In order to determine how these TFs are interacting during the neural induction and neural differentiation processes, and to complete our list of candidate genes involved, we decided to predict the putative transcription factor binding sites present in each enhancer found by ATACseq.

Cis-regulatory elements involved in Neural induction

In order to determine the cis-regulatory elements of our GRN, we analysed the transcription factor binding sites (TFBS) that are present in the 679 over-represented peaks in Activin-treated 11hpf ectodermal explants by using MATCH analysis on TRANSFAC database (minFP on vertebrate's non redundant database). A total of 7688 potential TFBS were identified through matrix comparison, and they were distributed according to the DNA binding domain type of the TF that recognize them (Figure 36).

The results presented in the distribution piechart show that a large quantity of such binding sites are bound by TFs containing C2H2 Zinc Finger domains (ZFC2H2, as KLF/SP, ZNFs, INSM, GLI, etc), Homeobox domains (as POU, VENT, PAIRED, NKX, etc), basic Helix-Loop-Helix domains (as BEN, MYOGENIN, etc) and High Mobility Group box domains (as SOX) (Figure 36).

In fact, the biggest group of transcription factors that are potentially interacting with such activated enhancers is the ZFC2H2 group, which is the largest and best-characterized class of proteins containing a Zinc Finger DNA binding domain. This group contains a great variety of TF sub-families as Zic, Klf, SP, Insm, Zeb and ZnFs. Indeed, many members of these subfamilies were also found to be over-expressed in our RNA-seq analyses in 11hpf ectodermal explants treated with Activin (Table 3 and Supplementary Table 1) and some of them (the orthologues of Snail, Zic, Gli1, Insm1, Zeb, Prdm8, Znf503 and Znf888) presented super-enhancers in their flanking genomic regions (Table 4). This suggest that these up-regulated TFs not only control the expression of target genes, but are also under the control of other TFs involved in neural fate acquisition.

In order to better understand the distribution of the whole set of trans-regulatory elements (here considered as specific TFs families) that are interacting with cis-regulatory elements (here considered as putative enhancers) found in Activin-treated ectodermal explants, we decided to focus on the first three major families found in this distribution pie chart. Additionally, we dissected the results corresponding to the enhancers containing SMAD binding sites to determine direct targets of Nodal. Indeed, Smad2/3 is the effector of the Nodal signaling pathway, which recruits a co-Smad (Smad4) into a multimeric complex that will translocate into the nucleus and bind to the promoter regions of direct target genes.

Klf, Zinc Finger and Sox TF families are neural induction regulators.

The results here obtained suggest that the orchestration of the neural induction process through the Nodal pathway implies a combination of a large set of transcription factors that will bind specific target genes during neural fate acquisition. As stated before, three gene sub-families are well over-represented: Zinc Finger factors, Sox-related factors and Krupel-like factors.

In amphioxus, the members of the Zinc finger (Znf) family have not been completely described.

Thus, the identified genes in this animal only correspond to genes with known orthologues in vertebrates. Here, the MATCH logarithm from TRANSFAC is not able to identify specific sites bound by amphioxus TFs, but it can identify the potential orthologous genes in vertebrates. As a result of this, the identification of vertebrate Znf333 might correspond to several members of the whole Znf sub-family (to be better identified yet). In this study, we have found a set of putative Znf genes, after blasting the corresponding sequence of the vertebrate Znf333 in the amphioxus genome, and we observed that most of these Znf candidates are up-regulated in ectodermal explants at 11hpf after Activin treatment (Supplementary Table 2). This supports the idea that this TF family is important in the control of the neural induction process. Interestingly, two putative Znf genes (Znf503, and Znf888) were not found after blasting the sequence corresponding to the vertebrate Znf333, even though they are located near super-enhancer regions

The presence of several Sox family members have been previously described in amphioxus, such as SoxB1a/SoxB1b/SoxB1c, SoxB2, SoxC, SoxD and SoxE. In fact, members of the SoxB subfamily (SoxB1a, SoxB1b, SoxB1c, SoxB2) were found to be up-regulated in ectodermal explants at 11hpf after treatment with Activin (Supplementary Table 3), and they are also known to be expressed in dorsal ectoderm during early gastrula stage, before getting restricted to the presumptive neural plate and neural tube at later stages. The striking similarity between the embryonic expression patterns of amphioxus SoxB genes with their respective vertebrate orthologues support the idea of a conserved neural role of this TF subfamily in chordates, and potential regulators of the neural induction process in cephalochordates. In conclusion, both ZnF and Sox families comprise a large number of genes that are up-regulated after Nodal activation, and seem to be regulating additional transcription factors downstream of Nodal signaling by binding their regulatory sequences at specific time-points.

On the other hand, the Krupel-like (Klf) family seems to be acting in a different way, when The Klf sub-family members were previously described as transcriptional repressors in vertebrates and, since the expression of the specific Klf1/2/4 is down-regulated during Nodalinduced neural fate acquisition, we hypothesized that this TF could be negatively regulating this process. Indeed, Klf1/2/4 could bind to the cis-regulatory elements of many actors involved in the neural induction process and only Nodal activation would allow the release of this global repression.

Considering the importance of these three TF sub-families, we integrated the following: Sox family, Znf family and specific Klf1/2/4 gene to the final pipeline used to design the GRN.

TFs whose expression is putatively activated by SMAD 2/3

All the experiments that were carried out during this project let to identify, though RNA-seq or ATAC-seq approach, a large set of downstream genes that are activated after Nodal activation.

However, such results do not provide a uniform information about the timing of activation of each TF involved in this process. In order to determine the direct targets of the Nodal pathway, we searched for ATAC-seq peaks containing Smad binding sites (Enhancers containing Smad binding sites, or SBS-E).

As pointed out before, 7688 putative TF binding sites were found in the ATAC-seq overrepresented peaks from Activin-treated 11hpf ectodermal explants. From these results, only 42 are binding sites for the Nodal downstream effector Smad (Figure 36). Our TRANSFAC analysis was able to detect Smad binding sites matches by comparing with a unique library matrix (V$SMAD4_Q6_01), therefore we assumed they are all putative binding sites for the Nodalmediated Smad2/3/4 complex. We have analyzed each of the SBS-E and affiliate them to their closest genes. Remarkably, from this list we could identify 39 genes presenting SBS-E (Supplementary table 5). From these results 8 are transcription factors thatare also up-regulated in ectodermal explants at 11hpf after Activin treatment (Supplementary table 6): FoxAB, Nr2f1, Hey1, Creb3l, Vent, Znf37, Pitx and Ost4. Additionally, we included Pax3/7, which is slightly downregulated and the node Irx since we found an SBS-E in an over-represented peak present in a locus containing three amphioxus-specific Irx paralogues (IrxA, IrxB and IrxC), which are closely related to vertebrate Irx1/6. The paralogues constituting this gene cluster were not found to be significantly up-regulated after Nodal activation in our RNA-seq data, suggesting that their regulation through SBS-E might be punctual to certain embryonic zones, or enhanced in later development.

Considering the TFs presenting SBS-E, and the results from previous pipelines (TFs presenting super-enhancers, and TF sub-families over represented in the distribution chart), we were able to draw a GRN that attempts to resume the main players during the neural induction process.

The Amphioxus Neural Induction Gene Regulatory Network

We have integrated the results obtained from the different workflow approaches that were carried out (Cis and Trans, Figure 34) into one final list of nodes. Here we summarize the results of the different pipelines. First, the identification of super-enhancers led to the selection of the following 25 TFs: Pitx,Neurogenin,Pax3/7,Vent,FoxB,FoxD,FoxAB,Vg1,Hey1,Otx,Twist,Zic,Znf503,Znf888,Gli,Gsc,Insm1,Nkx6.1,Nr2f1,Sim,SoxB2,Zeb,Ost4 and Prdm8.

After this, we analysed the TFBS from each Nodal-activated enhancer and determined the most and also presents a super-enhancer region. For these reasons, as it was previously stated for Irx, we integrated these whole sets of TFs in single nodes. Therefore, FoxAB, FoxB and FoxD constitute the node "Fox" in our GRN representation (Figure 37a,b). SoxB1 a/b/c , SoxB2, SoxC, SoxD and SoxE constitute the node "Sox". Finally, Znf333, Znf37, Znf503 and Znf888 constitute the node "Znf". In conclusion, we generated a final list containing 22 specific TFs (Pitx,Neurogenin,Pax3/7,Vent,Vg1,Hey1,Otx,Twist,Zic,Gli1,Gsc,Insm1,Nkx6.1,Nr2f1,Sim,Zeb,Prdm8,Ost4,Creb3l,Pou3f and Klf1/2/4) and 4 TF nodes (Irx, Fox, Sox and Znf).

over
Then, we established the direction of regulation/interaction among all these candidates by analyzing the TFBS present in their activated enhancers. Finally, we visually represented this interaction as a blue/white color gradient arrow, where the blue extreme represents the TF that is actively interacting with an enhancer, whereas the white extreme represents the TF that is regulated after this interaction (Figure 37a, 37b).

The non-linear organization of the GRN allows to clearly visualize the interaction between each member of the network. The TFs that are direct targets of Nodal (presenting SBS-E) are located in the upper part in white circles, whereas the indirect targets are located in the inferior part. All the arrows were counted (blue for arrows exiting each circle, and white for the arrow arriving to each circle) in order to quantify the interaction of each node (Figure 37a, 37b).

After integrating exclusive positive targets of Nodal, we generated a GRN design that exhibits the general TF machinery activated by the Nodal signaling pathway (Figure 37a). This representation allows us to propose that there is not a linear regulation of neural induction by Nodal. Hence, Nodal would be triggering neural fate acquisition through several downstream actors activated at the same time, and there would be no master gene positively controlling amphioxus neural induction. In this design, there is also a clear clusterization of blue arrows in specific nodes of the network, suggesting a wider activity for their corresponding TFs (Sox, Znf, Fox, Pou3f, Creb3l, and Vent1).

On the other hand, adding Klf to our previous GRN design offers a complementary understanding of the negative regulation of the neural induction process (Figure 37b). In fact, each of the nodes presented in this GRN (excepting Pou3f, whose locus was located into an incomplete scaffold)

have active enhancers containing KLF-binding sites. In consequence, KLF proteins might be acting on the whole set of TFs that were activated by Nodal.

As explained before, only Klf1/2/4 expression seems to be significantly affected by Nodal. In fact, Klf1/2/4 basal expression is relatively high in early amphioxus embryos, and it decreases in later stages (RNA-seq data base from control stages). Our model supports the hypothesis of a negative regulatory role of Klf 1/2/4 on Nodal targets. Here, Klf 1/2/4 would be silenced by Nodal as soon as neural induction takes place, thus releasing the whole set of TFs. At the same time, the whole GRN would be activated by Nodal so that neural fate can be acquired.

Validation of the Gene Regulatory Network

Our extensive work allowed us to propose a non-linear GRN orchestrating the neural induction process in amphioxus under the positive control of Nodal and negative control of Klf1/2/4. In order to validate this network, we assessed 3 specific points: The activity of the putative enhancers used in this study (through reporter assay), the non-linearity of the GRN (through constitutive repression), and the putative negative control of Klf1/2/4 (through transient overexpression). In amphioxus, this transient transgenesis experiment normally leads to mosaic embryosthis is why, a complete pattern can only be deducted by the integration of all the partial patterns found in each embryo. Considering this, we have tested the SBS-Es corresponding to 7 TFs present in the GRN (Creb3l, Pitx, Irx, Znf37, Hey1, Vent and Nr2f1), obtaining mosaic embryos that exhibit a variety of expression patterns driven by each injected enhancer (each of them was able to direct an expression pattern). Nonetheless, the final patterns were never found to be restricted to presumptive neural tissue or neural plate for any of the tested enhancers. For exemple, more than half of the embryos injected with Creb3l enhancer construction showed GFP expression in pro-neural territories at G4 stage (11hpf), whereas the rest of them exhibited GFP expression in the future paraxial mesoderm territory. At N2 stage, half of the embryos exhibited neural expression in the cerebral vesicle and some of them showed labeling in the neural plate, but none of these embryos exhibited an exclusive neural pattern. Our results show that the SBS-Es used in this study are functional enhancers and, therefore, the adopted approach to design our GRN based on ATAC-seq data is correct. Moreover, the non neurally restricted patterns found in this experiment suggest that the neural expression of a specific Nodal target gene might require the regulation through the combined activity of several regulatory regions. In this context, an additional transgenesis experiment was carried out, by using the activated enhancers of a classic neural marker: Neurogenin, in order to determine if individual enhancers activation is able, or not, to reproduce exclusive neural patterns.

Reporter validation of putative enhancers in Neurogenin-containing locus

Previous reporter vector experiments showed that the enhancers directly activated by Nodal signaling (SBS-Es) are not able to direct neurally restricted expression. In order to confirm if additional enhancers also trigger pleiotropic patterns when observed individually, we performed the same approach using the whole set of enhancers (5'1, 5'2; 3'1,3 '2, 3'3,3'4, 3'5, 3'6) in the vicinity of the well known neural marker Neurogenin (Figure 39). We observed that each injected embryo exhibits GFP expression driven by their respective enhancer, but none of the patterns obtained in this experiment reproduce the typical Neurogenin. expression pattern. It is known that enhancers are able to act in a synergic and additive manner towards a transcription factor for specific developmental processes. In this context, our results indicate that the specificity of a neural gene expression (i.e. Neurogenin) might be based on the multiple activation and combination of its total set of cis-regulatory regions, at a specific developmental time. Thus, the vision of a non-linear GRN directing the neural induction process fits with the results obtained with these reporter assays.

Functional analysis of Neurogenin, SoxB1a and Pou3f

The function of the GRNs nodes was tested through the overexpression of constitutive transcriptional repressor chimeras of three key TFs that are known to be classic neural markers in amphioxus: Neurogenin, SoxB1a and Pou3f. Indeed, knock-down techniques (morpholinos, shRNA, RNAi, …) are inefficient in B. lanceolatum and, although CRISPR/Cas9 system works, the percentage of mutations is still not sufficient to allow the analysis of phenotype in F0 embryos. Chimeras were constructed by fusing the full length of the TF coding sequence to the Engrailed repressor domain. The corresponding mRNAs were synthesized and later injected in unfertilized amphioxus eggs. Embryos were then fixed at different developmental stages for further in situ hybridization analyses with specific markers.

In embryos injected with SoxB1a and Pou3f chimera mRNAs, the expression of the neural markers Hu/Elav and Neurogenin was reduced compared to control (Figure 40). In addition, the presumptive neural territory in those embryos was also reduced. On the other hand, the injection of mRNA of the constituve repressor form of Neurogenin did not affect the expression of the neural marker Hu/Elav significantly. Nonetheless, the expression of the anterior marker Otx is absent in the cerebral vesicle of these injected embryos.

These transgenesis experiments strongly support the hypothesis that neural fate acquisition might not be controlled through a linear regulation, since the injection of each of the three chimeras resulted in a reduction of the neural territory but they were not able to completely abolish the formation of neural cells

Functional analysis of Klf1/2/4 through mRNA overexpression.

To functionally test the repressing activity proposed for Klf1/2/4, we synthetized its corresponding mRNA, and injected it into unfertilized eggs. After fertilization, we fixed the injected embryos at N1 and N2 stages. These embryos were delayed in their development and seemed to gastrulate abnormally (similarly to embryos where the BMP signal is inhibited by Dorsomorphin treatment). The expression of all the neural markers tested (Neurogenin, Sox2, Pou3f and Hu/Elav) was completely lost after Klf1/2/4 mRNA injection suggesting that no neural territory forms (Figure 41).

To better understand the phenotype of the injected embryos, we also looked at the expression of SoxB1a (pan-ectodermal and then neural plate marker) and the epidermal marker K1.

Interestingly, SoxB1a was expressed in a restricted ectodermal territory, corresponding the putative neural plate domain. On the other hand, K1 expression was observed in all the presumptive epidermis region but was absent in the presumptive neural plate territory.

Altogether these data suggest that in these embryos, the epidermal vs neural domains are defined.

However, only the pro-epidermal cells enter the differentiation program, whereas the presumptive neural cells are blocked in an unspecified pan-ectodermal state (SoxB1a expression). These results support the hypothesis that Klf1/2/4 negatively regulates the neural induction process by repressing the expression of the transcription factors that are activated by the Nodal signaling pathway. Additional RNA-seq data (supplementary table 4) shows that Klf1/2/4 expression decreases in Activin-treated ectodermal explants, suggesting that once Nodal expression increases in the developing embryo, Klf1/2/4 is silenced.

Discussion and

Concluding remark

The first part of this project was based on the results from the studies that were previously carried out in our laboratory, leading to the identification of Nodal as the main signal triggering neural fate acquisition in amphioxus. From this, my main goal was the identification of the genetic toolkit activated by Nodal signaling during the neural induction process. First, we determined the activation window of the Neural induction process since, until then, most of the results were biased by the observable earliest expression of Neurogenin (at 8-9 hpf approximately) [102,188].

In this context, activation or inhibition of the Nodal signaling pathway at several developmental time points have shown that only early embryos are sensitive to Nodal signal (Figure 28). In fact, early inhibition of Nodal impedes the formation of neural cells whereas late treatments do not affect neural fate acquisition, indicating that the neural induction is an early process in cephalochordates Considering that Nodal is an important signaling pathway regulating many developmental processes in the embryo, we decided to focus our work on ectodermal explants (previously obtained by microsurgery dissection of 8-cells stage blastomeres) at 11hpf, in order to get rid of the action of this pathway on mesendodermal cells at that specific timepoint. In this context, the results obtained through RNA-seq analysis from ectodermal explants allowed us to identify several candidate genes that are over-expressed after Nodal activation. On the other hand, the results obtained through ATAC-seq approach allowed us to draw the regulatory landscape underlying the neural induction process, by elucidating the cis and trans-regulatory actors. The results obtained from both approaches constituted a large amount of data and we only focused on part of them to identify a robust number of putative candidates involved in the regulation of the neural induction process. In consequence, we were able to identify multiple TF families that form the basis of the GRN underlying the neural induction process after activation of the Nodal signaling pathway. Each node of this GRN constitutes a specific gene that was determined by the proximity of their activated enhancers. In the case of "Fox", "Sox", "Znf" and "Irx"; they represent a set of genes that present active TFs (i.e. "Fox" includes the TFs FoxAB, FoxB and FoxD) after Nodal activation. On the other hand, Pou3f (here representing the whole Pou gene family) was included in this GRN design since a large percentage of TFBS were Pou-binding sites, suggesting that this gene might be regulating a set of genes previously activated by Activin.

We propose a non-linear GRN involved in neural induction process based on the results from ATAC-seq. The over-expression of constituve represor forms of three key nodes in this network,

Neurogenin, SoxB1a and Pou3f, has shown that the neural fate is probably not induced by a single gene in a straightforward manner, but instead by the combination of action of several genes at the same time. Regarding this point, the pleiotropic expression patterns exhibited by the reporter vectors containing enhancers with Smad-binding sites suggested that a single enhancer is not sufficient to direct a specific TF expression exclusively in the neural territory, but it might be necessary to activate the whole set of Nodal-activated enhancers of the GRN TFs in order to launch the neural fate program. In a similar way to Pou gene family, Klf family was identified as one of the main gene family regulating Nodal-activated TFs. After scanning the seven members of the Klf family in amphioxus, we found Klf1/2/4 to be down-regulated after Activin treatment, and more interestingly, its locus contains a negative super-enhancer region that is sensitive to Nodal activation. These characteristics makes Klf1/2/4 the most outstanding TF in this GRN. This gene is assumed to be a pluripotency marker that keeps cells into undifferentiated state before their dif ferentiation. In fact, the well known role of its vertebrate ortologue as re-programming TF in iPS cells suggests that Klf1/2/4 might also have a potential conserved role in cephalochordates. On the other hand, it was previously shown that Klf4 is able to reduce the availability of Neurogenins and to promote gliogenesis in mouse [187], supporting an additional conserved function in the regulation of neural fate across chordates.

In this study we found a massive presence of putative Klf binding sites in most of the Nodalactivated enhancers found by ATAC-seq, indicating that Klf1/2/4 might be a key gene regulating the whole GRN for neural induction activated by Nodal. Moreover, functional analysis through mRNA injection showed that its over-expression is able to completely abolish neural induction in amphioxus and promote stemnesses identity in pro-neural territories of the ectoderm. For this reason, the following part will be focused on discussing the potential role of Klf 1/2/4 in neural induction.

Klf 1/2/4 specifically affects neural cell differentiation

Functional analysis of Klf1/2/4 in amphioxus embryos has shown that when it is overexpressed the presumptive neuro-ectoderm stays in a state of pluripotency (expression of SoxB1a), without entering the differentiation process into neural cells (Figure 41). Remarkably, the effect of Klf1/2/4 over-expression does not affect the epidermal fate in the ectoderm (expression of K1), In contrast, single knock-down of Klf4 has no significant effects, whereas the triple knock-down of Klf4, Klf2, and Klf5 lead to rapid differentiation of ESCs [192] in mouse. This suggest a potential redundant function of different members of the Klf family when one of them is silenced.

suggesting
Indeed, it has also been showed that substituting Klf4 for Klf1, Klf2 or Klf5 can also allow iPSC generation [193]. In the case of amphioxus, not only Klf5 and Klf1/2/4 are down-regulated under Nodal activation, but also Klf3/8/12 and Klf15 (Chapter 3.4.3 and Supplementary Table 4),

suggesting that the combinatory down-regulation of Klf1/2/4 and other members of the KLF family might promote cell differentiation, as it was observed in mice.

In fact, the basal expression of Klf1/2/4 and Klf15 in early whole embryos and eggs is extremely low, whereas the expression of the rest of the Klf members is higher (Figure 42). Later, the expression of Klf1/2/4 increases until gastrula (11hpf aprox) and decreases subsequently (red line), whereas the expression of Klf5 and Klf6/7 increases during neurulation (yellow and blue lines). This observation suggests a potential role of Klf1/2/4 in gastrulating embryos whereas the other members of the Klf family could be mainly involved in the formation of the neural plate.

Concluding Remark

Our hypothesis suggests that the

Sorting of down-represented ATAC-seq peaks from ectodermal explants at 11hpf.

The main results obtained in this project were generated from the comparative experiments performed in ectodermal explants at 11hpf. From this set of data, only positive ATAC-seq peaks (over-represented enhancers/peaks after Nodal activation) were considered. It is, however, necessary to analyze the negative peaks obtained from such approach (down-represented enhancers/peaks after Nodal activation) in order to complement the list of negative regulators of our GRN. In addition to this approach, it will also be important to analyze the negative and positive peaks obtained in Activin-treated ectodermal explants at 19hpf, in order to discard late targets, and set a progressive GRN for the development and maturation of differentiated neurons.

Elaboration of a consensus GRN based on the results obtained through different bioinformatic pipelines

The GRN presented here is based on the direction of regulation between nodes, which were obtained through the prediction of TFBS undertaken with the Match TM tool available in the geneXplain platform. Such a bioinformatics tool integrates a user-friendly web interface with access to the TRANSFAC database. Such database comprises a collection of information of TFs and a fine library of positional weight matrices for prediction of potential TFBS. The group of matrices (profile) selected was "vertebrate_non_redundant" with the cut-off criteria "minFP" (Minimize false positives). It is important to point out that the matrices belonging to the selected profile show a lack of specificity for certain TFs, as it is the case for Neurogenin. In fact, we could not identify potential binding sequences for Neurogenin inside the group of matrices here selected (vertebrate_non_redundant), but we obtained a set of binding sites for other bHLH TFs as Myogenin, Hes, BEN-containing domain, etc. This suggests that some of the matrices used in this study might be masking results from different members of their corresponding TF families (i.e. Neurogenin and Myogenin belong to the class A of bHLH TF family).

This hypothesis was tested by performing a similar TFBS prediction approach in a small set of over-represented peaks , by using the MatInspector TM software tool, which is comprised into the Genomatix platform. This bioinformatics tool gives access to a large library of matrix descriptions from their own database ElDorado. We observed that the profiles used in this analyses comprise a wide set of matrices matching to several binding sites of specific bHLH TFs, as Neurogenin (Matrix V$NEUROG.01) or Neurogenin/NeuroD (Matrix V$NGN_NEUROD.01). In fact, some TFs of the GRN, as FoxAB, present enhancers containing Neurogenin-binding sites that were not previously detected through the Match TM software tool.

Moreover, the additional software tools for motifs discovery HOMER (Hypergeometric Optimization of Motif EnRichment), which is able to screen TRANSFAC and JASPAR databases under specific parameters, did not lead to the identification of specific Neurogenin binding sites.

This last software tool also offers the possibility to better constrain the parameters used for TFBS prediction and to decrease the false-positive rate. The results we obtained using HOMER slightly varied from the ones obtained through the Match TM tool. Indeed, the SOX and POU transcription factor families exhibit a robust position as major controllers of the downstream genes activated by Nodal. This observation contrasts with our previous results, where KLF, SOX and ZnF families appeared to be the main regulators of the neural induction process.

Altogether, these observations support the need of a comparative approach by using different bioinformatics pipelines in order to optimize the identification of potential TFBS, and consequently sharpen the structure of the GRN.

Animals (Embryos and ectodermal explants)

Adult specimens of the european amphioxus, Branchiostoma lanceolatum, were collected from sand samples retrieved in Argelès-sur-Mer, France [160]. They were separated in 5L aquaria with a water temperature of 16 to 17 C with a 14h/10h light/dark cycle. In order to induce spawning, males and females with mature gonads were selected, exposed to a 24h thermal shock at 23C, and isolated in plastic cups. After spawning, gametes were collected separately for in vitro fertilization. After 150 min post-fertilization, embryos reach the 8-cell stage. At that moment, micro-dissection was performed using human eyelash to separate micromeres from macromeres.

Micromeres were isolated in Petri dishes and incubated until the desired stage. Either embryos or ectodermal explants were incubated at 19C until 11hpf and 19phf.

Pharmacological treatments

Embryos and explants of Branchiostoma lanceolatum were diversely treated with Dorsomorphin 19hpf). Activin is able to dorsalize and neutralize embryos when treated early in development.

Recombinant amphioxus FGF1/2 protein was produced in our laboratory by using a specific expression vector. FGF1/2 is a signaling protein that binds the FGFR leading to the activation of the Nodal signaling pathway. Treatments were done in ectodermal explants at 3hpf and stopped at 11hpf or 19hpf. Results were not presented in this study.

RNA-seq and data analysis

RNA was extracted from embryos and explants at 5hpf, 11hpf and 19hpf using the RNeasy® Minikit (QIAGEN) and the RNeasy Microkit (Qiagen), respectively. An average of 100 embryos and 300 explants were used for each RNA extraction. They were stored at -80C and sent to the Centre for Genomic Regulation in Barcelone for sequencing through Illumina®. Studies were done in triplicate for the embryos at 5hpf and duplicate for the rest of the samples. The quality of the reads were evaluated by using the FastQC analysis to each of the sample files, through their corresponding Phred quality scores. The final results obtained from the sequencing were analysed

In the case of constitutive repressors (Neurogenin, SoxB1a and Pou3f), the complete coding sequence corresponding each target gene was cloned into a repressive Engrailed chimera vector or an activating VP16 chimera vector (pBluescript II -based backbones containing SP6 promoter). mRNA synthesis was undertaken using the mMESSAGE mMACHINE ® SP6 transcription kit from Invitrogen. On the other hand, Branchiostoma lanceolatum Klf1/2/4 was cloned into a pCS2+ expression vector from Addgene, and mRNA was synthetized with the same mMESSAGE transcription kit.

Enhancers cloning

The sequences corresponding putative genomic regulatory regions obtained after ATAC-seq analysis were directly extracted from the Branchiostoma lanceolatum genome browser, through their genomic position into the assembled scaffolds. Genomic DNA was extracted from adult animals using the DNeasy® Blood & Tissue Kit from QIAGEN. The genomic regions were amplified by PCR using gDNA and enhancer-specific primers with 15bp extensions (according to the established protocol of the In-Fusion® HD cloning kit from Clontech). The PCR products were cloned into a modified CMV/mCherry/GFP reporter vector (pBluescript II -based backbone) using the In-Fusion® HD cloning kit from Clontech. Vectors were finally linearized with restriction enzymes, then purified and injected into unfertilized eggs alongside mCherry mRNA as control for all injected eggs. After fertilization, transgenic embryos develop until 11hpf or 19hpf.

Whole mount in situ hybridization

Branchiostoma lanceolatum embryos at different developmental stages (at 5hpf, 11hpf and 19hpf) and explants (at 11hpf and 19hpf) were fixed in 4% paraformaldehyde diluted in EGTA-MOPS buffer, and stocked in ethanol 70%. DIG-labeled antisense RNA probes were synthesized from previously synthetized vectors, using Roche enzymes. In situ hybridization experiments were carried out in all samples, as previously described (Yu & Holland, 2009a;Somorjai et al, 2008). The images of Branchiostoma lanceolatum embryos and explants after in situ hybridization were taken using the high resolution CCD camera CoolSnap™ from Photometrics under an Olympus microscope.

Introduction

Forkhead box (Fox) transcription factors originated early during evolution and are specific to opisthokonts. They are present in fungi as well as in metazoans [START_REF] Mazet | An ancient Fox gene cluster in bilaterian animals[END_REF][START_REF] Larroux | Genesis and expansion of metazoan transcription factor gene classes[END_REF]Shimeld et al., 2010a) in which they play essential roles during embryonic development [START_REF] Carlsson | Forkhead transcription factors: key players in development and metabolism[END_REF]Tuteja and Kaestner, 2007a,b;[START_REF] Benayoun | Forkhead transcription factors: key players in health and disease[END_REF]. Fox proteins possess a helix-turn-helix DNA-binding domain called the forkhead domain which corresponds to a conserved region of approximately 110 amino acids [START_REF] Weigel | The forkhead genes, Foxc1 and Foxc2, regulate paraxial versus intermediate mesoderm cell fate[END_REF][START_REF] Clark | Co-crystal structure of the HNF-3/fork head DNA-recognition motif resembles histone H5[END_REF]. A molecular phylogeny-based classification of the Fox gene family allowed to propose its subdivision into 24 classes (ranged from FoxA to FoxS and including subfamilies that were recently subdivided: FoxJ (FoxJ1 and FoxJ2), FoxL (FoxL1 and FoxL2), and FoxN (FoxN1/4 and FoxN2/3) (Mazet et al., 2003). Many Fox gene losses or duplications occurred in different bilaterian clades, affecting different Fox classes. For example, FoxAB is found in cephalochordates and in the sea urchin but not in tunicates or vertebrates [START_REF] Tu | Sea urchin Forkhead gene family: phylogeny and embryonic expression[END_REF]Yu et al., 2008a), and families R and S are vertebrate-specific [START_REF] Wotton | Comparative genomics of vertebrate Fox cluster loci[END_REF]Shimeld et al., 2010b). Using phylogenetic analyses, it has been proposed that 22 Fox gene families were already present in the bilaterian ancestor (Shimeld et al., 2010b).

Cephalochordates (i.e., amphioxus) belong to the chordate phylum together with tunicates and their sister group, the vertebrates. They present morphological, developmental, and genomic characteristics that are proposed to be very similar to the ancestral state in the chordate clade, making amphioxus a key model system to understand chordate evolution [START_REF] Bertrand | Amphioxus FGF signaling predicts the acquisition of vertebrate morphological traits[END_REF]Escriva, 2011, 2014). Interestingly, it has been shown that amphioxus is the only living bilaterian possessing at least one member of each of the 22 Fox gene families proposed to have been present in Urbilateria (Yu et al., 2008a). Thus, the study of Fox genes in this cephalochordate may shed light on the functional evolutionary history of this transcription factor gene family. Past studies using genomic data from the Caribbean cephalochordate Branchiostoma floridae described the presence of 32 Fox genes in this species (Yu et al., 2008a)a n dt h e expression pattern of 11 of these genes was previously described: FoxAa and FoxAb (formerly named AmHNF3-1 and AmHNF3-2, respectively) [START_REF] Shimeld | Characterisation of amphioxus HNF-3 genes: conserved expression in the notochord and floor plate[END_REF], FoxB [START_REF] Mazet | The evolution of chordate neural segmentation[END_REF], FoxC [START_REF] Mazet | An ancient Fox gene cluster in bilaterian animals[END_REF], FoxD (Yu et al., 2002b), FoxE4 (Yu et al., 2002a), FoxF [START_REF] Mazet | An ancient Fox gene cluster in bilaterian animals[END_REF][START_REF] Onimaru | Development and evolution of the lateral plate mesoderm: comparative analysis of amphioxus and lamprey with implications for the acquisition of paired fins[END_REF]), FoxG (Toresson et al., 1998), FoxL1 (Mazet et al., 2006), FoxN1/4a (Bajoghli et al., 2009), FoxQ1 andFoxQ2 (Yu et al., 2003;[START_REF] Mazet | An ancient Fox gene cluster in bilaterian animals[END_REF]. In this work we searched for Fox sequences in the transcriptome of the Mediterranean amphioxus Branchiostoma lanceolatum. We found 28 Fox sequences and we describe here the spatiotemporal expression pattern of 13 Fox genes during embryonic development, including seven previously described in B. floridae and six for which expression was not known. We show that in B. lanceolatum some Fox genes exhibit ubiquitous expression as FoxK and FoxM,whiletheothersshow specific and dynamic expression patterns restricted to diverse embryonic territories. These expression patterns suggest that Fox genes are performing both general and specific functions during amphioxus embryonic development, most of them being probably ancestral in the chordate clade.

Materials and Methods

Phylogenetic Analysis

All reference sequences, except for B. lanceolatum,wereobtained from Genbank or from Fritzenwanker et al. (2014) The multiple alignment was performed only for the conserved Forkhead amino acid domain sequences using the MUSCLE module implemented in MEGA 6 and manually refined in its interface [START_REF] Tamura | MEGA6: molecular evolutionary genetics analysis version 6.0[END_REF]. The best fit substitution model for phylogenetic reconstruction was estimated using MEGA 6 [START_REF] Tamura | MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods[END_REF]. Bayesian inference (BI) tree was inferred using MrBayes 3.2 [START_REF] Ronquist | MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space[END_REF], with the model recommended by MEGA 6 under the Akaike information criterion (RtRev+Ŵ), at the CIPRES Science Gateway V. 3.1 [START_REF] Miller | A RESTful API for access to phylogenetic tools via the CIPRES science gateway[END_REF]. Two independent runs were performed, each with four chains and 1 million generations. A burn-in of 25% was used and a 50 majority-rule consensus tree was calculated forthe remaining trees.

Cloning and Expression Study

B. lanceolatum Fox sequences were recovered from its reference transcriptome (Oulion et al., 2012)byTBLASTNusingsequences from B. floridae as queries. Specific primers were then designed for RT-PCR amplification from total RNA. Primer sequences are as follow:

FoxA_a_5 ′ AAGTCGCCGGTGTACGAGATG FoxA_a_3 ′ GTATTATAGAGACGAAGGTTG FoxA_b_5 ′ CATTTCCTCAGAACAGACATG FoxA_b_3 ′ TCCTAAAGACTCCCAACAACA FoxAB_5 ′ CAGTGTGAGGTGAACATCATG FoxAB_3 ′ CGATTGACAGGTTGATAGAAC FoxB_5 ′ ACAACAGGACCCTGACTCGT FoxB_3 ′ GCATTCCCTGACGTCTTGA FoxC_5 ′ AACCGTCCCGTTTTCCTCATG FoxC_3 ′ CAGTTTTGATTCGTAAGGACT FoxD_5 ′ ACAGCTGTGGAGTGGACACTT FoxD_3 ′ CACGAGACATGTAAGTCTCCG FoxEa_5 ′ AACCAACCCCGTACCAGCATG FoxEa_3 ′ ATATGACACGGACACTGAACT FoxG_5 ′ ACGCACATTAGCACAGTTCG FoxG_3 ′ ACTTGACCCTGGCTTGACAC FoxJ1_5 ′ TACAGACAACTGTAAACCATG FoxJ1_3 ′ TTGTAATGCAGGGTGGGGCCT FoxK_5 ′ GGAAGGCGGAGTTGGACAATG FoxK_3 ′ CCGGACACGTCCTGCACCTGT FoxM_5 ′ AGGAGAGTGTGACAAACCATG FoxM_3 ′ TTCTCAGCTATTCAGTAATAC FoxN1/4a_5 ′ GCGCACCGAGTATCGTTCTGA FoxN1/4a_3 ′ ACATAGGTAGGACTATGTACT FoxN2/3_5 ′ CAGTAAACACGAGCAGACATG FoxN2/3_3 ′ AGCTGAAGACAATGATGATCC
A mix of total mRNA of B. lanceolatum extracted from embryos at different developmental stages was used as a template for retro-transcription. Amplification was performed using Advantage 2 Polymerase kit (Clontech) and a touch-down PCR program with annealing temperature ranging from 65 to 40 • C. Amplified fragments were cloned using the pGEM-T Easy system (Promega) and sub-cloned in pBluescript II KS+ for probe synthesis.

Whole Mount In situ Hybridization

Probes were synthesized using the DIG labeling system (Roche) after plasmid linearization with the appropriate enzymes. Ripe animals of B. lanceolatum were collected in Argelès-sur-Mer (France), and gametes were obtained by heat stimulation [START_REF] Fuentes | Preliminary observations on the spawning conditions of the European amphioxus (Branchiostoma lanceolatum) in captivity[END_REF](Fuentes et al., , 2007)). In vitro fertilization was undertaken in Petri dishes filled with filtered sea water. Fixation and whole mount in situ hybridization were performed as described in Somorjai et al. (2008).

Results

Molecular Phylogenetic Analysis of B. lanceolatum Fox Gene Sequences

We looked for Fox gene sequences in the reference transcriptome of B. lanceolatum (Oulion et al., 2012). The sequences that were recovered were used to conduct a phylogenetic tree reconstruction presented in Figure 1.W es h o w e dt h a t B. lanceolatum possesses at least 28 Fox genes, each of them being orthologous to one of the 32 genes described in B. floridae and corresponding to at least one member of each of the 22 families present in the bilaterian ancestor (Yu et al., 2008a). Specific duplications, that occurred in the cephalochordate clade at least in the ancestor of B. floridae and B. lanceolatum,g a v er i s et o three members in the FoxQ2 group (FoxQ2a, FoxQ2b, FoxQ2c), two members in the FoxN1/4 group (FoxN1/4a and FoxN1/4b), and two genes in the FoxE group (FoxEa and FoxEc). We then analyzed the expression pattern during B. lanceolatum embryonic development of 13 of these 28 Fox genes corresponding to those showing a higher expression level in the transcriptome (Oulion et al., 2012).

FoxAa and FoxAb

FoxAa (formerly named AmHNF3-1) [START_REF] Shimeld | Characterisation of amphioxus HNF-3 genes: conserved expression in the notochord and floor plate[END_REF] was first expressed at the gastrula stage in the anterior ventral endoderm and in the mesendodermal layer of the dorsal blastoporal lip (Figures 2A,B). At the late gastrula stage, we detected transcripts in the axial dorsal mesendoderm corresponding to the presumptive notochord territory, as well as in mesendoderm cells of the archenteron floor (Figures 2C,D). Expression in the axial mesoderm and endoderm persisted through mid-late neurula stage (Figures 2E,F). Later on, at late neurula stage before the mouth opens, the expression in the notochord was restricted to the most anterior and posterior tips of the embryo, while the endodermal expression was restricted to the middle region of the gut (Figure 2G). At the larva stage, the expression at the anterior tip of the notochord and in the tailbud was still observed and we detected a diffuse expression in the gut (Figure 2H).

FoxAb (formerly named AmHNF3-2)( Shimeld, 1997) expression was first detected at the gastrula stage as a weak signal in the mesendodermal part of the dorsal blastoporal lip (Figures 2I,J). At the late gastrula stage, we detected expression in the central paraxial mesoderm on both sides of the notochord anlagen (Figures 2K,L). At the mid-late neurula stage transcripts were detected in the neural tube, including the cerebral vesicle, and in the dorsal part of the endoderm (Figures 2M,N). At the late neurula stage, before the mouth opens, FoxAb was expressed in the neural tube and in the most anterior part of the pharynx. In the posterior region, expression was detected in the tailbud and in the dorsal midline of the gut (Figure 2O). At the larva stage, we observed expression in the pharynx, in the preoral pit, in the club-shaped gland and in the tailbud. At this stage, the expression in the neural tube gets restricted to some neurons and to the posterior part of the cerebral vesicle (Figure 2P and Figure S1A).

FoxAB

FoxAB transcripts were detected as a weak and ubiquitous signal from the eight-cell stage to the blastula stage (Figures 2Q,R). This ubiquitous expression was confirmed by the presence of reads in transcriptome analyses (data not shown). At the gastrula stage we observed a strong specific expression in the dorsal blastoporal lip, the amphioxus putative organizer (Figures 2S,T).

At the late gastrula stage, expression gets restricted to the presumptive notochord territory (Figures 2U,V). No expression could be detected by in situ hybridization in later stages.

FoxB

FoxB expression was first detected dorsally, both in the ectoderm and in the mesendoderm, as a weak signal in mid gastrula stage embryos (Figures 2W,X). Later on, in early neurula stage embryos, a signal could be observed in the neural plate on either side of the midline, as well as in two patches in the posterior paraxial mesendoderm (Figures 2Y,Z). During the late neurula stage, expression was detected in the most posterior paraxial mesoderm that give rise to the newly formed somites and in the neural tube posterior to the cerebral vesicle (Figures 2A' ,B'). Then, FoxB expression in the mesoderm faded away in late neurulae (Figure 2C') and get later restricted to the cerebral vesicle and to some neurons along the neural tube in larvae (Figure 2D' and Figure S1B).

FoxC

FoxC was expressed at the gastrula stage in the dorsal paraxial mesendoderm (Figures 3A,B). Later on, at the late gastrula stage, expression was detected in the region that gives rise to the three most anterior somites (Figures 3C,D). In mid-late neurulae, the transcripts remained all along the body in the somites and a new expression domain appeared in the anterior endoderm at the level where the first gill slit opens (Figure 3E). At the late neurula stage, the expression persisted in the pharynx and somites and was also detected in the club-shaped gland anlagen (Figures 3F,G). At the larva stage a diffuse expression was observed in the somites as well as in the preoral pit, in the club-shaped gland and in the first gill slit (Figure 3H and Figure S1C).

FoxD

FoxD transcripts were first detected at the gastrula stage in the dorsal blastoporal lip (Figures 3I,J). Then, at the late gastrula stage, FoxD was expressed in the dorsal axial mesendoderm, in part of the dorsal paraxial mesendoderm as two patches on both sides of the midline and in the anterior region of the neural plate (Figures 3K,L). At the mid-late neurula stage, the notochord and the somites, as well as the cerebral vesicle, were labeled (Figure 3M). At the late neurula stage, before the mouth opens, transcripts were detected in the paraxial somitic mesoderm, in the notochord, in the cerebral vesicle and in the posterior endoderm (Figures 3N,O). A faint labeling was also detected at this stage in the first gill slit and in the club-shaped gland anlagens. At the larva stage, we observed a low expression level in the cerebral vesicle, in the preoral pit, in the club-shaped gland, in the first gill slit, in the notochord and in the posterior part of the gut. We also observed an anterior to posterior gradient of expression in the somites (Figure 3P and Figure S1D).

FoxEa

FoxEa (formerly named FoxE4 in B. floridae) expression was first detected at early neurula stage in the antero-ventral mesendoderm (Figures 3Q,R). Later on, at the mid-late neurula stage, FoxEa transcripts were detected ventrally in the endoderm with a higher expression level on the right side of the pharynx (Figures 3S,T), and a slight expression domain in the posterior gut was also visible. At the late neurula stage, FoxEa transcripts remained ventrally in the pharyngeal endoderm on the right side (Figure 3U). Finally, at the larva stage, transcripts were detected in the club-shaped gland (Figure 3V and Figure S1E).

FoxG

FoxG expression was first observed at the neurula stage in the anterior region of the first somites (Figures 3W,Y). At the late neurula stage, FoxG was expressed in the anterior ventral region of the three most anterior somites (Figures 3X,Z). Later on, in late neurula before the mouth opens, a neural expression appeared in some individual neurons within the neural tube, while the expression observed in the first somites disappeared (Figure 3A'). This expression persisted in the larva stage embryos in which FoxG was also detected in some neurons of the cerebral vesicle (Figure 3B' and Figure S1F).

FoxJ1

FoxJ1 showed a dynamic expression pattern. Expression began during gastrulation and was detected in the ectoderm except the ectoderm around the blastopore (Figures 4A,B). Later on, at the late gastrula stage, this expression pattern persisted in the ectoderm that give rise to the epidermis (Figures 4C,D).

At the mid-late neurula stage, we detected transcripts in the neural tube while the expression in the epidermis was completely lost (Figures 4E,F). This neural tube expression was no more observed in late neurula stage embryos before the mouth opens (data not show), however at the larva stage we observed expression at the anterior tip of the embryo and in the pharynx at the level of the preoral pit and of the first gill slit (Figure 4G and Figure S1G).

FoxK

FoxK was ubiquitously expressed from the eight-cell stage to the blastula stage (Figures S2A,B). At the gastrula stage, the expression became restricted to the mesendoderm (Figures S2C,D), and by the late gastrula stage transcripts were detected mostly in the dorsal mesoderm (Figures S2E,F). At the mid-late neurula stage, we detected a stronger expression in the most anterior region of the embryo (Figures S2G,H). Transcripts were then detected in the whole embryo at the late neurula stage with a stronger expression in the anterior tip (Figures S2I,J). Finally, at the larva stage, we observed a ubiquitous expression with a higher level at the anterior tip and in the pharynx (Figure S2K).

FoxM

FoxM transcripts were detected ubiquitously during the whole embryonic development, from the eight-cell stage until the midlate neurula stage except in the epidermis (Figures S2L-S). Later on, at late neurula stage, FoxM expression could not be detected anymore by in situ hybridization (Figure S2T).

FoxN1/4a

Ubiquitous FoxN1/4a expression was detected from the eight-cell stage until the blastula stage (Figures 4H,I). At the gastrula stage, as i g n a lw a sd e t e c t e di nt h ea n t e r i o re c t o d e r m( Figures 4J,K).

Later on, at the early neurula stage, we observed transcripts in the anterior endoderm as well as in the axial central mesoderm ( Figures 4L,M). At the mid-late neurula stage, we detected three major expression domains: one anterior, at the level of the cerebral vesicle, a second one in the anterior ventral endoderm and a third one in the posterior mesoderm (Figure 4N). At the late neurula stage before the mouth opens, we observed expression in the anterior and posterior endoderm (Figure 4O). Finally, at the larva stage, we detected expression in the posterior region of the gut and in the anus (Figure 4P).

FoxN2/3

Ubiquitous expression of FoxN2/3 was observed from the eightcell stage (Figure 4Q)t ot h eb l a s t u l as t a g e( Figure 4R). Then, at the gastrula stage, the expression was restricted to the 4S,T). At the late gastrula stage, the expression remained strong in the mesendoderm but started to become lower in the ventral part (Figures 4U,V). By the mid-late neurula stage, FoxN2/3 transcripts were detected in the mesoderm and in the neural tube (Figure 4W). At the late neurula stage, before the mouth opens, the expression was mainly detected in the paraxial mesoderm (somites) and in the notochord. A new expression domain also appeared at this stage in the pharyngeal endoderm (Figures 4X,Y). At the larva stage, we did not detect any specific signal using in situ hybridization.

mesendoderm (Figures

Discussion Fox Genes Expression in Cephalochordate Species

The complete or partial embryonic expression patterns of FoxAa, FoxAb, FoxB, FoxC, FoxD, FoxEa, FoxG, and FoxN1/4a were previously described in B. floridae and/or B. belcheri [START_REF] Shimeld | Characterisation of amphioxus HNF-3 genes: conserved expression in the notochord and floor plate[END_REF][START_REF] Terazawa | Formation of the chordamesoderm in the amphioxus embryo: analysis with Brachyury and fork head/HNF-3 genes[END_REF][START_REF] Toresson | Conservation of BF-1 expression in amphioxus and zebrafishsuggests evolutionary ancestry of anterior cell types that contribute to the vertebrate telencephalon[END_REF][START_REF] Mazet | The evolution of chordate neural segmentation[END_REF]Yu et al., 2002a,b;[START_REF] Mazet | An ancient Fox gene cluster in bilaterian animals[END_REF][START_REF] Bajoghli | Evolution of genetic networks underlying the emergence of thymopoiesis in vertebrates[END_REF]. These genes overwhelmingly show a similar embryonic expression to what we observed in B. lanceolatum,a sw eh a v e previously noticed for other important developmental genes (Somorjai et al., 2008). However, our work brings some new information.

First, in contrast to what has been described in B. floridae,we showed that FoxAa and FoxAb have different expression patterns. Indeed, in B. floridae, FoxAb in situ hybridization data showed that it has a similar expression to FoxAa at early stages whereas expression was no more detected after the eight somites stage [START_REF] Shimeld | Characterisation of amphioxus HNF-3 genes: conserved expression in the notochord and floor plate[END_REF]. Here we showed that although both genes were expressed in the mesendodermal part of the dorsal blastoporal lip at the gastrula stage, the overall expression patterns are consistently different between the two genes and we observed a restricted expression of FoxAb from the gastrula to the larva stage. These discrepancies might be explained by the fact that the level of expression of FoxAb is very low. Indeed, staining of embryos hybridized to FoxAb took very long suggesting a low expression level. Thus, the staining time used in B. floridae might have been too short to detect expression in late stage embryos. Moreover, the expression we observed for FoxAa in B. lanceolatum is different from what was observed in B. floridae but similar to what has been described in B. belcheri [START_REF] Terazawa | Formation of the chordamesoderm in the amphioxus embryo: analysis with Brachyury and fork head/HNF-3 genes[END_REF]. Indeed, as in B. belcheri, FoxAa was not expressed in the central nervous system of B. lanceolatum.O n the other hand, FoxAb showed a very specific expression in the ventral part of the neural tube in neurula stage embryos, which has been proposed to be homologous to the vertebrate floor plate. Vertebrates have three FoxA group paralogous genes that are expressed in the organizer, the notochord, the floor plate and the endoderm [START_REF] Friedman | The Foxa family of transcription factors in development and metabolism[END_REF]. In Ciona [START_REF] Gregorio | The regulation of forkhead/HNF-3beta expression in the Ciona embryo[END_REF], Ci-fkh is also expressed in the notochord, the floor plate and the endoderm. The data we obtained in B. lanceolatum suggest that the expression of FoxA in the chordate ancestor was similar to what is observed in tunicates and that independent subfunctionalizations occurred in cephalochordates after specific gene duplication and in vertebrates after the two rounds of whole genome duplications.

Concerning FoxB, expression in B. floridae was first detected in neurulae with five somites [START_REF] Mazet | The evolution of chordate neural segmentation[END_REF]. Here we showed that in B. lanceolatum FoxB expression could be observed in gastrula embryos in the dorsal posterior mesendoderm and ectoderm. Then, in neurulae, we detected expression in the neural plate similar to B. floridae, as well as an expression in the most posterior somites that was not previously described. This expression in the neural plate/neural tube and in the lastly formed somites persisted until the late neurula stage. Interestingly, in amphioxus three different somitic populations have been described (Bertrand et al., 2011). The first, most anterior, population forms under the control of the FGF signal and the two posterior populations forms independently of the FGF signal. Several genes are expressed specifically in these three somitic populations but only one gene, Mox, [START_REF] Minguillon | The single amphioxus Mox gene: insights into the functional evolution of Mox genes, somites, and the asymmetry of amphioxus somitogenesis[END_REF] is expressed in the second and third populations. The present data suggest that FoxB also plays a role in the formation of these somitic population since it is also expressed in the two most-posterior somitic populations.

In B. floridae, FoxC has been described as being firstly expressed in the mesoderm of neurulae but its expression was described only in one developmental stage [START_REF] Mazet | An ancient Fox gene cluster in bilaterian animals[END_REF]. Here we showed that expression starts much earlier, at the gastrula stage, in the dorsal paraxial mesendoderm, the presumptive somitic mesoderm territory. Expression persisted in the paraxial mesoderm/somites until the larva stage, and at the late neurula stage we started to observe expression in the clubshaped gland anlagen and at the place where the first gill slit opens. These data suggest a major ancestral role of FoxC during somitogenesis which would have been conserved in vertebrates [START_REF] Kume | The murine winged helix transcription factors, Foxc1 and Foxc2, are both required for cardiovascular development and somitogenesis[END_REF][START_REF] Weigel | The forkhead genes, Foxc1 and Foxc2, regulate paraxial versus intermediate mesoderm cell fate[END_REF][START_REF] Wotton | Expression of FoxC, FoxF, FoxL1, and FoxQ1 genes in the dogfish Scyliorhinus canicula defines ancient and derived roles for Fox genes in vertebrate development[END_REF] and lost in tunicates in which FoxC is expressed in neural and palp cells [START_REF] Imai | Regulatory blueprint for a chordate embryo[END_REF].

FoxD and FoxEa expression in B. lanceolatum was very similar to previous descriptions in B. floridae (Yu et al., 2002a,b). However we noticed expression in some specific regions of the pharynx in late neurulae and larvae for FoxD,a n dat r a n s i e n t expression in mid-late and late neurula stage embryos in the posterior endoderm for FoxEa that were not described in the Caribbean species.

FoxG, previously known as Brain Factor 1 (BF-1), was described in B. floridae as a gene that is ventrally expressed in the cerebral vesicle and in the anterior-most portion of the first somite pair [START_REF] Toresson | Conservation of BF-1 expression in amphioxus and zebrafishsuggests evolutionary ancestry of anterior cell types that contribute to the vertebrate telencephalon[END_REF]. Our results showed a conserved expression pattern in the cerebral vesicle area in B. lanceolatum. However, mesoderm expression is not only limited to the first somite pair but the first three somite pairs exhibit the same pattern at the neurula stage suggesting that this gene might play a role during anterior somitogenesis. This result highlights the functional differences between the formation of the anterior somites which is under the control of the FGF signaling pathway and the formation of the most posterior somites which is not FGF-dependent (Bertrand et al., 2011). Moreover, expression is localized in the ventral part of these three most anterior somites which will give rise to the perivisceral coelom, suggesting a function of FoxG in the establishment of the somitic compartments.

FoxJ1 and the Formation of Motile Cilia

FoxJ1 orthologs were identified in many eumetazoans as well as in sponges [START_REF] Larroux | Developmental expression of transcription factor genes in a demosponge: insights into the origin of metazoan multicellularity[END_REF])andc hoanofla gellate s [START_REF] King | The genome of the choanoflagellate Monosiga brevicollis and the origin of metazoans[END_REF]. In vertebrates, FoxJ1 plays an essential role in the generation of motile cilia and in mediating Left/Right asymmetry [START_REF] Chen | Mutation of the mouse hepatocyte nuclear factor/forkhead homologue 4 gene resultsi n an absence of cilia and random left-right asymmetry[END_REF]Brody et al., 2000;Yu et al., 2008b). It has also recently been shown that misexpression of FoxJ1 from placozoans, echinoderms and platyhelminthes in zebrafish embryos induces the expression of ciliary genes, whereas the inactivation of FoxJ1 in the flatworm Schmidtea mediterranea impairs the normal differentiation of motile cilia, suggesting a conserved function in metazoans [START_REF] Vij | Evolutionarily ancient association of the FoxJ1 transcription factor with the motile ciliogenic program[END_REF]. This conserved function is also supported by the embryonic expression of FoxJ1 in different phyla [START_REF] Choi | Developmental expression of FoxJ1.2, FoxJ2, and FoxQ1 in Xenopus tropicalis[END_REF][START_REF] Tu | Sea urchin Forkhead gene family: phylogeny and embryonic expression[END_REF]Fritzenwanker et al., 2014). In B. lanceolatum,weshowed that FoxJ1 is first expressed in the ectoderm of the gastrulae, excluding the blastoporal region and the presumptive neural plate, at the time at which motile cilia start to grow. Then, in neurulae, expression was lost in the epidermis and appeared in the closed neural tube. At the larva stage, expression was restricted to the anterior tip of the animal and to the ciliated preoral pit and first gill slit. This expression pattern suggests that in amphioxus FoxJ1 might also play a role in the formation of motile cilia. However, other cells, like the epithelial gut cells, also harbor motile cilia and do not express FoxJ1,s u g g e s t i n g that other genes might also be implicated in ciliogenesis in these embryonic structures.

FoxAB

In B. lanceolatum, FoxAB was transiently expressed in the organizer at the gastrula stage and in the presumptive notochord later on. No expression could be detected in mid-neurulae or larvae. FoxAB family genes were described in hemichordates (Fritzenwanker et al., 2014), sea urchin [START_REF] Tu | Sea urchin Forkhead gene family: phylogeny and embryonic expression[END_REF]a n d cnidarians and are absent in vertebrates and tunicates, the two other chordate clades (Yu et al., 2008a). In the hemichordate Saccoglossus kowalevskii, FoxAB is expressed in the ectoderm and the mouth perforates through the ring expressing this gene in the ventral side (Fritzenwanker et al., 2014). In bryozoans, FoxAB also shows an ectodermal expression [START_REF] Fuchs | Gene expression in bryozoan larvae suggest a fundamental importance of pre-patterned blastemic cells in the bryozoan life-cycle[END_REF]. Therefore, it is still difficult to propose any scenario for the evolution of the function of FoxAB family genes in bilaterians. FoxAB could have been recruited for the patterning of the notochord field in the ancestor of chordates, but the absence of genes of this family in tunicates and vertebrates make this hypothesis unlikely.

FoxK and FoxM Ubiquitous Expression

We detected a ubiquitous expression of FoxK starting at the eight-cell stage until the larva stage. In other bilaterians data are scarce. In vertebrates, there are two paralogs in the FoxK family, FoxK1 and FoxK2. In mouse, the study of the function of FoxK1 during embryonic development was undertaken showing that the gene is involved in myogenic differentiation [START_REF] Bassel-Duby | Myocyte nuclear factor, a novel winged-helix transcription factor under both developmental and neural regulation in striated myocytes[END_REF]. In Ciona intestinalis [START_REF] Imai | Gene expression profiles of transcription factors and signaling molecules in the ascidian embryo: towards a comprehensive understanding of gene networks[END_REF]a s in the hemichordate S. kowalevskii (Fritzenwanker et al., 2014), the expression of FoxK is quite ubiquitous as observed for B. lanceolatum.F i n a l l y ,s t u d i e si nDrosophila have shown that FoxK is involved in the differentiation of midgut in the fly embryo [START_REF] Casas-Tinto | FoxK mediates TGF-beta signalling during midgut differentiation in flies[END_REF]. Altogether these data do not allow us to infer any putative ancestral function for FoxK family genesand further studies are required in different animal phyla.

FoxM expression is also ubiquitous in B. lanceolatum and was first detected as early as the eight-cell stage. Then the expression level continuously decreased while development proceeds and became undetectable by in situ hybridization at the late neurula stage. In Xenopus, FoxM1 is maternally expressed and transcripts are thereafter detected in the neuroectoderm [START_REF] Pohl | The Fox gene family in Xenopus laevis:FoxI2, FoxM1 and FoxP1 in early development[END_REF]. Moreover this gene has been shown to be important for early neuronal differentiation [START_REF] Ueno | FoxM1driven cell division is required for neuronal differentiation in early Xenopus embryos[END_REF]. In mouse, FoxM1 is expressed in dividing cells and knock-out animals exhibit embryonic lethal phenotype due to many malformations affecting different organs such as the liver, the heart, the lung, or the vasculature [START_REF] Kalin | Multiple faces of FoxM1 transcription factor: lessons from transgenic mouse models[END_REF]. As for FoxK, the data available up to now do not give us any indication on the putative ancestral function of genes belonging to the FoxM family.

FoxN1/4a and FoxN2/3 Expression

In all vertebrates studied so far, FoxN1 plays an essential role in thymus development [START_REF] Ma | Foxn1 maintains thymic epithelial cells to support T-cell development via mcm2 in zebrafish[END_REF][START_REF] Neves | Modulation of Bmp4 signalling in the epithelial-mesenchymal interactions that take place in early thymus and parathyroid development in avian embryos[END_REF][START_REF] Lee | Early development of the thymus in Xenopus laevis[END_REF][START_REF] Romano | FOXN1: a master regulator gene of thymic epithelial development program[END_REF]. Moreover, in mammals, FoxN1 is essential for hair formation whereas it is also expressed in chick during feather development [START_REF] Darnell | Developmental expression of chicken FOXN1 and putative target genes during feather development[END_REF]. Although mammal and fish FoxN1s are able to activate the expression of hair keratin genes, FoxN1/4 from amphioxus is not because its N-terminal region of the forkhead domain is different compared with vertebrates [START_REF] Schlake | A role for FoxN3 in the development of cranial cartilages and muscles in Xenopus laevis (Amphibia: Anura: Pipidae) with special emphasis on the novel rostral cartilages[END_REF]. On the other hand, FoxN4 is expressed in the nervous system, including retina, during vertebrate development [START_REF] Danilova | Expression of the winged helix/forkhead gene, foxn4, during zebrafish development[END_REF][START_REF] Kelly | Expression oft h e forkhead transcription factor FoxN4 in progenitor cells in the developing Xenopus laevis retina and brain[END_REF][START_REF] Boije | Forkheadbox N4 (FoxN4) triggers context-dependent differentiation in the developing chick retina and neural tube[END_REF]. Outside vertebrates, embryonic expression has been described in S. kowalevskii (Fritzenwanker et al., 2014)and in a single developmetal stage of B. floridae [START_REF] Bajoghli | Evolution of genetic networks underlying the emergence of thymopoiesis in vertebrates[END_REF]. In the hemichordate, expression of FoxN1/4 is ubiquitous during early development and is thereafter observed in the ectoderm. In B. lanceolatum, the expression of FoxN1/4a was very dynamic with a maternal ubiquitous expression followed by restricted expression in the ectoderm at the gastrula stage, in the endoderm and axial mesoderm in neurulae, in the cerebral vesicle, the pharynx and the posterior somites later on, and, finally, in the posterior gut of the larvae. These data suggest that FoxN1 and FoxN4 probably acquired new functions in vertebrates, and analysis of the expression of FoxN1/4 family genes in tunicates will be needed to better understand this point. Interestingly, the gut of amphioxus larva and adult is considered as a major organ for immunity and FoxN1/4a might, as vertebrates FoxN1, play a role in the control of immune system function in amphioxus. However, further functional studies are required to test this hypothesis.

In vertebrates, FoxN3 is important for craniofacial and eye development [START_REF] Schuff | FoxN3 is required for craniofacial and eye development of Xenopus laevis[END_REF][START_REF] Samaan | Foxn3 is essential for craniofacial development in mice and a putative candidate involved in human congenital craniofacial defects[END_REF]Schmidt et al., 2011). In Xenopus, FoxN3 is expressed in neural crest and eye field whereas FoxN2 is expressed early in the eye field and then in branchial arches, retina and vagal ganglion [START_REF] Schuff | Temporal and spatial expression patterns of FoxN genes in Xenopus laevis embryos[END_REF]. In mouse, FoxN2 is expressed in craniofacial, limb, nervous system and somitic tissues [START_REF] Tribioli | The murine fork head gene Foxn2 is expressed in craniofacial, limb, CNS and somitic tissues during embryogenesis[END_REF]. In Ciona intestinalis, expression of FoxN2/3 is quite ubiquitous during early development and becomes more intense in the sensory vesicle, the mesenchyme, the notochord and the palps after gastrulation [START_REF] Imai | Gene expression profiles of transcription factors and signaling molecules in the ascidian embryo: towards a comprehensive understanding of gene networks[END_REF]. In sea urchin FoxN2/3 is expressed in the non-skeletogenic mesoderm and, later on, in the endoderm and it has been shown that FoxN2/3 function is important for ingression and for the expression of genes coding for proteins of the skeletal matrix [START_REF] Rho | The control of foxN2/3 expression in sea urchin embryos and its function in the skeletogenic gene regulatory network[END_REF]. Here, we show that FoxN2/3 in amphioxus was ubiquitously expressed at early stages. Then, at the gastrula stage, its expression was restricted to the endomesoderm and later on we observed a specific expression in the somites. Altogether, this suggests a conserved role of FoxN2/3 in the development of mesoderm in deuterostomes, although genes of this family seem to have acquired specific functions in each chordate lineage.

Conclusions

Analyzing the expression of Fox genes in the Mediterranean amphioxus, B. lanceolatum showed us several points. First, as previously described for other gene families (Somorjai et al., 2008), the expression of orthologous genes in different amphioxus species shows a high degree of stasis. However, differences may be found that can easily be explained by variation in experimental sensitivity. And, second, the comparative analyzes of the expression of amphioxus Fox genes with other metazoans and particularly chordates have shown a high degree of conservation for some genes (e.g., FoxC, FoxD), but also divergent patterns in others (e.g., FoxM, FoxN1/4a). This indicates that Fox genes were necessary for essential functions in metazoans but they were also instrumental for the evolution of new functions. Further studies in amphioxus and other metazoans, and particularly functional studies, will be extremely important in the future to establish the complete picture of Fox genes expression and function and their role in the evolution of animals.

T he first developmental step in the formation of the vertebrate central nervous system (CNS) is called neural induction. It is the instructive process by which naive ectodermal cells are committed to a neural fate. The concept of neural induction was established by Hilde Mangold and Hans Spemann. They showed that the dorsal blastopore lip of a newt gastrula, when grafted to the ventral side of a host gastrula, was able to induce the formation of a Siamese twin embryo in which the secondary CNS developed from the host and not from the graft1 . This embryonic territory with inductive capacities was called the organizer and many studies have been carried out to understand the nature of the neural inductive signals coming from this structure. In the first molecular model of neural induction, called the 'default model' , it was proposed that ectodermal cells become epidermal when exposed to bone morphogenetic protein (BMP) signals and neural when deprived of BMP and of any other signals 2 . In the early vertebrate embryo, the BMP signalling pathway is active in the ventral region, whereas the dorsal organizer produces BMP antagonists that act as neural inducers 3-5 . However, this model has been challenged by various studies suggesting that fibroblast growth factor (FGF) signalling also contributes directly to neural induction in Xenopus and chick 6-11 .

If the mechanisms controlling neural induction in vertebrates are subject to debate, their evolutionary origins are even more obscure. In tunicates, the sister group of vertebrates, the embryo lacks an organizer and inhibition of the BMP signal is not required for the formation of the CNS 12 , whereas the FGF signal is indispensable 13 . Therefore, understanding how the CNS develops in cephalochordates (that is, amphioxus), the sister group of all remaining chordates, may shed light on the ancestral mechanisms controlling neural induction in this lineage. Graft experiments 14 and gene expression data 15 have suggested that the amphioxus dorsal blastopore lip could be homologous to the vertebrate organizer. Additionally, it has been shown that activation of the BMP signalling pathway ventralizes the amphioxus embryo, leading to loss of the neural plate 15-17 . However, experiments that have been carried out to inhibit this signal have resulted in a modest expansion of axial neural plate gene expression, calling into question the applicability of the default model 18 . On the other hand, inhibition of the FGF signalling pathway does not suppress neural induction 19 , supporting the idea that FGF is not unconditionally required. Notably, activation of the Nodal-Activin pathway in amphioxus leads to complete dorsalization of the embryo in which the whole ectoderm expresses neural genes 16 . However, the precise mode of action of Nodal in ectoderm neuralization remains to be described. In vertebrates, Nodal is a key signal produced by the organizer, which acts as a mesendoderm inducer and controls gastrulation movements 20 . Although these early functions of Nodal signalling have been extensively studied, its putative role during neural induction remains to be addressed.

The study of neural induction in amphioxus has been hampered by the lack of appropriate experimental setups. To overcome this fundamental problem, we developed microsurgery to obtain naive ectodermal explants. Using graft experiments combined with molecular analyses, we show that the dorsal blastopore lip of amphioxus is a functional organizer able to promote the formation of a secondary body axis and the acquisition of neural fate by naive ectodermal cells. We also demonstrate that Nodal-Activin is the main neural induction signal originating from the organizer in amphioxus. Finally, we show that this pathway also participates in neural induction in Xenopus.

Amphioxus organizer

Although previous studies 14,15 have suggested that the dorsal blastopore lip of amphioxus may be homologous to the vertebrate organizer, there was no direct evidence demonstrating that it is able to trigger neural induction. To address this question, we first reproduced old grafting experiments 14 . We grafted the dorsal lip of the ARTICLES NATURE ECOLOGY & EVOLUTION blastopore of early gastrula (G2) stage embryos onto the ventral side of the archenteron of host embryos (Fig. 1a). Grafted embryos showed partial secondary axis formation (Fig. 1a), as it has previously been described 14 , or complete secondary axis development (Fig. 1a). We then explored the putative neural inductive capacity of the dorsal blastopore lip on naive ectoderm. Ectoderm explanted from G1 gastrulae (gastrula explant) developed into blastula-shaped hollow balls consisting of a single cell layer, or of an outer single cell layer associated with an inner cell mass, as it has previously been observed 21 . The inner cell mass acquired a mesodermal fate as indicated by Brachyury expression (Fig. 1b). The external cell layer developed entirely into epidermis as shown by the expression of Keratin 1 (K1, Fig. 1b and Supplementary Fig. 1). Moreover, no Neurogenin expression could be detected (Fig. 1b), regardless of the presence or absence of Brachyury-positive cells, showing that gastrula explants developed by default into epidermis. To ascertain the inductive capacities of the organizer, we grafted the mesendodermal part of the dorsal blastopore lip of fluorescent embryos onto gastrula explants (Fig. 1c). In grafted gastrula explants, Neurogenin was broadly expressed exclusively in explant cells, indicating that host cells received a neuralinducing signal produced by the graft (Fig. 1c). By contrast, we did not observe Neurogenin expression when we recombined gastrula explants and the ventral blastopore lip (Fig. 1d).

BMP signal

In cephalochordates, the BMP pathway is active very early during embryogenesis, as indicated by a nuclear phosphorylated pSmad1/5/8 signal that is observed before the blastula stage 18 . Ectopic activation of this pathway leads to a complete ventralization of the embryo in which the whole ectoderm becomes epidermis 15,17 , whereas repression of BMP signalling through the application of dorsomorphin, a chemical inhibitor of BMP receptors, at the blastula stage, only induces a modest expansion of neural markers 18 . Given that nuclear pSmad1/5/8 labelling was reported at cleavage stages 18 , we reasoned that earlier dorsomorphin application was required to obtain more penetrant effects. When the drug was applied at the cleavage stage (3.5 h post fertilization (hpf) at 19 °C), embryos kept a gastrula shape, with no contact between mesendoderm and ectoderm. Dorsomorphin-treated embryos displayed a completely dorsalized mesendoderm, as revealed by the radial expression of dorsal markers (Nodal, Vg1 and Zic (Fig. 2a,b)). Strikingly, the ectoderm responded differently. Indeed, although the expression of the ventral ectoderm marker EvxA was lost, there was no expansion of the expression of the dorsal ectoderm markers towards the ventral region (Fig. 2a,b). In fact, expression of these genes was lost in the entire ectoderm, which also failed to express markers of epidermal (K1) or neural (Neurogenin, Hu/Elav) fates (Fig. 2a,b). However, the ectoderm expressed SoxB1a, ruling out a non-specific blockage of transcription. In control embryos, SoxB1a is first expressed throughout the ectoderm, until the onset of gastrulation, and is later restricted to the neural plate (Fig. 2a and Supplementary Fig. 2). Therefore, after dorsomorphin treatment, the ectoderm showed an expression profile that is similar to uncommitted ectoderm: SoxB1a-positive and Neurogenin-and K1-negative. To verify that dorsomorphin efficiently inhibited the BMP pathway, we undertook double treatments with recombinant zebrafish (z)BMP4 protein. In these embryos, K1 expression was not recovered, indicating that in our assays dorsomorphin completely abolished BMP activity (Fig. 2c). Together these data show that inhibiting the BMP signalling pathway at early developmental stages leads to dorsalization of the mesendoderm and that it blocks ectodermal cell fate commitment.

Nodal-Activin signal and interaction with FGF and BMP

Nodal signalling is active before the blastula stage in amphioxus, as indicated by the zygotic expression of Nodal and its target Lefty at cleavage stage 22 . Early ectopic activation of the Nodal-Activin signalling pathway through recombinant Activin treatment induces embryo dorsalization 15 . In these conditions, the whole anterior ectoderm expresses Otx, whereas the whole posterior ectoderm expresses Wnt3, suggesting complete neuralization 16 . On the other hand, inhibition of Nodal-Activin signalling using SB505124, an inhibitor of the Alk4, Alk5 and Alk7 receptors, induces ventralization, and consequently the loss of neural structures 16 . We confirmed that in Activin-treated embryos the ectoderm was entirely neuralized as indicated by the expression of the pan-neural

ARTICLES NATURE ECOLOGY & EVOLUTION

markers Neurogenin and Hu/Elav, and by the complete loss of K1 expression (Fig. 3). We have previously shown that embryos in which the FGF signalling pathway is inhibited still displayed ectodermal Neurogenin expression 19 . We asked whether FGF signalling was involved in Nodal-Activin-mediated neuralization. Addition of the FGF receptor inhibitor SU5402 to Activin-treated embryos did not suppress Neurogenin expression at the G4 stage (Fig. 3). By contrast, at the N2 stage, neural gene expression was lost in the anterior ectoderm (Fig. 3). Conversely, K1 expression was recovered in the anterior ectoderm (Fig. 3), indicating a change of fate in the anterior ectodermal region from neural to epidermal. The Nodal-Activin signalling pathway is therefore able to completely neuralize the ectoderm independently of the FGF signal, which is, however, probably required for the maintenance of the anterior neural fate and/or the patterning of the anterior neural tissue.

We tested whether neuralization by Nodal-Activin pathway in amphioxus involved BMP pathway inhibition by first analysing nuclear pSmad1/5/8. As expected, no labelling could be detected in dorsomorphin-treated neurula (Supplementary Fig. 3a). By contrast, pSmad1/5/8 nuclear staining was observed in the ectoderm of Activin-treated embryos (Supplementary Fig. 3a), suggesting that the intracellular cascade activated by BMP was not interrupted by Activin. We then analysed the effects of dorsomorphin and Activin treatments at a global scale using an RNA-sequencing (RNA-seq) approach. Transcriptome analyses supported the specificity of the treatments and reinforced the previous conclusions based on in situ hybridization experiments: Activin induced neuralization, and dorsomorphin inhibited the differentiation of ectoderm cells, whereas it induced dorsalization of the mesendoderm (Supplementary Fig. 3b). We also undertook double treatments with Activin and zBMP4 recombinant proteins. Treated embryos displayed a phenotype similar to what was observed after Activin and SU5402 treatment (Supplementary Fig. 3c). The whole ectoderm expressed Neurogenin at the G4 stage, but at the N2 stage this expression was lost in the anterior region (Supplementary Fig. 3c), suggesting that BMP inhibition is required for the maintenance of the anterior neural territory. Next, we tested the ability of the Nodal-Activin pathway to neuralize the ectoderm of dorsomorphin-treated embryos. Double Activin and dorsomorphin treatment often led to exogastrulation and the whole ectoderm always expressed Neurogenin (Supplementary Fig. 3c). This result demonstrates that the uncommitted ectoderm of dorsomorphin-treated embryos can resume neural differentiation. Together, our data strongly suggest that Nodal-Activin is able to neuralize the amphioxus ectoderm independently of BMP signalling.

ARTICLES NATURE ECOLOGY & EVOLUTION

Ectodermal explants and grafts

In whole-embryo assays, ectodermal cells are exposed to both endogenous signals produced by the organizer and the mesendoderm, and to exogenous inducers or inhibitors. This multiplicity of signals complicates interpretations. To overcome this issue, we undertook experiments on ectodermal explants. In addition to gastrula explants, we prepared explants grown from animal blastomeres (Supplementary Fig. 4), dissected at the eight-cell stage (blastomere explant), in order to test the effect of our reagents at an earlier stage. Similar to gastrula explants, ectoderm from blastomere explants always developed into epidermis (Fig. 4a and Supplementary Fig. 5). When we inhibited the BMP signal in gastrula explants and blastomere explants, K1 expression was lost (Fig. 4a), but no expression of Neurogenin was detected (Fig. 4a), as it was observed in whole embryos (Fig. 2a). To test which signals could trigger neural induction in explants, we used treatments with Activin, FGF1/2, or both Activin and FGF1/2. In Activin and in FGF1/2-treated gastrula explants, neural identity was promoted only in a subset of cells (Fig. 4a). However, the double Activin and FGF1/2 treatment induced Neurogenin expression in a broader region, and caused near-complete loss of K1 expression (Fig. 4a). Notably, these effects were more pronounced when using blastomere explants, consistent with our experiments on the time window of neural induction in the whole embryo (Supplementary Fig. 6). Therefore, Activin treatment alone or with FGF1/2 induced expression of Neurogenin in the whole blastomere explants and complete loss of K1 expression (Fig. 4a). These results were similar regardless of the presence or absence of Brachyury-positive inner cells, showing that neuralization was independent of the presence of mesoderm. Therefore, both FGF and Nodal-Activin pathways are able to induce neural fate in ectodermal explants, probably through complementary mechanisms.

To test the putative epistatic relationships between these two signals, we undertook double treatments with Activin and SU5402 or FGF1/2 and SB505124. When treatments were applied to gastrula explants, Neurogenin was still expressed in a restricted region of the explants (Fig. 4b). By contrast, in blastomere explants, Neurogenin expression mostly disappeared after FGF1/2-SB505124 application, whereas it persisted in Activin-SU5402-treated explants (Fig. 4b). This again indicates a primary role for the Nodal-Activin and a secondary role for the FGF signalling pathway.

Finally, because we showed that the dorsal blastoporal lip was able to induce neural tissue formation in ectodermal explants (Fig. 1), we tested which signal could be responsible for this induction. We applied FGF-and Nodal-Activin-signalling pathway inhibitors to grafted ectoderm explants. We found that inhibition of the FGF signal did not prevent neural tissue formation, whereas inhibition of the Nodal-Activin signal abrogated Neurogenin expression (Fig. 4c). Overall these data strongly suggest that in amphioxus the organizer triggers the first step of neuroectoderm development through the Nodal-Activin signal.

Nodal signal in Xenopus

We decided to test whether, in the classical vertebrate model Xenopus Nodal signalling could also induce neural tissue. Because Nodal is involved in inducing the Spemann organizer 23 , its role as a direct neural inducer is difficult to test. Our previous work has shown that the response of Xenopus embryonic cells to Nodal signals changes with time 24 . Thus, we reasoned that treatment with Nodal after the stage when embryonic cells are able to become mesendoderm in response to this signal could allow us to evaluate its potential as a neural inducer. We found that in whole embryos or ectoderm explants, the application of the recombinant Nodal protein at mid-gastrula stage 11 was unable to induce ectopic expression of organizer (gsc, chordin) or mesendoderm markers (xbra (also known as T), xnot-2 (also known as not), sox17, mixer) (Fig. 5a and Supplementary Fig. 7a). However, Nodal induced ectopic neural tissue expression of sox2 and foxd5 in both whole embryos and ectoderm explants (Fig. 5b and Supplementary Fig. 7a). Conversely, the epidermal marker k81 was strongly repressed (Supplementary Fig. 7a). Furthermore, direct neural induction in ectoderm explants was also achieved through the expression of an inducible form of activated smad2 (ref. 25 ), when induced at stage 11 (Fig. 5c and Supplementary Fig. 7b). The induced neural tissue had an anterior character as revealed by otx2 expression (Supplementary Fig. 7b), and was stable as revealed by the presence of ectopic neurons expressing n-tubulin at the tailbud stage (Fig. 5b).

We then tested whether the effect of Nodal on neural induction was achieved through inhibition or competition with the BMP signal. As has previously been shown 11 , injection of zBMP4 protein into embryos at stage 11 did not expand the epidermal tissue, whereas injection of Nodal, or co-injection of Nodal and zBMP4, caused the expansion of the neural plate (Fig. 5d). Moreover, in the three conditions, we observed prominent pSmad1 nuclear staining in the ectoderm (Fig. 5e), demonstrating that nodal does not induce neural tissue through BMP inhibition. Finally, combining cycloheximide and Activin treatments on animal caps, we showed that the early neural regulator foxd5 (refs 11,26 ), but not sox2, is an immediate early target of the Nodal signalling pathway (Fig. 5f and Supplementary Fig 7c), which is consistent with the recent finding that smad2/3 binds to an active enhancer of this gene at stage 10.5 but not at blastula stage 9 (ref. 27 ).

Next, we asked whether Nodal-Activin signalling was required for neural induction in Xenopus. When the inhibitors of Nodal-Activin receptors, SB505124 and SB431542, were applied to stage 11 embryos, morphogenesis proceeded normally, but neural tissue development was altered as revealed by the significant down-regulation of foxd5, sox2 and n-tubulin expression (Supplementary Fig. 8). We also injected a dominant-negative form of the Nodal-Activin receptor Alk4 (DN-Alk4) in the presumptive neural ectoderm in eight-cell embryos in order to potently inhibit the Nodal-Activin pathway from the earliest possible stage, while avoiding perturbations of the organizer mesoderm. In these embryos, chordin expression was maintained, whereas foxd5, sox2 and n-tubulin expression

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was strongly down-regulated in injected ectodermal cells (Fig. 6a,b). Notably, injection of recombinant Noggin could neuralize control ectoderm cells but not cells that received DN-Alk4, further demonstrating the BMP-independent role of the Nodal-Activin pathway (Fig. 6c). We conclude that in standard assays in Xenopus, Nodal behaves as a bona fide neural inducer, as it is capable of directly activating neural markers in the absence of organizer mesoderm, is required for neural tissue development and functions without interfering with the BMP signal.

Discussion

Although previous embryological studies 14 and gene expression data 15 have suggested that the dorsal blastopore lip of amphioxus has the same properties as the vertebrate organizer, no direct evidence for its capacity to induce neural fate has been reported. By using microsurgery techniques we demonstrate that the amphioxus dorsal blastopore lip is indeed equivalent to the vertebrate dorsal organizer. It can induce a secondary nervous system when grafted into a host embryo and convert ectodermal cells from an epidermal to neural identity.

The results we present here also suggest that the default model does not account for neural induction in amphioxus. Although BMP inhibition is required for neural tissue to emerge, it is not sufficient, and instructive cues must be delivered. Indeed, both in whole embryos and in explants, inhibition of BMP signalling led to the maintenance of an undifferentiated state of the ectoderm. At first glance, this result appears different from what has been published when the same approach was used at later stages in whole embryos 18 . Indeed, Kozmikova and co-workers observed a modest lateral expansion of the specific neural plate markers Brn1/2/4 and SoxB1c, whereas SoxB1a expression became widespread in the non-neural ectoderm. The effects caused by BMP inhibition in their study are in fact consistent with our own observations using earlier treatment. We suggest, however, that late BMP inhibition probably did not fully prevent neural induction in this assay and that the increased expression of the neural markers Brn1/2/4 and SoxB1c reflects the conversion of the neural plate border territory into axial neural ectoderm, although this hypothesis remains to be tested.

We also provide evidence that both Nodal-Activin and FGF signals are implicated in different steps of CNS formation in amphioxus. Together, our data suggest that Nodal-Activin is the main neural-inducing signal originating from the organizer, whereas FGF is involved in the maintenance and/or patterning of the anterior neural territory. Notably, we also show that the activity of Nodal-Activin on neural induction is independent of the presence of mesodermal cells. Likewise, we report that neuralization of ectoderm by Nodal in Xenopus can occur independently of the presence

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of organizer mesoderm. Moreover, neuralization by Nodal-Activin is maintained in the presence of active BMP signalling in both amphioxus and Xenopus.

Our results also have implications for the evolution of the molecular control of neural induction. First, we propose that the default model does not account for neural induction throughout the chordate lineage. Indeed, although BMP signal inhibition is necessary for neural induction in all vertebrates 3 , BMP plays no role in this process in tunicates 12 and BMP inhibition appears insufficient to trigger neural induction in chick and amphioxus, although it may be required to maintain the anterior neural territory in both species 9 . The situation is different in the tunicate Ciona, where FGF is the main neural inducer 13 , whereas Nodal is required for posterior neural tube formation and for the specification of trunk epidermal sensory neurons 28-30 . Concerning vertebrates, Nodal activity has been shown to be required to prevent precocious neural

ARTICLES NATURE ECOLOGY & EVOLUTION

differentiation in the mouse pluripotent epiblast 31 . Likewise, concomitant inhibition of smad1 and smad2 was proposed as a necessary condition for neural induction in Xenopus 32 . By contrast, the activity of Smad2/Smad3 seems to be required for neural induction in zebrafish 33 , raising the possibility that multiple phases of intervention of this pathway may complicate interpretations. Here, we show that Nodal is a bona fide neural inducer in Xenopus. We also show that the mesendoderm and neural inductive capacities of Nodal could be uncoupled. Thus, our assay provides a new and valuable technical framework to work out the mechanisms of neural induction in this animal. Multiple Nodal ligands are expressed in the Spemann organizer, possibly contributing directly to neural induction 24,34 . Supporting this idea, we show here that in two assays designed to preserve organizer formation, Nodal pathway inhibition caused severe loss of neural marker gene expression. Collectively, our data suggest that the implication of Nodal-Activin signalling in the first step of CNS formation might be ancestral in chordates. Notably, it has recently been shown that Nodal is also able to trigger secondary body axis formation and neural fate commitment in the sea urchin 35 , pushing back the possible ancestral role of this signal in neural induction to the base of the deuterostome lineage. In conclusion, we suggest that neural induction can be triggered by BMP inhibition, by FGF and by Nodal signalling but that the precise spatio-temporal contribution of each pathway may vary across chordate lineages.

Methods

Obtaining embryos, microdissection and grat experiments. Branchiostoma lanceolatum adults were collected at the Racou beach in Argelès-sur-Mer (France). Spawning was induced as previously described 36,37 . Staging is according to Hirakow and Kajita 38,39 . For explantation experiments, embryos were fertilized and kept in scratched Petri dishes. Gastrula-stage explant preparation was carried out on embryos that were not previously dechorionated except when gastrula explants were prepared for grat experiments. To maintain the chorion surrounding the embryo immobile, the bottom of the Petri dish was cut with a microscalpel to create two or three stripes. When the chorion was immobilized, the position of the embryo inside the chorion was stabilized (with the antero-posterior axis parallel to the bottom of the Petri dish) and gastrula explants were obtained using dissection perpendicular to the Petri dish with a microscalpel. For blastomere explant preparation and grating, the embryos were manually dechorionated just before the irst cell division by projection against the borders of an agarose-coated Petri dish (0.8% agarose in 0.2 μ m iltered sea water) until microdissection. Embryos were dissected with an eyelash. For grat experiments, ater microdissection, hosts and grats were gently put into contact and were let stationary for one hour, the time needed for at least one cell division to occur. To avoid any efect of the agarose coat on the molecules used for treatments, all the treatments were carried out in scratched Petri dishes. For grat experiments using luorescent embryos, Dextran (10,000 MW) coupled to Texas Red (Invitrogen) was injected in unfertilized eggs. Eggs obtained from NASCO Xenopus laevis females were fertilized in vitro, dejellied, cultured, staged and injected as previously described 10,40 .

Embryo and explant treatments. Amphioxus embryos, explants or grafts were treated with the following molecules: dorsomorphin (35 μ M for whole embryos and 10 μ M for explants, prepared as in ref. 41 , Sigma-Aldrich), Activin (50 ng ml -1 , human recombinant protein, R&D), zBMP4 (250 ng ml -1 , zebrafish recombinant protein, R&D), SB505124 (50 μ M, dissolved in dimethyl sulfoxide (DMSO), Sigma-Aldrich), FGF1/2 (2 μ g mL -1 , B. lanceolatum recombinant protein), SU5402 (50 μ M, dissolved in DMSO, Calbiochem). The concentrations used were defined after pilot experiments using the following concentration ranges of drug or recombinant proteins: dorsomorphin: 10-50 μ M, Activin: 10-100 ng ml -1 , zBMP4: 50-250 ng ml -1 , SB505124: 25-100 μ M, FGF1/2: 500-2 μ g ml -1 , SU5402: 25-100 μ M. Whole embryos were treated at 3.5 hpf at 19 °C except when specified. Explants and grafted explants were directly treated following the explantation or graft procedure. For time-window experiments, embryos were washed at least four times after treatment to ensure that no drugs/ recombinant proteins were left in the culture medium. Xenopus embryos were injected in the blastocoel with 10 ng mouse recombinant Nodal protein (R&D), 3.5 ng zebrafish recombinant BMP4 protein (R&D), or 30 ng recombinant human Noggin (R&D). DN-Alk4 and GR-t-Smad2 (ref. 25 ) mRNAs were synthesized with the Ambion mMessage mMachine kit. Eight-cell embryos were injected with 3 ng of DN-Alk4 in one of the two dorsal-animal blastomere.

  . It has been reported that Smad2-Smad4 complexes are not able to bind DNA alone, and hence require other transcription factors for DNA binding, in contrast to Smad3-Smad4 complexes which are capable of directly binding DNA. Nodal signaling can be antagonized by soluble inhibitors of the Lefty subclass of TGF-β factors, Lefty1 and Lefty2, by directly binding to Nodal or interfering with the activity of its co-receptors TDGF1/CFC1. Additionally, two members of the DAN family, CER1 (Cerberus) and DAND5, are thought to inhibit Nodal signaling by sequestering the ligand [114].

  ), FoxB, FoxG, Collier/Olfactory/Early B-cell factor (Coe), Huntingtin (Htt), Single-minded (Sim), Neural cell adhesion molecule (NCAM) and Synaptotagmin (Syt).

  vertebrates[183]. The cadherin of the adhesion G protein coupled receptor (Celsr1/2) also seems to be involved in neural progenitor fate decision during the development of the cerebral cortex in humans[184]. Finally, KDM3 (Histone H3 lysine 9 Demethylase) has been described to facilitate the Neurog2 chromatin accessibility during neuronal transcription in Xenopus[185].

  is a method that allows to identify open chromatin regions and potential regulatory sequences. This technique is based on the use of the hyperactive Tn5 transposase that cut and ligate adapters for high-throughput sequencing at regions of increased accessibility. After the transposition reaction, amplification and sequencing, the over-represented regions are mapped in the genome which allows to define a regulatory landscape based on peaks that represent open active regions of chromatin[186]. We have performed ATAC-seq experiments in embryos and ectodermal explants at 11hpf and 19hpf, previously treated with dorsomorphin or Activin, and in corresponding control embryos/explants. The results can be visualized in the Branchiostoma lanceolatum genome browser at USCS (follow the link at http://amphiencode.github.io/). This represent a large amount of data and, in consequence, we decided to focus on the results obtained in 11hpf ectodermal explants treated with Activin for this project.

3 Table 4 :

 34 List of genes presenting active super-enhancers in ectodermal explants treated with Activin at 11hpf. a. List of transcription factors; b. List of additional genes; c. List of unknown genes here referred with their annotation number. All genes are organized according to the number of peaks in their respective cluster.

1 .

 1 Reporter validation of putative enhancers containing Smad binding sites.In order to show that the SBS-Es used in this study are effectively driving the expression of their respective TFs during neural induction, we decided to inject a set of reporter vectors containing such putative enhancers sequences. Therefore, we amplified and cloned each SBS-E into a vector containing the amphioxus β-actin minimal promoter upstream of the GFP coding sequence and flanked by Tol2 transposase recognition sites. Each final vector was injected in non-fertilized eggs together with the mRNA of Tol2. After fertilization, the injected embryos were let to develop and finally fixed at 11hpf and 19hpf. GFP expression driven by the target enhancer was analyzed by in situ hybridization (Figure38 a,b).

  In this work, we have only studied the function of few nodes of the GRN (SoxB1a, Pou3f and Neurogenin) through constitutive repressor forms overexpression. The objective would be in a first time to use the same approach for the other nodes of the GRN, focusing on the nodes which are putative direct target of Smad. Since most of the classic knock-down techniques are not efficient in amphioxus (shRNA, siRNA, morpholino), it will be crucial to develop new approaches with the purpose of blocking the expression of all the nodes that have been presented in this GRN. In this context, KO through CRISPR/Cas9 gene editing is promising and should allow us to have a clearer view of the function of each node.

( 35 µM

 35 for whole embryos and 10 µM for explants, Sigma), Puromycin (50 ng/ml for only 30 minutes each treatment), SB-505124 (50 ng/mL for several periods of time), Activin (50 ng/mL, human recombinant protein) or recombinant amphioxus FGF1/2 (2 µg/mL).Dorsomorphin(6-(4-(2-(Piperidin-1-yl)ethoxy)phenyl)-3-(pyridin-4-yl)pyrazolo[1,5-a] pyrimidine) is a BMP signaling inhibitor which targets the type I receptors ALK2, ALK3 and ALK6. Treatments were carried out in whole embryos and ectodermal explants. Doses in explants were lower in comparison to embryos, because Dorsomorphin affects their whole morphological integrity. In fact, explants treated to higher doses do not keep a tissue-like structure but as dissociated cells. Treatment was done, in each case, at 3hpf and continued until the desired stage (11hpf or 19hpf) in whole embryos, or ectodermal explants. Puromycin (3′-[α-Amino-p-methoxyhydrocinnamamido] -3′-deoxy-N,N-dimethyladenosine dihydrochloride) inhibits protein synthesis by causing premature chain termination and leading to premature termination of translation. Treatments were only carried out in whole embryos at 2h30m after fertilization, for only 30 minutes. After this, embryos were washed and treated to specific treatments. SB-505124 (2-( 5-Benzo [1,3] dioxol -5 -yl -2-tert-butyl-3H-imidazol-4-yl ) -6methylpyridine hydrochloride hydrate) is a selective inhibitor of TGF-β type I receptors ALK5, ALK4 and ALK7, therefore blocking the activation of the nodal signaling pathway through Smad2/3. It does not inhibit ALK1, 2, 3, or 6 (these receptors are exclusively blocked by Dorsomorphin). Treatments were carried out in whole embryos, starting at 3hpf and washed at a desired time point. SB505124 is able to ventralize the embryos when treated early in development (until 6-7hpf). Recombinant human Activin is a protein that bind to heteromeric receptor complexes of TGF-b type I (Act RI-A and Act RI-B) and a type II (Act RII-A and Act RII-B) serine-threonine kinase receptors, activating the Nodal signaling pathway. Treatments were carried out in whole embryos or ectodermal explants with equal doses, starting at 3hpf until the desired stage (5hpf, 11hpf or

FIGURE 1 |FIGURE 2 |

 12 FIGURE 1 | Phylogenetic analysis of B. lanceolatum Fox genes. Unrooted 50 majority-rule consensus Bayesian inference tree based on the amino acid sequences of the forkhead domain. Posterior probablilities are shown at each node. The different paralogy groups are coloredinpinkorlight

FIGURE 3 |

 3 FIGURE 3 | Expression of B. lanceolatum FoxC, FoxD, FoxEa, and FoxG. In all the panels except (B,J), anterior is to the left. In lateral and blastoporal views dorsal is to the top. FoxC expression pattern (A-H). Gastrula lateral (A) and blastoporal (B) views. The double arrowhead indicates the expression in the paraxial mesoderm. Late gastrula lateral (C) and dorsal (D) views. The arrowheads marks the region that will give rise to the three most anterior somites. In mid-late neurula lateral view (E) the arrow indicates a new expression domain in the anterior endoderm. Late neurula dorsal (F) and lateral (G) views. The arrow marks the expression domain in the pharynx. Larva lateral view (H). FoxD expression pattern (I-P).Gastrula lateral (I) and blasporal (J) views. Late gastrula lateral (K) and dorsal (L) views. The arrow indicates the expression in the anterior region of the neural

FIGURE 4 |

 4 FIGURE 4 | Expression of B. lanceolatum FoxJ1, FoxN1/4a,and FoxN2/3. In all the panels except (B, H, I, K, Q, R, T) anterior is to the left. In lateral and blastoporal views dorsal is to the top. FoxJ1 expression pattern (A-G).Gastrulalateral(A) and blasporal (B) views. Late gastrula lateral (C) and dorsal (D) views. Mid-late neurula lateral (E) and dorsal (F) views. In the larva lateral view (G) the bracket indicates the pharyngeal region. FoxN1/4a

Figure 1 |

 1 Figure 1 | The dorsal blastoporal lip of amphioxus is homologous to the vertebrate organizer. a, Graft experiments were carried out by dissecting and inserting the blastopore lip of a G2 gastrula-stage embryo into the ventral archenteron of a host embryo at the same developmental stage. Some embryos showed a partial double axis as indicated using the neural marker Netrin. Other embryos formed a complete double axis as indicated by expression of the notochordal marker Brachyury and by immunostaining of axons against acetylated α -tubulin. The arrowhead indicates the position of the transverse section and the asterisk the ectopic expression of Netrin. b, Ectodermal explants were obtained through microdissection of the animal pole of G1 gastrula (gastrula explants). Half of the ectodermal explants showed an inner cell mass expressing the mesoderm marker Brachyury. The external cell layer expressed the epidermal marker K1, whereas no expression of the neural marker Neurogenin was detected. c, Graft of a gastrula explant onto a fluorescently labelled (Texas Red) dorsal blastoporal lip induced the expression of Neurogenin exclusively in host cells. Green signal corresponds to auto-fluorescence. d, Negative control grafts of the mesendoderm part of the ventral blastopore lip onto gastrula explants did not induce Neurogenin expression. Anterior is to the left. Scale bars, 50 and 10 μ m for the transverse section in a. Numbers correspond to the number of embryos showing similar labelling out of the total number of embryos.

Figure 2 |

 2 Figure 2 | The role of BMP in ectodermal cell-fate commitment. a, Expression of Nodal, Vg1, Zic, EvxA, SoxB1a, K1, Neurogenin and Hu/Elav in G1, G4 and N2 stage control embryos and in dorsomorphin-treated N2 stage embryos. The black arrowhead indicates the early ectodermal Neurogenin expression domain in control embryos. b, Schematic partial representation of the results presented in a. c, Expression of K1 and Neurogenin at the N2 stage in control embryos and embryos treated with both dorsomorphin and zBMP4. All in situ hybridization images are side views with anterior towards the left. Scale bars, 50 μ m.

Figure 3 |

 3 Figure 3 | Role of Nodal-Activin and FGF signalling pathways in ectoderm specification. Expression at G4 and N2 stages of Neurogenin, Hu/Elav, K1 and Otx in control embryos, in embryos treated with recombinant Activin, and in embryos treated with both Activin and SU5402. All in situ hybridization images are side views with the anterior towards the left. Enlargements of the anterior region (white box) of N2 stage embryos are shown below the pictures of whole embryos to highlight Otx expression in the ectoderm. Scale bar, 50 μ m.

Figure 4 |

 4 Figure 4 | Nodal-Activin is the main signal triggering neural induction. a, Expression of K1 and Neurogenin in control, dorsomorphin-treated, Activintreated, FGF1/2-treated or Activin and FGF1/2-treated gastrula explants and blastomere explants. b, Expression of Neurogenin in FGF1/2-and SB505124treated and Activin-and SU5402-treated gastrula explants and blastomere explants. c, Neurogenin expression induction by the graft of the dorsal blastoporal lip on gastrula explant is lost after SB505124 treatment in most cases, but maintained after SU5402 treatment. The number of explants showing the presented expression pattern out of the total number of explants is indicated. Scale bar, 50 μ m.

Figure 5 |

 5 Figure 5 | Nodal induces neural tissue in Xenopus. a, Expression of gsc, xbra and xnot-2 at early neurula stage 13 in control embryos and in embryos injected with recombinant Nodal at stage 10 or 11. Dorsal views for gsc and xnot-2, ventral views for xbra. Scale bar, 200 µ m. b, Expression of sox2 and foxd5 (left: front view; right: dorsal view) and n-tubulin (ectopic neurons indicated by a white asterisk) in control and nodal-injected embryos. Scale bar, 200 µ m. c, Embryos were injected with GR-t-Smad2 mRNA, animal caps were explanted at the early gastrula stage, and induced or not with dexamethasone at stage 11 and processed for sox2 in situ hybridization. Scale bar, 200 µ m. d, Expression of sox2 and k81 in control embryos and in embryos injected with zBMP4, nodal or both recombinant proteins. Scale bar, 250 μ m. e, Confocal images of pSmad1 immunostaining and nuclear DAPI staining in control embryos and in embryos injected with zBMP4, Nodal or both recombinant proteins. Scale bar, 50 μ m. f, In situ hybridization of sox2 and foxd5 in animal caps treated with cycloheximide (CHX), Activin or both. Scale bar, 200 μ m. The number of embryos showing the phenotype displayed out of the total number of embryos analysed is indicated.

Figure 6 |

 6 Figure 6 | Nodal-Activin signaling is required within the ectoderm for neural induction in Xenopus. a, Expression of chordin, sox2 and foxd5 at stage 10 in control embryos and in embryos injected with DN-Alk4 mRNA in dorsal animal cells. Vegetal view for chordin and dorsal view for sox2 and foxd5. b, Expression of chordin at stage 13 and n-tubulin at stage 15 and 25 in control embryos and in embryos injected with DN-Alk4 mRNA in dorsal animal cells. Dorsal view for chordin and n-tubulin at stage 15 and stage 25 (left) and lateral view for n-tubulin at stage 25 (right). c, Expression of sox2 at stage 10 in control embryos and in embryos injected with DN-Alk4 mRNA in dorsal animal cells. Recombinant Noggin was injected in the blastocoele at stage 8 to induce neural tissue. Embryos are shown in dorsal view. In all cases, embryos were injected with fixable fluorescein lysine dextran and revealed by immunostaining (orange). The number of embryos showing the phenotype displayed out of the total number of embryos analysed is indicated. Scale bars, 250 μ m.

Table 1 : Results from DESeq2 analysis of RNA-seq data from Activin-treated whole embryo versus control

 1

		Corresponding to	BaseMean	Log2FoldChange	Padj
	ContigAmph618	Pitx-isoform2	62,76544	1,94134	6,38E-13
	ContigAmph5752	Lefty	1298,80850	1,88598	2,12E-19
		BL01930
	ContigAmph26407	(BL11242*)	1474,82741	1,66394	1,59E-14
	ContigAmph4746	PCED1B	830,36498	1,59614	3,18E-12
		BL01930
	ContigAmph18508	(BL11242*)	707,61778	1,57534	3,14E-12
	ContigAmph83137	BL11242*	450,07106	1,53925	3,14E-12
	ContigAmph95131	Pitx-isoform1	37,12681	1,49863	2,37E-07
	ContigAmph15228	BL01555	268,45884	1,39379	6,47E-08
	ContigAmph9543	BL11242*	225,30468	1,31389	4,79E-06
	ContigAmph27946	BL11242*	81,09125	1,21675	0,0001
	ContigAmph28185	BL11242*	70,70695	1,18592	0,0004
	ContigAmph10738	VG1	776,70559	1,18039	1,94E-06
	ContigAmph372	Neurogenin	64,20411	1,09291	0,0030
	5hpf. (log2FC >1,0; Padj <0,1). Code BL numbers correspond to the amphioxus annotation number, when transcript
	has not been determined or characterized. Transcripts are organized according to their Log2FC value.

Table 2 : Results from DESeq2 analyses from whole embryos treated with Puromycin/Activin vs control at 5hpf

 2

		Corresponding		Log2FoldChan
		to	BaseMean	ge	Padj
	ContigAmph561	CDH17	52.1552267209	1.2683282054	0.0176674457
	ContigAmph69282	BL53474	45.7964257351	1.211256115	0.0464467607
	ContigAmph3628	BL24826	203.224466656	1.1761782908	0.0439947531
	ContigAmph2536	SDK1/2	103.8492869647	1.1435363678	0.0357939471
	ContigAmph22641	BL83871	75.2844789221	1.0212701946	0.0853121601
	ContigAmph17465	BL21735	179.046955366	0.9978648868	0.0462830648
	ContigAmph54793	AKAP1	415.6140714348	0.8823904716	0.0549528424
	ContigAmph10947	BL00583	1107.5132454696	0.860661164	0.0459339071
	ContigAmph8006	CACHD1	1726.1976074345 0.8228211136	0.0138449854
	ContigAmph15756	RNF145	302.3276779984	0.8223708966	0.0530834047
	ContigAmph17375	BL06780	943.1603621786	0.8184422142	0.05572642
	ContigAmph5297	KDM3A	1770.5028827365 0.7929070524	0.0602222451
	ContigAmph4496	AUTS2/AUTSL 710.5487257259	0.7875128525	0.036288215
	ContigAmph12206	GPR12	919.3024038	0.7734390979	0.0342000089
	ContigAmph4143	EPHA	428.8760530926	0.7664395073	0.0647949036
	ContigAmph26690	SSX2IP	1060.5907164189	0.756533968	0.0864091533
	ContigAmph22385	CELSR1/2	1665.3364106724 0.7544413512	0.0435048472
	ContigAmph7035	BL02905	1648.6251047067 0.7500505719	0.0872386609

(log2FC>0,[START_REF] Bachiller | The Organizer Factors Chordin and Noggin Are Required for Mouse Forebrain Development[END_REF] Padj <0,1)

. The final data was obtained by eliminating the transcripts found to be up-regulated in Puromycin vs control analysis. Code BL number correspond to the amphioxus annotation number, when transcript has not been determined or characterized.

  AccessibleChromatin with high throughput sequencing (ATAC-seq) with the purpose of identifying open chromatin regions that might contain active enhancers during neural induction after Nodal activation.

	Sema1	411,0651325	4,712270382	9,83E-22
	Usp48	435,8079592	4,691030747	4,00E-23
	Sgs-3-like	142,2513628	4,689408958	1,12E-14
	Klhl24	3106,113673	4,688998053	6,51E-31
	Protein C10-like	325,4671274	4,687290563	1,18E-21
	Snai-like	Basemean 120,1313486	Log2FC 4,615960438	Padj 1,60E-12
	Pitx-isoform2	232,7673683	7,507398641	1,53E-19
	Pitx-isoform1	426,7488076	7,192433669	5,18E-28
	Prdm8	140,4157017	7,188901498	5,30E-14
	Insm1	119,7332854	6,990903458	1,59E-13
	Neurogenin	2241,065989	6,774087165	7,23E-42
	Smoc1	1679,117874	6,658683327	1,23E-54
	Lefty	2910,080918	6,123228298	1,95E-09
	Ski-like	235,7825783	6,069451292	4,47E-22
	Neurexin4	8840,656562	5,890309099	2,02E-54
	Cdr2l	267,7395036	5,766417597	7,00E-19
	Dynlt1	1257,65872	5,751059291	4,25E-06
	Chordin	774,3308495	5,553010975	5,90E-26
	Ski	205,0503013	5,531319208	1,01E-21
	Mucin5B	251,6301298	5,526555941	1,32E-21
	Nvl	380,9637854	5,496989936	1,03E-23
	Vg1	714,7592006	5,369501854	4,46E-27
	Fzd4	542,0621306	5,252092777	9,77E-22
	Septin5	110,124916	5,192999044	1,49E-10
	FoxAB	946,9347033	5,159318816	1,63E-05
	Ski-like	672,7192561	5,149688431	2,20E-32
	Abcc4	226,2746767	5,080266002	9,78E-22
	Auts2	224,4661048	5,070796786	4,72E-22
	Ptdss1	123,8524602	4,987918221	4,64E-13
	Crim1	780,1250685	4,910114599	8,38E-18
	Cnr3-like	1515,819849	4,785190545	2,31E-28
	Pced1B	461,5326031	4,748110222	7,82E-25

Table 3 : Results from DESeq2 analyses from ectodermal explants at 11hpf treated with Activin vs control

 3 (log2FC>4,5; Padj <0,1; Basemean>100). Table only contains identified genes in B. lanceolatum. Unknown genes were not included. Gene names are organized according to their Log2FC value. Orange color highlight genes that were identified in previous analyses (at 5hpf).

  comparing to the rest of ZnF and Sox factors. In amphioxus, this family is composed by 7 members whose expression levels react differently to Nodal activation. In fact, Activin treatment leads to the down-regulation of Klf15 Klf1/2/4, Klf3/8/12 and Klf5 in ectodermal explants at 11hpf, whereas Klf9, Klf6/7 and Klf10 are up-regulated (Supplementary table4). From all the members of the Klf family, only Klf1/2/4 present clustered peaks (1 putative super-enhancer) that is sensitive to Nodal activation in ectodermal explants (in this case, ATAC-seq peaks in its vicinity are under-represented in Activin-treated 11hpf ectodermal explants). Strikingly, Klf binding sites are virtually present in all the 679 enhancers that are activated after Activin treatment, indicating that this gene is strongly involved in regulating the expression of other target genes that are downstream of the Nodal signaling pathway.

  represented TF sub-families: Kruper-like factors (Specifically Kl/2/4), Sox-related factors (SoxB1 a/b/c , SoxB2, SoxC, SoxD and SoxE) and Zinc finger factors (Putative orthologues of ZnF333). Finally, we associated each SBS-E to their closest gene and found the following 10

TFs

: FoxAB, Nr2f1, Hey1, Creb3l, Vent, Znf37, Pitx, Ost4, Pax3/7

and Irx. Additionally, we added the classic neural markers Pou3f (Brn1/2/4, as representative homeobox gene expressed in neural plate) to this list as a complementary resource suggested by the results obtained from our collaborators at CABD in Sevilla (Supplementary table

7

). Some TFs were redundantly found in more than one pipeline; as FoxAB, which present a super-enhancer region containing a Smadbinding site; whereas others like SoxB2 are part of a whole TF sub-family (Sox-related factors)

  an exclusive role in neural fate acquisition/silencing. Interestingly, Klf family came on major public attention after the experiments carried out by Takahashi and Yamada in 2006, as one of the TFs involved in pluripotency re-programming. In fact, induced pluripotent stem cells

(iPSCs) were obtained from adult mouse fibroblasts after introducing four factors, Oct3

/4, Sox2, and Klf4, under ES cell culture conditions [190]

. After this, additional experiments have shown that Klf4 overexpression prevents embryonic stem cells (ESCs) from differentiation in rodents, thus promoting ESC self-renewal

[191]

. This results support our hypothesis of a conserved role of Klf4 (or Klf1/2/4 in cephalochordates) in pluripotency maintenance, and a potential role in preventing the cells from entering cell differentiation.

Results

Frontiers in Ecology and Evolution | www.frontiersin.org

July 2015 | Volume 3 | Article 80

Biologie Intégrative des Organismes Marins (BIOM), Observatoire Océanologique, Sorbonne Universités, UPMC University Paris 06, CNRS, Banyulssur-Mer 66650, France

.2 Aix Marseille Univ, CNRS, IBDM, Marseille 13288,

France.3 Centre for Genomic Regulation, Barcelona Institute of Science and Technology (BIST), Barcelona

08003, Spain.4 Universitat Pompeu Fabra (UPF), Barcelona 08003, Spain. Laurent Kodjabachian, Hector Escriva and Stephanie Bertrand jointly supervised this work. *e-mail: laurent.kodjabachian@univ-amu.fr; hescriva@obs-banyuls.fr; stephanie.bertrand@obs-banyuls.fr

© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

NATURE ECOLOGY & EVOLUTION | VOL 1 | AUGUST 2017 | 1192-1200 | www.nature.com/natecolevol

Acknowledgements

This work would not have been successfully carried out without the infinite patience and invaluable academic support of my thesis supervisors Stèphanie Bertrand and Héctor Escrivà, who have guided me throughout my PhD studies. I am also very grateful to all the members of the team "Evolution and Development of Chordates", especially to Lucie Subirana and Kevin Magre for their continuous assistance with transcriptomic data analyses and enhancer cloning. I would also like to express my gratitude to Lydvina Meister, and former team members Josselyn Demouy and Daniel Aldea, for providing a great working environment during the sleepless months of the amphioxus spawning-season. I am especially indebted to Dr. José Luis Gomez-Skarmeta for methodological advice referring ATAC-seq experiments, Alvaro Ponce Cabrera for his help with Peak calling, and Dr. Manuel Irimia for his support with transcriptomic analysis from ectodermal explants. I am grateful to all of those with whom I have had the pleasure to work during these years at the Observatoire Oceanologique de Banyuls-sur-mer: Sebastien Darras, Agnes Roure, Yann Le Petillon, Ronan Lagadec, Rafath Chowdury, all the colleagues from BIOM, LECOB, LOMIC, LBBM, and all the personnel of the Laboratoire Arago and the Biodiversarium.

Finally, nobody has been more important to me in the pursuit of my goals than the members of my family. I would like to thank my mother Techi, whose sacrifice and love guided me to where I am now. I would also like to thank my friends for their eager and loving support, especially to Fabio, Sagir, Ivana, Aurora, Gabo, Rodo, Johann and Lucie 2.0. Thank you for those long distance conversations in the moments of more need and loneliness.

Acknowledgments

DA holds a fellowship from CONICYT "Becas Chile." Part of this study was supported by the EXOMOD grant from the CNRS.

Acknowledgements

The laboratory of H.E. was supported by the CNRS and the ANR-16-CE12-0008-01 and S.B. was supported by the Institut Universitaire de France. This project was supported in L.K. 's laboratory by ANR BSV2-021-02 and by Fondation ARC. M.I. is supported by an ERC Starting Grant (grant agreement ERC-StG-LS2-637591) and the Spanish Ministry of Economy and Competitiveness ('Centro de Excelencia Severo Ochoa 2013-2017' , SEV-2012-0208 to the CRG). Some of the Xenopus experiments were performed in the PiCSL-FBI core facility (IBDM, AMU-Marseille), member of the France-BioImaging national research infrastructure. Some of the amphioxus experiments were carried out on the Cytometry and Imaging Platform of the Observatoire Océanologique de Banyuls-sur-Mer. RNA sequencing was performed at the CRG Genomics facility. We thank S. Darras for technical help and M. Belgacem for amphioxus FGF1/2 in vitro production.

Data availability. Accession numbers of sequences used for in situ hybridization probe synthesis and the sequences of primers used for quantitative PCR analysis are given in Supplementary Tables 1,2. RNA-seq raw reads are available at the BioProject database (accession number PRJNA354850).

Perspectives 115

During this project, our team has identified some experiments that need to be aimed in the future in order to better elucidate the neural induction process triggered by Nodal. In this context, the following perspectives are proposed as complementary approaches to this work.

Determination of the function and regulation of Klf1/2/4 though in-vitro assay.

To better understand the function of Klf1/2/4 protein, and to confirm our hypothesis on its role during neural induction, several experiments need to be undertaken. First, we need to test wether Klf1/2/4 is able to bind the putative binding sites found in this study in the over-represented ATAC-seq peaks. This could first be achieved by ElectroMobility Shift Assayel shift assay (EMSA). Once the binding site are validated in vitro, transfection assays in cell culture should be undertaken to test for the transcriptional activation/repression function of Klf1/2/4 using reporter construction bearing these binding sites. To go further into the function of Klf1/2/4 in the embryo context, injection of mRNA of constitutive activator and/or repressor forms of Klf1/2/4 should be undertaken. In addition, it would also be important to functionally test the other members of the Klf family, in order to determine the role of each Klf in amphioxus.

Functional test of enhancers by combining them in the same reporter vector.

Our vector constructions have lead us to evaluate if the enhancers we have identified in this study were real. Indeed, all the expression patterns directed by each of such enhancers validated the approach that was undertaken. On the other hand, the fact of obtaining non-neural patterns directed by enhancers that regulate classic neural genes, as Neurogenin, suggest that the combination of their full set of enhancers might be required in order to reproduce a robust neural pattern during amphioxus neural development. In order to test this, it is necessary to know the expression patterns of each of the TFs drawn in our GRN through ISH, in order to identify exclusive neural markers from this gene list. Finally, a similar reporter vector analysis of the enhancers corresponding to such neural markers (as the one carried out in Neurogenin's enhancers) will let us determine the necessity of constructing vectors containing the whole set of enhancers regulating their corresponding target gene in order to recapitulate a exclusive neural expression pattern.

Determination of the dynamic processes involved in neural induction through scRNAseq.

The final hypothesis of this project suggests a fundamental role of pluripotency factors in the neural induction process of amphioxus. The stemness release mechanism we propose is taking place after Nodal activation would be a progressive process that needs to be tracked in time and space. We propose to use single-cell RNAseq (scRNAseq) approach in whole embryo and in ectodermal explants both in wild-type and Activin-treated conditions. This will allow us to follow the cell state during the whole process of neural induction and neural differenciation and to clearly define if there is indeed a sequential loss of stemness, and which are the actors implicated in such a process.

Materials and methods

through a differential gene expression analysis performed on ABiMS-Galaxy platform. The sequence data were aligned to the amphioxus BraFlo1 reference genome using Bowtie2 aligment, and the differentially expressed genes were obtained by using the DESeq2 package. Genes with adjusted p-values less than 0.01 were considered significant. The cutoff for the FoldChange was set from 2 to -2 for further studies.

ATAC-seq library and data analysis

Among 50-80 embryos and 80-100 explants were transferred in a 1.5ml tube, respectively, in duplicates (Either treated samples or controls). After a one-minute centrifugation at 13,000 rpm, seawater was carefully removed. 50 µl of cold lysis buffer pH 7.4,10 mM NaCl, 3 mM MgCl2, 0.1% Igepal) were added and cells were lysed by gentle pipetting. While 25 µl of the lysate was centrifuged at 500 g for 10 minutes at 4°C, the other 25 µl were used to count nuclei after DNA labeling with DAPI (between 20,000 to 75,000 nuclei were used per transposition reaction). The supernatant was removed, the nuclei resuspended in the reaction mix (25 µl 2x TD buffer (Illumina), 2.5 µL Tn5 transposase (Illumina), 22.5 µL nuclease free H2O) and incubated at 37°C for 30 minutes. Following transposition, 3 µl of 3M AcoNa (pH5.3) were added to the reaction to adjust the pH, and the DNA was purified using the MinElute PCR purification Kit (Qiagen), following the manufacturer's instructions. The transposed DNA was eluted in 10 µL elution buffer preheated at 37°C.

To amplify the library, the following components were combined: 10 µL of transposed DNA, 10 µL nuclease free H2O, 2.5 µL Nextera PCR primer 1 59 (25 µM), 2.5 µL Nextera PCR primer 2 59 (25 µM) and 25 µL NEBNext® high-fidelity 2x PCR master mix (NEB). We used the following conditions for PCR amplification: 72°C for 5 minutes, 98°C for 30 seconds, followed by 13 cycles at 98°C for 10 seconds, 63°C for 30 seconds and 72°C for 1 minute. Following PCR amplification, 3 µl of 3M AcoNa (pH5.3) were added to the reaction to adjust the pH, and the library was purified using the MinElute PCR purification Kit (Qiagen), following the manufacturer's instructions using 20 µL of elution buffer preheated at 37°C. Sequencing was performed, producing an average of 60 million 50-nt paired-end reads per sample. Results obtained from the sequencing were analysed by the Gomez-Skarmeta laboratory in Sevilla 123 (CABD), to generate a list of under-represented and over-represented peaks. These peaks were finally deposited into the European Amphioxus UCSC Genome Browser website (http://genomeeuro.ucsc.edu).

Motifs analysis

Putative enhancer genomic sequences were obtained from the UCSC Browser website. The nucleotide sequences ranged from -100 to +100 bp relative to the peak center of the ATAC-seq data. To identify putative binding sites of DNA-binding proteins, the sequences were scanned with MATCH program inside the GeneXplain GmbH v4.9 platform using vertebrate position weight matrices from the TRANSFAC database (vertebrate_non_redundant). The parameters were established according to the best cut-off estimation for minimizing false positive rates (minFP) in extended genomic DNA sequences. Parallel analyses were carried out in collaboration with the CABD in Sevilla, using a suite of tools for Motif Discovery: HOMER (Hypergeometric Optimization of Motif EnRichment), considering a Pvalue <0.01. Motif analysis was done in whole embryos and ectodermal explants treated with Activin or Dorsomorphin. Results corresponding to 11hpf ectodermal explants treated with Activin are present in Supplementary Table 7.

cDNA cloning

All the sequences for classic neural markers were identified in the Branchiostoma lanceolatum assembled transcriptome, through BLASTn using Branchiostoma floridae and Branchiostoma belcheri sequences, or through tBLASTn, using the Homo sapiens and several vertebrates' sequences. RNA was extracted from embryos at different developmental stages using the RNeasy® Minikit from QIAGEN and cDNA was synthesized using the M-MLV reverse transcription kit from Promega. Next, neural markers and putative target genes were amplified from the cDNA by polymerase chain reaction (PCR), using gene-specific primers. The PCR products were cloned into the pGEM®T Easy Vector System from Promega, and sub-cloned into a pBks plasmid.

Supplementary

In this chapter, I included the articles that were part of my thesis project. In the first article, "Expression of Fox genes in the cephalochordate Branchiostoma lanceolatum", we analysed the expression pattern of several Fox genes during amphioxus development, as well as the phylogenetic relationships of Fox amphioxus genes within the Fox family that is composed by 24 classes. Fox genes as FoxAb, FoxD, FoxG and FoxJ present punctual neural expression patterns during the formation of the neural plate, and FoxAB is expressed in the dorsal blastorporal lip (amphioxus organizer) in early gastrulae stage. The expression is these genes are not neural specific, since they are also expressed in other territories, as somites and notochord. Here I worked with Aldea in producing data from ISH and phylogenetics analyses.

The second article "Nodal-Activin pathway is a conserved neural induction signal in chordates" recapitulates the base on which my thesis project was built. We demonstrated that Nodal is the instructive signal that triggers neural fate, and we showed enough evidence that Nodal and BMP Supplementary tables containing complementary RNA-seq data, and a list of enhancers containing SMAD binding sites, are also available in this chapter.

Supplementary Material

The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fevo.

2015.00080

Conflict of Interest Statement: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2015 Aldea, Leon, Bertrand and Escriva. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with theseterms.

ARTICLES NATURE ECOLOGY & EVOLUTION

alkaline phosphatase antibody and iodonitrotetrazolium/5-bromo-4-chloro-3indolyl phosphate substrate, Roche) was performed to reveal injected territories in fixed embryos. Two-cell embryos were injected with 400 pg of GR-t-Smad2 mRNA, animal caps were explanted at the early gastrula stage 10 and treated with dexamethasone at 10 μ g ml -1 . Negative controls were treated with 1% ethanol. Cycloheximide treatment (10 μ g ml -1 ) was started 1 h before to the addition of Activin (5 ng ml -1 , human recombinant protein, R&D) to avoid any delay in action, and treatment was continued for 2 h at 18 °C. SB505124 (Sigma-Aldrich) and SB431542 (Sigma-Aldrich) were dissolved in DMSO, diluted 1:100 in 0.1× MBS (final concentration 200 and 800 μ M, respectively) and applied to gastrula stage 11 embryos. Control embryos were treated with 1% DMSO (Sigma-Aldrich) diluted in 0.1× MBS.

In situ hybridization, immunostaining and quantitative RT-PCR. Amphioxus in situ hybridization was performed as previously described 42 and unless mentioned otherwise were carried out with at least 12 embryos, all of which the same phenotype. Amphioxus anti-tubulin immunostaining was carried out as described in ref. 19 using a primary antibody against acetylated tubulin (Sigma-Aldrich T6793, 1:500), and a secondary antibody (1:500) coupled to fluorescein. Immunostaining against pSmad1/5/8 was carried out using a rabbit polyclonal anti-pSmad1/5/8 primary antibody (Cell Signaling 9511 L, 1:150) and a secondary antibody (1:500) coupled to FITC. Images were processed in ImageJ using the parallel iterative deconvolution 2D plug-in. Xenopus embryos or animal caps were processed for in situ hybridization as described in ref. 11 . For quantitative RT-PCR, total RNA was extracted with the RNeasy mini kit (Qiagen), cDNA was synthesized using the SuperScript II reverse transcriptase (Invitrogen), and amplifications were performed in the presence of SYBRGreen (Invitrogen) on an iQ5 machine (Bio-Rad). Immunostaining against pSmad1 in Xenopus was carried out as described in ref. 43 using a rabbit polyclonal anti-pSmad1/5/8 primary antibody (Cell Signaling 9511 L, 1:100) and a secondary antibody (1:500) coupled to Alexa561. Accession numbers and primer sequences are given in Supplementary Tables 1,2.

RNA-seq analysis. Embryos from two females were separated in three independent batches for each female: control, dorsomorphin-treated and Activin-treated. Embryos were collected from each batch at the G4 (half of the embryos) and N2 stages and frozen in liquid nitrogen. RNA extraction was performed using the RNeasy mini kit (Qiagen). Sequencing was done at the CRG Genomics Unit and raw reads submitted to the BioProject database (accession number PRJNA354850). The clean sequencing reads from each sample were mapped to a Branchiostoma lanceolatum transcriptome assembly 44 using the software Bowtie2 with default parameters 45 . To calculate and normalize the mapped read counts, we used the software RSEM (http://deweylab.biostat.wisc. edu/rsem/) 46 . We analysed the differential expression between control and treated embryos for selected genes and generated a heatmap using Morpheus (https://software.broadinstitute.org/morpheus/).

Statement that all experiments were performed in accordance with relevant guidelines and regulations. All the experiments were performed following the Directive 2010/63/EU of the European parliament and of the council of 22 September 2010 on the protection of animals used for scientific purposes. All Xenopus experiments were approved by the 'Direction départementale de la Protection des Populations, Pôle Alimentation, Santé Animale, Environnement, des Bouches du Rhône' (agreement number F 13 055 21). Ripe adults from the Mediterranean invertebrate amphioxus species (B. lanceolatum) were collected at the Racou beach near Argelès-sur-Mer, France, (latitude 42° 32′ 53′ ′ N and longitude 3° 3′ 27′ ′ E) with specific permission from the Prefect of Region Provence Alpes Côte d' Azur.

Author contributions

Conceptualization of this study was done by Y.L.P., L.K., H.E. and S.B.; the study was carried out by Y.L.P., G.L., P.S., M.C., A.L., L.S., M.I., H.E. and S.B.; writing of the original draft was done by Y.L.P., L.K., H.E. and S.B.; funding was acquired by M.I., L.K., H.E. and S.B.; this study was supervised by L.K., H.E. and S.B.

Competing interests

The authors declare no competing financial interests.

Pitx-isoform1

Supplementary Table 1: RNA-seq analysis of all the TFs presenting superenhancers. Results were selected from the DESeq2 analyses from ectodermal explants at 11hpf treated with Activin vs control.