; Helene

Mathieu Hugo L Marc

Mariana Mathias

Nataly, Sandrine, Elsa Laetitia Stephan

Sandra

Nacho

Eleonora Fanny

Brieuc, Sheree, Medhi, Amandine, Valeria Tatiana Perrine

A Phillipe

Jean-Loius Christian

Estrella Carlos

Daniel Aldea

Lucie Subirana

Celine Keime

Jose Luis Gomez-Skarmeta

Sylvain Marcellini

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

Hector Escriva
email: hescriva@obs-banyuls.fr

Preface

"Nothing in biology makes sense except in the light of evolution" Theodosius Dobzhansky [START_REF] Dobzhansky | Nothing in Biology Makes Sense Except in the Light of Evolution[END_REF] After obtaining its own division in the Society for Integrative and Comparative Biology (SICB), the Evolutionary Developmental Biology (Evo-Devo) emerged officially as a discipline in 1999. However, the combination between developmental biology and evolutionary biology took place at least one decade before, when biologists began to look at and compare the expression patterns of developmental genes in different organisms. Unexpectedly, when they analyzed the expression of Hox genes at early stages in embryos of invertebrates (fruit fly) and vertebrates (mice), they found a conserved expression pattern in the anteroposterior axis among these animals. This was the beginning of fruitful decades for this "new discipline". In general terms, the Evo-Devo tries to unravel the evolutionary scenarios in which, from a unique ancestor, the appearance of all the morphological/anatomical characteristics and shapes observable today occurred.

The body of vertebrates is characterized by a highly specialized anterior structure called "the head". First attempts to understand the evolutionary origin of the vertebrates' head date from the beginning of the 19 th century. At that time, the discussion was taken under the framework of comparative embryology and the first hypotheses were postulated. Unravelling how novelties arise during evolution is one of the major tasks in Evo-Devo, thus in the last decades efforts to understand the origin of the vertebrates' head have brought new hypotheses to the scenario but many questions still remain to be fully clarified.

Since the appearance of the "new genomic era" in the early 2000s, many highthroughput tools have been developed and other classical tools have been improved. Thus, taking advantage of these new technologies it seems more than ever necessary to unravel the origin of the vertebrates' head (nothing in evolution makes sense except in the light of these new technologies). Under this framework, in the following pages, I will present my work using a cephalochordate (amphioxus) as an animal model to shed light on the origin of the vertebrates' head. In other words, in the context of the Evo-Devo discipline, through a comparative approach between amphioxus and vertebrates, and using both classical developmental biology and recent high-throughput techniques, I tried here to make one more step in the understanding of the evolutionary changes that precluded the evolution of the vertebrate's head.

Introduction

Unlike the other chordates (i.e. urochordates and cephalochordates) the vertebrates possess an extremely specialized structure in the anterior part of their body called "the head". This structure is composed by skeletal structures, muscles, primary sensory organs (vision, taste, smell, hearing and balance), and a complex organ (the brain) that process the information coming from outside. The arising of this novelty (the head) over more than 500 million years ago paced the transition between a filter-feeding to a predator life-style, and supposed the appearance of the first vertebrates. Even if no demonstration exists, it is extensively accepted that the last common ancestor of all chordates possessed its body completely segmented from the most anterior to the most posterior part of the body. However, the head of the vertebrates is unsegmented, even if some nerves appear as segments. At contrary to the trunk of the vertebrates, where the mesoderm shows a clear segmentation brought by the somites (structures derived from the paraxial mesoderm). In this first part of the introduction I will present you the three major postulated hypotheses about the origin of the vertebrates' head during the last centuries, as well as supporting data or controversial points for each of these hypotheses.

These three hypotheses are:

 Segmentalist hypothesis (it claims that the body -the head and the trunk-of vertebrates is formed by the same segmental process)  Non segmentalist hypothesis (it claims that the head is formed by a process different from the trunk)  The "New Head" hypothesis (it claims that the head is a completely new structure originated thank to the appearance of neural crest cells and placodes)

HYPOTHESES FOR THE ORIGIN OF THE VERTEBRATES' HEAD

Segmentalist Hypothesis

We can attribute the first ideas about the origin of the vertebrates' head to Goethe, who inspired by the thoughts of the German philosopher Immanuel Kant, proposed a model where the body of all living animals consists of equivalent segments (vertebrae), and the skull represents a modified part of these segments (vertebral theory) (Figure 1) [START_REF] Goethe | Das Schädelgrüt aus sechs Wirbelknochen aufgebaut[END_REF]. The idea of an "archetype" proposed by Goethe and from which any type of living animals could be derived by simple modifications was, later on, modified by the British zoologist Owen, who proposed an extreme "archetype" for vertebrates, where the final drawing showed an animal that possesses different characteristics coming from derived and ancestral traits. In this way Owen proposed that the formation of the anterior portion of the body (head) is similar to the posterior one (trunk), meaning that the whole body is formed in a metameric fashion (repeated segments) (Figure 1) [START_REF] Owen | The Principal Forms of the Skeleton and of the Teeth[END_REF]. The vertebral theory of Goethe. Goethe proposed that similarly to the vertebrae of the body, the skeleton of the mammalian skull is segmented. Then the different bones of the head would represent five head segments (1 to 5), (modified from [START_REF] Jollie | Segmentation of the vertebrate head[END_REF]). (B) The vertebrate archetype of Owen, who proposed that the anterior portion of the vertebrate body is similar to the posterior part of the vertebrate body (trunk), (modified from [START_REF] Owen | The Principal Forms of the Skeleton and of the Teeth[END_REF]). Abbreviations: ns, nasal; fr, frontal; par, parietal; ip, interval parietal; so, supraoccipital; eo, exoccipital; cv, cervical; as, aliphenoid; os, orbitosphenoid. Decades later, at the end of the 19 th century, Balfour described for the first time "head cavities" in shark embryos. In fact, he found three pairs of cavities (premandibular, mandibular and hyoid), that he compared with the somitic coeloms (cavities) of the trunk [START_REF] Balfour | A preliminary account of the development of the elasmobranch fishes[END_REF]Balfour, 1876). At the beginning of the 20 th century, Koltzoff and Damas described head somites during the development of lamprey embryos (the earliest divergent group of vertebrates), supporting the idea of a segmented origin of the head (Damas, 1944;Koltzoff, 1902). At the same time, Goodrich, who was one of the major proponents of a segmental structure of the head, proposed the presence of primary mesodermal segments in the head comparable to the somitic segments found in the trunk. He also claimed that the head was segmented into eight units, which represent a primitive condition of jawed vertebrates, and that the ancestor of all vertebrates was an amphioxus-like creature (Goodrich, 1918;Goodrich, 1930) (Figure 2). Notably, the body of extant amphioxus is completely segmented from the most anterior to the most posterior part, and possesses a similar morphology to vertebrates but much simpler. These reasons pushed zoologists at that time to think that amphioxus represented the primitive state of all vertebrates. In sum, unlike Goethe and Owen, who considered only skeletal elements in their hypotheses, Goodrich's model integrated nerves, muscles, and pharyngeal elements into one single metamere. In the head region, each compartment contains a nerve, a head somite, and a branchial arch. (B) When the skeletal and peripheral nervous systems are removed from the scheme, it is clear that Goodrich based his model on the segmentation of the paraxial mesoderm. Abbreviations: pm, premandibular cavity; mm, mandibular cavity; hm, hyoid cavity; ot, otic vesicle; pv, Platt's vesicle; s0-7, somites. Both figures were modified from (Goodrich, 1918;Kuratani et al., 1999).

Non Segmentalist Hypothesis

In contrast with the segmental view of the head, the "non segmentalist" movement, which arose late in the 19th century, proposed that the head of vertebrates was formed by a different segmental process of that of the trunk. Thus, Froriep [START_REF] Froriep | Entwicklungsgeschichte des Kopfes [part 1][END_REF][START_REF] Froriep | Entwicklungsgeschichte des Kopfes [part 2][END_REF] put in manifest his doubts about the homology between the preotic and postotic cavities and the most posterior trunk somites observed in lamprey embryos. Later, Kingsbury and Adelmann proposed that the components of the vertebrate head such as neuromeres (segments of the central nervous system), somitomeres (segments of paraxial mesoderm), and branchiomeres (segments of branchial arches), should not be integrated into single series of units (a single metamere), but as separated developmental processes [START_REF] Kingsbury | The developmental origin of the notochord[END_REF][START_REF] Kingsbury | Branchiomerism and the theory of head segmentation[END_REF][START_REF] Kingsbury | The morphological plan of the head[END_REF]. In 1972, Romer proposed a dual segmental theory, claiming that the segmental process forming the body somites must be considered as an independent process of that of the gills (pharyngeal arches) (Figure 3). In the same work, Romer also proposed that during the evolution the "somatic or active" part of the embryo, (i.e. most part of the muscles, bones and central nervous system) have been trying to gain the control of the "visceral or passive" part of the embryo, (i.e. the digestive tract and its appendages) [START_REF] Romer | The vertebrate as a dual animal-somatic and visceral[END_REF]. In this way, Romer also claimed that the chordates evolved from a primitive sessile arm-feeder (passive animal) to a tunicate. Then the free-swimming larva of the tunicate underwent secondary evolutionary events giving rise to a primitive filter-feeding vertebrate (active animal) [START_REF] Romer | The vertebrate as a dual animal-somatic and visceral[END_REF]. Nowadays, tunicates are not considered in the discussion of the origin of the vertebrates' head since they have apparently lost muscular somites and their phylogenetic position has been revised (see below). In the segmental hypothesis of Goodrich the vertebrate's head is assumed to contain only one type of segmentation that involves metamerism of paraxial mesodermal segments and pharyngeal arches. (B) Non segmentalist theory assumes independent patterns of metamerism for somites and pharyngeal arches questioning the presence of segments in the mesoderm of the head. Modified from [START_REF] Kuratani | Evolutionary developmental biology and vertebrate head segmentation: A perspective from developmental constraint[END_REF].

One of the most controversial points between both hypotheses (segmentalist/non segmentalist) is the existence or not of the "cephalic somitomeres" (i.e. segments in the cephalic mesoderm). The "cephalic somitomeres" were described for the first time in 1980s as bulges in the cephalic mesoderm of chick embryos [START_REF] Anderson | The influence of the metameric pattern in the mesoderm on migration of cranial neural crest cells in the chick embryo[END_REF][START_REF] Jacobson | Somitomeres: mesodermal segments of vertebrate embryos[END_REF][START_REF] Jacobson | Morphogenesis of the head of a newt: mesodermal segments, neuromeres, and distribution of neural crest[END_REF][START_REF] Meier | Development of the chick embryo mesoblast. Formation of the embryonic axis and establishment of the metameric pattern[END_REF][START_REF] Meier | Morphogenesis of the cranial segments and distribution of neural crest in the embryos of the snapping turtle, Chelydra serpentina[END_REF][START_REF] Meier | Metameric pattern development in the embryonic axis of the mouse. I. Differentiation of the cranial segments[END_REF]. Nevertheless, since then, there has been no clear evidence, at a molecular or cell lineage levels, about the existence of these "cephalic somitomeres" [START_REF] Freund | The metameric pattern of the head mesoderm-does it exist?[END_REF]Jouve et al., 2002). Thus, from a morphological level, any clear metameric pattern is observed in the head mesoderm that suggests the presence of the "cephalic somitomeres". Conversely, a segmental pattern is observed in the trunk due to the metameric formation of the somites. Similarly, a segmental pattern is observed in the cranial nerves due to rhombomeres and the pharyngeal pouches. [START_REF] Begbie | Induction of the epibranchial placodes[END_REF][START_REF] Begbie | Integration between the epibranchial placodes and the hindbrain[END_REF][START_REF] Kuratani | Rhombomere transplantation repatterns the segmental organization of cranial nerves and reveals cell-autonomous expression of a homeodomain protein[END_REF]. Altogether, this lead to Kuratani et al. to propose that the "cephalic somitomeres" rather than a real metameric process, could be a regionalization of the mesoderm into several domains induced by some other embryonic structures in the vicinities [START_REF] Horigome | Development of cephalic neural crest cells in embryos of Lampetra japonica, with special reference to the evolution of the jaw[END_REF]Kuratani et al., 1999). Nevertheless, new molecular data shows the expression of the segmental gene c-hairy in chicken embryos indicating an oscillatory pattern with two pulses entering into the cephalic mesoderm. The premandibular mesoderm (prechordal mesoderm) representing one wave and the rest of the head mesoderm representing the second wave (Jouve et al., 2002). Taken together, this suggests that the entire head mesoderm of vertebrates might be formed by two segments, in opposition to the hypothetical number of head mesoderm segments (seven or eight) assumed in the hypothetical vertebrate ancestor [START_REF] Holland | Embryonic development of heads, skeletons and amphioxus: Edwin S. Goodrich revisited[END_REF] (Figure 4). (Jouve et al., 2002). Each oscillation is numbered together with the mesodermal part generated after that oscillation. Note that there are only two oscillations in the head mesoderm, one for the premandibular mesoderm, and the other for the rest of the cephalic mesoderm. Abbreviation: (pmm) premandibular mesoderm or prechordal mesoderm, (cm) cephalic mesoderm, (som) somite. Modified from [START_REF] Kuratani | Craniofacial development and the evolution of the vertebrates: the old problems on a new background[END_REF].

Another controversial point between these two hypotheses is the presence of head cavities described in lampreys (Damas, 1944;Koltzoff, 1902). Their observation suggested that head mesoderm was segmented in the ancestral vertebrate. However, thanks to the development of scanning electron microscopy techniques in the last decades, it was possible to understand the head mesoderm morphology of lampreys in a much better way. Thus, Kuratani's laboratory was able to show in lamprey embryos that (i) there are no overt head cavities in the premandibular, mandibular, or hyoid mesoderm and (ii) the developmental sequence of the head mesoderm in lamprey embryos is completely different from that of trunk somites (Kuratani et al., 1999). Cyclostomes, to which belong lampreys and hagfishes, are the most early divergent group of vertebrates. Therefore, the results of Kuratani's laboratory suggest that the head cavities (premandibular, mandibular and hyoid cavities) observed in sharks are probably derived and not ancestral features.

The "New Head" Hypothesis

In 1983, a new hypothesis for the origin of the vertebrates' head was postulated by Northcutt and Gans [START_REF] Gans | Neural crest and the origin of vertebrates: a new head[END_REF]. Thus, thanks to the discovery and description of new cell populations as the neural crest and neurogenic placodes, they realized that these cell populations together with an unsegmented head mesoderm (derived from the lateral plate mesoderm) are exclusive to vertebrates and that during the evolution these structures played a crucial role to switch from filter feeding to active predation. Thus, they proposed that the rostral head of vertebrates is a completely new structure and that the neural crest and neurogenic placodes evolved from the epidermal nerve plexus of ancestral deuterostomes [START_REF] Gans | Neural crest and the origin of vertebrates: a new head[END_REF][START_REF] Northcutt | The genesis of neural crest and epidermal placodes: a reinterpretation of vertebrate origins[END_REF]. Recently, Northcutt has rejected their last claim, proposing that neural crest and neurogenic placodes evolved due to the rearrangement of germ layers in the blastulae of the ancestral deuterostome that gave rise to the chordates [START_REF] Northcutt | The new head hypothesis revisited[END_REF]. To better understand the "new head" hypothesis, the neural crest cells and neurogenic placodes derivatives are examined in more details below.

Neural Crest Cells

Firstly described in 1868 by the Swiss Embryologist Wilhelm His, the neural crest cells (NCCs) are a migratory group of cells derived from the ectodermal tissue and deepithelialized during embryogenesis. The NCCs were initially associated with the origins of neurons and ganglia, until the 1890s when Julia Platt demonstrated their role in the formation of the visceral cartilage of the head and in the teeth of the mud puppy Necturus [START_REF] Hall | The neural crest and neural crest cells: discovery and significance for theories of embryonic organization[END_REF][START_REF] Platt | The development of the cartilaginous skull and of the branchial and hypoglossal musculature in Necturus[END_REF][START_REF] Trainor | Origins and plasticity of neural crest cells and their roles in jaw and craniofacial evolution[END_REF]. However, this breakthrough was controversial and not completely accepted until 50 years later [START_REF] Horstadius | The neural crest[END_REF].

Nowadays, we have a more detailed view about NCCs and their derivatives. Molecularly, the NCCs are characterized by the expression of a set of transcription factor genes as AP2, Snail1/2, FoxD3 and SoxE [START_REF] Green | Evolution of vertebrates as viewed from the crest[END_REF]. In addition, and thanks to the initial work leaded by Le Douarin using chicken/quail chimeras [START_REF] Le Douarin | The neural crest[END_REF], it was possible to follow the derivatives of the NCCs. Thus, it is known that NCCs generate the peripheral nervous system (PNS), establishing the connection between the central nervous system (CNS) and the periphery. The melanocytes of the body also derive from NCCs, as the mesenchymal cells that are able to differentiate into connective tissue, adipose tissue, bone, cartilage and into cells forming the wall of blood vessels [START_REF] Dupin | The contribution of the neural crest to the vertebrate body[END_REF]. Regarding the contribution of the NCCs to the formation of the cranium and facial skeleton structures, it was established that most of these craniofacial structures in vertebrates are derived from cephalic NCCs [START_REF] Couly | The triple origin of skull in higher vertebrates: a study in quail-chick chimeras[END_REF] and that only the posterior part of the neurocranium (the portion of the skull that covers the brain) has a mesodermal origin (Figure 5) [START_REF] Couly | The developmental fate of the cephalic mesoderm in quail-chick chimeras[END_REF][START_REF] Couly | The triple origin of skull in higher vertebrates: a study in quail-chick chimeras[END_REF]. Additionally, while head muscles are derived mostly from cranial mesoderm (it will be treated later) their connective cells and attached tendons derive from cephalic NCCs [START_REF] Grenier | Relationship between neural crest cells and cranial mesoderm during head muscle development[END_REF]. [START_REF] Portman | Ernfiihrüng in die vergleichende Morphologie der Wirbeltier[END_REF], the vertebrate (gnathostome) skull is assumed to be composed of the cartilaginous neurocranium (light blue), the viscerocranium (light green), and the dermatocranium (brown). (B) Neural-crest-derived elements have been colored in red, and the mesodermal elements in blue, based on several cell-labelling and molecular genetic experiments reported by [START_REF] Couly | The triple origin of skull in higher vertebrates: a study in quail-chick chimeras[END_REF][START_REF] Le Lievre | Participation of neural crest-derived cells in the genesis of the skull in birds[END_REF][START_REF] Le Lievre | Mesenchymal derivatives of the neural crest: analysis of chimaeric quail and chick embryos[END_REF][START_REF] Morriss-Kay | Derivation of the mammalian skull vault[END_REF][START_REF] Noden | Craniofacial development: new views on old problems[END_REF]. (C) Distribution of the cephalic mesoderm (blue) and crest-derived ectomesenchyme (red) in the chicken pharyngula by [START_REF] Noden | Interactions and fates of avian craniofacial mesenchyme[END_REF]. (D) Results from chicken experiments were extrapolated to the human perinatal skull. Abbreviations: dc, dermatocranium; eth, ethmoidal region of the neurocranium; hy, hyoid arch; md, mandibular arch; mo, mouth; n, notochord; ncr, neurocranium; occ, occipital; ph, pharynx; pma, premandibular arch; vcr, viscerocranium; ver, vertebrae or vertebral column.

Neurogenic Placodes

In vertebrates the neurogenic placodes give rise to part of the cranial sensory apparatus (vision, taste, smell, balance and hearing). It has been established that all placodes arise from a common precursor territory, the preplacodal ectoderm (PPE) located around the anterior neural plate and neural crest, a region characterized by the expression of the transcription factor genes of the Six1/2 and Six4/5 families and of their coactivators from the Eya family [START_REF] Schlosser | Induction and specification of cranial placodes[END_REF]. The different derivatives of placodes are as follows: (i) the adenohypophyseal placode, which gives rise to the anterior pituitary, (ii) the olfactory placodes that generate the chemosensory neurons of the olfactory epithelium and the vomeronasal organ, (iii) the lens placodes that will form the lens of the eyes, (iv) the profundal and trigeminal placodes, which generate somatosensory neurons sensing temperature, touch and pain in the head, (v) the lateral line placodes that generate mechanosensory hair cells to detect movement in the water (these placodes have been lost in amniotes), (vi) the otic placodes that generate mechanosensory hair cells to detect auditory stimuli and, (vii) the epibranchial placodes, which form viscerosensory neurons (Figure 6) (Schlosser, 2015). In amniotes, profundal and trigeminal placodes are commonly referred to as ophthalamic and maxillomandibular placodes of the trigeminal nerve, respectively. Modified from [START_REF] Streit | Early development of the cranial sensory nervous system: from a common field to individual placodes[END_REF]. (B) Cranial placodes in a tailbud stage Xenopus embryo. Modified from [START_REF] Schlosser | Development of neurogenic placodes in Xenopus laevis[END_REF]. 1.1.3.3 The "New Head" hypothesis and the Brain As Gans and Northcutt observed 30 years ago, the contribution of the NCCs and neurogenic placodes to the formation of the head is tremendous, indicating that these vertebrate novelties have clearly played a crucial role for the appearance of the head. Interestingly, some facial structures do not develop from these tissues. These structures are the head muscles (their origin will be treated later). Additionally, in their work, Gans and Northcutt argued the difficulty to incorporate into the classical segmentalist views a NCCs origin for the skull, since segmentalists claimed a metameric process for the entire vertebrate body and the NCCs contribute only to the anterior (head) and not to the posterior (trunk) skeletal parts of the vertebrate body. Thus they discussed that it was unlikely that the vertebrate's head originated through modifications of the same processes that pattern segments in the trunk, i.e. the metameric idea of Goodrich.

Another point that they discussed is the fact that there is no homologue of the brain in either the cephalochordates or the urochordates. Therefore, in vertebrates, the sister group of both, the forebrain and midbrain would represent a neomorphic structure [START_REF] Gans | Neural crest and the origin of vertebrates: a new head[END_REF][START_REF] Northcutt | The new head hypothesis revisited[END_REF]. Nevertheless, more recent molecular and transmission electron microscopy (TEM) studies have revealed that indeed the cephalochordates present an homologue of the vertebrate midbrain [START_REF] Schubert | A retinoic acid-Hox hierarchy controls both anterior/posterior patterning and neuronal specification in the developing central nervous system of the cephalochordate amphioxus[END_REF] and an homologue of the vertebrate diencephalon (posterior part of the forebrain) [START_REF] Lacalli | The infundibular balance organ in amphioxus larvae and related aspects of cerebral vesicle organization[END_REF][START_REF] Lacalli | Basic features of the ancestral chordate brain: a protochordate perspective[END_REF]. Thus, the only neomorphic structure in vertebrates is the telencephalon (the anterior part of the forebrain) [START_REF] Holland | The origin and evolution of chordate nervous systems[END_REF]. Moreover, an homologous genetic program for patterning the brain in vertebrates is present in the hemichordate Saccoglossus kowalevskii (that belongs to the ambulacraria, the sister group of chordates) and partly in the invertebrate chordates (i.e. cephalochordates and urochordates), suggesting that these animals have not completely retained the basal genetic program patterning the brain, and that indeed the genetic program to build the entire brain is more ancient that previously thought [START_REF] Holland | Evolution of bilaterian central nervous systems: a single origin?[END_REF][START_REF] Pani | Ancient deuterostome origins of vertebrate brain signalling centres[END_REF].

Final remarks

The vertebrates' head is one of the most complex structures of all animals. During the last centuries, different works have contributed to increase our knowledge about its development in different vertebrate species (mice, frog, lamprey, and chicken). Nevertheless, its evolutionary origin still remains unclear. Firstly, hypotheses on the origin of the vertebrates' head claimed that the entire body of vertebrates, including the head, is formed by the same segmental process. Contrary to the segmental vision of the head, the non segmentalist movement claimed that the formation of the head and the trunk are independent processes. In the early 1980s, Gans and Northcutt highlighted that neural crest cells, neurogenic placodes and unsegmented head mesoderm are unique innovations of vertebrates and are linked to the origin of the vertebrates' head, and established the "new head" hypothesis. Thanks to the development of better microscopy techniques, in the 1990s, Kuratani showed that the cyclostomes, the most basally divergent group of vertebrates, do not possess head cavities, indicating that i) the origin of head mesoderm was probably unsegmented and ii) head cavities in sharks are probably a derived feature in Chondrichthyes. Therefore, even if the question of the origin of the vertebrates' head seems to be blurry, the data presented so far allow us to conclude that (i) there is a widely acceptance that the ancestor of all chordates was completely segmented all along the body (ii) the vertebrates' head is formed by a completely different process from the truncal segmentation (iii) neural crest cells and neurogenic placodes form most of the facial structures of the vertebrates' head and (iv) that the brain do not represent a unique novelty of the vertebrates since the genetic program for building a brain was already present in hemichordates.

Importantly the anterior mesoderm, which give rise some head structures in vertebrates (mostly muscles, it will be treated later), is an unsegmented tissue. Thus the evolutionary question arising from this observation is how an unsegmented anterior mesoderm arose from a hypothetical ancestor of all chordates possessing all its body segmented? Three different scenarios emerge to answer this question:

(i)
The addition of a completely new structure in the most anterior part of the body, a "new head". (ii)

The loss of anterior mesoderm segmentation (iii)

The loss of anterior mesoderm with a secondary acquisition of head mesoderm

The first scenario, the Gans and Northcutt scenario, supposes the appearance of a completely new structure, notably a complex structure including both the crane and the brain. This hypothesis seems unlikely because the cephalochordates (the most basally divergent group among chordates) possess homologous structures to the vertebrates' brain. Therefore, to distinguish between the last two scenarios, the use of an invertebrate chordate for comparative studies shedding light about the origin of the vertebrates' head seems an excellent choice. Below, I introduce the cephalochordate amphioxus as an animal model for these comparative studies and I explain why amphioxus is the best model to try to answer our question about the origin of the vertebrate's head.

AMPHIOXUS AS A MODEL

Identification and Description of Amphioxus

In 1774, the German zoologist Peter Simon Pallas described and classified a new mollusk that he called "Limax lanceolaris" (Limax = slug ; lanceolaris = lancet); it was the first description of amphioxus (Figure 7) [START_REF] Pallas | Limax lanceolatus. p. 19 + Plate I[END_REF]. Decades later, in 1834, Gabriel Costa, an Italian zoologist, recognized this "mollusk" as being closer to the vertebrates and renamed it as "Branchiostoma lubricus", because of its "mouth gills" (branchio = gills; stoma = mouth); this mouth gills were in fact the oral cirri of amphioxus. Thus, the name Branchiostoma remained as the Linnaean name of the genus [START_REF] Costa | Cenni zoologici, ossia descrizione somaria delle specie nuove di animali discoperti in diverse contrade del regno nell'anno 1834[END_REF]. In 1836, William Yarell described in Branchiostoma lubricus a structure characteristic of all chordates, the notochord. This structure in amphioxus extends all along the body (from the most anterior to the most posterior part), this is why amphioxus was named as cephalochordate (kephalé = head; khordé = chord). At the same time the name amphioxus was first used to designate cephalochordates (from the Greek; amphioxus = pointed on both sides) (Yarrell, 1836). The phylum Cephalochordata is composed by three genus; Branchiostoma, Asymmetron and Epigonichthys [START_REF] Poss | Lancelets (Cephalochordata: Branchiostomatidae): how many species are valid? Israel[END_REF]. All of them possess characteristics shared with all chordates (urochordates and vertebrates). Thus the cephalochordates possess a dorsal hollowed neural tube, a dorsal notochord, pharyngeal gill slits, segmented muscles, ventral gut, mouth, and anus (Figure 8). Amphioxus also possesses some structures shared with vertebrates as the endostyle and preoral pit that are homologous to the thyroid gland and to the adenohypophysis, respectively. On the other hand, amphioxus lacks some essential vertebrate structures, as an internal skeleton, neural crest cells (NCCs), placodes and paired sense organs (Bertrand and Escriva, 2011).

Phylogenetic Position and Amphioxus Species

Based on morphological characteristics and phylogenetic studies using the ribosomal ribonucleic acid coding gene sequences (rRNA) [START_REF] Winchell | Evaluating hypotheses of deuterostome phylogeny and chordate evolution with new LSU and SSU ribosomal DNA data[END_REF], cephalochordates were considered as the sister group of vertebrates for a long-time. However, recent genome-scale studies have shown that urochordates and not cephalochordates are the closest phylum to vertebrates [START_REF] Delsuc | Tunicates and not cephalochordates are the closest living relatives of vertebrates[END_REF][START_REF] Delsuc | Additional molecular support for the new chordate phylogeny[END_REF][START_REF] Putnam | The amphioxus genome and the evolution of the chordate karyotype[END_REF]. Additionally, new studies have identified in urochordates a type of migratory neural crestlike cells similar to the vertebrate neural crest cells, a typical characteristic of the vertebrates and absent in amphioxus [START_REF] Jeffery | Migratory neural crest-like cells form body pigmentation in a urochordate embryo[END_REF]. Thus, the new phylogenetic tree of life places the phylum Cephalochordata as the most basally divergent group among chordates (Figure 9). New genomic studies allowed to place cephalochordates (amphioxus) at the base of the chordates whereas the urochordates (i.e. Ciona) get placed as the sister group of vertebrates [START_REF] Delsuc | Tunicates and not cephalochordates are the closest living relatives of vertebrates[END_REF]. Two whole genome duplications occurred after the divergence of urochordates (2R). Another whole genome duplication took place during the evolution of the teleosts (3R). Times of phylogenetic divergence are not to scale, and the tree branches are intended only to depict general relationships. Modified from [START_REF] Aguinaldo | Evidence for a clade of nematodes, arthropods and other moulting animals[END_REF][START_REF] De Rosa | Hox genes in brachiopods and priapulids and protostome evolution[END_REF] In their study based on morphological characteristics, Poss and Boschung identified and characterized at least 23 different species from the genus Branchiostoma, and 7 from the genus Epigonichthys, showing that amphioxus has colonized all the seas except for the Arctic and Antarctic oceans (there is no study showing the contrary). They described a worldwide repartition of amphioxus living in shallow waters in the Mediterranean or Caribbean sea, and Pacific, Atlantic or Indian oceans (Figure 10) [START_REF] Poss | Lancelets (Cephalochordata: Branchiostomatidae): how many species are valid? Israel[END_REF]. Recent molecular analyses suggest that they might in fact be more species than described previously at least in the genus Branchiostoma and Asymmetron. Thereby, Branchiostoma belcheri has been subdivided into three different species, Branchiostoma belcheri, Branchiostoma japonicum and Branchiostoma tsingtauense [START_REF] Zhang | Branchiostoma japonicum and B. belcheri are distinct lancelets (Cephalochordata) in Xiamen waters in China[END_REF], and Asymmetron lucayanum also seems to be a multispecies clade (Kon et al 2006). Thus, new molecular analyses accompanied by new field collection seem to be needed to clarify the number of species and their phylogenetic relationships within the phylum Cephalochordata. Modified from [START_REF] Poss | Lancelets (Cephalochordata: Branchiostomatidae): how many species are valid? Israel[END_REF].

Environment

Amphioxus is a filter-feeder animal that usually lives in shallow waters burrowed into the sand leaving only its mouth outside the sediment for filtering sea water. Amphioxus lives in tropical and temperate sea waters with a preference for coarse sand as sediment. However, some species like for example the Caribbean species Branchiostoma floridae can be found in thinner sand [START_REF] Desdevises | A snapshot of the population structure of Branchiostoma lanceolatum in the Racou beach, France, during its spawning season[END_REF][START_REF] Gosselck | Age structure, growth and weight of Branchiostoma senegalense (Acrania, Branchiostomidae) off North-West Africa[END_REF][START_REF] Webb | The ecology of Lagos lagoon. IV. On the reactions of Branchiostoma nigeriense Webb to its environment[END_REF]. So far, only one species living in deep waters (229 meters depth) has been identified and called Asymmetron inferum [START_REF] Kon | Phylogenetic position of a whale-fall lancelet (Cephalochordata) inferred from whole mitochondrial genome sequences[END_REF].

Reproduction and Life Cycle

All the species in the phylum Cephalochordata are gonochoric and reproduce sexually by external fertilization. The spawning season of different amphioxus species usually corresponds to the spring-summer season and it spans during three to six months per year depending on the species. For instance, the spawning season for B. belcheri and B. lanceolatum takes place during two or three months, whereas for B. floridae it lasts almost five months. The increase of the temperature of the sea water during the spring-summer triggers the spawning in most of the amphioxus species. A. lucayanum is an exception since it is able to spawn during two different periods of the year, one during the summer and the other one during the autumn [START_REF] Holland | Laboratory spawning and development of the Bahama lancelet, Asymmetron lucayanum (cephalochordata): fertilization through feeding larvae[END_REF]. Normally, after the sunset, amphioxus swim up into the water column and release their gametes, coming back afterwards into the sand. 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. Another case is represented by A. lucayanum, that is apparently influenced by the lunar cycle, thus most of the population tends to spawn the day after the full moon [START_REF] Holland | Laboratory spawning and development of the Bahama lancelet, Asymmetron lucayanum (cephalochordata): fertilization through feeding larvae[END_REF]. After fertilization, the embryos develop and form a larva with a planktonic life style until they reach metamorphosis. The length of the planktonic period depends on each species. Thus in the case of B. lanceolatum this period takes 2-3 months and for B. floridae 2-3 weeks. At the end of metamorphosis, the juvenile become benthonic and goes into the sand where it continues growing until it reaches the adult stage (Bertrand and Escriva, 2011). Regarding the lifespan of amphioxus, again it depends on the species. Thus, it has been published that B. floridae can live between 2-3 years and B. lanceolatum between 5-8 years (Bertrand and Escriva, 2011;[START_REF] Futch | Nearshore marine ecology at Hutchinson Island, Florida: 1971-1974: IV. lancelets and fishes[END_REF].

Embryonic Development

The embryonic development of the genus Branchiostoma has been very well described and studied for more than 150 years [START_REF] Cerfontaine | Recherches sur le development de l'amphioxus[END_REF][START_REF] Conklin | The embryology of amphioxus[END_REF][START_REF] Hatschek | Amphioxus and its Development[END_REF][START_REF] Kowalevsky | Entwickelungsgeschichte des Amphioxus lanceolatus[END_REF][START_REF] Kowalevsky | Weitere studienuber die entwicklungsgechichte des Amphioxus lanceolatus, etc[END_REF][START_REF] Wilson | On multiple and partial development in amphioxus[END_REF][START_REF] Wilson | Amphioxus and the mosaic theory of development[END_REF]. Regarding the genus Epigonichthys, there is no study describing its embryonic development. For the genus Asymmetron, there is only one study where the authors show that the embryonic development of A. lucayanum is similar to what is observed in the genus Branchiostoma, finding differences only at the beginning of the larva stage [START_REF] Holland | Laboratory spawning and development of the Bahama lancelet, Asymmetron lucayanum (cephalochordata): fertilization through feeding larvae[END_REF]. During my research project, I used the species B. lanceolatum as an animal model, therefore the embryonic stages described in this work correspond to the embryonic development of this species at 19 °C (Bertrand and Escriva, 2011;Fuentes et al., 2007;Fuentes et al., 2004).

From fertilization until the gastrula stage, in particular during gastrulation, the development of amphioxus is similar to the one of invertebrate deuterostomes (i.e. a hollow blastula invaginates to form a gastrula in a similar manner to the sea urchin). However, the gastrula stage could be considered as a transition state towards a vertebrate-like development, since at the end of this stage the formation of characteristic structures of chordates as the notochord, the neural tube and the somites starts. The embryonic development of amphioxus is carried out as follows.

Fecundation to blastula stage

Once the spermatozoid has fertilized the oocyte, the chorion or the fertilization membrane raises and coats the zygote avoiding polyspermy and protecting the zygote from the exterior. The next phase, called cleavage, corresponds to the segmentation of the zygote. Thus, the first division occurs after 90 minutes, then after seven synchronic divisions the blastula stage is reached, this stage is characterized by the presence of a blastocoel, an internal cavity without communication with the exterior. During the blastula stage, it is possible to identify the cells that will give rise to the mesendoderm and ectoderm, being the mesendodermal cells bigger than the ectodermal cells. This observation is based on morphological description or cell lineage labelling, but not on molecular identification [START_REF] Holland | Early development of cephalochordates (amphioxus). Wiley interdisciplinary reviews[END_REF].

Gastrulation

The beginning of gastrulation corresponds to a flattening of the blastula at the vegetal pole where the cells will become mesendodermal cells. Then, a movement of invagination of the vegetal pole into the blastocoel is observed, until the vegetal pole touches the animal pole (Figure 11). In amphioxus, during gastrulation, a second movement is observed, that is the involution of some cells at the level of the blastoporal lip. At the end of gastrulation, two germ layers are formed: (i) one internal layer called mesendoderm that will give rise to the endoderm in the ventral part, and to the mesoderm in the dorsal part; (ii) and one external layer, the ectoderm, that will give rise two different tissues, the epidermis in the anterior and ventral part, and the neural plate (neuroectoderm) in the dorsal region of the gastrula (Figure 11).

Figure 11. Two germ layers are formed during gastrulation in amphioxus. In this schematic representation the fate map of the different tissues and their movements during gastrulation are depicted. The presumptive ectoderm (blue), endoderm (yellow), neural plate (green), notochord (orange) and the paraxial mesoderm that will give rise to the somites (light orange) are depicted during gastrulation. Lateral views for all except for blastula stage and blastoporal view of gastrula. Abbreviations: (An) animal pole, (Veg) vegetal pole, (D) dorsal part, (V) ventral part, (A) anterior part, (P) posterior part of the embryo. Modified from [START_REF] Holland | Early development of cephalochordates (amphioxus). Wiley interdisciplinary reviews[END_REF].

Neurulation to metamorphosis

The neurula stage starts by the flattening of the dorsal part of the gastrula forming the neural plate. Then major events are observed. At the level of the ectoderm, the epidermal part completely detaches from the neural plate and cells fuse in the dorsal midline. On the other hand, the edges of the neural plate begin to fold until they fuse to form the neural tube. Regarding the dorsal mesoderm, it is possible to differentiate three regions, the axial mesoderm (central position), that will give rise to the notochord, and the paraxial mesoderm (both sides of the axial mesoderm), that will give rise to the anterior somites. At the same time, the blastopore begins to close posteriorly. Then, the embryo elongates through the addition of new structures in the posterior part produced by the tailbud, which derives from the blastoporal lips, until the larval stage (Figure 12). Figure 12. Neurulation in amphioxus. In this schematic representation it is possible to observe the events occurring during neurulation in amphioxus. At late gastrula stage the mesoderm (red) extends along the antero-posterior axis, and the paraxial mesoderm (red) and neural plate (dark blue) are differentiated from the axial mesoderm and non-neural ectoderm, respectively. At early neurula stage the mesoderm separates by constriction laterally to form somites and middorsally to form the notochord. The epidermal ectoderm spreads over the neural plate. At neurula and late neurula stages the somites are completely formed and spreads laterally and ventrally to form the body coelom. The neural tube is formed through the dorsal folding of the lateral edges of the neural plate until they fuse at the midline. Modified from [START_REF] Langeland | An amphioxus snail gene: expression in paraxial mesoderm and neural plate suggests a conserved role in patterning the chordate embryo[END_REF].

At the end of the neurula stage (subdivided into; early neurula (N1), mid neurula (N2), and late neurula (N3) stages) the first signs of pharyngeal enlargement are observed [START_REF] Hirakow | Electron microscopic study of the development of amphioxus, Branchiostoma belcheri tsingtauense: the neurula and larva. Kaibogaku zasshi[END_REF]. From this moment the embryo enters into the so-called premouth stage, which is characterized by the development of structures such as the pharynx, mouth and digestive tube. Once these structures are developed the larval stage begins. The planktonic larvae remain growing and adding new somites at the posterior part and gill slits in the pharyngeal region. The number of gill slits that are formed before metamorphosis is different for each amphioxus species, but in the case of B. lanceolatum metamorphosis occurs after the formation of 13-15 gill slits which takes between 2-3 months. During the larval stage, the gill slits are formed asymmetrycally as well as 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 (half-somite) than the right-side somites. Finally, during the metamorphosis, the mouth gets positioned rostrally. The gill slits are duplicated into two rows in a first time, and then one of these rows migrates to the left side. Moreover, the metapleural folds develop and cover the pharynx forming the atrium. The intestine gets also regionalized and the hepatic caecum is formed (Figure 13). (Bertrand and Escriva, 2011).

Amphioxus as an Animal Model in EVO-DEVO

Amphioxus has always been considered a fascinating animal model for answering evolutionary questions. Thus, with the recent phylogenetic data, amphioxus is now occupying an interesting phylogenetic position as the most basally divergent group among chordates [START_REF] Delsuc | Tunicates and not cephalochordates are the closest living relatives of vertebrates[END_REF]. Moreover, fossil records dating from 520 million years ago (Pikaia gracilens from the middle Cambrian found in Burges Shale, and fossil records from the lower Cambrian, in particular Yunnanozoon, Haikouichthys and Myllokunmingia found in Chengjiang, China) show morphological characteristics (Figure 14) that could be considered similar to those harbored by amphioxus (Figure 8). Even if the phylogenetic position of some fossil records is still controversial, it is likely that the ancestor of all chordates possessed an amphioxus-like body-plan, making amphioxus the only living animal with a high resemblance to the hypothetical chordate ancestor. [START_REF] Bertrand | Amphioxus: how to become a vertebrate][END_REF]. (C) Pikaia gracilens found in Burges Shale in Canada, modified from [START_REF] Long | The rise of fishes: 500 million years of evolution[END_REF].

During the last decades the advances in whole genome analyses showed that amphioxus possess a "simple" genome [START_REF] Putnam | The amphioxus genome and the evolution of the chordate karyotype[END_REF]. Thus, as it was proposed by Ohno [START_REF] Ohno | Evolution by Gene Duplication[END_REF], and confirmed later by [START_REF] Dehal | Two rounds of whole genome duplication in the ancestral vertebrate[END_REF] two rounds of whole genome duplications occurred during the evolutionary history of vertebrates (three in teleosts) (Figure 9) [START_REF] Jaillon | Genome duplication in the teleost fish Tetraodon nigroviridis reveals the early vertebrate proto-karyotype[END_REF][START_REF] Meyer | Gene and genome duplications in vertebrates: the one-to-four (-to-eight in fish) rule and the evolution of novel gene functions[END_REF][START_REF] Taylor | Genome duplication, a trait shared by 22000 species of ray-finned fish[END_REF]). What still remains controversial is the precise timing of these duplications. In any case, the duplications happened after the divergence of cephalochordates and urochordates [START_REF] Putnam | The amphioxus genome and the evolution of the chordate karyotype[END_REF] and before the divergence of chondrichtyans [START_REF] Robinson-Rechavi | Phylogenetic dating and characterization of gene duplications in vertebrates: the cartilaginous fish reference[END_REF]. Unfortunately, the complete genome sequence of a key animal to understand genome duplications in vertebrates, the lamprey, was not able to completely demonstrate the exact timing of the two rounds of genome duplications, even if it was suggested that both occurred before the cyclostomes divergence [START_REF] Smith | Sequencing of the sea lamprey (Petromyzon marinus) genome provides insights into vertebrate evolution[END_REF]. An example of the result of the whole genome duplications is represented by the Hox clusters in vertebrates. Thus, there is a unique Hox cluster in amphioxus [START_REF] Garcia-Fernandez | Archetypal organization of the amphioxus Hox gene cluster[END_REF] compared to four clusters in mammals, and for each specific Hox gene in amphioxus there are between one and four Hox orthologue genes in most vertebrates (Figure 15). Nevertheless, as any other animal, amphioxus has its own evolutionary history and possesses 15 Hox genes instead of the 14 assumed to have been present in the chordate ancestor, suggesting that a specific duplication of one Hox gene occurred in amphioxus (Holland et al., 2008a). The fact that amphioxus possess a "simple" genome represents an advantage to understand the evolution of the function of different signalling pathways. For instance in amphioxus there is only one FGF receptor and 8 ligands compared with the four FGF receptors and 22 ligands in vertebrates (Oulion et al., 2012b). The simple inhibition of the FGF receptor in amphioxus can show us its direct role during the embryonic development of amphioxus (Bertrand et al., 2011). On the contrary, in vertebrates, the multiple FGF receptors and ligands makes more complicated the interpretation of the results of their inhibition during development. [START_REF] Ohno | Evolution by Gene Duplication[END_REF]) (2R), there are four Hox clusters (HoxA to HoxD) in vertebrates. In amphioxus there is only one Hox cluster since it diverged before the 2R. The genes Mox and Evx flanking the Hox cluster in amphioxus and vertebrates confirmed the synteny. Also it is possible to observe the losses of certain Hox genes in vertebrates. Modified from [START_REF] David | How Hox genes can shed light on the place of echinoderms among the deuterostomes[END_REF].

Even if several species form the phylum Cephalochordata, only three or four of them are used for Evo-Devo studies. The most used species are the Mediterranean species B. lanceolatum, the Caribbean specie B. floridae and the Asian species B. belcheri. Importantly, for these species the genome and transcriptome are publicly available [START_REF] Huang | Decelerated genome evolution in modern vertebrates revealed by analysis of multiple lancelet genomes[END_REF][START_REF] Mou | EST analysis of mRNAs expressed in neurula of Chinese amphioxus[END_REF]Oulion et al., 2012a;[START_REF] Putnam | The amphioxus genome and the evolution of the chordate karyotype[END_REF]. Additionally, a few studies have also been undertaken after the correct classification of the different Asian species, so today we can find literature for Branchiostoma japonicum and Branchiostoma tsingtauense and more interestingly in a species from a different genus, Asymmetron lucayanum [START_REF] Holland | The origin and evolution of chordate nervous systems[END_REF]. Regarding the possibility to use amphioxus as a model for evo-devo studies, a lot has been done during the last 30 years. Thus, during the summer time (from May to August), for the Mediterranean species B. lanceolatum, it has been shown that an increase of 3-4 °C of the water temperature during 36 hours can trigger spawning of the animals in captivity, making it possible to obtain embryos every night (Fuentes et al., 2007;Fuentes et al., 2004). On the contrary, for the species B. belcheri and B. floridae, the induction of spawning has been less studied, and it is only possible to obtain embryos during the natural field spawning nights.

Besides in situ hybridization as the classical experimental technique, amphioxus offers the possibility to interfere with several signaling pathways by using pharmacological treatments directly added to the seawater in which the embryos develop (Bertrand and Escriva, 2011). Performing immunohistochemistry staining is also possible in amphioxus using specific antibodies or heterologous antibodies used in vertebrates designed against conserved epitopes (Figure 16). Finally, concerning our ability to modify gene function, although microinjections in eggs of amphioxus were established 10 years ago, a lot of improvements are still needed. Indeed, mRNA injection allows overexpression of a given gene in all the amphioxus species, but knock-down is only efficiently working using morpholino antisense oligonucleotides injection in the Caribbean species B. floridae [START_REF] Holland | Analyses of gene function in amphioxus embryos by microinjection of mRNAs and morpholino oligonucleotides[END_REF]. In addition, recently for the Asian specie B. belcheri, a new method to induce direct deletions, mutations or insertions in the genome have been reported, thus the transcription activator-like effector nuclease (TALEN) method seems to be effective in amphioxus as in vertebrates (zebrafish, frog, rat, mouse) [START_REF] Li | Mutagenesis at specific genomic loci of amphioxus Branchiostoma belcheri using TALEN method[END_REF]. Finally, microinjections of plasmids can also be used in several species to obtain transient mosaic transgenic embryos. (H-J) Immunohistochemistry labelling using antibodies against phosphorylated histone H3 (H, I) and acetylated tubulin (J) in embryos at gastrula (I) and late neurula (H, J) stages. (K-L) Pharmacological treatment using the inhibitor of the fibroblast growth factors (FGFs) signaling pathway SU5402. In control larva the expression of Brachyury is restricted to the tailbud (K), whereas in treated larva the expression is observed throughout the entire notochord which elongated during the treatment period (L). (M) Transient transgenic amphioxus obtained by microinjection of the reporter plasmid p339_hsp70-GFP. Figure extracted from (Bertrand and Escriva, 2011).

Final remarks

Firstly described more than 200 years ago, amphioxus called the attention of researchers because of its morphologically simple characteristics resembling a vertebrate, but at the same time lacking some essential features as limbs, internal skeleton, neural crest cells or neurogenic placodes among others. In the last decades, and supported by phylogenetic data, amphioxus was positioned as the most basally divergent group among chordates. In addition, even if the interpretations based on fossil records are always controversial, it is extensively accepted that the ancestor of all chordates possessed an amphioxus-like morphology with a body completely segmented. Due to the effort of several laboratories around the world, amphioxus has emerged as a new animal model for the study of the invertebrate-chordate to vertebrate transition. Importantly, in the European species B. lanceolatum, the spawning can be controlled by a temperature shock allowing us to get embryos every night during the natural spawning season of this species. In addition, several molecular tools have been implemented (microinjection of unfertilized eggs, pharmacological treatments, in situ hybridization or high throughput analyses among others). Undoubtedly, a lot has to be done in the future (CRISPR/Cas9 technology, the possibility to get embryos all year long or to complete the life cycle in captivity). Nonetheless the tools available today allow us to use amphioxus as a good approach to understand how novelties arose in vertebrates.

The body of amphioxus shows a clear segmentation given by the somites (structures derived from the paraxial mesoderm) along the antero-posterior axis. Moreover, the most anterior part of amphioxus lacks all the structures that define a vertebrate head, such as sensory paired organs (eyes, noise or ears), a complex brain, skeletal elements or particularly, an unsegmented mesoderm. Indeed, it is believed that the loss of segmented somites in the anterior part of the vertebrate embryo during evolution allowed to release the developmental constrains imposed by the somites and the formation of new structures. Thus, functional elucidation of the development of anterior somitogenesis in amphioxus may shed light on differences with vertebrates that explain the evolution of the head. Are all the somites of amphioxus formed by the same segmental process? Are there any differences between the anterior and posterior somites? Is the genetic program for the formation of somites similar between vertebrates and cephalochordates? These are the questions that will be treated in the next chapters.

SOMITOGENESIS

The most conspicuous part of the amphioxus body are the muscles. In amphioxus, muscles extend from the most anterior to the most posterior part of the body and give to amphioxus the ability to generate undulatory movements to escape from predators or to change their position into the sand. The muscles in amphioxus derive exclusively from the somites, therefore in the following pages I will show the morphological, developmental and genetic programs known to be involved in somitogenesis and myogenesis in amphioxus and vertebrates. Importantly, since facial muscles in vertebrates do not develop from somites, I will also treat in a separate chapter the formation of facial muscle structures in vertebrates. Finally, I will treat the differences between anterior and posterior somitogenesis in amphioxus and I will discuss all these data in the context of the origin of the vertebrates' head.

Somitogenesis in vertebrates and amphioxus: a morphological description

In vertebrates, somites develop from the mesenchyme present in the tailbud region called presomitic mesoderm (PSM), in the most posterior region of the embryo. Somites form as spherical epithelial structures, derived from this presomitic mesoderm (PSM) via a mesenchymal-to-epithelial transition (Hubaud and Pourquie, 2014;[START_REF] Yabe | Molecular mechanism for cyclic generation of somites: Lessons from mice and zebrafish[END_REF]. Once the somites are formed, they are compartmentalized. Thus, the ventral portion of the somite is de-epithelialized to form the mesenchymal sclerotome, the dorsal portion called dermomyotome remains as an epithelial sheet and the myotome arise later between the sclerotome and the dermomyotome by delamination of its edges (Figure 17a-b). Each of these compartments will give rise to different tissues. Hence, the sclerotome will give rise to the axial skeleton, the dermomyotome to the dorsal dermis and skeletal muscles and the myotome to the skeletal muscle precursors [START_REF] Brent | Developmental regulation of somite derivatives: muscle, cartilage and tendon[END_REF].

In amphioxus, somites can be divided morphologically in two classes: the most anterior 8-10 somite pairs, pinch off by enterocoely from dorsolateral grooves of the archenteron at early neurula (N1) stage as a single mesodermal layer. And then, at the beginning of late neurula stage (N3), the new somites are formed by schizocoely one at a time, by budding off from the epithelium that surround the neurenteric canal, allowing the elongation of the body in the posterior part (Figure 18) (Beaster-Jones et al., 2008;Mansfield et al., 2015;Schubert et al., 2001b). Thus, the posterior somites of amphioxus unlike those in vertebrates that are generated by budding off from the PSM, are derived directly from an epithelium (i.e. amphioxus lacks a PSM region in its posterior part of the body), and do not undergo a mesenchymal-to-epithelial transition. Then, amphioxus somites are divided into myotome (medial) and non-myotome (lateral) compartments. The myotome will give rise to the myomeres that will constitute the body musculature. The non-myotome compartment is divided in dermomyotome (external cell layer), presumptive lateral plate and sclerotome. The dermomyotome will give rise to the dermis, connective tissues, and the fin box (a segmented structure formed dorsal to the neural tube). The presumptive lateral plate will give rise to the perivisceral coelom. Thus, while in vertebrates the sclerotome gives rise to structures as cartilages or bones, amphioxus do not possess such structures, instead the sclerotome in amphioxus will give rise to the mesothelium that encloses the sclerocoel separating the myotomes from other structures as notochord and neural tube (Figure 17c) (Mansfield et al., 2015;[START_REF] Scaal | Somite compartments in anamniotes[END_REF].

Molecular Control of Somitogenesis

The clock and wavefront model

If the precise anatomical structures of vertebrate's tailbuds vary from one species to the other, the global mechanism of posterior elongation is similar, and the molecular control of this process seems to be conserved. Indeed, it has been shown that FGF, Wnt and retinoic acid (RA) signaling pathways are interacting in all vertebrates to allow a harmonious elongation of the posterior body structures even if the fine interactions between these pathways are still not completely elucidated [START_REF] Aulehla | Signaling gradients during paraxial mesoderm development[END_REF]Hubaud and Pourquie, 2014;[START_REF] Krol | Evolutionary plasticity of segmentation clock networks[END_REF].

Different studies on the elongation process have shown that the function of FGF signaling is both to maintain a proliferative zone formed by pluripotent cells in the most posterior part of the embryo, thereby allowing growth of the axis, and to control the timing of cell differentiation. Indeed, FGFs ligands have been shown to inhibit cell differentiation in the caudal part of the embryo [START_REF] Boulet | Signaling by FGF4 and FGF8 is required for axial elongation of the mouse embryo[END_REF][START_REF] Dubrulle | fgf8 mRNA decay establishes a gradient that couples axial elongation to patterning in the vertebrate embryo[END_REF]. Thus, the attenuation of the FGF signal is required for mesodermal differentiation as well as for positioning the segment boundaries of the nascent somites in the mesoderm [START_REF] Akiyama | An anterior limit of FGF/Erk signal activity marks the earliest future somite boundary in zebrafish[END_REF]. But absence of the FGF signal is not sufficient to induce cell differentiation, and another signal is needed. This signal is RA, coming from the anterior already formed somites [START_REF] Maden | Abnormalities of somite development in the absence of retinoic acid[END_REF]. Thus, RA attenuates FGF signaling in the paraxial mesoderm, where it also controls somite boundary position. In fact it has been proposed that FGF and RA form opposing signals that downregulate each other [START_REF] Del Corral | Opposing FGF and retinoid pathways control ventral neural pattern, neuronal differentiation, and segmentation during body axis extension[END_REF][START_REF] Kumar | Retinoic acid controls body axis extension by directly repressing Fgf8 transcription[END_REF]. Many data also demonstrate the implication of Wnt signaling in the posterior elongation of vertebrate embryos. One of the roles of Wnts is to balance the opposing FGF/RA signals [START_REF] Olivera-Martinez | Wnt signals provide a timing mechanism for the FGF-retinoid differentiation switch during vertebrate body axis extension[END_REF]. Indeed, some Wnts expressed in the caudal part of the embryo can activate FGF signaling. Wnts also act in concert with FGFs to maintain the cells of the proliferative zone in an undifferentiated state, whereas more anteriorly, where FGF signaling is absent, Wnts can activate RA signaling by increasing Raldh2 expression (an enzyme of RA synthesis) [START_REF] Aulehla | Wnt3a plays a major role in the segmentation clock controlling somitogenesis[END_REF][START_REF] Aulehla | A beta-catenin gradient links the clock and wavefront systems in mouse embryo segmentation[END_REF][START_REF] Del Corral | Opposing FGF and retinoid pathways control ventral neural pattern, neuronal differentiation, and segmentation during body axis extension[END_REF][START_REF] Dunty | Wnt3a/beta-catenin signaling controls posterior body development by coordinating mesoderm formation and segmentation[END_REF][START_REF] Vermot | Retinoic acid coordinates somitogenesis and left-right patterning in vertebrate embryos[END_REF][START_REF] Zhao | Effect of retinoic acid signaling on Wnt/beta-catenin and FGF signaling during body axis extension[END_REF] This model in which FGF, Wnt and RA interact allows both cell proliferation and regionalization of the extending embryonic axis. Indeed, regionalization of the mesoderm is concomitant with a segmentation process giving rise to the somites. In this case, FGF, Wnt and RA signals, as well as Notch, interact through the so-called "clock and wavefront model" [START_REF] Cooke | A clock and wavefront model for control of the number of repeated structures during animal morphogenesis[END_REF][START_REF] Dequeant | A complex oscillating network of signaling genes underlies the mouse segmentation clock[END_REF]Hubaud and Pourquie, 2014). This interaction permits the synchronized activation of segmentation genes in the presomitic mesoderm (PSM) in response to a periodical signal emitted by a mechanism acting as a segmentation clock. So, sustained oscillation occurs in the FGF, Wnt, and Notch signaling pathways mainly due to negative feedback processes (mediated by Lunatic fringe in the Notch pathway, by Axin2 in the Wnt pathway, and by MKP3/Dusp6 in the FGF pathway) [START_REF] Aulehla | Wnt3a plays a major role in the segmentation clock controlling somitogenesis[END_REF][START_REF] Dequeant | A complex oscillating network of signaling genes underlies the mouse segmentation clock[END_REF][START_REF] Ferjentsik | Notch is a critical component of the mouse somitogenesis oscillator and is essential for the formation of the somites[END_REF]. Oscillations start at the most posterior part of the presomitic mesoderm and at its anterior part, the opposition of the FGF and RA pathways define a border, the so-called wavefront, where the oscillations end, and the tissue is subdivided into a prospective somite with rostro-caudal polarity [START_REF] Del Corral | Opposing FGF and retinoid pathways control ventral neural pattern, neuronal differentiation, and segmentation during body axis extension[END_REF][START_REF] Kumar | Retinoic acid controls body axis extension by directly repressing Fgf8 transcription[END_REF]. Thus, the opposition of the FGF and RA pathways defines the position of the future somite boundary during the process of segmentation. The final segmentation step is the establishment of a morphological somite boundary which involves a mesenchymal-to-epithelial transition. Moreover, RA signal, besides its role in the differentiation and patterning of the emerging somites, is also required for the synchronous and symmetrical left-right development of somites [START_REF] Vermot | Retinoic acid coordinates somitogenesis and left-right patterning in vertebrate embryos[END_REF].

In addition to FGF, Wnt and RA other genes, mainly controlled by these signals, play important roles in all vertebrates studied so far suggesting their conserved role across vertebrates. For example, the segmentation clock is defined by at least one member of the HES/HER family, (Hubaud and Pourquie, 2014). Thus, the oscillatory expression of the segmentation gene Hairy1, an effector of the Notch pathway, patterns the PSM [START_REF] Palmeirim | Avian hairy gene expression identifies a molecular clock linked to vertebrate segmentation and somitogenesis[END_REF]. Additionally, when FGF signal is inhibited the posterior expression of the master regulator for the segmentation clock Hes7 disappear [START_REF] Niwa | The initiation and propagation of Hes7 oscillation are cooperatively regulated by Fgf and notch signaling in the somite segmentation clock[END_REF]. Moreover, Axin2 a negative regulator of the WNT pathway shows oscillatory expression in the PSM. Additionally, mesoderm posterior (MESP) genes are master regulators of the segmental program that forms the future segment boundary. They are activated immediately after the determination front, as a bilateral stripe. Indeed, inactivation of Mesp1 and Mesp2 genes in the PSM blocks segment formation in mouse [START_REF] Oginuma | Identification of presomitic mesoderm (PSM)-specific Mesp1 enhancer and generation of a PSM-specific Mesp1/Mesp2-null mouse using BAC-based rescue technology[END_REF]. Interestingly, Mesp2 is activated by Tbx6-mediated Notch signaling and repressed by the FGF signal [START_REF] Kageyama | Oscillatory gene expression and somitogenesis[END_REF] (Figure 19).

Taken together, all these data show an extraordinary complex mechanism controlling posterior elongation and somite formation in vertebrates. Moreover, even if conserved among vertebrates, slight differences exist between different vertebrate species, what complicates even more the whole picture. But despite the complexity of the system, a striking cross-talk between the FGF, WNT, Notch and RA signaling pathways is fundamental for the process. Then, the question that arises is the evolutionary origin of such mechanisms. In other words, is the molecular core for the control of vertebrates somitogenesis conserved in non-vertebrate chordates?

Genetic control of somitogenesis in amphioxus: does it exist a clock and a wavefront?

In all vertebrates studied so far the "clock and wavefront model" appears as a conserved mechanism for the formation of somites. In amphioxus, gene expression of most of the genes involved in the "clock and wavefront model" have been described suggesting that these genes might be playing a similar role in amphioxus. Thus, it is possible to classify the genes involved in somitogenesis of amphioxus in two groups (i) those putatively involved in keeping a presomitic mesoderm, and (ii) those putatively involved in segmental processes. Belonging to the first group and expressed in the posterior mesoderm at gastrula stage and in the tail bud at neural stage are Paraxis, Lcx, Pbx, Axin and OligoA (Beaster-Jones et al., 2008). In the second group and expressed as stripes are Delta (Rasmussen et al., 2007), Notch [START_REF] Holland | Characterization and developmental expression of the amphioxus homolog of Notch (AmphiNotch): evolutionary conservation of multiple expression domains in amphioxus and vertebrates[END_REF], Tbx15/18 /22, Hey1, NeuroD/atonal-related, Pcdh𝛿2-17/18, Uncx4.1 (Beaster-Jones et al., 2008), HairyB, HairyC and HairyD (Minguillon et al., 2003), IrxB [START_REF] Kaltenbach | Developmental expression of the three iroquois genes of amphioxus (BfIrxA, BfIrxB, and BfIrxC) with special attention to the gastrula organizer and anteroposterior boundaries in the central nervous system[END_REF], Ripply [START_REF] Li | Expression of a novel somiteformation-related gene, AmphiSom, during amphioxus development[END_REF], and Six1/2 (Kozmik et al., 2007). Interestingly, the first 8-10 somites which form by enterocoely, express a set of genes that are uniquely expressed in these somites and not in those derived from the tail bud. These genes are: Engrailed, a segmentation gene in flies, with detectable expression starting from early neurula stage (N1) [START_REF] Holland | Sequence and embryonic expression of the amphioxus engrailed gene (AmphiEn): the metameric pattern of transcription resembles that of its segment-polarity homolog in Drosophila[END_REF]; HairyB, a modulator of Delta-Notch signaling during segmentation of vertebrate somites [START_REF] Gibb | The segmentation clock mechanism moves up a notch[END_REF], with a detectable expression from gastrula stage (Minguillon et al., 2003); Pbx, a homeodomain containing cofactor for Hox genes [START_REF] Laurent | PBX proteins: much more than Hox cofactors[END_REF]; and OligA, probably also involved in neurogenesis in amphioxus as it was shown in vertebrates [START_REF] Bronchain | The olig family: phylogenetic analysis and early gene expression in Xenopus tropicalis[END_REF]. From these genes, only Engrailed and HairyB are expressed in the paraxial mesoderm as stripes, and might be playing a specific role during the segmentation of the anterior 8-10 somites (Figure 20) (Beaster-Jones et al., 2008). Moreover, in addition to the enterocoelic and schyzocoelic somites, several data suggest an additional division of the amphioxus somites, particularly within the anterior enterocoelic ones. Ontogenetically, the most anterior of these enterocoelic somites form simultaneously, whereas the most posterior form sequentially. Additionally, gene expression also differentiates these two somitic regions. Indeed, Mox is never expressed in the most anterior simultaneously formed somites, suggesting a functional difference with the posterior somites [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]. In addition, in our laboratory we have shown that inhibition of FGF or MAPK pathways at the blastula stage induces a complete loss of the most anterior enterocoelic somites, whereas formation of all the most posterior somites (enterocoelic and schyzocoelic) is independent of these two pathways (Bertrand et al., 2011). Therefore, we clearly establish the presence of three different somitic populations in amphioxus: (i) the most anterior enterocoelic, FGF-sensitive, Moxnegative, and Engrailed-positive somites; (ii) the posterior enterocoelic, FGF-insensitive, Mox-and Engrailed positive somites; and (iii) the posterior schizocoelic, FGF-insensitive, Engrailed-negative, and Mox-, Axin-, Lcx-, and Paraxis-positive somites (Figure 21). Concerning the "wavefront", in vertebrates, as I have explained before, it is characterized by an expression gradient of members of the FGF, RA and WNT families. In amphioxus, transcripts of Wnts1, [START_REF]1 Anterior somitogenesis in amphioxus sheds lights on the origin of the vertebrates[END_REF]5,6, and 8 are expressed in the posterior mesoderm at the gastrula and neurula stages and in the larval tail bud [START_REF] Holland | Developmental expression of AmphiWnt1, an amphioxus gene in the Wnt1/wingless subfamily[END_REF][START_REF] Holland | Nuclear beta-catenin promotes non-neural ectoderm and posterior cell fates in amphioxus embryos[END_REF][START_REF] Schubert | Characterization of amphioxus AmphiWnt8: insights into the evolution of patterning of the embryonic dorsoventral axis[END_REF]Schubert et al., 2001b) suggesting that members of the WNT family could play a role in maintaining an undifferentiated state of the presomitic mesendoderm before the segmentation. However, the small distance (10uM) in which these genes are expressed make difficult to determine whether there is or not a gradient of expression. Regarding the role of FGF signal, it was already shown that this signal is only involved in the formation of the most anterior somites and not in the posterior somites (Bertrand et al., 2011). Additionally, any study testing the role of the RA signal in amphioxus somitogenesis have been performed, leaving the question open about its ancestral role at the base of the chordates.

Taking together these known data concerning amphioxus somitogenesis, it is quite probable that the "clock and wavefront" system was selected specifically in vertebrates in parallel to the development of more complex somite-derived structures but that it was not required for somitogenesis in the ancestor of chordates. In addition, the specific role of FGF signal in the formation of the most-anterior somites in amphioxus also suggests that functional evolution of FGF signal was instrumental for the appearance of the vertebrates' head (Bertrand et al., 2011), something which has been the fundamental question of my PhD work, and will be discussed in depth later.

MYOGENESIS

In vertebrates, all the truncal muscles derive from the somites, and are controlled by a group of genes called the PSEDN network (comprising Pax-Six-Eya and Dach). Remarkably, the facial muscles possess a different developmental origin and are controlled by different master genes. Thus, in the following pages I will describe the development and genetic control of the trunk and facial muscles in vertebrates and I will compare these data with some known data of amphioxus myogenesis.

Genetic control of truncal myogenesis in vertebrates and amphioxus

1.4.1. 1 Truncal myogenesis in vertebrates: the Pax-Six-Eya-Dach network.

The conserved Pax-Six-Eya-Dach network (PSEDN) has been implicated in a variety of developmental processes in eyes, muscles, endocrine glands, placodes and pharyngeal pouches across bilaterians [START_REF] Heanue | Synergistic regulation of vertebrate muscle development by Dach2, Eya2, and Six1, homologs of genes required for Drosophila eye formation[END_REF]Kozmik et al., 2007;Schlosser, 2015). Interestingly, in vertebrates these network acts upstream of myogenic related factor genes (MRFs). Thus, Pax3 and Pax7, two well-known myogenic genes, are expressed in the somites already formed, playing a direct role in the induction of myogenic cells, where Pax3 plays a major role during primary myogenesis and Pax7 in later phases of myogenesis [START_REF] Maroto | Ectopic Pax-3 activates MyoD and Myf-5 expression in embryonic mesoderm and neural tissue[END_REF][START_REF] Seale | Pax7 is required for the specification of myogenic satellite cells[END_REF]. Double mutant mice Six1 -/-Six4 -/-and Eya1 -/-Eya2 -/-show a loss of expression of Pax3 in the hypaxial dermomyotome demonstrating that SIX transcription factors and their coactivators, control the expression of Pax3 in the hypaxial dermomyotome (Grifone et al., 2007;Grifone et al., 2005). Additionally, studies have shown that SIX proteins can bind transcriptional regulatory sequences of Myog and Myf5, in conjunction with PAX3 protein in the epaxial dermomyotome, suggesting that both SIX and PAX3 can act in parallel to activate the myogenic program (Figure 22) (Giordani et al., 2007). Concerning the last member of the network, Dachschund2 (Dach2), it plays a role together with Eya2 to regulate myogenic differentiation. Additionally, Eya2 acts together with Six1 to regulate myogenesis [START_REF] Heanue | Synergistic regulation of vertebrate muscle development by Dach2, Eya2, and Six1, homologs of genes required for Drosophila eye formation[END_REF], showing a conserved role of the PSEDN. Interestingly, and unlike vertebrates, where the complete segmentation of the somites occurs before the myogenic determination, in amphioxus, it seems that the onset of myogenesis and the segmental process are coupled (i.e. genes as Delta, Notch, Tbx15/18/22, Hey1, Uncx4.1, HairyB, C and D, IrxB and Ripply are expressed almost at the same time as genes involved in myogenic determination). Indeed, classical myogenic factors such as MRF1 (Myogenic Regulatory Factor 1) and MRF2, which belong to a family of muscle-specific basic helix-loop-helix (bHLH) transcription factors and orthologues of MyoD, are associated in amphioxus with myogenic determination and are expressed in the paraxial mesoderm at the gastrula stage, later on at neurula stage MRF1 is expressed in all the somites, whereas the MRF2 expression become more restricted to the posterior somites. Finally at larva stage there is no longer expression of these transcription factors (Schubert et al., 2003).

Concerning the PSDEN network in amphioxus, orthologues of these genes show a dynamic expression pattern. Thus, at gastrula stage Pax3/7 is expressed in the axial and paraxial mesoderm. At early neurula stage (N1) the expression is still detected in the paraxial mesoderm, and also new domains of expression are observed in the neural plate and endoderm. At mid-neurula stage (N2) a conspicuous signal is detected in the posterior axial mesoderm (the nascent notochord), meanwhile the expression in the paraxial mesoderm begins to fade away except in the most anterior somites. At late neurula stage expression in the somites is no longer detectable except in the wall of the first somites on the left side (Figure 23a-e) (Holland et al., 1999). In amphioxus, the SIX family genes (specifically Six1/2 and Six4/5) and their cofactor Eya are expressed in the paraxial mesoderm at the gastrula stage. Later, at mid-neurula stage Six1/2, Six4/5 are expressed in all the presumptive somites excepting the most posterior (newly formed somites) where an expression of Eya is observed. At late neurula stage, when the somites start to be formed by schizocoely from the tailbud, Six1/2 is expressed in all the somites, whereas Six4/5 and Eya are only expressed in the most posterior two pairs. Finally at early larva stage Six1/2, Six4/5 and Eya are expressed in the last three posterior somites (Figure 23f-r) (Kozmik et al., 2007). Concerning Dach, the homologue of Dach2 in vertebrates, it is firstly expressed at late gastrula in the paraxial mesoderm, and this expression is detected in the forming somites until neurula stage (8-10 somites) when the somites are still formed by enterocoely. Later, the expression of Dach is no longer detected (Figure 23s-v) [START_REF] Candiani | Cloning and developmental expression of amphioxus Dachschund[END_REF]. Up to date, any functional analyses of the PSEDN network have been done in vivo in amphioxus. However, in vitro it has been shown the protein-protein interaction between Six-Eya proteins as in other animals. But any interaction has been detected between Dach and Eya proteins as in Drosophila and some vertebrates like chicken [START_REF] Chen | Dachshund and eyes absent proteins form a complex and function synergistically to induce ectopic eye development in Drosophila[END_REF][START_REF] Heanue | Synergistic regulation of vertebrate muscle development by Dach2, Eya2, and Six1, homologs of genes required for Drosophila eye formation[END_REF]Kozmik et al., 2007). Certainly, unravelling the specific role of the PSEDN network in myogenesis in amphioxus, could shed some light about the ancestral role of this network in the formation of muscles in the ancient chordates. [START_REF] Candiani | Cloning and developmental expression of amphioxus Dachschund[END_REF]Holland et al., 1999;Kozmik et al., 2007).

Formation of Facial Muscles in Vertebrates

Facial muscles in vertebrates

The head of vertebrates comprises at least six different groups of muscles with different developmental origins. Thus, the extra-ocular muscles (EOM) derive from the prechordal mesoderm whereas the masticatory and facial expression muscles both derive from the paraxial head mesoderm (Diogo et al., 2015). Other cell types also derive from the paraxial head mesoderm as the cardiomyocytes [START_REF] Kelly | The arterial pole of the mouse heart forms from Fgf10-expressing cells in pharyngeal mesoderm[END_REF][START_REF] Mjaatvedt | The outflow tract of the heart is recruited from a novel heart-forming field[END_REF][START_REF] Waldo | Conotruncal myocardium arises from a secondary heart field[END_REF], the posterior part of the neurocranium, and angiogenic cells (Figure 24) [START_REF] Couly | The developmental fate of the cephalic mesoderm in quail-chick chimeras[END_REF][START_REF] Couly | The triple origin of skull in higher vertebrates: a study in quail-chick chimeras[END_REF][START_REF] Evans | Spatial relations between avian craniofacial neural crest and paraxial mesoderm cells[END_REF][START_REF] Hacker | A distinct developmental programme for the cranial paraxial mesoderm in the chick embryo[END_REF][START_REF] Noden | The embryonic origins of avian cephalic and cervical muscles and associated connective tissues[END_REF]. The other muscles that compose the head as the tongue and neck muscles (hypobranchial and cucullaris muscle groups) possess an embryonic origin that is still debated and might be derived mostly from cells provided by the most anterior somites or/and from the paraxial head mesoderm [START_REF] Birchmeier | Genes that control the development of migrating muscle precursor cells[END_REF][START_REF] Czajkowski | Divergent and conserved roles of Dll1 signaling in development of craniofacial and trunk muscle[END_REF][START_REF] Harel | Distinct origins and genetic programs of head muscle satellite cells[END_REF][START_REF] Huang | Molecular and cellular analysis of embryonic avian tongue development[END_REF][START_REF] Piekarski | Muscular derivatives of the cranialmost somites revealed by long-term fate mapping in the Mexican axolotl (Ambystoma mexicanum)[END_REF][START_REF] Theis | The occipital lateral plate mesoderm is a novel source for vertebrate neck musculature[END_REF]. Thus, only three groups of muscles that compose the head are completely derived from the cranial mesoderm which comprises the prechordal mesoderm and the paraxial head mesoderm, these muscles are the EOM, the masticatory and the facial expression muscles.

Genetic programs for facial muscles formation in vertebrates

The activation of the head myogenesis program is different from the truncal myogenesis program. Nevertheless, in both cases the myogenic regulatory factors (MRFs), determine muscle identity and/or promote muscle differentiation [START_REF] Buckingham | Myogenic progenitor cells and skeletal myogenesis in vertebrates[END_REF][START_REF] Sambasivan | Skeletal muscle stem cell birth and properties[END_REF]. The different MRF genes (e.g., Myf5, Mrf4, MyoD and Myogenin) are able to trigger the differentiation of muscles in both head and trunk [START_REF] Molkentin | Defining the regulatory networks for muscle development[END_REF]. However, the upstream regulators required for the activation of the trunk muscle program are different from those required for the head muscle program. Thus, genes as Tbx1, Pitx2, Tcf21 (also known as Capsulin), Msc (MyoR) and Lhx2 represent the upstream regulators in the head [START_REF] Bothe | Extrinsic versus intrinsic cues in avian paraxial mesoderm patterning and differentiation[END_REF]Bryson-Richardson and Currie, 2008;[START_REF] Grifone | Heartening news for head muscle development[END_REF]Sambasivan et al., 2011;[START_REF] Tzahor | Heart and craniofacial muscle development: a new developmental theme of distinct myogenic fields[END_REF][START_REF] Tzahor | Head muscle development[END_REF][START_REF] Tzahor | Pharyngeal mesoderm development during embryogenesis: implications for both heart and head myogenesis[END_REF]. Indeed, Tbx1, a T-box transcription factor, has an important role regulating the expression of Myf5 and MyoD in all branchiomeric muscles [START_REF] Dastjerdi | Tbx1 regulation of myogenic differentiation in the limb and cranial mesoderm[END_REF][START_REF] Grifone | Properties of branchiomeric and somite-derived muscle development in Tbx1 mutant embryos[END_REF][START_REF] Kelly | The del22q11.2 candidate gene Tbx1 regulates branchiomeric myogenesis[END_REF]. Pitx2, a bicoid-related homeodomain transcription factor, has a crucial role in specifying EOMs and it is a key factor in the development of branchiomeric muscles [START_REF] Dong | Pitx2 promotes development of splanchnic mesoderm-derived branchiomeric muscle[END_REF][START_REF] Kitamura | Mouse Pitx2 deficiency leads to anomalies of the ventral body wall, heart, extra-and periocular mesoderm and right pulmonary isomerism[END_REF][START_REF] Shih | Cranial muscle defects of Pitx2 mutants result from specification defects in the first branchial arch[END_REF]. Both Tbx1 and Pitx2 might act in a cooperative way activating the same target genes, explaining why myogenesis is still observed in branchiomeric muscles of double mutant mice Tbx1:Myf5 [START_REF] Nowotschin | Tbx1 affects asymmetric cardiac morphogenesis by regulating Pitx2 in the secondary heart field[END_REF][START_REF] Sambasivan | Distinct regulatory cascades govern extraocular and pharyngeal arch muscle progenitor cell fates[END_REF]. Moreover, Tcf21 and Msc, both bHLH transcriptional repressors, are required by Myf5 to activate the muscle program in the first mandibular arch [START_REF] Lu | Control of facial muscle development by MyoR and capsulin[END_REF]. Finally, Lhx2, a LIM-homeobox transcription factor, has been identified as a new player in the development of pharyngeal and cardiac muscle formation (Figure 25) [START_REF] Harel | Pharyngeal mesoderm regulatory network controls cardiac and head muscle morphogenesis[END_REF]. In the case of the trunk program, as was shown before, myogenesis is controlled by the PSEDN network. Concerning the role of PSEDN factors in head muscles formation, it has been shown that the expression of Pax3 is absent in head muscle stem/progenitor cells [START_REF] Hacker | A distinct developmental programme for the cranial paraxial mesoderm in the chick embryo[END_REF][START_REF] Sambasivan | Distinct regulatory cascades govern extraocular and pharyngeal arch muscle progenitor cell fates[END_REF][START_REF] Tajbakhsh | Redefining the genetic hierarchies controlling skeletal myogenesis: Pax-3 and Myf-5 act upstream of MyoD[END_REF]. Interestingly, Six1, a critical factor in trunk myogenesis, has been shown to be implicated in head muscle development in zebrafish [START_REF] Lin | The transcription factor Six1a plays an essential role in the craniofacial myogenesis of zebrafish[END_REF] and is expressed in the anterior head mesoderm [START_REF] Bothe | Dynamic control of head mesoderm patterning[END_REF]. Thus, suggesting different roles for Pax and Six proteins in the formation of head muscles.

Other regulatory signals playing a role in head and trunk myogenesis

Other signaling pathways also play important roles in myogenesis and also differ between the head and trunk myogenesis. For example, bone morphogenetic protein (BMP) and Wnt/β-catenin signaling pathways appear as inhibitors of myogenesis in the head paraxial mesoderm, and the inhibitors of these signaling pathways (e.g. Noggin, Gremlin and Frzb) are inducers of head myogenesis [START_REF] Tzahor | Antagonists of Wnt and BMP signaling promote the formation of vertebrate head muscle[END_REF]. On the contrary, the Wnt signal emanating from the dorsal neural tube plays an inductive role in truncal myogenesis [START_REF] Munsterberg | Combinatorial signaling by Sonic hedgehog and Wnt family members induces myogenic bHLH gene expression in the somite[END_REF][START_REF] Stern | Myogenesis in paraxial mesoderm: preferential induction by dorsal neural tube and by cells expressing Wnt-1[END_REF][START_REF] Tajbakhsh | Differential activation of Myf5 and MyoD by different Wnts in explants of mouse paraxial mesoderm and the later activation of myogenesis in the absence of Myf5[END_REF]. The role of the Fibroblast Growth Factor (FGF) signaling pathway in myogenesis has not completely been clarified. Thus, FGF signal induces the expression of myogenic genes in the primary myotome triggering the appearance of muscle progenitors [START_REF] Delfini | The timing of emergence of muscle progenitors is controlled by an FGF/ERK/SNAIL1 pathway[END_REF]. Moreover, in fish fgf8 is required for MyoD expression in the somites [START_REF] Groves | Fgf8 drives myogenic progression of a novel lateral fast muscle fibre population in zebrafish[END_REF][START_REF] Reifers | Fgf8 is mutated in zebrafish acerebellar (ace) mutants and is required for maintenance of midbrain-hindbrain boundary development and somitogenesis[END_REF]. However, it has been shown that in some myoblast cultures, the FGF signal induce differentiation, whereas in other cultures FGF signal acts as a repressor [START_REF] Clegg | Growth factor control of skeletal muscle differentiation: commitment to terminal differentiation occurs in G1 phase and is repressed by fibroblast growth factor[END_REF][START_REF] Olwin | Repression of myogenic differentiation by aFGF, bFGF, and K-FGF is dependent on cellular heparan sulfate[END_REF][START_REF] Seed | Clonal analysis of vertebrate myogenesis. VIII. Fibroblasts growth factor (FGF)-dependent and FGF-independent muscle colony types during chick wing development[END_REF]. Regarding the role of the FGF signal in the head, experiments using FGF8 loaded beads grafted into the head mesoderm of chick embryos resulted in the downregulation of Myf5 and Pitx2 expression and in the upregulation of the expression of MyoR and Tbx1. Beads loaded with the FGF signaling inhibitory molecule SU5402 slightly upregulated Pitx2 and suppressed MyoR and Tbx1 expression. In addition, the inhibition of FGF signaling pathway using retinoic acid resulted in the downregulation of MyoR and Tbx1 expression [START_REF] Bothe | Dynamic control of head mesoderm patterning[END_REF][START_REF] Von Scheven | Neural tube derived signals and Fgf8 act antagonistically to specify eye versus mandibular arch muscles[END_REF]. Thus, these experiments suggest a direct role of FGF signaling for the muscle master regulators Tbx1 and Pitx2, activating Tbx1 and suppressing Pitx2 expression in the head mesoderm. Finally, the ligand of the Notch pathway, Delta 1 (Dll1) is not required in the head myogenic progenitors neither for satellite cell population nor for Pax7 expression, this is in an opposite way to its role in somites-derived muscle (Figure 25) [START_REF] Czajkowski | Divergent and conserved roles of Dll1 signaling in development of craniofacial and trunk muscle[END_REF].

Orthologues of vertebrate head muscles formation in amphioxus

As discussed earlier, the upstream regulators of the myogenic program in the vertebrate's head are different from those of the trunk. In amphioxus, to date, there is no study describing the expression pattern of neither Lhx2 nor Msc (MyoR) (although orthologous of these genes are found in the genome of amphioxus (personal information)). The LIM-homeobox family (i.e. Lhx1/5, Lhx3/4, Lmx, Lhx2/9 (Apterous), Lhx6/8 (Arrowhead) and Islet) play a conserved role in neuronal specification in metazoans [START_REF] Srivastava | Early evolution of the LIM homeobox gene family[END_REF]. In amphioxus, members of the LIM-homeobox family as Lhx3, Islet, and Lim1/5 (Lhx1/5) might be involved in processes such as hindbrain segmentation or neuronal specification but not in myogenesis [START_REF] Jackman | islet reveals segmentation in the Amphioxus hindbrain homolog[END_REF][START_REF] Langeland | An amphioxus LIM-homeobox gene, AmphiLim1/5, expressed early in the invaginating organizer region and later in differentiating cells of the kidney and central nervous system[END_REF]Wang et al., 2002). Presumably Lhx2 in amphioxus could be involved in neural specification. The expression of orthologues of upregulators of the myogenic head program in vertebrates as Tbx1 or Pitx2, have been described in amphioxus. Thus, Tbx1/10 is expressed in the endoderm of the gill slits and in the ventral part of the first 10-12 somites at the neurula stage (Figure 26a-b) (Mahadevan et al., 2004). In amphioxus, Pitx (the orthologue of Pitx1, 2 and 3 from vertebrates) is expressed in the left side of the mesoderm, ectoderm and endoderm at the neurula stage suggesting a role in left-right asymmetry patterning, as it is the case for Pitx2 in vertebrates. However, no expression is detected in somites during their formation (Figure 26c-e) (Boorman and Shimeld, 2002). Thus, orthologues of the upstream regulators of the myogenic program of the head in vertebrates, are not expressed at early stages in amphioxus, suggesting that they are not involved in the determination of the most anterior somites in amphioxus. (Boorman and Shimeld, 2002;Mahadevan et al., 2004)

Final remarks

As we have seen, a major difference exists in the control of myogenesis in the truncal and head muscles in vertebrates. The muscles of the head are triggered by different master regulators such as Tbx1, Pitx2, Lhx2, Tcf21, and Msc, whereas for the truncal muscles the Pax-Six-Eya-Dach network have a major role activating the muscle differentiation program. Nevertheless, for both the head and truncal muscles the differentiation program is triggered by members of the same gene family, the MRF genes. In amphioxus, it seems that the muscles differentiation program for the whole body is activated by the Pax-Six-Eya-Dach network. However, to date there is no study showing the specific role of this network in the formation of the muscle and somites in amphioxus.

Concerning somitogenesis, in vertebrates it has been very well studied, thus the "clock and wavefront" model seems to be valid for all the vertebrates studied so far. Hence, whether or not this model is conserved across chordates, brought different laboratories to perform comparative studies using amphioxus as a model system. In their studies, they look at the gene expression pattern of several genes involved in the "clock and wavefront" model. Notably, several genes involved in the segmental process in vertebrates are expressed in amphioxus posterior somites, suggesting a conserved function of these genes in both vertebrates and amphioxus. Also, they found that members of the WNT family are expressed in the tail bud of amphioxus suggesting a similar role in amphioxus as in vertebrates (keeping an undifferentiated state of the mesendoderm). However, it is difficult to determine whether there is or not a gradient, due to the tiny length of the amphioxus tailbud compared with the PSM of vertebrates. Concerning the role of retinoic acid, even if different studies have been published on the role of retinoic acid during amphioxus development [START_REF] Escriva | The retinoic acid signaling pathway regulates anterior/posterior patterning in the nerve cord and pharynx of amphioxus, a chordate lacking neural crest[END_REF][START_REF] Schubert | Retinoic acid influences anteroposterior positioning of epidermal sensory neurons and their gene expression in a developing chordate (amphioxus)[END_REF][START_REF] Schubert | Retinoic acid signaling acts via Hox1 to establish the posterior limit of the pharynx in the chordate amphioxus[END_REF], any functional study has been performed to test its role during amphioxus somitogenesis, leaving this question open.

Finally, although the FGF signaling pathway is not required for posterior somitogenesis in amphioxus as it is the case in vertebrates, recent studies performed in our laboratory have shown a direct role of this signal for the formation of the anterior somites. This observation has extraordinary evolutionary implications, since it suggests that changes in the function of FGF signaling played a crucial role during evolution for the origin of the vertebrates' head. In the next chapter, I will discuss the implications of this assumption.

FGF SIGNALING PATHWAY AND ANTERIOR SOMITOGENESIS IN AMPHIOXUS

FGF signaling pathway

Firstly identified in vertebrates 40 years ago, the Fibroblast Growth Factors (FGFs) form a family of extracellular signaling peptides that are crucial to activate different processes during the development of many metazoan organisms [START_REF] Itoh | Fibroblast growth factors: from molecular evolution to roles in development, metabolism and disease[END_REF]. In the presence of heparin sulphate proteoglycan (HSPG), FGFs act through binding to a dimeric form of its tyrosine kinase receptor (FGFR), triggering the transphosphorylation and activation of the intracellular TK domain of the receptor and finally activate several intracellular cascades (i.e. Ras/MAPK, PI3K/Akt and PLCγ/PKC). In vertebrates, because of the whole genome duplications (2R or 3R) there are between 22 to 27 FGFs and 4 FGFRs (Oulion et al., 2012b). It seems that the activation of Erk1/2 MAP kinases is a common response for all the 4 FGFRs whereas p38 and Jun kinases could be activated in a cell-type specific manner (Figure 27) [START_REF] Mason | Initiation to end point: the multiple roles of fibroblast growth factors in neural development[END_REF]. In contrast with these complexity in vertebrates, amphioxus only possesses eight FGFs and one FGFR (Oulion et al., 2012b). phosphatidylinositol-4-phosphate, (PKB) protein kinase B, (PLCγ) phospholipase Cγ, (SOS) son of sevenless, (TK) tyrosine kinase. Modified from [START_REF] Mason | Initiation to end point: the multiple roles of fibroblast growth factors in neural development[END_REF].

Expression of FGFs in amphioxus

In vertebrates, fgf8 and fgf4 play important roles in somitogenesis. Indeed, deleting the expression of these genes leads to the loss of expression of most PSM genes, including cycling genes, Wnt pathway genes and markers of undifferentiated PSM, suggesting a major role for fgf8 and fgf4 to keep an undifferentiated state of the PSM [START_REF] Boulet | Signaling by FGF4 and FGF8 is required for axial elongation of the mouse embryo[END_REF][START_REF] Naiche | FGF4 and FGF8 comprise the wavefront activity that controls somitogenesis[END_REF][START_REF] Niwa | Different types of oscillations in Notch and Fgf signaling regulate the spatiotemporal periodicity of somitogenesis[END_REF]. In amphioxus, it has been shown a dynamic expression during embryonic development for five (FGF8/17/18, FGF9/16/20, FGFA, FGFE and FGFC) of the eight FGFs described (Bertrand et al., 2011). Interestingly, FGF8/17/18, the orthologue of fgf8 of vertebrates, is expressed in the posterior dorsal mesendoderm at the gastrula stage. Later on, the expression in the mesoderm fades away rapidly and is no longer visible at midneurula stage (N2). The orthologue of fgf4 of vertebrates in amphioxus is FGFE and its expression is detected by in situ hybridization only at the midlate neurula stage (N2) in the first left somite. The other FGF ligands do not show any expression in the mesendoderm that will give rise the posterior somites (Figure 28). Taken together, these results suggest that FGF signal is not playing a crucial role for the formation of the posterior somites. Finally, in amphioxus, FGFR is expressed ubiquitously at all developmental stages except in the epidermis, and with a higher expression level in the mesoderm (Bertrand et al., 2011).

FGF signaling pathway controls anterior somitogenesis in amphioxus

Inhibition of the FGF signaling pathway using the FGFR inhibitor SU5402 at the blastula stage in amphioxus embryos leads to a loss of the anterior somites. Indeed, embryos treated with this inhibitor showed a loss of expression in the anterior paraxial mesoderm of genes as Brachyury2 (involved in mesoderm formation and differentiation of the notochord (Holland et al., 1995)), Delta (involved in segmentation in vertebrates (Rasmussen et al., 2007)), Snail (establishes a muscle/notochord boundary in Ciona [START_REF] Fujiwara | The snail repressor establishes a muscle/notochord boundary in the Ciona embryo[END_REF]), and MRF1 (determined muscle identity and/or muscle differentiation [START_REF] Buckingham | Myogenic progenitor cells and skeletal myogenesis in vertebrates[END_REF]).

Interestingly, this treatment does not affect the expression of genes such as Neurogenin (neuron differentiation), Chordin (axial dorsal mesendoderm marker) and Nodal (in amphioxus at gastrula stage it is expressed in the paraxial mesoderm [START_REF] Yu | An amphioxus nodal gene (AmphiNodal) with early symmetrical expression in the organizer and mesoderm and later asymmetrical expression associated with left-right axis formation[END_REF]) suggesting that Nodal pathway is not under the control of the FGF signalling pathway (Figure 29). In addition, the work performed in our laboratory showed that for the formation of the anterior somites, the FGF signal acts through the Ras/MAPK pathway. Thus, embryos treated at the same stages with the MAPK pathway inhibitor U0126 have a similar phenotype as the embryos treated with the inhibitor SU5402 (Bertrand et al., 2011) (Figure 29). In the same work, my colleagues showed that formation of the posterior somites is not controlled by the FGF signal. In fact, treatments at blastula stage with SU5402 do not affect the formation of the posterior somites. Moreover, later treatments (late gastrula stage) do not inhibit the formation of any somite, anterior nor posterior, although their morphology look impaired. Thus, these results indicated a crucial role of the FGF signal before the gastrula stage for the formation of the anterior somites (Bertrand et al., 2011). . Embryos treated at blastula stage were fixed at the late gastrula stage (A-G''), at the midneurula stage (H, H' and I, I'), or at the premouth stage (J-L and J'-L'). Embryos treated at late gastrula stage were fixed at premouth (M,M'). A-A'', E-E'', and G-G'' are blastopore views. B-B'', C-C'', D-D'', F-F'', H'H', I-I' and J'-M' are dorsal views. J-M are lateral views. The most anterior limit of MRF1 and MLC is labeled by a black arrow. Anterior is to the left in dorsal and lateral views, and dorsal is to the top in side and blastoporal views. Extracted from (Bertrand et al., 2011).

Final remarks

It is tempting to speculate that in order to evolve a new structure, it is necessary to first lose the ancestral functional structure. Particularly, in the case of the evolution of the vertebrate's head, we can imagine that loss of the anterior somites in the ancestor of vertebrates liberated the developmental constraints imposed by these structures allowing the appearance of new structures. In other words, the appearance of the head in vertebrates had to be preceded by the loss of anterior somites but preserving the structures developed from the ectoderm and endoderm. As I have shown in this chapter, the formation of the most anterior somites in amphioxus is controlled by the FGF signal, and the inhibition of the FGF signal does not affect other germ layers as the ectoderm or endoderm. Thus, an interesting hypothesis has been suggested, in which functional evolution of the FGF signal during early development in the ancestor of vertebrates, played a major role in the evolution of the head through the loss of anterior somites.

Finally, these first studies in amphioxus, both about anterior and posterior somitogenesis, opened many questions. Some of these questions are:

1) Is there a role for RA in somitogenesis in amphioxus? If this role exists, is there any opposition between RA/FGF signaling pathways in amphioxus somitogenesis as in vertebrates? Does RA play a role in the control of the amphioxus asymmetric somites?

2) Why the anterior somites but not the posterior somites are controlled by the FGF signal? How the boundary between the anterior FGF-sentive somites and the posterior FGF-insensitive somites is established along the antero-posterior axis of amphioxus?

3) Since in vertebrates, different master genes trigger the expression of the MRF genes in the head and in the trunk, can we expect in amphioxus any difference between the anterior and posterior somites regarding genes that control the expression of MRF genes?

And finally, probably one of the most important questions would be:

4) Which gene regulatory network is triggered by the FGF signaling for the control of anterior somitogenesis in amphioxus?

Introduction of Article 1

In this first article, I tried to understand the implication of different classical signals, playing a role in vertebrates' somitogenesis, such as FGF, RA, Wnt or Nodal, in the control of amphioxus somitogenesis. I also tried to understand whether a functional crosstalk exists in amphioxus between the FGF signal and Hox genes to control the limit of the anterior FGFsensitive somites. Therefore, the Hox genes (that encode transcription factors) are known for their role in patterning the antero-posterior body axis [START_REF] Lewis | A gene complex controlling segmentation in Drosophila[END_REF]. Thus, in vertebrates, Hox genes play a critical role in the patterning of the axial skeleton [START_REF] Wellik | Hox patterning of the vertebrate axial skeleton[END_REF]. Hox genes are organized in clusters and are expressed in a collinear way so that the Hox genes located at the 3' end within the cluster are expressed before and more anteriorly and those at 5' end are expressed later and more posteriorly along the anteroposterior axis of the embryonic trunk. In vertebrates, because of the two rounds of whole genome duplication there are four Hox gene clusters [START_REF] Holland | Hox genes and chordate evolution[END_REF]. Interestingly, changes in the expression of the Hox genes can lead to shifts in the normal development of the embryo. Thus, for instance, retinoic acid (RA) added ectopically, activates Hox expression anteriorly inducing a posteriorization of the embryo and changes in the axial pattern [START_REF] Kessel | Respecification of vertebral identities by retinoic acid[END_REF]. Amphioxus possesses only one Hox cluster (from Hox1 to Hox15), showing also a collinear expression (Holland et al., 2008a;[START_REF] Pascual-Anaya | Broken colinearity of the amphioxus Hox cluster[END_REF]. Remarkably, in amphioxus, the posterior limit of the anterior-most somites (the FGF sensitive somites) coincides with the anterior limit of expression of Hox1 (Figure 30) (Bertrand et al., 2011;[START_REF] Wada | Colinear and segmental expression of amphioxus Hox genes[END_REF]. Therefore, it might be possible that the anterior Hox1 limit could act as a frontier to differentiate between the anterior-most FGF-sensitive somites, and the posterior FGF-insensitive somites. In amphioxus, RA can activate the expression of Hox genes and like in vertebrates the embryo shows a posteriorization after RA treatment. Moreover, embryos treated with the RAR antagonist BMS009 show an anteriorization [START_REF] Escriva | The retinoic acid signaling pathway regulates anterior/posterior patterning in the nerve cord and pharynx of amphioxus, a chordate lacking neural crest[END_REF]. Thus, to test if Hox genes are acting as a frontier between these two types of somites (FGFsensitive and insensitive somites), we shifted the expression of Hox1 anteriorly or posteriorly with RA or the RAR antagonist, respectively. Then, we expected two possible outcomes;

1) The limit between FGF-sensitive and insensitive somites moves forward or backward, meaning that anterior Hox genes define the limit between FGF-sensitive and FGF-insensitives somites.

2) The limit between FGF-sensitive and insensitive somites do not moves, meaning that anterior Hox genes do not define the limit between FGF-sensitive and FGFinsensitives somites. Our results showed that the limit between the FGF-sensitive and FGF-insensitive somites is not controlled by Hox genes. Furthermore, in order to better understand the evolution of somitogenesis, we also tried to decipher the role of RA and FGF during the formation of the posterior somites. In vertebrates, posterior somitogenesis is controlled in part through the opposition between the posterior FGF/Wnt proliferation signal and the most anterior RA differentiation signal. We were able to show that it is not the case in amphioxus in which RA and FGF signals are not implicated in posterior somitogenesis and in which both signals do not seem to interact through a negative crosstalk. We also showed that the asymmetry of amphioxus somitogenesis is controlled by the Nodal signal in amphioxus and that this left/right asymmetry signal cannot be buffered by RA in contrary to vertebrates.

Introduction Article 2

Next-generation sequencing (NGS) technology has improved largely the studies in different fields of biology. Specially, this technology has facilitated the use of non-model organisms in the Evo-Devo field [START_REF] Da Fonseca | Next-generation biology: Sequencing and data analysis approaches for non-model organisms[END_REF]. In this second paper we took advantage of this new technology to shed light on how FGF signaling controls the formation of the anterior somites in amphioxus. We previously showed that treatment with the inhibitor of the FGF receptor (SU5402) at the blastula stage (5hpf) (early treatment) results in the loss of the most anterior somites. However, embryos treated at the late gastrula stage (14hpf) (late treatment) possess all the somites although they form incorrectly (Bertrand et al., 2011). Thus, to uncover the genes that are under the FGF control for the formation of the anterior somites, we performed a comparative RNA-seq analysis between RNAs extracted from early and late treated embryos. A list of candidate genes controlling somitogenesis was hence obtained and we confirmed their putative role in anterior somitogenesis by undertaking in situ hybridization.

From the list of genes that were specifically downregulated after early treatment and were expressed in the paraxial mesoderm, we chose several transcription factors for undertaking functional analyses. As knock-down techniques are not available in B. lanceolatum, we decided to construct constitutive activator or repressor forms of these transcription factors. To do so, we fused the repressor domain Engrailed or the activator domain VP16 with the DNA-binding domain of the different transcription factors, as previously described [START_REF] Mayor | A novel function for the Xslug gene: control of dorsal mesendoderm development by repressing BMP-4[END_REF]. The microinjection of the mRNA coding for these chimeras into the unfertilized eggs of amphioxus was performed as described by Yu et al. (Yu et al., 2004) and then after the fertilization, phenotypes were analyzed.

All the obtained data indicate that the FGF signal seems to act through the Ets family factor ER81/Erm/Pea3 to control anterior somitogenesis. Moreover, Six1/2 would be the master gene controlling anterior somites formation whereas Pax3/7 would be implicated in the control of the formation of the posterior ones. These results have an important value in the context of the existing debate about the evolution of the head of vertebrates. Indeed, amphioxus anterior and posterior somitogenesis are under the control of genes that are orthologues of those controlling trunk myogenesis in vertebrates. Moreover, orthologues of the genes controlling head mesoderm myogenesis in vertebrates (Tbx1 and Pitx2) are not expressed in amphioxus at the good time and place, suggesting that vertebrates secondarily recruited these genes for the control of head myogenesis. All this work is presented as a manuscript and additional data and general conclusions of my work will be treated in the last chapter of the thesis.

Anterior somitogenesis in amphioxus sheds lights on the origin of the vertebrates' head

Title: Anterior somitogenesis in amphioxus sheds light on the origin of the vertebrates' head

Introduction

The vertebrates' head appeared more than 500 million years ago. This body structure is a sophisticated novelty, and its appearance is associated with the transition from a filterfeeding to a predator life-style. Indeed, the head concentrates sensory organs and neural structures that coordinate movements by integrating sensorial stimuli to efficiently capture preys with specialized mandibular structures. In the last decades, embryological studies helped to unravel the developmental origin of the different components of the head. Thus, it is known that most of these structures derive from the neural crest cells (NCCs), the neurogenic placodes and the cranial mesoderm (CM) (Diogo et al., 2015;Le Douarin and Dupin, 2012;Schlosser, 2008Schlosser, , 2015)). While some studies suggest an early origin of the neural crest cells and placodes in non-vertebrate chordates (Bronner, 2015;Schlosser, 2015), the evolutionary origin of the cranial mesoderm remains poorly understood.

An important difference between the head and the trunk mesoderm in vertebrates is that the trunk is segmented into transitional epithelial somites during embryonic development, while segmentation of the CM is still debated. Historically, various authors considered the vertebrates' head mesoderm as segmented (revised in (Holland et al., 2008b)) similar to the anterior paraxial mesoderm of amphioxus. Indeed, although the exact anatomy of the chordate ancestor is unknown, it is extensively assumed that it possessed a completely segmented body from the most anterior to the most posterior part, similar to extant cephalochordates.

Particularly, the presence of head cavities in elasmobranch (rays and shark) embryos has been used as a strong argument for the segmentation of head mesoderm in vertebrates (Balfour, 1876). This led Goodrich to propose that mesoderm, branchial arches and cranial nerves were integrated into single metameres (Goodrich, 1918;Goodrich, 1930). This hypothesis led some authors to study early divergent vertebrates such as lampreys as a proxy for the intermediate organism between amphioxus and gnathostomes. The first studies in lampreys also described head cavities and suggested they were homologues of shark head cavities (Damas, 1944;Koltzoff, 1902). However, later studies have shown that real head cavities are absent in lampreys and regional segmentation in the lamprey's head may be induced by adjacent structures rather than being the result of a real segmentation (Kuratani et al., 1999). Thus head cavities would represent a gnathostome synapomorphy (revised in (Kuratani, 2008)).

Molecular data in chick embryos show that CM is formed after two waves of expression of the segmental gene hairy (Jouve et al., 2002), indicating that the CM is formed at best by two segments. Thus, the first wave would correspond to the prechordal cranial mesoderm (PCM) that gives rise to the extra-ocular muscles (EOM) and the second one would correspond to the entire cranial paraxial mesoderm (CPM) that gives rise to the masticatory and facial expression muscles (Diogo et al., 2015;Sambasivan et al., 2011). However, besides the diverse embryological origins of putative head segments (if any) in vertebrates and expression of segmental genes, muscle formation in the head and muscle formation in the trunk are controlled by different master genes in vertebrates. Therefore, Pitx2 plays a crucial role in the formation of the head muscles derived from both the prechordal cranial mesoderm (PCM) and the cranial paraxial mesoderm (CPM), whereas Tbx1 play a pivotal role in the formation of the muscles derived from the CPM (Sambasivan et al., 2011). Conversely, Pax3 plays a critical role in the formation of truncal muscles and is expressed in the forming somites of vertebrates (Buckingham and Relaix, 2007;Schubert et al., 2001a). Moreover, members of the SIX family together with their cofactors EYA are involved in the formation of truncal muscles in vertebrates (Grifone et al., 2007;Grifone et al., 2005).

The phylum Chordata comprises the vertebrates, urochordates (or tunicates) and cephalochordates (Bertrand and Escriva, 2011). In the sister group of vertebrates, the urochordates, somites have been secondarily lost, whereas in the cephalochordate amphioxus somites are present all along the antero-posterior axis of the body. These somites in amphioxus give rise to the muscles of the body, to the ventral mesoderm and to the dermis and connective tissues (Mansfield et al., 2015). In amphioxus, the anterior 8-10 somites pinch off from dorsolateral furrows in the wall of the archenteron by enterocoely and then posterior somites are added directly one at a time from the epithelium of the taildbud by schizocoely (Beaster-Jones et al., 2008;Mansfield et al., 2015). Remarkably, it has been shown that the FGF signal plays a pivotal role in the formation of the three most anterior somites of amphioxus (Bertrand et al., 2011). This result together with some gene expression patterns indicate that the body of amphioxus could be divided into three different compartments, (i) the three most anterior enterocoelic, FGF-sensitive, Mox-negative, and Engrailed-positive somites; (ii) the posterior enterocoelic, FGF-insensitive, Mox-and Engrailed-positive somites; and (iii) the posterior schizocoelic, FGF-insensitive, Engrailed-negative, and Mox-, Axin-, Lcx-, and Paraxis-positive somites. Despite these differences between amphioxus and vertebrates, conservation of gene expression between cephalochordates and gnathostomes has been used as a support for the head segmentation hypothesis in vertebrates (Holland et al., 2008b), particularly for Gbx and anterior Hox or Tbx15/18/22, Six1/2, Tbx1/10 and Delta. However, most of these genes are expressed posterior to the putative homologues of head mesoderm segments (e.g. Hox), or late during embryonic development, after the three most anterior somites have formed (e.g. Tbx1/10).

In this work we hypothesized that, if the vertebrate head mesoderm is homologue to the three most anterior somites of amphioxus, the control of myogenesis should be conserved. Thus, we functionally studied the master genes controlling anterior somitogenesis in amphioxus which are downstream of the FGF signal. We first performed a comparative RNAseq analysis revealing several transcription factors whose expression is controlled by FGF only during the formation of the most anterior somites in amphioxus, and whose vertebrate orthologues are involved in trunk myogenesis. Functional analyses using constitutive repressors of these transcription factors, such as the FGF target ER81/Erm/PEA3 or Six1/2, show that they are pivotal for the formation of the most anterior somites in amphioxus.

Conversely, the transcription factor Pax3/7 shows a central role in the formation of posterior somites. Overall, our work reveals that the body of amphioxus could be divided in two ancient compartments, where the most anterior FGF-sensitive somites are Six1/2 dependent whereas the posterior FGF-insensitive somites are Pax3/7 dependent. These results show that head muscles in vertebrates are formed using gene regulatory networks that are not implicated in anterior somitogenesis in amphioxus, and that anterior muscles in amphioxus are formed using similar gene regulatory networks as trunk muscles in vertebrates. Altogether our results allow us to suggest that the vertebrate ancestor lost its anterior somites through changes in the FGF function during early development, and that muscles of the head were secondarily gained during evolution through the recruitment of other transcription factors.

Material and Methods

Animals, embryo collection, drug treatments

Ripe adults from the Mediterranean amphioxus species (Branchiostoma lanceolatum) were collected at the Racou beach near Argelès-sur-Mer, France, (latitude 42° 32' 53" N and longitude 3° 03' 27" E) with a specific permission delivered by the Prefect of Region Provence Alpes Côte d'Azur. Branchiostoma lanceolatum is not a protected species. Gametes were collected by heat stimulation as previously described (Fuentes et al., 2007;Fuentes et al., 2004). Prior to pharmacological treatments, and before hatching, embryos were transferred to new Petri dishes with a known final volume of sea water. SU5402 (Calbiochem 572631) was dissolved in dimethylsulfoxide (DMSO) at 10 -2 M and added to cultures of embryos at a final concentration of 25 µM at the blastula stage (5 hours post fertilization (hpf) at 19°C) or at the gastrula stage (15,5hpf at 19°C). Control embryos were raised simultaneously with equivalent concentrations of DMSO in filtered sea water. Embryos were either fixed in PFA4%-MOPS as previously described (Holland et al., 1996) or frozen in liquid nitrogen.

Sequencing and RNA-seq analysis

Total RNA was extracted from embryos 3, 6 or 9 hours post treatment (hpt) as described in supplementary figure 1 using the RNeasy Plus Mini Kit (QIAGEN) after disrupting and homogenizing the sample with the TissueLyser (QIAGEN). Samples were sequenced using Illumina (1x54 bases) technology at the "Plateforme BIOPUCES et SEQUENÇAGE", IGBMC, Illkirch, France. Transcripts were mapped onto the reference transcriptome (Oulion et al., 2012a) with bwa v0.6.1 (Li and Durbin, 2010), and following the subsequent set of parameters; a seed of 27 bases, with a maximum of 2 differences in the seed, 4 mismatched allowed, a maximum of 4 gap extensions and default parameters for all others. The read counts were normalized across libraries with the method proposed by Anders and Huber and implemented in DESeq v1.10.1. (Anders and Huber, 2010). Resulting pvalues were adjusted for multiple testing by using the Benjamini and Hochberg (1995) method (Benjamini and Hochberg, 1995). Contigs were then clustered by temporal expression profile using the STEM software (Ernst and Bar-Joseph, 2006). A False Discovery Rate (FRD) <0,05 and Log2 Fold Change > 1 was chosen for analysis of significantly differentially expressed contigs between SU5402-treated and control embryos in early and late treatments. lanceolatum using the following primers Forward: 5'-CTTGTACACGGGGTCCTCTC-3' and Reverse: 5'-GGAGACAAACCGCTCTCTTG-3'. The sequence was confirmed by Sanger sequencing. Microinjections of plasmids and mRNA were carried out as described in Yu et al. (Yu et al., 2004).

.

Results

Comparative RNA-seq analyses reveal candidate genes downstream of the FGF signal during anterior somitogenesis

It has been previously shown that inhibition of the FGF signal during early development in amphioxus induces the loss of the most anterior somites (i.e. treatment with the FGFR inhibitor SU5402 at blastula stage (5 hpf at 19°C)). However, embryos treated with SU5402 at late gastrula stage (15,5 hpf at 19°C) do not lose the most anterior somites. These results indicate a specific role of the FGF signaling pathway during early development for the formation of the most anterior somites (Bertrand et al., 2011).

In order to understand how the FGF signal controls the formation of these somites, we performed a comparative RNA-seq analysis among transcriptomes of control versus treated embryos at blastula (FGF sensitive somitogenesis) and gastrula (FGF non sensitive somitogenesis) stages. Moreover, we undertook a dynamic analysis by sampling embryos at 3, 6 and 9 hours post treatment (hpt) in both cases (Fig. sup. 1). These analyses reveal an important number of contigs which expression is controlled directly or indirectly by the FGF signal at both developmental stages. Thus, in total the expression of 1677 and 2716 contigs were downregulated or upregulated during the early phase of development, respectively (i.e. blastula stage), whereas the expression of 1001 and 478 contigs were downregulated or upregulated during the later phase, respectively (i.e. gastrula) (Fig. 1a).

We took advantage of the dynamic expression profile following the treatments for each contig (see Fig. sup. 1) to select putative master genes in the hierarchy between the FGF signal and the observed phenotype. For this purpose, we clustered the contigs according to their temporal expression profile and we selected contigs that were downregulated at 3hpt after early treatment but not at 6 or 9 hpt nor after late stage treatment (Fig. 1b). A detailed analysis of these contigs reveals that many of them correspond to genes already described as being implicated in amphioxus somitogenesis (Beaster-Jones et al., 2008), but also to orthologues of genes involved in myogenesis or somitogenesis in vertebrates (Table 1).

Additionally, we identified genes that were not described previously in amphioxus and that might have a role in somitogenesis such as an orthologue of Lim domain only protein 4 gene (LMO4), which in vertebrates acts as a cofactor of Snail (Ferronha et al., 2013) and which is expressed in somites in mice and zebrafish (Kenny et al., 1998;Lane et al., 2002), or the orthologue of the Smad interacting protein 1 gene (SIP1), that has been recently shown to be involved in vertebrate somitogenesis (Kok et al., 2010;Maruhashi et al., 2005).

From this list of genes, we choose to concentrate further on orthologues of 24 genes described in vertebrates as having a role in somitogenesis or myogenesis, on genes highly downregulated, and on genes with an unknown role in amphioxus (Table 1). To validate our list of candidates as possible master genes at the top of the cascade downstream of FGF for the formation of the most anterior somites, we performed in situ hybridization on both control and treated embryos before the first somites form at gastrula (11 hpf at 19°C) and late gastrula (14 hpf at 19°C) stages. Thus, we broadly observed a downregulation of the expression in the paraxial mesoderm at gastrula and late gastrula stages when embryos were treated with SU5402 at the blastula stage (5hpf) (Fig. 2 and Fig. sup. 2) confirming the data obtained by RNA-seq. As previously described (Bertrand et al., 2011), the effectors of the FGF signaling pathway Er81/Erm/Pea3 and Dusp6/7/9 were strongly downregulated after treatment (Fig. 2 a-h). Additionally, the expression of myogenic genes as Six1/2, Six4/5, Eya, Pax3/7, FoxC, MRF2 and Mef2 was lost in the paraxial mesoderm that gives rise to the anterior somites (Fig. 2 i-j'). Interestingly, whereas the expression in the paraxial mesoderm is downregulated for genes such as Six4/5 or Eya, the expression in the axial mesoderm remains unaffected (Fig. Snail and its putative cofactor LMO4 was downregulated in the paraxial mesoderm (Fig. 2 q''-x''). Remarkably, the expression domain in the neural plate of Snail remains unaltered as previously mentioned (Bertrand et al., 2011) (Fig. sup. 2 r'',t''). Likewise, the expression in the paraxial mesoderm of Twist1/2 and SIP1 was downregulated, whereas the expression in the axial mesoderm was unaffected (Fig. 2 y''-f ''' and Fig. sup. 2 y''-f'''). Concerning members of the Gli superfamily, Zic showed a mild downregulation in the paraxial mesoderm (Fig. 2 g'''-j''') whereas Gli expression was highly downregulated in the paraxial mesoderm after treatment (Fig. 2 k'''-n'''). Frizzled4, a receptor of the Wnt signaling pathway, was also downregulated in the paraxial mesoderm (Fig. 2 o'''-r'''). For the other 68 genes selected, we failed to observe any specific signal using in situ hybridization or they were not expressed in the anterior paraxial mesoderm (Table 1).

The FGF signal acts through ER81/Erm/Pea3 for the formation of the most anterior somites

In our previous work we showed that the FGF signal acts through the Ras/MAPK pathway for the formation of the anterior somites in amphioxus (Bertrand and Escriva, 2011). Indeed, embryos treated at 5 hpf with the inhibitor of the ERK1/2 pathway (U0126) present a similar phenotype to the embryos treated with the inhibitor of the FGF receptor (SU5402) (Bertrand et al., 2011). From the variety of intracellular effectors of the FGF signaling pathway acting in the Ras/MAPK cascade (Wasylyk et al., 1998), our comparative RNA-seq data confirmed our previous finding showing the downregulation of the gene coding for the Ets family transcription factor ER81/Erm/PEA3 (Bertrand et al., 2011). These results led us to propose ER81/Erm/PEA3 as the candidate gene linking the FGF signal and the control of anterior somitogenesis. To investigate the specific role of ER81/Erm/PEA3 in the control of anterior somitogenesis in amphioxus, we generated protein chimeras by fusing the Engrailed repressor domain (Jaynes and O'Farrell, 1991) or the VP16 activation domain (Friedman et al., 1988) to the DNA binding domain (DBD) of ER81/Erm/PEA3 (Fig. 3a). We then microinjected the mRNA coding for these two chimeras into amphioxus unfertilized eggs.

Remarkably, we found that embryos injected with mRNA coding for Engrailed-ER81/Erm/PEA3 (Eng-EEP) lost the most anterior somites (Fig. 3c-d), whereas embryos injected with the mRNA coding for VP16-ER81/Erm/PEA3 did not show any observable phenotype alteration (Fig. sup. 3 a-b) when compared to control embryos at the late neurula stage (Fig. 3 b-c). This result suggests that the FGF signal acts through its nuclear effector ER81/Erm/PEA3 for the formation of the most anterior somites in amphioxus.

Six1/2 and Pax3/7 play different roles in amphioxus somitogenesis along the anteroposterior axis

Among all the candidate genes highlighted by the transcriptomic approach, two of them called our attention, Six1/2 and Pax3/7, because of their known implication in trunk myogenesis in vertebrates (revised in (Buckingham and Rigby, 2014)). To investigate the putative role of these transcription factors (Six1/2 and Pax3/7) in amphioxus we modified their in vivo function through microinjection in unfertilized eggs of mRNAs coding for the following protein chimeras: Engrailed-Six1/2 (Eng-Six1/2), Engrailed-Pax3/7 (Eng-Pax3/7), VP16-Six1/2 (VP16-Six1/2) and VP16-Pax3/7 (VP16-Pax3/7) (Fig. 3a). Embryos injected with mRNAs coding for VP16-Six1/2 or VP16-Pax3/7 did not show any observable phenotypic alteration (Fig. sup. 3 c-f). However, embryos injected with the mRNA coding for the constitutive repressor Eng-Six1/2 lost the most anterior somites (Fig. 3 e-f) and embryos injected with the mRNA coding for Eng-Pax3/7 were shorter than controls with an observed expression of the muscle gene marker MLC (Myosin Light Chain) in the anterior paraxial mesoderm but not in the posterior part of the body. This suggests that these embryos failed to form the posterior somites (Fig. 3 g-h). We then wanted to corroborate this result through the microinjection of both the constitutive repressor forms of Eng-Six1/2 and Eng-Pax3/7.

Embryos microinjected with Eng-Six1/2 and Eng-Pax3/7 were shortened and both the most anterior somites and the most posterior somites were not forming. However, the muscle marker gene MLC was still expressed in the central somites (Fig. 3 i). Likewise, embryos microinjected with Eng-Pax3/7 and then treated with the FGFR inhibitor SU5402 presented a similar phenotype (Fig. 3 j).

Functional relationship between Six1/2, Pax3/7 and ER81/Erm/PEA3 during anterior somitogenesis in amphioxus

The constitutive repression of ER81/Erm/PEA3 and Six1/2 target genes induced the loss of anterior somites whereas the repression of Pax3/7 target genes induced the loss of posterior somites. Remarkably, these three genes are coexpressed in the paraxial mesoderm during amphioxus embryogenesis (Fig. 4 a-c), and we decided to study the possible hierarchical relationships between them. For this purpose we analyzed the expression of each of these genes at the late gastrula stage when the paraxial mesoderm is already committed to the formation of the most anterior somites and at the late neurula stage when the anterior somites are already formed, in embryos injected with each constitutive repressor chimera. Thus, embryos injected with the constitutive repressor form of ER81/Erm/PEA3 showed a loss of Six1/2, Pax3/7 and MRF2 (Myogenic Regulatory Factor) expression (Fig. 4 f-h). This phenotype is similar to that obtained in embryos treated with SU5402 (see above). However, embryos injected with the constitutive repressor form of Six1/2 expressed ER81/Erm/PEA3 normally (Fig. 4 i), but Six1/2 (using a probe for the 3'UTR), Pax3/7 and MRF2 were downregulated in the paraxial mesoderm next to the invaginating mesoderm (Fig. 4 j-l). Then, embryos injected with the constitutive repressor form of Pax3/7 showed a normal endogenous expression of ER81/Erm/PEA3, Six1/2, and MRF2, consistent with our previous observations where anterior somites are formed normally in embryos microinjected with the Eng-Pax3/7 mRNA (Fig. 4 m-p).

We then studied the phenotype induced by the different microinjections at late neurula stage, when the anterior-most somites are already formed and posterior somites are added one-at-time in wild type embryos. Embryos injected with Eng-EEP, which loses the anteriormost somites as previously shown (Fig. 3 c-d), exhibited a loss of expression in the most anterior somites territory of Six1/2 and FoxC, a transcription factor related to myogenesis and mesoderm specification (Aldea et al., 2015;Amin et al., 2010;Andrikou et al., 2013) (Fig. 5 l,n). Pax3/7 is not expressed in the anterior somites at this developmental stage in wild type embryos. However, it is expressed in the neural tube, and this expression domain was maintained in Eng-EEP injected embryos or in SU5402 treated embryos (Fig. 5 c,h,m).

Likewise, embryos injected with Eng-Six1/2 did not show any signal in the most anterior somites using the probes Six1/2 and FoxC (Fig. 5 q, s), whereas the expression of the genes ER81/Erm/PEA3 and Pax3/7 (Fig. 5 p,r) seemed not to be altered. On the other hand, when we examined the expression of ER81/Erm/PEA3, Six1/2, and FoxC in embryos injected with Eng-Pax3/7, we detected transcripts of ER81/Erm/PEA3, Six1/2 and FoxC in the formed somites (i.e. anterior somites) (Fig. 5 u-x).

As we have shown, inhibition of the FGF signal at the blastula stage inhibits the formation of the anterior-most somites but does not affect the formation of the notochord in amphioxus (Bertrand et al., 2011). Indeed, genes that are normally expressed in both axial and paraxial mesoderm, such as Twist1/2 or SIP1, were downregulated in the paraxial mesoderm in SU5402 treated embryos, whereas the expression in the axial mesoderm remained unaffected (Fig. sup. 2b''', f'''). To test if the notochord was well-formed in embryos injected with the chimeras we used Brachyury2 as a marker gene (Holland et al., 1995). In a similar way to embryos treated with SU5402, embryos injected with Eng-EEP or with Eng-Six1/2 showed Brachyury2 (Bra2) expression along the antero-posterior axis of the embryo (Fig. 5 e,j,o,t). Remarkably, in Eng-Pax3/7 injected embryos, Bra2 was very weakly expressed in both the notochord and the posterior tailbud (Fig. 5 y), in agreement with the proposed role of Pax3/7 in the formation of the notochord and posterior somites in amphioxus (Holland et al., 1999).

ER81/Erm/PEA3 response elements are present in cis-regulatory regions of genes implicated in myogenesis

Gene expression is controlled through cis-regulatory elements both in time and space.

Moreover, changes in these elements were instrumental in the origin and evolution of morphological novelties in eukaryotes (Acemel et al., 2016;Wray et al., 2003). While discovery of these elements was extremely difficult and laborious in the past, recent advances in high-throughput technologies now allow their easy identification. Thus the Assay for transposase-Accessible Chromatin with high-throughput sequencing (ATAC-seq) technology allows to map the chromatin accessibility genome-wide, including open chromatin regions and enhancers (Buenrostro et al., 2015). We took advantage of this technique to search for open chromatin regions in the proximities of candidate transcription factors playing a role in somitogenesis during early development. Since we are interested in anterior somitogenesis, we performed ATAC-seq on amphioxus embryos at three different developmental stages: 8hpf (at 19°C, gastrula stage when the paraxial mesoderm is already committed to the formation of anterior somites), 15hpf (at 19°C, late gastrula stage, when segmentation by enterocoely of anterior somites starts) and 36hpf (at 19°C, late neurula stage, when anterior somites are already formed and posterior elongation starts adding somites in the tailbud region by schizocoely). Interestingly, we found a peak that is "turned on" at 8hpf and for which the accessibility to the chromatin is no longer observable at 15hpf and 36hpf (Fig. 6 a). This peak is located in the first intron of the gene Zic (one of the downregulated candidate genes (Table 1)). Moreover, the genomic sequence in which this ATAC-seq peak is localized possesses an Ets-family transcription factor DNA binding site. We cloned 1 Kb of genomic DNA harboring this putative enhancer in the 5' region of a construct containing the minimal promotor of amphioxus beta-actin (Feng et al., 2014) upstream of the GFP reporter gene (Fig. 6b) and flanked by the two Tol2 integration sites. Then, we microinjected this construction together with the mRNA coding for the Tol2 transposase into unfertilized amphioxus eggs.

After fertilization, embryos were fixed at 10 hpf. To better observe the GFP expression we performed in situ hybridization using a RNA probe against GFP. Remarkably, the GFP expression recapitulates the normal expression pattern of Zic (Fig. 6 c-f). A similar approach led us to find another genomic region in the proximity of Six1/2 (Fig. 6 g), where a potential DNA binding site for Ets family transcription factors is also present. This region harbors an ATAC-seq peak at 8hpf and 15hpf. Further analysis with a reporter plasmid displaying this putative sequence must be undertaken to corroborate the function of the putative enhancer in vivo.

Discussion

Amphioxus somites are divided into three ontogenetically and functionally different groups

Based on anatomic and functional data, previous studies showed that amphioxus somites can be divided into three different groups: (i) the most anterior enterocoelic, FGFsensitive, Mox-negative, and Engrailed-positive somites; (ii) the posterior enterocoelic, FGFinsensitive, Mox-and Engrailed-positive somites; and (iii) the posterior schizocoelic, FGFinsensitive, Engrailed-negative, and Mox-, Axin-, Lcx-, and Paraxis-positive somites (Bertrand et al., 2011). Here, we studied the implication of the FGF signal in the control of the most-anterior somites and we confirmed this three-part division. Moreover, we demonstrate that the most anterior somites are FGF signalling dependent for their formation through a gene regulatory cascade in which ER81/Erm/PEA3 and Six1/2 are major players, whereas the posterior schizocoelic somites are Pax3/7 dependent. The question of the regulatory mechanisms controlling the formation of the posterior enterocoelic somites, which are still formed even when Six1/2 and Pax3/7 target genes are repressed (see Fig.

Anterior mesoderm segmentation and myogenesis are concomitant processes controlled by the FGF signal in amphioxus

The FGF signal plays an essential role in vertebrate somitogenesis during the posterior elongation process as part of the clock and wavefront system (Hubaud and Pourquie, 2014).

In amphioxus, we have recently shown that, in contrary to vertebrates, the FGF signal does not play a significant role during posterior elongation and addition of new somites from the tailbud (Bertrand et al., 2015) whereas, it plays a pivotal role for the formation of the most anterior somites (Bertrand et al., 2011). How the FGF signal controls anterior but not posterior somitogenesis in amphioxus was an evident question we tried to answer through a comparative transcriptomic approach. The results obtained pointed out an important amount of genes whose expression is directly or indirectly controlled by the FGF signal specifically during anterior somitogenesis.

In amphioxus, embryonic development is relatively fast, and the first somites can already be observed as early as 17 hpf. However, the paraxial mesoderm is already committed to form the anterior somites at the gastrula stage (11 hpf). Consequently, we observed that most of the genes that emerged as early targets of the FGF signaling pathway during early somitogenesis in our comparative RNA-seq analysis were related with both the presomitic mesoderm segmentation and myogenesis processes in vertebrates. Thus, we observed that orthologues of genes implicated in vertebrate myogenesis such as Six1/2, Six4/5, Eya, Pax3/7, FoxC, MRF2 or Mef2, which are expressed before segmentation (Aldea et al., 2015;Kozmik et al., 2007;Schubert et al., 2003), as well as orthologues of genes involved in vertebrate segmentation such as Delta or their Hairy effectors, Ripply, Hey1/2, Uncx4.1 and Tbx15/18/22, which are also expressed before segmentation (Beaster-Jones et al., 2008;Minguillon et al., 2003;Rasmussen et al., 2007), were downregulated in the paraxial mesoderm (Fig. 2). Moreover, other orthologues of genes involved in vertebrate somitogenesis such as Gli, or genes that have not been described before in amphioxus, with putative roles in somitogenesis, such as LMO4 or SIP1, were also downregulated in the paraxial mesoderm. Interestingly, Snail a transcription factor involved in epithelial-tomesenchymal transition also shows a downregulation in the paraxial mesoderm but not in the neural plate. Taken together, these data confirm the essential role of the FGF signal in the control of anterior somitogenesis during early development in amphioxus. In addition, contrary to vertebrate somitogenesis where segmentation occurs before myogenesis (Bryson-Richardson and Currie, 2008), both processes seem to be coupled during amphioxus anterior somitogenesis.

FGF signaling controls anterior somitogenesis/myogenesis in amphioxus directly through its effector gene ER81/Erm/PEA3

In zebrafish and chick, Erm and Pea3 are the effectors of the FGF signal during somitogenesis (Brent and Tabin, 2004;Raible and Brand, 2001). It has been shown in a previous study (Bertrand et al., 2011), and confirmed by our RNA-seq approach, that the amphioxus orthologue of these genes, ER81/Erm/PEA3, is completely downregulated following inhibition of the FGF signal. These data suggested that in amphioxus ER81/Erm/PEA3 could be the effector of the FGF signal during the control of anterior somitogenesis. To functionally test this hypothesis, we injected the mRNA coding for the constitutive repressor form of ER81/Erm/PEA3 (Eng-EEP). We observed that the embryos lost the most anterior somites similarly to embryos treated with SU5402 supporting our proposition.

We then used an ATAC-seq approach in order to find putative enhancers that would be active during early development of amphioxus and containing Ets family transcription factor binding sites. We found that amphioxus Zic contains a peak in its first intron with a clear Ets family transcription factor binding site that could represent an active enhancer. Moreover, this peak is specific of early development since it is only present at 8hpf and disappears in later stages. Thus, we performed transient transgenesis in amphioxus by microinjecting a reporter plasmid in which expression of GFP is controlled by the putative enhancer of Zic. Our results clearly show that this enhancer directs gene expression to the paraxial mesoderm at the gastrula stage. In vertebrates, Zic genes are involved in different processes during embryogenesis such as neuroectodermal development, neural crest induction, somite segmentation and myogenesis (Houtmeyers et al., 2013). Particularly, in mice, Zic2 and Zic3 play an essential role in paraxial mesoderm segmentation (Inoue et al., 2007). Moreover, Zic1 and Zic2 act together with Pax3 and Gli2 to activate Myf5, a key transcription factor for myogenesis in mice (Himeda et al., 2013;Pan et al., 2011). Similarly, in ascidians, Macho-1, that encodes a transcription factor of the Zic family, plays a central role in muscledetermination (Imai et al., 2004;Sawada et al., 2005). In amphioxus, Zic is expressed in the most anterior somites, in the posterior tailbud and during neural development (Gostling and Shimeld, 2003) and our data suggests that it could be implicated in anterior somitogenesis through an activation by the FGF-ER81/Erm/PEA3 cascade.

Anterior an posterior somitogenesis in amphioxus are controlled by different regulatory logics involving the same players

In vertebrates, SIX1 is essential for myogenesis as highlighted by the impaired somitogenesis phenotype observed in Six1 -/-mice mutants (Relaix et al., 2013). In addition, it has been shown that SIX1 acts together with SIX4 to activate Myf5 and participates to the direct transcriptional activation of the key transcription factor gene in myogenesis of vertebrates, Myod (Giordani et al., 2007;Grifone et al., 2005;Relaix et al., 2013;Wu et al., 2014). Another key player of myogenesis in vertebrates is PAX3. In mice, Pax3 is expressed in the presomitic mesoderm and then in all newly formed somites (Schubert et al., 2001a) and Pax3 mutants show impaired somitogenesis and trunk muscle formation (Tremblay et al., 1998). Interestingly, PAX3 binds together with SIX1/4 and its EYA cofactors to the enhancer that drives the early expression of Myf5 in the hypaxial somites (Daubas and Buckingham, 2013).

In amphioxus, Six1/2 expression is first detected at the gastrula stage in the invaginating mesendoderm and in the paraxial mesoderm. Then, starting from early neurula stage, transcripts are detected in all presumptive somites excepting the two most posterior ones (Kozmik et al., 2007). Expression of Pax3/7 in amphioxus is quite dynamic. Thus, it is first detected at the early gastrula stage in the axial and paraxial mesoderm and later on, at the neurula stage, in the paraxial mesoderm, in the endoderm, in the lateral edges of the neural plate and in the nascent notochord (Holland et al., 1999). According to their gene expression pattern and their role in vertebrates, it has been proposed that Six1/2 and Pax3/7 might play crucial roles in myogenesis and/or somitogenesis in amphioxus (Holland et al., 1999;Kozmik et al., 2007). However, no functional study supporting this proposition has been performed until now.

Here, in the comparative transcriptomic approach, we showed that, when the FGF signal is blocked, both Six1/2 and Pax3/7 are highly downregulated, specifically during early development. This result, together with the known role of these genes in vertebrates led us to functionally study their implication during early development in amphioxus. Our experimental approach consisted in the overexpression of constitutive repressor chimeras of each transcription factor in the embryo and the study of the induced phenotype.

Injection of Eng-Six1/2 induces the loss of anterior somites, a similar phenotype as embryos treated with the FGFR inhibitor SU5402, indicating its direct role in the control of anterior somitogenesis in amphioxus. However, injection of Eng-Pax3/7 does not induce the loss of the anterior somites but the embryos do not form the posterior somites. These results show that formation of anterior and posterior somites in amphioxus are controlled differently, with Six1/2 as a master gene for anterior somitogenesis and Pax3/7 for posterior somitogenesis.

Moreover, we were able to establish a functional hierarchy among these genes and the FGF effector ER81/Erm/PEA3. Thus, repression of target genes of ER81/Erm/PEA3 (through injection of Eng-EEP induces loss of gene expression in the paraxial mesoderm of amphioxus gastrula for Six1/2, Pax3/7 and MRF2, demonstrating the high position of this gene in the cascade controlling anterior somitogenesis. However, while repression of Six1/2 target genes induces the loss of anterior somites and the loss of Pax3/7 and MRF2 expression in the paraxial mesoderm, the expression of ER81/Erm/PEA3 is not lost, confirming the higher position of ER81/Erm/PEA3 in the cascade. Conversely, repression of target genes of Pax3/7 does not induce the loss of ER81/Erm/PEA3, Six1/2 nor MRF2 expression in the gastrula but posterior somites fail to form as well as posterior notochord, suggesting that Pax3/7 is the master gene controlling the formation of these two structures (Fig. 3 e-f).

anterior somitogenesis changed, inducing the loss of anterior somites and liberating the developmental constraints imposed by them in this part of the body. Secondarily, de novo muscles appeared which formation was regulated by new regulatory cascades (Pitx2 and Tbx1) that were coopted for this purpose. (b,d,f,h). Orthologues of genes that in vertebrates are involved in myogenesis as Six1/2 (i-l), Six4/5 (m-p), Eya (q-t), Pax3/7 (u-x), FoxC (y-b'), MRF2 (c'-f') and Mef2 (g'-j') are downregulated in the paraxial mesoderm after SU5402 treatment (j,l,n,p,r,t,v,x,z,b',d',f',h',j'). Orthologues of genes that in vertebrates are involved in somite segmentation as Delta (k'-n'), HairyB (o'-r'), HairyC (s'-v'), HairyD (w'-z'), Ripply (a''-d''), Hey1/2 (e''-h''), Uncx4.1 (i''-l''), Tbx15/18/22 (m''-p'') are expressed in stripes in control embryos whereas this pattern is lost after SU5402 treatment. Snail (q''-t'') and the Snail co-factor LMO4 (u''-x'') expression is lost in the paraxial mesoderm following SU5402 treatment. Similarly for Twist1/2 (y''-b''') and Smad interacting protein 1 (SPI1) (c'''-f''') is observed a downregulation in the paraxial mesoderm but not in the axial mesoderm. Zic is mildly downregulated after treatment (g'''-j'''), whereas Gli shows a strong downregulation (k'''-n'''). Finally, Frizzled4 (Fz4) is downregulated in the paraxial mesoderm of treated embryos (o'''-r'''). Scale bar = 100 μm. (c,d) and Eng-Six1/2 (e,f) injected embryos whereas in Eng-Pax3/7 injected embryos the posterior somites were absent (g,h). In Eng-Six1/2+Eng-Pax3/7 (i) injected embryos and in Eng-Pax3/7 injected embryos and then treated with SU5402 at blastula stage (j) the most anterior somites as well as the most posterior somites were lost whereas the middle somites (3) were still formed. Lateral views for a, c, e and g. Dorsal views for b, d, f, h, i, and j. Scale bar = 250 μm. g,i) whereas the expression of Pax3/7 and Bra2 was similar to control embryos (h,j). The expression of Six1/2 and FoxC was lost in the most anterior somites region in embryos injected with Eng-EEP or Eng-Six1/2 (l,n,q,s) whereas the expression of Pax3/7 and Bra2 was not affected (m,o,r,t). In Eng-Pax3/7 injected embryos only the anterior somites are formed and a downregulation of the expression of Brachyury is observed in the notochord and tailbud (u-y) observed. Lateral views in all panels and anterior is to the left in all embryos. Scale bar = 250 μm.

General discussion and additional data

Hox genes, FGF signal and the anterior somitogenesis in amphioxus

In the first part of this work, I was interested in investigating the relationship between Hox genes and the FGF signal in the formation of the anterior somites in amphioxus. This because the most anterior limit of expression of Hox1 surprisingly matches with the posterior limit of the FGF-sensitive somites (Bertrand et al., 2011;[START_REF] Wada | Colinear and segmental expression of amphioxus Hox genes[END_REF]. This leads us to think that a functional relationship between both signals could exist, with the anterior limit of Hox genes expression acting as a boundary between the FGF-sensitive and FGF-insensitive somites. Moreover, it was previously proposed that nested expression of Hox genes and Gbx, a gene belonging to the Homeobox family [START_REF] Castro | A Gbx homeobox gene in amphioxus: insights into ancestry of the ANTP class and evolution of the midbrain/hindbrain boundary[END_REF], at early gastrula stage, establishes the position of the anterior somites (Holland et al., 2008b). Certainly, anterior somites are established at early gastrula, when boundaries of the future somites are indicated by the expression of genes such as Delta, their effectors Hairy, Ripply, Tbx15/18/22, Uncx4.1, Hey1/2 or Six1/2 (Article 2) (Beaster-Jones et al., 2008). Nevertheless, our finding shows that Hox genes do not define this boundary (Article 1). Indeed, anterior somitogenesis is not affected by the inhibition or activation of the level of RA signal, a known molecule that controls Hox expression in vertebrates as in amphioxus [START_REF] Escriva | The retinoic acid signaling pathway regulates anterior/posterior patterning in the nerve cord and pharynx of amphioxus, a chordate lacking neural crest[END_REF]. Thus, anterior somites are formed independently of the Hox code.

Interestingly, our RNA-seq data reveals that Hox1 and Gbx were downregulated at early stages of development in amphioxus when embryos were treated with SU5402 (Article 2, Table 1), suggesting that they are controlled by the FGF signal. Hox1 and Gbx are not only expressed in the mesoderm, but also in neuroectodermal and endodermal tissues [START_REF] Castro | A Gbx homeobox gene in amphioxus: insights into ancestry of the ANTP class and evolution of the midbrain/hindbrain boundary[END_REF][START_REF] Wada | Colinear and segmental expression of amphioxus Hox genes[END_REF]. Therefore, it might be possible that at early stages of development this downregulation corresponds to a specific control of the FGF signal over the Hox genes for neural development. Indeed, Hox genes play major roles in the patterning of the central nervous system (CNS) in vertebrates as in amphioxus [START_REF] Maden | Retinoid signalling in the development of the central nervous system[END_REF][START_REF] Rhinn | Retinoic acid signalling during development[END_REF][START_REF] Schubert | A retinoic acid-Hox hierarchy controls both anterior/posterior patterning and neuronal specification in the developing central nervous system of the cephalochordate amphioxus[END_REF]. Moreover, these observations are supported by the fact that genes involved in neural developmental processes (i.e. Neurogenin, FezF, CBFA2T1 among other genes with expression domains in the neuroectoderm at early stages) were also downregulated in SU5402 treated embryos at the blastula stage (5hpf). In addition, recent works in our laboratory show the inductive role of the FGF signal for the formation of the anterior neural plate. This work shows how the anterior neural tissue is not maintained when the FGF signal is inhibited. Altogether, these observations are supported by our data from the RNA-seq analysis and corroborate the recent work performed in our laboratory about the neural induction process in amphioxus (unpublished data).

RNA-seq analysis and the role of the FGF signal in anterior somitogenesis

Our comparative RNA-seq analysis allows us to suggest many conclusions. First of all, we observed how the expression of the different genes fluctuates between the different points at early treatment (3hpt, 6hpt and 9hpt) and late treatment (3hpt, 6hpt and 9hpt) (Table 1). We used STEM software to cluster genes given their expression profiles. From these profiles, we concentrated on genes showing a downregulation at early stages after early treatment and for which no expression modification was observed after late treatment (Fig. 1b, Article 2). We believed this profile should be observed for genes that were putatively under the control of the FGF signal at early stages of development but that were not controlled by this signaling pathway afterwards. We confirmed this by in situ hybridization. Indeed, genes that showed a downregulation in the paraxial mesoderm after the treatment at the blastula stage (early treatment) (Fig. 2, Article 2), did not show any change when embryos were treated at 15,5hpf (late treatment) (Figure 31). Thus, we detected genes highly downregulated (Log2 Fold Change lower than -1,5 and p-value < 0,05) just after early treatment (3hpt) as ER81/Erm/PEA3, Pax3/7, Tbx15/18 [START_REF] Chen | Dachshund and eyes absent proteins form a complex and function synergistically to induce ectopic eye development in Drosophila[END_REF]Ripply,MRF1,Iroquois B,Iroquois C,Orthopedia,Nkx6,Gli,Plecktrin5/7/9,CBFA2T1,DDR2,Trauco,Caleuche,Pincoya,WDrepeat49,AnkirinBR1,C2orf81 homolog and Arx. To validate the RNA-seq results, we performed in situ hybridization for all of them and deciphering their precise expression patterns. In addition, we were really interested in determining the expression pattern of unknown genes in amphioxus as Trauco (Contig 71939), Caleuche (Contig 6363) or Pincoya (Contig 32885) among others, because they showed a high response to the FGF signal inhibition just after treatment (3hpt) (Table 1, Article 2). However, we failed to observe any signal by in situ hybridization for these last genes. Later, we selected genes highly downregulated at 6hpt from the early treatment (Log2 Fold Change lower than -1,5 and p-value < 0,05) such as Six1/2, MyosinIX, Hey1/2, FoxAa, Snail, Nk2, Dmbx, Lhx2, Lim1/5, SIP1, Neurogenin, BMP3/3b, Gremlin, Gbx, FezF, Ash, Jagged1, and Jagged2. Finally, other genes were chosen manually to perform in situ hybridization based on their Log2 Fold Change, or their known role in myogenesis or segmental processes (those genes were Dusp6/7/9, Six4/5, Eya, Twist1/2, MYLK, Mnx, SoxE, Fyve domain, RS-like, and BIGH). In the second article (Article 2) we highlighted the genes putatively under the control of the FGF signal and with known roles in vertebrate myogenesis (Six1/2, Six4/5, Eya, Pax3/7, FoxC, MRFs genes, Mef2), vertebrate segmentation process (Delta, theirs cofactors Hairy, Ripply, Hey1/2, Uncx4.1, Tbx15/18/22) and numerous other genes expressed in the paraxial mesoderm (Snail, its hypothetical cofactor LMO4, Twist1/2, SIP1, Zic, Gli, Frizzled4). Thus, RNA-seq together with in situ hybridization data show clearly how the expression of genes involved in anterior somitogenesis in amphioxus is abolished at early stages (notably observed in the Fig. 2, Paper 2). Indeed stripes of gene expression indicating the boundaries of the future somites are no longer visible after the treatment at blastula stage (early treatment) (Fig. 2, Article 2). Remarkably, genes with multiple expression domains as paraxial mesoderm, neural plate or axial mesoderm (i.e. Snail, Twist1/2, SIP1 among others) presented a specific downregulation of the expression in the paraxial mesoderm but not in the other territories.

Additionally, embryos treated with SU5402 develop a normal notochord as we observed at the late neurula stage, where the notochord extends all along the anteroposterior axis (Fig. 5j, Paper 2). Thus, FGF signal acts exclusively in the specification of the anterior paraxial mesoderm that gives rise to the most anterior somites in amphioxus. Similarly, in Xenopus, the FGF signal maintains a positive feed-back with Brachyury, being necessary for the initiation of its transcription and also plays a pivotal role in the establishment of the paraxial mesoderm but is not needed for the establishment of the axial mesoderm [START_REF] Fletcher | The role of FGF signaling in the establishment and maintenance of mesodermal gene expression in Xenopus[END_REF]. In ascidians, the sister group of vertebrates, FGF signal is required for mesenchyme, notochord and secondary muscle development [START_REF] Kim | Role of the FGF and MEK signaling pathway in the ascidian embryo[END_REF][START_REF] Kim | An FGF signal from endoderm and localized factors in the posterior-vegetal egg cytoplasm pattern the mesodermal tissues in the ascidian embryo[END_REF]. Hemichordates, together with echinoderms, represent the sister group of chordates called ambulacraria. In the hemichordate Saccoglossus kowalevskii, the ligand FGF8/17/18 induces mesoderm from endomesoderm [START_REF] Green | FGF signaling induces mesoderm in the hemichordate Saccoglossus kowalevskii[END_REF]. The authors suggested that FGF signal could have been required for the formation of the whole mesoderm in the ancestor of all deuterostomes, followed by a secondary loss of FGFdependency in different cell populations and within the different deuterostome groups [START_REF] Green | FGF signaling induces mesoderm in the hemichordate Saccoglossus kowalevskii[END_REF]. Thus, in amphioxus, the role of the FGF signal for mesoderm induction was conserved in the anterior paraxial mesoderm and lost in the posterior paraxial mesoderm that is formed independently of the FGF signal.

In amphioxus, even if there are no functional studies demonstrating the role of FGF8/17/18, its expression pattern suggests that it could act as the ligand of FGFR to trigger and induce the anterior paraxial mesoderm in amphioxus (Bertrand et al., 2011). Indeed, FGF8/17/18 is expressed at the gastrula stage in the dorsal posterior mesendoderm. Later on, at early neurula, transcripts are detected in the posterior part of the dorsal mesendoderm. Then FGF8/17/18 is no longer expressed in mesodermal tissues. Remarkably, in embryos treated with SU5402 no downregulation of FGF8/17/18 expression could be detected (data not shown). Additionally, other FGF genes (i.e. FGF9/16/20, FGFA, FGFE, and FGFC) are not expressed in mesodermal tissues at early stages when anterior somites are specified, supporting the fact that FGF8/17/18 could be the ligand that binds the FGF receptor to control this process.

Interestingly, the expression pattern of ER81/Erm/PEA3, one of the effectors of the FGF signal that is activated by the MAPK pathway [START_REF] Munchberg | The Xenopus Ets transcription factor XER81 is a target of the FGF signaling pathway[END_REF][START_REF] Roussigne | Divergence in regulation of the PEA3 family of ETS transcription factors[END_REF] overlaps with the early expression of FGF8/17/18 (Bertrand et al., 2011). Thus, ER81/Erm/PEA3 starts to be expressed at the gastrula stage in the dorsal mesendoderm. Later on, at the late neurula stage, transcripts are highly expressed in the mesoderm and, thereafter, by the premouth stage, expression is conspicuous in the pharyngeal endoderm, in the anterior tip of the embryo and in the neural tube along the antero-posterior axis of the embryo (Figure 32). Moreover, our RNA-seq data showed a strong downregulation of this gene expression suggesting its direct role as an effector of the FGF signal in amphioxus for the formation of anterior somites. To test the role of specific key transcription factors in the formation of anterior somites in amphioxus we constructed protein chimeras, as described in Article 2. Thus, most of the embryos injected with the constitutive repressor form of ER81/Erm/PEA3 showed a phenotype similar to embryos treated with SU5402 (Fig 3c-d, Article2). Moreover, embryos injected with the constitutive activator form of ER81/Erm/PEA3 and then treated with the inhibitor of the FGF signal at blastula stage, showed a mild rescue of the anterior somites formation (data not shown). However, for embryos injected with Eng-EEP, we also observed a range of different phenotypes, with embryos lacking MLC expression (Figure 33) or Brachyury expression in the notochord (Figure 33) at late stages. Since ER81/Erm/PEA3 responds to the FGF signal, we propose that, during early development this transcription factor acts as the effector of the FGF signal for the formation of the anterior somites. This result is supported by our observations at late gastrula stage in embryos injected with Eng-ER81/Erm/PEA3 in which the expression of the genes Six1/2, MRF2 and Pax3/7 was similar to the expression in embryos treated with SU5402 (Fig. 4, Article 2). Then, at later stages ER81/Erm/PEA3 could be acting through another pathway than FGF signal, which can explain the strong phenotype observed in some injected embryos (Figure 33). Altogether, these data demonstrate the link between the FGF signal and its effector (ER81/Erm/PEA3) for the formation of anterior somites in amphioxus at early stages and suggest that ER81/Erm/PEA3 could also be necessary at later stages for other embryonic processes.

Possible role of upregulated genes in anterior somitogenesis

Interestingly, our comparative RNA-seq analysis reveals that at early treatment 2716 contigs were upregulated compared to the 1677 downregulated contigs (Fig. 1, Article 2). During my thesis I was mainly focused on the study of downregulated genes, because they are genes that putatively control expression of other genes implicated in anterior somitogenesis. Thus, we did not undertake a deep study of upregulated genes. However, two genes drawn our attention because they were highly upregulated (Table 1, Article 2). FoxEa, which in amphioxus is expressed in the pharyngeal endoderm (Figure 3, Annex Article 1) and which function could have played an important role in thyroid gland evolution [START_REF] Mazet | The Fox and the thyroid: the amphioxus perspective[END_REF], and Scratch, which is expressed in the lateral sides of the amphioxus neural tube (Figure 34) and which in vertebrates has a neural-specific role [START_REF] Dam | Neuron-specific expression of scratch genes during early zebrafish development[END_REF][START_REF] Nakakura | Mammalian Scratch: a neural-specific Snail family transcriptional repressor[END_REF]. When we performed in situ hybridization for these genes in wild type embryos, they were not expressed at early stages when anterior somites start to be formed. In addition, in situ hybridization in treated embryos did not show any clear upregulation of their expression (data not shown). Thus, no conclusion can be drawn from these data and further analysis will be necessary to define the role of the upregulated genes in the formation of the anterior somites in amphioxus. Are these genes implicated in the formation of the endoderm or the axial mesoderm? One important question remaining is the fact that the axial mesoderm does not disappear after FGF signal inhibition and there is no indication on what do the paraxial mesoderm cells of the gastrula become. Do they become axial mesoderm, do they become endoderm? Further analyses using cell lineage approaches will be necessary to answer this question.

Six1/2 and Pax3/7 control somitogenesis in amphioxus

Our results suggest that ER81/Erm/PEA3 is the effector of the FGF signal for the formation of the anterior somites. So, during my work, the next step was to investigate the downstream target genes of this transcription factor for the formation of the anterior somites in amphioxus. As we showed in Article 2, we observed that Six1/2 plays a crucial role in the formation of the FGF-sensitive somites and that Pax3/7 is necessary for the formation of the posterior somites that form by schizocoely in amphioxus (Fig. 3, Article 2). The SIX family includes the genes Six1/2, Six4/5 and Six3/6 and they act together with their cofactor Eya in different developmental processes (Kozmik et al., 2007). Our RNA-seq and in situ hybridization data show that Six1/2, Six4/5 and Eya are under the control of the FGF signal at earlies stages. In vertebrates, Six1/2 acts in conjunction with Six4/5 to activate the myogenic program (Daubas and Buckingham, 2013;[START_REF] Santolini | MyoD reprogramming requires Six1 and Six4 homeoproteins: genome-wide cis-regulatory module analysis[END_REF]. In amphioxus, coincidently at gastrula stage the expression pattern of Six1/2 and Six4/5 overlap, but then from early neurula stage Six4/5 start to be expressed only in the most posterior forming somites, whereas Six1/2 is expressed in all the somites (Kozmik et al., 2007). In mice, it has been shown that Six4 knockout (KO) do not show developmental defects, while Six1 KO (Grifone et al., 2005) show developmental defects. However, the double KO Six4:Six1 exhibits stronger developmental defects than Six1 KO. Differential DNA-binding specificity explains this difference in vertebrates [START_REF] Ando | Slc12a2 is a direct target of two closely related homeobox proteins, Six1 and Six4[END_REF]. Therefore, it could be interesting to investigate the role of Six4/5 in the development of amphioxus and test whether Six4/5 plays an essential role in the formation of anterior somites in amphioxus or not.

The development is finely controlled by different signaling pathways, which in turn control gene expression at the right time and place. During the last years, studies of cisregulatory elements (CREs) have demonstrated their important role in this control. Thus, CREs are able to drive the expression of a specific gene in different tissues and at different time. In vertebrates, it has been shown that different CREs located upstream of the starting site of Six1 are able to drive the differential expression pattern of this gene during development [START_REF] Sato | Regulation of Six1 expression by evolutionarily conserved enhancers in tetrapods[END_REF]. Thus, they observed specific enhancer activity in somites, cranial mesoderm, endoderm, notochord or cranial placodes covering almost all the tissues in which Six1 is expressed [START_REF] Sato | Regulation of Six1 expression by evolutionarily conserved enhancers in tetrapods[END_REF]. We performed an ATAC-seq analysis to investigate the open chromatin regions in the genome of amphioxus at three different developmental stages. When we looked at the genetic landscape surrounding the gene Six1/2, we observed the presence of open chromatin territories in the region located upstream of this gene (Fig. 6,Paper2). Thus, it could be interesting to dissect and test the role of these putative enhancers in vivo. In the Article 2, we showed how a specific genomic region was able to drive the expression of Zic, validating this kind of approaches.

Certainly, an interesting experiment to perform in the future could be a ChIP-seq using a specific antibody for ER81/Erm/PEA3 to corroborate our findings and search for new candidate genes involved in anterior somitogenesis. Moreover, this could help us better understand our observations that some embryos injected with Eng-EEP completely loss the expression of MLC, and therefore both axial and paraxial mesoderm.

FGF signal and vertebrates head mesoderm

In chick embryos, patterning of the head mesoderm depends on the antagonistic roles of the FGF and BMP signaling pathways together with RA as a suppressor signal [START_REF] Bothe | Dynamic control of head mesoderm patterning[END_REF]. These signals in turn control the expression of master genes such as Pitx2, Tbx1, Alx4, and MyoR [START_REF] Bothe | The molecular setup of the avian head mesoderm and its implication for craniofacial myogenesis[END_REF]. Moreover, it has been shown that the head mesoderm in chick embryos is regionalized into two territories. An anterior territory marked by the expression of Pitx2 and a posterior territory, adjacent to the first one, where muscles start to be formed following the expression of Tbx1. Interestingly, FGF signal is essential to activate the posterior head mesoderm expression of Tbx1 [START_REF] Abu-Issa | Fgf8 is required for pharyngeal arch and cardiovascular development in the mouse[END_REF][START_REF] Bothe | Dynamic control of head mesoderm patterning[END_REF]. There is no study showing which signaling pathway triggers the expression of Pitx2 in the anterior territory of the head mesoderm, nevertheless it has been shown that low levels of the FGF signal induce the expression of Pitx2 in the anterior territory of the head mesoderm [START_REF] Bothe | Dynamic control of head mesoderm patterning[END_REF]. In amphioxus, Tbx1/10 is expressed starting from the late neurula stage in the ventral part of the first 8-10 somites (Mahadevan et al., 2004). Besides, Pitx is involved in the left-right asymmetry and is expressed only in the left side of the ectoderm, mesoderm and endoderm starting from early neurula stage (Boorman and Shimeld, 2002). From our RNA-seq data of the early treatment experiment Pitx is upregulated at 6hpt and Tbx1/10 expression is not modified after FGF signal inhibition (Table 1, Article 2). Recent studies of Tbx1/10 knock-down in amphioxus were performed through morpholino injection (Koop et al., 2014). The authors observed a smaller pharynx and fused gill slit, however they did not notice any difference in the 8-10 anterior somites (Koop et al., 2014). Altogether, these data allow us to propose that Tbx1, together with Pitx2 were co-opted in vertebrates to control cranial myogenesis when the head mesoderm appeared secondarily.

Our results under the context of the evolution of the vertebrates' head

Functional changes in signaling pathways have been instrumental in the appearance of morphological novelties [START_REF] Pires-Dasilva | The evolution of signalling pathways in animal development[END_REF][START_REF] Wagner | Evolutionary novelties[END_REF]. For instance, in vertebrates, ectopic activation of the RA signal leads to the truncation of the rostral part of the embryo [START_REF] Holder | Retinoic acid modifies development of the midbrain-hindbrain border and affects cranial ganglion formation in zebrafish embryos[END_REF][START_REF] Kuratani | Rostral truncation of a cyclostome, Lampetra japonica, induced by all-trans retinoic acid defines the head/trunk interface of the vertebrate body[END_REF][START_REF] Morriss-Kay | Effects of retinoic acid excess on expression of Hox-2.9 and Krox-20 and on morphological segmentation in the hindbrain of mouse embryos[END_REF][START_REF] Papalopulu | Retinoic acid causes abnormal development and segmental patterning of the anterior hindbrain in Xenopus embryos[END_REF]. Moreover, Kuratani and colleagues observed, in lampreys embryos, severe phenotypes after treatment with RA which induce extension of anterior segments from the most anterior to the most posterior part of the embryo [START_REF] Kuratani | Rostral truncation of a cyclostome, Lampetra japonica, induced by all-trans retinoic acid defines the head/trunk interface of the vertebrate body[END_REF]. Furthermore, simple inhibition of the FGF signal leads to the loss of anterior segments in amphioxus (Bertrand et al., 2011). Comparative gene expression patterns between lampreys, gnathostomes and amphioxus, led segmentalists to propose that anterior somites of amphioxus evolved into the existent head mesoderm of vertebrates (Holland et al., 2008b). However, their ideas are only based on observations and no functional analyses were undertaken to test their hypothesis. This work, together with previous publications of our laboratory, has deciphered the role of the FGF signal in the formation of the most anterior somites (this work) (Bertrand et al., 2011). Remarkably, we demonstrated that Pax3/7 is necessary for the formation of the posterior somites of amphioxus (those formed by schizocoely). In lamprey, Pax3/7 is not expressed in the head mesoderm (that is located anterior to the otic vesicle) but is expressed in somite-derived skeletal muscles [START_REF] Kusakabe | Expression and interaction of muscle-related genes in the lamprey imply the evolutionary scenario for vertebrate skeletal muscle, in association with the acquisition of the neck and fins[END_REF]. Likewise, in gnathostomes, Pax3 is expressed in all the somites but not in the head mesoderm (Schubert et al., 2001a). Additionally, Six1/2 is essential for the formation of the FGF-sensitive somites in amphioxus (Fig 3, Article 2). In vertebrates, Six1 plays a pivotal role in myogenesis (Buckingham and Rigby, 2014). Moreover, Six1 is expressed in the posterior head mesoderm and pharyngeal pouches in contrary to Pax3/7 that is not expressed in the head mesoderm [START_REF] Sato | Regulation of Six1 expression by evolutionarily conserved enhancers in tetrapods[END_REF][START_REF] Zou | Patterning of the third pharyngeal pouch into thymus/parathyroid by Six and Eya1[END_REF]. Thus, is possible to imagine an ancient compartmentalization, where Pax3/7 sensitives-somites conserved its role in the trunk of the vertebrates, whereas at the place of the anterior Six1/2 sensitives-somites the unsegmented head mesoderm emerged but conserving the expression of Six1/2 as is still observed in anterior structures of extant vertebrates [START_REF] Guo | A Tbx1-Six1/Eya1-Fgf8 genetic pathway controls mammalian cardiovascular and craniofacial morphogenesis[END_REF][START_REF] Lin | The transcription factor Six1a plays an essential role in the craniofacial myogenesis of zebrafish[END_REF]Relaix et al., 2013).

Altogether, these data allow us to propose that if the body of the ancestor of all chordates was completely segmented as it is widely accepted, then changes in the FGF signal led to the loss of the anterior paraxial mesoderm, relaxing developmental constraints in the most anterior part of the embryo and allowing the acquisition of the unsegmented head mesoderm. This new head mesoderm co-opted Pitx as the master gene for the formation of head muscles in the anterior part, and Tbx1 in the posterior part adjacent to the forming somites. FGF signal activates Tbx1 and suppresses Pitx2 expression in head mesoderm of chick embryos [START_REF] Bothe | Dynamic control of head mesoderm patterning[END_REF], then we could imagine that the loss of anterior FGF signal facilitated the co-option of Pitx in the anterior head mesoderm. We observed in our RNA-seq analysis that Tbx1/10 is downregulated after late treatment with SU5402 (Table 1, Article 2), suggesting a putative control of Tbx1/10 by FGF in amphioxus at late stages. All these changes were accompanied by the two rounds of whole genome duplication (2RWGD) [START_REF] Ohno | Evolution by Gene Duplication[END_REF] that brought not only new genes to this scenario, but also new cis-regulatory elements to control gene expression in specific tissues (Figure 35).

Annex Articles

In this section I have included two articles that were part of my work during my thesis, although they are not completely related with the central topic of my research, which is the origin of the vertebrates' head. In the first article "Expression of Fox genes in the cephalochordate Branchiostoma lanceolatum", we analyzed 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 (ranging from FoxA to FoxS). Importantly, Fox genes are involved in pivotal developmental processes, and they are present in fungi as in metazoans. Finally, a comparative analysis within the chordates supports a well-conserved expression domain for some Fox genes but also some divergent expression domains. This suggests that functional evolution of some Fox genes was essential for the evolution of new characters.

The second article entitled "A single three-dimensional chromatin compartment in amphioxus indicates a stepwise evolution of vertebrate Hox bimodal regulation" shows that the amphioxus How cluster is contained within a topologically associating domain (TAD) while in vertebrates, the Hox cluster is surrounded by two TADs located at the 5' and 3' regions of the Hox genes. These TADs in vertebrates play essential roles in the control of Hox gene expression in the during limb development. Our results in amphioxus suggest a stepwise evolution in the bimodal control of the Hox genes in vertebrates in which no TADs existed in pre-chordate metazoans, a single TAD appeared in amphioxus in which long range regulatory elements acquired specific functions in the control of Hox gene expression and then a second TAD appeared in vertebrates together with the bimodal control of Hox gene expression in the limbs.

Expression of Fox genes in the cephalochordate

Branchiostoma lanceolatum

A single three-dimensional chromatin compartment in amphioxus indicates a stepwise evolution of vertebrate Hox bimodal regulation

Figure 1 .

 1 Figure 1. Early hypotheses of head segmentation. (A)The vertebral theory of Goethe. Goethe proposed that similarly to the vertebrae of the body, the skeleton of the mammalian skull is segmented. Then the different bones of the head would represent five head segments (1 to 5), (modified from[START_REF] Jollie | Segmentation of the vertebrate head[END_REF]). (B) The vertebrate archetype of Owen, who proposed that the anterior portion of the vertebrate body is similar to the posterior part of the vertebrate body (trunk), (modified from[START_REF] Owen | The Principal Forms of the Skeleton and of the Teeth[END_REF]).Abbreviations: ns, nasal; fr, frontal; par, parietal; ip, interval parietal; so, supraoccipital; eo, exoccipital; cv, cervical; as, aliphenoid; os, orbitosphenoid.

Figure 2 .

 2 Figure 2. The head metamerism theory of Goodrich. (A) In the head region, each compartment contains a nerve, a head somite, and a branchial arch. (B) When the skeletal and peripheral nervous systems are removed from the scheme, it is clear that Goodrich based his model on the segmentation of the paraxial mesoderm. Abbreviations: pm, premandibular cavity; mm, mandibular cavity; hm, hyoid cavity; ot, otic vesicle; pv, Platt's vesicle; s0-7, somites. Both figures were modified from(Goodrich, 1918; Kuratani et al., 1999).

Figure 3 .

 3 Figure 3. Segmental/Non segmental theories. (A) In the segmental hypothesis of Goodrich the vertebrate's head is assumed to contain only one type of segmentation that involves metamerism of paraxial mesodermal segments and pharyngeal arches. (B) Non segmentalist theory assumes independent patterns of metamerism for somites and pharyngeal arches questioning the presence of segments in the mesoderm of the head. Modified from[START_REF] Kuratani | Evolutionary developmental biology and vertebrate head segmentation: A perspective from developmental constraint[END_REF].

Figure 4 .

 4 Figure 4. c-hairy expression in chick embryos and somitomeres. The morphological pattern of somitomeres in the chick embryo is shown on the right, as a simplified illustration. Hypothetical somitomeres are numbered. On the left is shown the oscillating expression of the gene c-hairy 1, in the early chick embryo, based on(Jouve et al., 2002). Each oscillation is numbered together with the mesodermal part generated after that oscillation. Note that there are only two oscillations in the head mesoderm, one for the premandibular mesoderm, and the other for the rest of the cephalic mesoderm. Abbreviation: (pmm) premandibular mesoderm or prechordal mesoderm, (cm) cephalic mesoderm, (som) somite. Modified from[START_REF] Kuratani | Craniofacial development and the evolution of the vertebrates: the old problems on a new background[END_REF].

Figure 5 .

 5 Figure 5. Neural crest cells and their skeletal derivatives in the vertebrate cranium. (A) According to the original scheme[START_REF] Portman | Ernfiihrüng in die vergleichende Morphologie der Wirbeltier[END_REF], the vertebrate (gnathostome) skull is assumed to be composed of the cartilaginous neurocranium (light blue), the viscerocranium (light green), and the dermatocranium (brown). (B) Neural-crest-derived elements have been colored in red, and the mesodermal elements in blue, based on several cell-labelling and molecular genetic experiments reported by[START_REF] Couly | The triple origin of skull in higher vertebrates: a study in quail-chick chimeras[END_REF][START_REF] Le Lievre | Participation of neural crest-derived cells in the genesis of the skull in birds[END_REF][START_REF] Le Lievre | Mesenchymal derivatives of the neural crest: analysis of chimaeric quail and chick embryos[END_REF][START_REF] Morriss-Kay | Derivation of the mammalian skull vault[END_REF][START_REF] Noden | Craniofacial development: new views on old problems[END_REF]. (C) Distribution of the cephalic mesoderm (blue) and crest-derived ectomesenchyme (red) in the chicken pharyngula by[START_REF] Noden | Interactions and fates of avian craniofacial mesenchyme[END_REF]. (D) Results from chicken experiments were extrapolated to the human perinatal skull. Abbreviations: dc, dermatocranium; eth, ethmoidal region of the neurocranium; hy, hyoid arch; md, mandibular arch; mo, mouth; n, notochord; ncr, neurocranium; occ, occipital; ph, pharynx; pma, premandibular arch; vcr, viscerocranium; ver, vertebrae or vertebral column.

Figure 6 .

 6 Figure 6. The cranial placodes of vertebrates. (A) Cranial placodes in a 10-13 somite stage chick embryo.In amniotes, profundal and trigeminal placodes are commonly referred to as ophthalamic and maxillomandibular placodes of the trigeminal nerve, respectively. Modified from[START_REF] Streit | Early development of the cranial sensory nervous system: from a common field to individual placodes[END_REF]. (B) Cranial placodes in a tailbud stage Xenopus embryo. Modified from[START_REF] Schlosser | Development of neurogenic placodes in Xenopus laevis[END_REF].

Figure 7 .

 7 Figure 7. Limax lanceolaris. The first description of amphioxus was made by Peter Simon Pallas in 1774 classifying amphioxus as a "mollusk" (Pallas, 1774). A zoom of the drawing (dotted red rectangle) shows an enlargement of the amphioxus drawing.

Figure 8 .

 8 Figure 8. Amphioxus basic anatomy. (A) Schematic view of amphioxus basic anatomy. The most important morphological characteristics are depicted. These include the dorsal hollow neural tube, the dorsal notochord, the intestine and segmented muscles. The schema depicts an amphioxus burrowed in the sand. Anterior is to the top (B) Branchiostoma lanceolatum adult individual. It is possible to observe the segmented muscles and gonads (white). Anterior is to the left.

Figure 9 .

 9 Figure 9. Selected animals from Bilateria showing its three main branches Ecdysozoa, Lophotrochozoa and Deuterostomia (Hydra as outgroup).New genomic studies allowed to place cephalochordates (amphioxus) at the base of the chordates whereas the urochordates (i.e. Ciona) get placed as the sister group of vertebrates[START_REF] Delsuc | Tunicates and not cephalochordates are the closest living relatives of vertebrates[END_REF]. Two whole genome duplications occurred after the divergence of urochordates (2R). Another whole genome duplication took place during the evolution of the teleosts (3R). Times of phylogenetic divergence are not to scale, and the tree branches are intended only to depict general relationships. Modified from[START_REF] Aguinaldo | Evidence for a clade of nematodes, arthropods and other moulting animals[END_REF][START_REF] De Rosa | Hox genes in brachiopods and priapulids and protostome evolution[END_REF]

Figure 10 .

 10 Figure 10. Amphioxus global distribution. Representation of the 23 species from the genus Branchiostoma and of the 7 species from the genus Epigonichthys described by Poss and Boschung.Modified from[START_REF] Poss | Lancelets (Cephalochordata: Branchiostomatidae): how many species are valid? Israel[END_REF].

Figure 13 .

 13 Figure 13. Embryonic development and metamorphosis of amphioxus. Photographs of the most representative embryonic and larval stages of amphioxus are presented (B. lanceolatum). The embryos were grown at 19°C. (A-C) Early stages of development including gastrula stage. (D-F) Neurulation stages. (G-J) Larval stages. Scale bar 50 μm. Modified from(Bertrand and Escriva, 2011).

Figure 14 .

 14 Figure 14. Fossil records from the Cambrian. (A) Yunnanozoon livium, found in 1984 in Chengjiang in China. (B) Haikouella lanceolata, collection from professor Jun Yuan Chen. Scale bar: 1 cm, modified from[START_REF] Bertrand | Amphioxus: how to become a vertebrate][END_REF]. (C) Pikaia gracilens found in Burges Shale in Canada, modified from[START_REF] Long | The rise of fishes: 500 million years of evolution[END_REF].

Figure 15 .

 15 Figure 15. Hox clusters in mammals and amphioxus. Due to the two rounds of whole genome duplications proposed by[START_REF] Ohno | Evolution by Gene Duplication[END_REF]) (2R), there are four Hox clusters (HoxA to HoxD) in vertebrates. In amphioxus there is only one Hox cluster since it diverged before the 2R. The genes Mox and Evx flanking the Hox cluster in amphioxus and vertebrates confirmed the synteny. Also it is possible to observe the losses of certain Hox genes in vertebrates. Modified from[START_REF] David | How Hox genes can shed light on the place of echinoderms among the deuterostomes[END_REF].

Figure 16 .

 16 Figure 16. Experimental approaches developed during the last 30 years in amphioxus. (A-G) In situ hybridization showing the expression pattern of key developmental genes as Delta (A-C), Neurogenin (D-E), Netrin (F) and Brachuyry (G, K, L) at different stages as gastrula (A-D), neurula (B, C, E, F), and late neurula (G). (H-J) Immunohistochemistry labelling using antibodies against phosphorylated histone H3 (H, I) and acetylated tubulin (J) in embryos at gastrula (I) and late neurula (H, J) stages. (K-L) Pharmacological treatment using the inhibitor of the fibroblast growth factors (FGFs) signaling pathway SU5402. In control larva the expression of Brachyury is restricted to the tailbud (K), whereas

Figure 17 .

 17 Figure 17. Somites development in amphioxus and vertebrates. A, B and C shows early (left panels), mid (middle panels) and late (right panels) stages for development and organization of the somites in anamniote vertebrates (e.g. fishes, amphibians), amniote vertebrates (mammals) and amphioxus.The schema shows somites unbent from their true chevron-or W-shape and for simplicity the ribs and ventral muscles were omitted (and in anamiotes, the myoseptal cells), which are derived from somites and migrate ventrally into the lateral plate mesoderm. Abbreviations: (CDM) central dermomyotome, (DC) dermal cells, (DM) dermomyotome, (ECL) external cell layer, (EP) epidermis, (FBM) fin box mesothelium, (MY) myotome, (NT) neural tube, (NO) notochord, (PLP) presumptive lateral plate, (PVM) perivisceral mesothelium, (SL) sclerocoel, (SCM) scleromesothelium, (SO) somitocoel, (SC) sclerotome, (M) trunk muscle. Modified from(Mansfield et al., 2015)

Figure 19 .

 19 Figure 19. The clock and wavefront model. (A) In vertebrates the somites bud off from the anterior PSM periodically and sequentially. (B) In chick embryos one somite is formed approximately every 90 minutes, the oscillatory expression of the segmentation gene c-Hairy1 is shown in different colors, the expression of individual cells (boxed in red) shows how the cells turn on and off the expression of a gene in synchrony, resulting in apparent waves of gene expression across the PSM. (C)The wavefront is characterized by opposite gradients of the signaling pathways, Wnt (light blue) and FGF (green) in the posterior tailbud and RA (purple) more anteriorly. The segmentation clock is represented on the right side of the scheme, showing that the oscillations slow down as they reach the anterior part of the PSM. Wnt activity seems to act as a pacemaker mechanism to regulate the periodicity of cyclic gene oscillations. Prospective somites in the PSM are numbered with S0 being the forming somite and the somite next to form labelled S -I and being already patterned in its rostral (A) and caudal (P) portion. The somite recently formed is numbered as S I. Modified from[START_REF] Gibb | The segmentation clock mechanism moves up a notch[END_REF].

Figure 20 .

 20 Figure 20. Scheme of segmental gene expression during amphioxus somitogenesis. Gene expression at midneurula stage (N2) in the anterior somites is on the left. Gene expression in the tail bud and the nascent posterior somites is on the right. Only HairyB, Pbx, OligoA and Engrailed are expressed in the enterocoelic somites (8-10 anterior somites). Expression of Axin, Lcx and Paraxis in the posterior presomitic mesoderm suggests a role to keep an undifferentiated state of the mesoderm. Vertebrate orthologues of Tbx15/18/22, Delta, Notch, Hey1, Uncx4.1, HairyC, HairyD, Pcdh𝛿2-17/18, IrxA, NeuroD/atonal-related, Six1/2 and Ripply are involved in the segmental process suggesting a common role in amphioxus. Modified from (Beaster-Jones et al., 2008).

Figure 21 .

 21 Figure 21. Posterior somite budding is not dependent on FGF signaling. The different somitic regions are depicted with different colors. The three most anterior somites formed by enterocoely are FGFsensitive, whereas the posterior somites are FGF-insensitive. Modified from(Bertrand et al., 2011).

Figure 22 .

 22 Figure 22. Myogenic induction in vertebrates. Schema of the signaling pathways and genetic interactions during the myogenic induction in vertebrate somites. In the epaxial muscles, PAX3, MYF5 and MYF6 induce MyoD expression independently. By contrast, in the hypaxial dermomyotome, PAX3 induces expression of Myf5 directly, which in turn activates MyoD expression. WNT1 signalling from the dorsal neural tube induces myogenesis through direct activation of Myf5, whereas Wnt7a expression from the dorsal ectoderm preferentially activates MyoD. Hedgehog signalling pathway (SHH) also regulates myogenesis through the maintenance of Myf5 expression. Six1 and Six4 regulates Myf6 expression in the epaxial dermomyotome and together with their cofactors (Eya1 and Eya2) induce the expression of Pax3 in the hypaxial dermomyotome. Modified from (Bryson-Richardson and Currie, 2008).

Figure 23 .

 23 Figure 23. Expression pattern of the PSEDN in representatives stages of amphioxus. Lateral views for A, D, E, F, I, J, L, N, P, T and V. Blastoporal view for B. Dorsal view for C, G, H, K, O, and S. Crosssections for J, M, Q and R. In all the panels (excepting cross-sections) anterior is to the left. (A-E) Pax3/7 expression pattern. At early gastrula is observed a conspicuous expression in the axial and paraxial mesoderm (A-B), later on the expression is observed in the paraxial mesoderm (arrows in C). At mid-neurula stage is observed an expression in the nascent notochord (F-I) Six1/2 expression pattern. First detected in the paraxial mesoderm and dorsomedially in the mesendoderm at gastrula stage (F), later from late gastrula transcripts are detected in all the somites except in the most posterior pair and in the pharyngeal endoderm (J-M) Six4/5 expression pattern. At early gastrula stage Six4/5 is detected in the ectoderm and dorsomedially in the mesendoderm (J), later at late neurula stage expression is detected in the nascent notochord, and in the somites except in the most posterior pair (K,L) a section through a is observed in M showing an expression in the notochord and posterior somites (N-R) Eya expression pattern. First detected in the dorsomeadilly mesendoderm at early gastrula stage (N), transcripts of Eya are later detected in the most posterior somites in late neurula and premouth stages (O, P). Cross-section in (a) and (b) shows the expression in the most anterior somites (S-V) Dach expression pattern. Transcripts of Dach are first detected at early neurula in the paraxial mesoderm and endoderm (arrow) (S). Cross-section through (d) shows mainly the expression of Dach in the dorsal part of the somites. At neurula stage Dach is detected in the first five somites and the forming somites. Later on Dach is no longer detected in the somites. Modified from[START_REF] Candiani | Cloning and developmental expression of amphioxus Dachschund[END_REF] Holland et al., 1999; Kozmik et al., 2007).

Figure 24 .

 24 Figure 24. Head mesoderm and its derivatives. (A) Schema of a hypothetical 15-somites vertebrate embryo (dorsal view). The mesoderm that will give rise to the muscles along the embryo are colored according to the legend. Based on fate mapping and gene expression, the different subdivisions of the cranial mesoderm (head mesoderm) and its derivatives are shown (upright). (B) Schema of the vertebrate head muscles and their origins (legend). Abbreviations: (PCM) prechordal mesoderm, (LPM) lateral plate mesoderm, (EOMs) extra-ocular muscles, (CPM) cranial paraxial mesoderm, (CLM) cranial lateral mesoderm, (PA) pharyngeal arches. Modified from(Sambasivan et al., 2011).

Figure 25 .

 25 Figure 25. Regulatory signals and genetic programs involved in muscle development in head and trunk. (A) Different studies have highlighted the diverse roles played by the intercellular signaling pathways such as the Wnt/β-catenin, BMP or FGF signals in the control of head and trunk myogenesis. In addition, the gene regulatory network for the activation of the myogenic program is completely different in head and trunk. Thus, transcription factors as Tbx1, Pitx2, Msc (MyoR) and Tcf21 are upstream of MRFs during head myogenesis, whereas transcription factors as Pax3, Six1,4 and the cofactors of the last Eya1,2 are key regulators of trunk myogenesis. Modified from (Bryson-Richardson and Currie, 2008). (B) In the branchial arches Pitx2 and Tbx1 act at the top of the cascade in the myogenic program. They turn on the transcription factors Tcf21 and Msc that bind cisregulatory regions of Myf5 (ECR-1) or MyoD (DRR and PRR). Modified from[START_REF] Moncaut | Musculin and TCF21 coordinate the maintenance of myogenic regulatory factor expression levels during mouse craniofacial development[END_REF].

Figure 26 .

 26 Figure 26. Expression pattern of Tbx1/10 and Pitx in amphioxus. (A-B) First detected at mid-neurula stage, Tbx1/10 transcripts are observed in the ventral half of the somites and in the ventral branchial arch mesoderm and endoderm. At larva stage transcripts are detected in the first three branchial arch whereas expression in the somites is no longer observed (C-E) Pitx expression is observed in the left anterior mesoderm, ectoderm and endoderm at neurula stage, this expression continues through the development of amphioxus suggesting a role in the left-right asymmetry of amphioxus. Modified from(Boorman and Shimeld, 2002; Mahadevan et al., 2004)

Figure 27 .

 27 Figure 27. Schematic representation of the FGF signaling pathway. Once FGFs interact with their receptor they trigger a multiplicity of intracellular signaling cascades (i.e. Ras/MAPK, PI3K/Akt and PLCγ/PKC). The Erk-MAP kinase pathway has been most widely implicated in FGF developmental functions to date and is activated by Ras downstream of an FRS2-SOS-Grb2 complex. Abbreviations: (CAM) cell adhesion molecule, (CREB) cyclic AMP response element binding protein, (FLRT) fibronectine leucine-rich transmembrane proteins, (FRS) FGF receptor substrate, (HSPG) heparin sulphate proteoglycan, (Ig) immunoglobulin, (IP3) inositol triphosphate, (MAPK) mitogen-activated protein kinase, (MPK) MAPK phosphatase, (PI3K) phosphatidylinositol-3-kinase, (PIP4)

Figure 28 .

 28 Figure 28. FGF8/17/18, FGFE and FGFR expression in amphioxus. (A-G) Dynamic expression of FGF8/17/18 in amphioxus. (A,B) posterior dorsal mesendoderm expression at the gastrula stage. This expression fades at early neurula stage (C). Then a transient expression is observed in the cerebral vesicle (D). (E,F). Pharyngeal expression is observed at late neurula (E), premounth (F) and larva stage (G). (H-J) FGFE, the amphioxus orthologue of fgf4 in vertebrates, shows an expression restricted to the first left somite at the mid neurula stage (H). At the larva stage, the expression is observed in the neural tube, the gut, and the club-shaped gland. (K-Q) (K) Gastrula stage embryo showing expression in the anterior mesendoderm. (L,M) At early neurula stage expression is observed in the paraxial mesoderm. (N,O) At midlate neurula stage expression is observed in the mesoderm, particularly in the most anterior and most posterior somites. (P) In premouth stage embryo, expression is observed in the notochord, the posterior somites and the anterior pharyngeal endoderm. (Q) Finally, at larva stage, expression is observed in the notochord and the anterior pharyngeal endoderm. Anterior is to the left in all embryos. Blastoporal view (E), dorsal views (M,O), lateral views for the rest. Modified from(Bertrand et al., 2011).

Figure 29 .

 29 Figure 29. FGF and MAPK signaling pathway inhibition induce the loss of the most anterior somites. Expression patterns by whole-mount in situ hybridization of Neurogenin(A-A''), Chordin (B-B''), Brachuyry2 (C-C''), Delta (D-D''), Snail (E-E''), MRF1 (F-F''), Nodal (G-G''),and MLC (H'-L') after treatments with SU5402 (50 μm) or with U0126 (25 μm). Embryos treated at blastula stage were fixed at the late gastrula stage (A-G''), at the midneurula stage (H, H' and I, I'), or at the premouth stage (J-L and J'-L'). Embryos treated at late gastrula stage were fixed at premouth (M,M'). A-A'', E-E'', and G-G'' are blastopore views. B-B'', C-C'', D-D'', F-F'', H'H', I-I' and J'-M' are dorsal views. J-M are lateral views. The most anterior limit of MRF1 and MLC is labeled by a black arrow. Anterior is to the left in dorsal and lateral views, and dorsal is to the top in side and blastoporal views. Extracted from(Bertrand et al., 2011).

Figure 30 .

 30 Figure30. Hox1 expression in a premouth embryo. Anterior limit of Hox1 coincides with the posterior limit of FGF-sensitive somites. Modified from[START_REF] Pascual-Anaya | Broken colinearity of the amphioxus Hox cluster[END_REF]

  sup. 2 p,t), suggesting that different regulatory signals control their expression in axial and paraxial mesoderm. Furthermore, embryos treated with SU5402 lose the anterior stripe of expression of the segmentation genes Delta, HairyB, HairyC, HairyD, Ripply, Hey1/2, Uncx4.1 and Tbx15/18/22 (Fig. 2 k'-p''). Moreover, the expression of the transcription factor

Figure 1 .

 1 Figure1. Comparative RNA-seq analysis. (a) Venn diagram comparing the sets of significantly differentially expressed contigs between SU5402-treated and control embryos in early and late treatments. The total number of upregulated contigs and the total number of downregulated contigs are indicated in red and green, respectively. A False Discovery Rate (FRD) <0,05 and Log2 Fold Change > 1 was chosen for this analysis. (b) Line graph showing the profile of contigs highly downregulated at 3 hpt after early treatment, and which expression is not affected at later stages or after late treatment. Fold change is showed relative to control and expressed in Log2.

Figure 2 .

 2 Figure 2.In situ hybridization of candidate gene in control and SU5402-treated embryos at 11 hpf and 14 hpf of. Dorsal views in all panels. Vertebrate orthologues of effectors of the FGF signaling pathway as ER81/Erm/Pea3 (EEP) (a-d) and Dusp6/7/9 (e-h) are downregulated in treated embryos(b,d,f,h). Orthologues of genes that in vertebrates are involved in myogenesis as Six1/2 (i-l), Six4/5 (m-p), Eya (q-t), Pax3/7 (u-x), FoxC (y-b'), MRF2 (c'-f') and Mef2 (g'-j') are downregulated in the paraxial mesoderm after SU5402 treatment(j,l,n,p,r,t,v,x,z,b',d',f',h',j'). Orthologues of genes that in vertebrates are involved in somite segmentation as Delta (k'-n'), HairyB (o'-r'), HairyC (s'-v'), HairyD (w'-z'), Ripply (a''-d''), Hey1/2 (e''-h''), Uncx4.1 (i''-l''), Tbx15/18/22 (m''-p'') are expressed in stripes in control embryos whereas this pattern is lost after SU5402 treatment. Snail (q''-t'') and the Snail co-factor LMO4 (u''-x'') expression is lost in the paraxial mesoderm following SU5402 treatment. Similarly for Twist1/2 (y''-b''') and Smad interacting protein 1 (SPI1) (c'''-f''') is observed a downregulation in the paraxial mesoderm but not in the axial mesoderm. Zic is mildly downregulated after treatment (g'''-j'''), whereas Gli shows a strong downregulation (k'''-n'''). Finally, Frizzled4 (Fz4) is downregulated in the paraxial mesoderm of treated embryos (o'''-r'''). Scale bar = 100 μm.

Figure 3 .

 3 Figure 3. Role of ER81/Erm/Pea3, Six1/2 and Pax3/7 in amphioxus development. (A) Chimeras were constructed by fusing the Engrailed repressor domain at the N-terminal extremity of the full-length or DNA binding domain of ER81/Erm/PEA3, Six1/2 or Pax3/7. (B) In situ hybridization of MLC (Myosin Light Chain) in control and treated embryos fixed at the late neurula stage. Control embryos showed somites from the most anterior to the most posterior part of the body (a,b). Anterior somites were lost (double head arrows) in Eng-EEP (c,d) and Eng-Six1/2 (e,f) injected embryos whereas in Eng-Pax3/7 injected embryos the posterior somites were absent (g,h). In Eng-Six1/2+Eng-Pax3/7 (i) injected embryos and in Eng-Pax3/7 injected embryos and then treated with SU5402 at blastula stage (j) the most anterior somites as well as the most posterior somites were lost whereas the middle somites (3) were still formed. Lateral views for a, c, e and g. Dorsal views for b, d, f, h, i, and j. Scale bar = 250 μm.

Figure 4 .

 4 Figure 4. Relationships between ER81/Erm/PEA3, Six1/2 and Pax3/7 during anterior somitogenesis. Expression at the late gastrula stage of ER81/Erm/PEA3, Six1/2, Pax3/7 and MRF2 using coding regions or 3'UTR probes in control embryos (a-d), and embryos injected with mRNA of Eng-EEP (e-h), Eng-Six1/2 (i-l) or Eng-Pax3/7 (m-p). Compared with the expression in the paraxial mesoderm of Six1/2 (arrow) and Pax3/7 (arrowhead) (b,c), in EEP-Eng injected embryos this expression is clearly downregulated for Six1/2 (f) and Pax3/7 (double arrowhead) (g), as well in Six1/2-Eng injected embryos is observed a downregulation of the expression for Six1/2 (double arrow) (j) and Pax3/7 (double arrowhead) (k). Dorsal views in all panels. Scale bar = 100 μm.

Figure 5 .

 5 Figure 5. Expression of ER81/Erm/PEA3, Six1/2, Pax3/7, FoxC and Bra2 at the late neurula stage.(a-e) Expression pattern in control embryos. A loss of ER81/Erm/PEA3, Six1/2 and FoxC expression (arrows) in the most anterior somites region was observed in SU5402 treated-embryos (f,g,i) whereas the expression of Pax3/7 and Bra2 was similar to control embryos (h,j). The expression of Six1/2 and FoxC was lost in the most anterior somites region in embryos injected with Eng-EEP or Eng-Six1/2 (l,n,q,s) whereas the expression of Pax3/7 and Bra2 was not affected (m,o,r,t). In Eng-Pax3/7 injected embryos only the anterior somites are formed and a downregulation of the expression of Brachyury is observed in the notochord and tailbud (u-y) observed. Lateral views in all panels and anterior is to the left in all embryos. Scale bar = 250 μm.

Figure 6 .

 6 Figure 6. Putative cis-regulatory regions implicated in the control of anterior somitogenesis in amphioxus. (a) Genomic landscape of Zic and ATAC-seq profiles at 8hpf, 15hpf and 36hpf. The putative regulatory region that was further tested is framed. (b) Construction used to test the Zic regulatory region. (c,d) GFP in situ hybridization in embryos injected with the reporter construct. (e, f) Endogenous Zic expression. (c, e) are lateral views. (d, f) are blastoporal views. (g) Genomic landscape of Six1/2 and ATAC-seq profiles at 8hpf, 15hpf and 36hpf. Putative regulatory region is framed. Scale bar = 100 μm (c-f) for in situ hybridization. Scale bar in Kb in each figure for the genomic size.

Figure 1 Figure 3

 13 Figure 1

Figure 6

 6 Figure 6

  Figure supplementary 1

Figure 31 .

 31 Figure 31. Expression pattern of some early downregulated genes in embryos treated with SU5402 at late stage. In all the panels we observe that expression of the genes that did not change between control and SU5402-treated embryos at late stage 15,5hpf. Embryos were fixed 3 hours post treatment (hpt). (A-B) Gli; (C-D) Lim1/5; (E-F) Pax3/7; (G-H) Ripply; (I-J) Eya; (K-L) Frizzled4; (M-N) Six1/2; (O-P) Snail; (Q-R) Dbmx; (S-T) Delta; (U-V) ER81/Erm/PEA3; (W-X) SLIM. Dorsal views for all the panels. Anterior part of the embryo to the left. Scale bar = 100 μm.

Figure 32 .

 32 Figure 32. Expression pattern of ER81/Erm/PEA3. (A-B) Gastrula stage, (C-D) Early neurula stage, (E-F) Late neurula stage, (G) Premouth stage. Blastoporal view in A, lateral view in B, D, F and G and dorsal view in C and E. Anterior part of the embryo to the left.

Figure 33 .

 33 Figure 33. Embryos injected with the constative repressor form of ER81/Erm/PEA3 (EEP) and fixed at late neurula stage. The different phenotypes obtained when we injected Eng-EEP are showed in this panel. We used Brachyury and MLC as markers. We observed several phenotypes, from mild to strong. A strong phenotype in some embryos that completely lost the mesoderm (Embryo 1 and 4), having only undifferentiated mesendermal cells at the posterior tip of the embryo (A-B). We also observed a majority of embryos that lost the anterior somites (embryos 5 and 6). Embryo 2 shows a partial formation of the notochord and in embryo 3 the notochord is observed all along the anteroposterior body axis. Lateral views A, C, E. Dorsal views B, D, F, G, H, and I. Anterior part of the embryo is to the left. Scale bar = 250 μm.

Figure 34 .

 34 Figure 34. Expression pattern of Scratch during amphioxus development. (A-B) Late-neurula stage (N3), (C) Premounth stage, (D) Larva stage. Dorsal view in A, lateral view in B, D and F. Anterior part of the embryo is to the left.

Figure 35 .

 35 Figure 35. Proposed evolutionary scenario. It is extensively accepted that the last common ancestor of chordates possessed a completely segmented body (ancestral state). Then, changes in the FGF signal led to the loss of anterior somites releasing anterior developmental constraints and therefore allowing the appearance of a new head mesoderm through the recruitment of Tbx1 and Pitx2 as master genes.

Table 1 (early treatment) LATE TREATMENT

 1

	EARLY		log2(SU5402_	log2(SU5402_	log2(SU5402_
	TREATMENT		early_3h/contr ol_early_3h)	early_6h/contr ol_early_6h)	early_9h/contr ol_early_9h)
	Gene	Contig
	FGFRL	ContigAmph185	-0,75022	-1,01257	-0,47420
	ER81/Erm/Pea3	ContigAmph721	-1,56461	-1,79781	-1,40944
	Dusp6/7/9	ContigAmph532	-0,99657	-1,31484	-1,50884
	Six1/2	ContigAmph22225	0,05092	-3,31653	-2,15307
	Six4/5	ContigAmph11898	-1,22021	-0,66773	-0,09167
	Eya	ContigAmph11987	-0,61321	-0,82563	-0,45499
	Pax3/7	ContigAmph722	-1,73175	-0,73943	0,00035
	MRF1 (MyoD)	ContigAmph20	-2,57887	-5,02267	-4,03844
	MRF2	ContigAmph48667	-1,58165	-2,27308	-4,66284
	MRF2+	ContigAmph48666	Inf	-Inf	-3,70710
	nMRF	ContigAmph23548	-1,83499	-1,56040	-3,17398
	Mef2	ContigAmph68834	-1,26712	-0,45172	-0,74420
	MyosinIX	ContigAmph25887	2,21487	-1,71201	0,55038
	Tropomyosin	ContigAmph13883	-1,06364	-1,43108	-1,52932
	Myosin M	ContigAmph3928	-1,20789	1,40176	0,08484
	Megf10 (multi-epidermal
	growth factor)	ContigAmph5426	-0,84379	-1,4073	0,53060
	Delta	ContigAmph545	-0,78555	-1,33571	-1,30155
	HairyA	ContigAmph69139	-0,32616	-1,32118	-1,47409
	HairyB	ContigAmph23802	0,24193	-0,81341	-1,13287
	HairyC	ContigAmph13984	-0,29795	-0,94975	-1,08168
	HairyD	ContigAmph26026	0,42427	-0,25837	-1,10155
	Tbx15/18/22	ContigAmph10784	-1,79183	-1,11924	0,21992
	Tbx2/3	ContigAmph25992	-1,25959	-0,23618	-0,19686
	Ripply	ContigAmph12500	-1,70922	-3,10483	-3,46246
	Uncx4.1	ContigAmph16193	0,37002	-0,86587	-2,55917
	Hey1/2	ContigAmph10502	1,00286	-1,83256	-3,77714
	FoxAa	ContigAmph13681	0,06526	-1,90987	0,00446
	FoxAb	ContigAmph605	-1,09346	-0,47433	0,39692
	FoxD	ContigAmph16311	-1,21079	-1,05958	-1,44851
	FoxK	ContigAmph8300	-1,03081	-0,71772	-0,32742
	FoxM	ContigAmph8796	-1,44343	0,42414	0,64578
	FoxN1/4a	ContigAmph96	-1,28591	-1,39333	2,64616
	FoxJ1	ContigAmph7689	-1,21796	-0,30612	0,74905
	FoxN2/3	ContigAmph7898	-1,07121	-0,10349	0,20278
	Iroquois A	ContigAmph3136	-0,72778	-1,39670	-1,67986
	Iroquois B	ContigAmph190	-1,50283	0,37380	-0,12389

Acknowledgments

Acknowledgements D.A. holds a fellowship from "Becas Chile". S.B. is supported by the Institut Universitaire de France. H.E. laboratory was supported by the Agence National de la Recherche (ANR BLAN 1716 01). We would like to thank Angela Nieto and Jose Manuel Mingot for providing the pcDNA3-spacer-GFP-NX plasmid and Naohito Takatori for providing the Tol2 system vectors.

Iroquois C ContigAmph20930 -0,49631 -0,79893 -0,16339 Snail ContigAmph197 -0,74459 -0,71977 -0,43198 LMO4 ContigAmph6103 -0,57331 -0,65750 -0,67496 Twist1/2 ContigAmph6309 -0,83667 -0,93456 -0,54575 Nk2 ContigAmph16821 -0,70484 -1,74418 -1,26544 Nkx6 ContigAmph12366 -0,66370 -0,17798 -0,10810 MYLK (myosin light chain kinase, smooth muscle) ContigAmph6257 -0,72112 0,35196 1,45288 Mnx ContigAmph26310 -0,43697 -0,41574 -0,20361 Dmbx ContigAmph81202 -0,60131 -0,37863 0,36055 Lhx2 ContigAmph13034 -1,39335 -2,91994 -1,17088 Lim1/5 ContigAmph27865 -0,54496 -1,15735 -0,20104 Gli ContigAmph6177 -0,86631 -0,60416 -0,17213 Zic ContigAmph13605 -0,62629 -0,54165 -0,31076 SIP1 (smad interacting protein 1) ContigAmph10806 -1,00105 -0,59389 0,05705 Hedgehog protein ContigAmph85576 -0,09886 -0,46778 -0,30100 Neurogenin ContigAmph372 -0,58766 -1,17254 -0,17740 BMP3/3b ContigAmph6959 -0,07497 -0,22288 -0,48451 Gremlim ContigAmph14171 -0,16347 0,13195 0,96367 Gbx ContigAmph272 -0,62605 -0,55233 0,07332 Orthopedia ContigAmph7952 -0,11142 -0,43097 -0,49430 CBFA2T1 ContigAmph18897 -0,27458 -0,58582 -0,03539 FezF ContigAmph11 -0,74738 -0,85548 -0,42916 Ash ContigAmph2086 0,33137 -0,58692 -0,02093 SoxE ContigAmph589 -0,47233 -0,56939 -0,32775 Frizzled4 ContigAmph6161 -0,28670 -0,22734 -0,13469 Frizzled5/8 ContigAmph30949 0,89175 0,15306 -0,20587 FoxC ContigAmph25978 -0,45362 -0,40519 -0,21133 Jagged1 ContigAmph5697 0,87788 -0,20664 -0,73096 Jagged2 ContigAmph5698 0,97711 -0,16317 -0,71654 Hox2 ContigAmph34651 -1,37347 -1,71724 -1,00728 Hox1 ContigAmph13529 -0,28290 -0,32049 0,07783 Cdx ContigAmph13490 -0,43771 -0,47840 0,00503 EphrinA ContigAmph852 -0,36585 -0,20883 0,13685 zinc finger FYVE domain ContigAmph19894 0,12568 -0,08577 -0,07575 DDR2 (discoidin domaincontaining receptor) ContigAmph8142 -1,04730 -0,39516 0,06935 recombining binding protein suppressor of hairless ContigAmph4612 -0,54059 -0,24352 0,17370 SFMBT2 (Scm-like with four MBT domains protein 1) ContigAmph33445 -0,39933 -0,32113 -0,37411 TGF-B (transforming growth factor beta receptor type 3 precursor) ContigAmph19500 -0,41427 -0,26066 0,11028 Patched protein like ContigAmph5682 -0,43257 -0,10653 0,34510 Plecktrin5/7/9 ContigAmph17242 -0,24745 -0,06351 0,16132 RS-like (rolling stone-like) ContigAmph21895 -0,34099 0,06819 -0,00649 Uncharacterized protein (Trauco) ContigAmph71939 -0,14778 -0,50688 1,00230 Zinc finger protein (Caleuche) ContigAmph6363 -0,79437 -1,28931 -0,20981 F-box only protein 32 (Pincoya) ContigAmph32885 -0,78725 -0,40273 -0,61728 WD repeat 49 ContigAmph1857 0,196526 0,01251 0,00767 AnkirinBR1 ContigAmph24453 -0,46296 -0,13931 0,15490 Ankirin-notch ContigAmph8400 -0,67667 0,02464 0,28382 Ataxin ContigAmph3989 -0,32558 -0,34553 0,17824 Coe ContigAmph1527 -1,02104 -0,57769 -0,20524 Mdp ContigAmph11791 -0,37071 -0,42159 -0,10674 Arx ContigAmph7953 -0,41841 -0,08032 -0,17481 KREMEN ContigAmph1046 -0,17690 -0,53029 -0,35302 4andhalfLIM domain protein 2 ContigAmph17569 0,584313 0,13634 0,22598 4andhalfLIM domain protein 5 ContigAmph15489 -0,72203 -1,06985 -0,59169 BIGH (transforming growth factor-beta-induced protein ig-3) ContigAmph44472 0,12063 -0,40541 0,35041 C2orf81 homolog ContigAmph12430 0,05603 -0,03099 -0,29486 Pitx ContigAmph618 -0,67810 -0,84858 0,02582 Tbx1/10 ContigAmph12871 -0,86406 -1,18742 -1,32082 FoxEa ContigAmph10961 -0,71653 -0,94467 -0,83185 Scratch ContigAmph19102 -0,17393 -1,03846 -0,14243 Table 1 (late treatment)

Evolution of the role of RA and FGF signals in the control of somitogenesis in Chordates Abstract

The appearance of new body structures during animal evolution is a central question in Evolutionary Developmental Biology (Evo-Devo). Particularly, the origin of the vertebrates' head has attracted the attention of researchers for many years. In this context, one striking question concerns the evolution of the unsegmented mesoderm of the vertebrates' head from a completely segmented amphioxus-like ancestor. It is extensively assumed that paraxial mesoderm of the hypothetical chordate ancestor, as that of extant cephalochordates, was segmented from its most anterior to its most posterior part. Interestingly, it has been shown that formation of the anterior-most somites in the cephalochordate amphioxus is controlled by the FGF signaling, suggesting that functional evolution of this signaling pathway during early development in the vertebrate ancestor could have played a key role in the loss of mesoderm segmentation in the vertebrate's head.

Here, using a comparative RNA-seq approach, we looked for "putative genes", controlled by the FGF signal during early development in amphioxus, playing a role in the control of anterior somitogenesis. Then, we functionally tested whether some of these genes directly control anterior somitogenesis in amphioxus. Our results show a functional compartmentalization of the paraxial mesoderm in amphioxus, where Six1/2 and the FGF effector ER81/Erm/PEA3 are major players in the anterior somitogenesis in amphioxus while Pax3/7 controls posterior somitogenesis. This regulatory cascade in amphioxus resembles that for the control of trunk somitogenesis in vertebrates and diverges from the gene cascades controlling the formation of the vertebrate head muscles. Our results strengthen the hypothesis that changes in the FGF function during early development were instrumental for the loss of anterior somites, releasing developmental constraints in the anterior part of the embryo and allowing a secondary acquisition of head muscles in the ancestor of vertebrates.

Cloning and in situ hybridization

For B. lanceolatum genes not previously published, sequences were recovered from its reference transcriptome (Oulion et al., 2012a) by TBLASTN using sequences from B. floridae as queries. Specific primers were then designed for RT-PCR amplification of partial coding regions. We also designed specific primers to amplify the 3'UTR regions of ER81/Erm/PEA3, Six1/2 and Pax3/7. The complete list of primers used is presented in Table 2. A mix of total RNA 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. All the clones were confirmed by Sanger sequencing. For GFP, probe was synthesized from a pcDNA3-spacer-GFP-NX plasmid (gift from Angela Nieto and Jose Manuel Mingot). Whole mount in situ hybridizations were performed as described in Somorjai et al. (2008) (Somorjai et al., 2008).

Constructs, in vitro mRNA synthesis and microinjection

All the vectors were constructed using the pCS2+ expression vector backbone.

Constitutive activator forms of Pax3/7 (VP16-Pax3/7), Six1/2 (VP16-Six1/2) and ER81/Erm/Pea3 (VP16-EEP) were created by fusing the coding sequence of the 81 aa activation domain of VP16 protein (Friedman et al., 1988) belcheri (Feng et al., 2014). We then cloned our putative enhancers from genomic DNA of B.

Amphioxus somitogenesis sheds light on the evolution of the vertebrate's head

Skeletal muscle determination and differentiation in vertebrates is driven by members of the myogenic regulatory family (MRF), such as MyoD, Myf5, or MRF4. These genes are implicated in the control of myogenesis both in the head and the trunk. However, their expression is controlled by different upstream regulatory cascades in these two body regions.

In the trunk, it has been shown that Pax3 and members of the SIX family together with their cofactors EYA, are major regulators of myogenesis, while in the head, Pitx2 plays a crucial role in the formation of the head muscles derived from both the prechordal cranial mesoderm (PCM) and the cranial paraxial mesoderm (CPM), whereas Tbx1 play a pivotal role in the formation of the muscles derived from the CPM (Sambasivan et al., 2011).

Here, we have shown that amphioxus anterior somitogenesis is controlled by the FGF signal through its effector ER81/Erm/PEA3, which in turn controls Six1/2 expression. Moreover, we have shown a compartmentalization of the amphioxus somites along the anteroposterior axis. Only the most anterior somites are controlled by Six1/2 and the FGF signal, while posterior somites are controlled by Pax3/7, which probably also plays a minor role in anterior somitogenesis since it is expressed in the paraxial mesoderm of the amphioxus gastrulae. Interestingly, Pitx and Tbx1/10 are not expressed during early development of amphioxus when the most anterior somites form. Indeed, Pitx is expressed asymmetrically starting at the midneurula stage in somites and endoderm suggesting it is implicated in left/right axis patterning (Boorman and Shimeld, 2002) whereas Tbx1/10 is expressed in the ventral part of the somites but at late stages, when the anterior somites are already formed (Mahadevan et al., 2004). Moreover, injection of Tbx1/10 morpholinos does not seem to affect somitogenesis, but mainly lead to the formation of a shorter pharynx (Koop et al., 2014). Altogether these results indicate that muscles of the head of vertebrates are not formed using the same program as anterior muscles in amphioxus.

In the context of the intense debate about the origin of the vertebrate's head, our results are particularly interesting. Indeed, since amphioxus anterior somitogenesis is controlled by a similar regulatory cascade as trunk myogenesis in vertebrates, and major regulators of head myogenesis in vertebrates do not play a similar role in amphioxus, it is tempting to propose the following evolutionary scenario. An amphioxus-like vertebrate ancestor, completely segmented from its most anterior to its most posterior part, formed its anterior somites using a FGF-ER81/Erm/PEA3-Six1/2 gene regulatory cascade and its most posterior somites using a Pax3/7 regulatory cascade. Then, during evolution, the role of the FGF signal in the control of Table 2. Primers for all the genes that were cloned. Name of each gene and forward and reverse sequence for each of these primers.

Supplementary figure legends

Supplementary figure 1. Experimental approach for the comparative RNA-seq analysis. Embryos were treated with SU5402, an inhibitor of the FGF receptor at 5 hpf (at 19°C, blastula stage) and at 15,5 hpf (at 19°C, late gastrula stage). Total RNA extraction was undertaken 3h, 6h and 9h after either early or late treatment, and sequenced using Illumina technology. hpf: hours post fertilization, (E3, E6, E9): total RNA extraction for early treatment, (L3, L6, L9): total RNA extraction for late treatment. 2. In situ hybridization of downregulated genes at 11 hpf and 14 hpf in control and SU5402-treated embryos. Blastoporal views in all panels. Vertebrate orthologues of effectors of the FGF signalling pathway as ER81/Erm/Pea3 (EEP) (a-d) and Dusp6/7/9 (e-h) are downregulated in treated embryos (b,d,f,h). Orthologues of genes that in vertebrates are involved in myogenesis as Six1/2 (i-l), Six4/5 (m-p), Eya (q-t), Pax3/7 (u-x), FoxC (y-b'), MRF2 (c'-f') and Mef2 (g'-j') are downregulated in the paraxial mesoderm after SU5402 treatment (j,l,n,p,r,t,v,x,z,b',d',f',h',j'). Orthologues of genes that in vertebrates are involved in somite segmentation as Delta (k'-n'), HairyB (o'-r'), HairyC (s'-v'), HairyD (w'z'), Ripply (a''-d''), Hey1/2 (e''-h''), Uncx4.1 (i''-l''), Tbx15/18/22 (m''-p'') are expressed in stripes in control embryos whereas this pattern is lost after SU5402 treatment. Snail (q''-t'') and the Snail co-factor LMO4 (u''-x'') expression is lost in the paraxial mesoderm following SU5402 treatment. Similarly for Twist1/2 (y''-b''') and Smad interacting protein 1 (SPI1) (c'''-f''') is observed a downregulation in the paraxial mesoderm but not in the axial mesoderm. Zic is mildly downregulated after treatment (g'''-j'''), whereas Gli shows a strong downregulation (k'''-n'''). Finally, Frizzled4 (Fz4) is downregulated in the paraxial mesoderm of treated embryos (o'''-r'''). Scale bar=100 μm.

Supplementary figure