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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) (Pallas, 1774). 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 (Costa, 1834). 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).
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.
The phylum Cephalochordata is composed by three genus; Branchiostoma, Asymmetron and Epigonichthys (Poss and Boschung, 1996). 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).
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.
Phylogenetic Position and Amphioxus Species
Based on morphological characteristics and phylogenetic studies using the ribosomal ribonucleic acid coding gene sequences (rRNA) (Winchell et al., 2002), 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 (Delsuc et al., 2006; Delsuc et al., 2008; Putnam et al., 2008). Additionally, new studies have identified in urochordates a type of migratory neural crest-like cells similar to the vertebrate neural crest cells, a typical characteristic of the vertebrates and absent in amphioxus (Jeffery et al., 2004). Thus, the new phylogenetic tree of life places the phylum Cephalochordata as the most basally divergent group among chordates (Figure 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 (Delsuc et al., 2006). 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 (Aguinaldo et al., 1997; de Rosa et al., 1999)
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) (Poss and Boschung, 1996). 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 (Zhang et al., 2006), 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.
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 (Poss and Boschung, 1996).
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 (Desdevises et al., 2011; Gosselck and Spittler, 1979; Webb and Hill, 1958). So far, only one species living in deep waters (229 meters depth) has been identified and called Asymmetron inferum (Kon et al., 2007).
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 (Holland and Holland, 2010). 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 (Holland and Holland, 2010). 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; Futch and Dwinell, 1977).
Embryonic Development
The embryonic development of the genus Branchiostoma has been very well described and studied for more than 150 years (Cerfontaine, 1906; Conklin, 1932; Hatschek, 1893; Kowalevsky, 1867, 1876; Wilson, 1892, 1893). 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 (Holland and Holland, 2010). 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 (Holland and Onai, 2012).
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 (Holland and Onai, 2012).
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 (Langeland et al., 1998).
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 (Hirakow and Kajita, 1994). 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).
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).
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 (Delsuc et al., 2006). 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.
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 (Bertrand et al., 2007). (C) Pikaia gracilens found in Burges Shale in Canada, modified from (Long, 1995).
During the last decades the advances in whole genome analyses showed that amphioxus possess a “simple” genome (Putnam et al., 2008). Thus, as it was proposed by Ohno (Ohno, 1970), and confirmed later by Dehal et al. (Dehal and Boore, 2005) two rounds of whole genome duplications occurred during the evolutionary history of vertebrates (three in teleosts) (Figure 9) (Jaillon et al., 2004; Meyer and Schartl, 1999; Taylor et al., 2003)). What still remains controversial is the precise timing of these duplications. In any case, the duplications happened after the divergence of cephalochordates and urochordates (Putnam et al., 2008) and before the divergence of chondrichtyans (Robinson-Rechavi et al., 2004). 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 (Smith et al., 2013). 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 (Garcia-Fernandez and Holland, 1994) 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.
Figure 15. Hox clusters in mammals and amphioxus. Due to the two rounds of whole genome duplications proposed by (Ohno, 1970) (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 (David and Mooi, 2014).
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 (Huang et al., 2014; Mou et al., 2002; Oulion et al., 2012a; Putnam et al., 2008). 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 (Holland et al., 2015). 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 (Holland and Onai, 2011). 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) (Li et al., 2014). Finally, microinjections of plasmids can also be used in several species to obtain transient mosaic transgenic embryos.
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 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; Yabe and Takada, 2016). 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 (Brent and Tabin, 2002).
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; Scaal and Wiegreffe, 2006).
Table of contents :
1. Introduction
1.1 HYPOTHESES FOR THE ORIGIN OF THE VERTEBRATES’ HEAD
1.1.1 Segmentalist Hypothesis
1.1.2 Non Segmentalist Hypothesis
1.1.3 The “New Head” Hypothesis
1.1.4 Final remarks
1.2 AMPHIOXUS AS A MODEL
1.2.1 Identification and Description of Amphioxus
1.2.2 Phylogenetic Position and Amphioxus Species
1.2.3 Environment
1.2.4 Reproduction and Life Cycle
1.2.4 Embryonic Development
1.2.5 Amphioxus as an Animal Model in EVO-DEVO
1.2.6 Final remarks
1.3 SOMITOGENESIS
1.3.1 Somitogenesis in vertebrates and amphioxus: a morphological description
1.3.2 Molecular Control of Somitogenesis
1.4 MYOGENESIS
1.4.1 Genetic control of truncal myogenesis in vertebrates and amphioxus
1.4.2 Formation of Facial Muscles in Vertebrates
1.4.3 Orthologues of vertebrate head muscles formation in amphioxus
1.4.4 Final remarks
1.5 FGF SIGNALING PATHWAY AND ANTERIOR SOMITOGENESIS IN AMPHIOXUS
1.5.1 FGF signaling pathway
1.5.2 Expression of FGFs in amphioxus
1.5.3 FGF signaling pathway controls anterior somitogenesis in amphioxus
1.5.4 Final remarks
2. Introduction of Article
2.1 Evolution of the role of RA and FGF signals in the control of somitogenesis in Chordates
3. Introduction Article
3.1 Anterior somitogenesis in amphioxus sheds lights on the origin of the vertebrates’ head
4. General discussion and additional data
4.1 Hox genes, FGF signal and the anterior somitogenesis in amphioxus
4.2 RNA-seq analysis and the role of the FGF signal in anterior somitogenesis
4.3 Possible role of upregulated genes in anterior somitogenesis
4.4 Six1/2 and Pax3/7 control somitogenesis in amphioxus
4.5 FGF signal and vertebrates head mesoderm
4.6 Our results under the context of the evolution of the vertebrates’ head
5. Annex Articles
5.1 Expression of Fox genes in the cephalochordate Branchiostoma lanceolatum
5.2 A single three-dimensional chromatin compartment in amphioxus indicates a stepwise evolution of vertebrate Hox bimodal regulation
6. Bibliography
