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Abstract Background Fibroblast Growth Factors (FGF) and their receptors are well known for having major implications in cell signalling controlling embryonic development. Recently, a gene coding for a protein closely related to FGFRs (Fibroblast Growth Factor Receptors) called FGFR5 or FGFR-like 1 (FGFRL1), has been described in vertebrates. An orthologous gene was also found in the cephalochordate amphioxus, but no orthologous genes were found by the authors in other non-vertebrate species, even if a FGFRL1 gene was identified in the sea urchin genome, as well as a closely related gene, named nou-darake, in the planarian Dugesia japonica. These intriguing data of a deuterostome-specific gene that might be implicated in FGF signalling prompted us to search for putative FGFRL1 orthologues in the completely sequenced genomes of metazoans. Results We found FGFRL1 genes in the cnidarian Nematostella vectensis as well as in many bilaterian species. Our analysis also shows that FGFRL1 orthologous genes are linked in the genome with other members of the FGF signalling pathway from cnidarians to bilaterians (distance < 10 Mb). To better understand the implication of FGFRL1 genes in chordate embryonic development, we have analyzed expression patterns of the amphioxus and the mouse genes by whole mount in situ hybridization. We show that some homologous expression territories can be defined, and we propose that FGFRL1 and FGF8/17/18 were already co-expressed in the pharyngeal endoderm in the ancestor of chordates. Conclusion Our work sheds light on the existence of a putative FGF signalling pathway actor present in the ancestor of probably all metazoans, the function of which has received little attention until now.

Abstract Background The origin of nuclear receptors (NRs) and the question whether the ancestral NR was a liganded or an unliganded transcription factor has been recently debated. To obtain insight into the evolution of the ligand binding ability of estrogen receptors (ER), we comparatively characterized the ER from the protochordate amphioxus (Branchiostoma floridae), and the ER from lamprey (Petromyzon marinus), a basal vertebrate. Results Extensive phylogenetic studies as well as signature analysis allowed us to confirm that the amphioxus ER (amphiER) and the lamprey ER (lampER) belong to the ER group. LampER behaves as a "classical" vertebrate ER, as it binds to specific DNA Estrogen Responsive Elements (EREs), and is activated by estradiol (E2), the classical ER natural ligand. In contrast, we found that although amphiER binds EREs, it is unable to bind E2 and to activate transcription in response to E2. Among the 7 natural and synthetic ER ligands tested as well as a large repertoire of 14 cholesterol derivatives, only Bisphenol A (an endocrine disruptor with estrogenic activity) bound to amphiER, suggesting that a ligand binding pocket exists within the receptor. Parsimony analysis considering all available ER sequences suggest that the ancestral ER was not able to bind E2 and that this ability evolved specifically in the vertebrate lineage. This result does not support a previous analysis based on ancestral sequence reconstruction that proposed the ancestral steroid receptor to bind estradiol. We show that biased taxonomic sampling can alter the calculation of ancestral sequence and that the previous result might stem from a high proportion of vertebrate ERs in the dataset used to compute the ancestral sequence. Conclusion Taken together, our results highlight the importance of comparative experimental approaches vs ancestral reconstructions for the evolutionary study of endocrine systems: comparative analysis of extant ERs suggests that the ancestral ER did not bind estradiol and that it gained the ability to be regulated by estradiol specifically in the vertebrate lineage, before lamprey split.

Regulation of gene expression is arguably the main mechanism underlying the phenotypic diversity of tissues within and between species. Here we assembled an extensive transcriptomic dataset covering 8 tissues across 20 bilaterian species and performed analyses using a symmetric phylogeny that allowed the combined and parallel investigation of gene expression evolution between vertebrates and insects. We specifically focused on widely conserved ancestral genes, identifying strong cores of pan-bilaterian tissue-specific genes and even larger groups that diverged to define vertebrate and insect tissues. Systematic inferences of tissue-specificity gains and losses show that nearly half of all ancestral genes have been recruited into tissue-specific transcriptomes. This occurred during both ancient and, especially, recent bilaterian evolution, with several gains being associated with the emergence of unique phenotypes (for example, novel cell types). Such pervasive evolution of tissue specificity was linked to gene duplication coupled with expression specialization of one of the copies, revealing an unappreciated prolonged effect of whole-genome duplications on recent vertebrate evolution., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

The emergence of new structures can often be linked to the evolution of novel cell types that follows the rewiring of developmental gene regulatory subnetworks. Vertebrates are characterized by a complex body plan compared to the other chordate clades and the question remains of whether and how the emergence of vertebrate morphological innovations can be related to the appearance of new embryonic cell populations. We previously proposed, by studying mesoderm development in the cephalochordate amphioxus, a scenario for the evolution of the vertebrate head mesoderm. To further test this scenario at the cell population level, we used scRNA-seq to construct a cell atlas of the amphioxus neurula, stage at which the main mesodermal compartments are specified. Our data allowed us to validate the presence of a prechordal-plate like territory in amphioxus. Additionally, the transcriptomic profile of somite cell populations supports the homology between specific territories of amphioxus somites and vertebrate cranial/pharyngeal and lateral plate mesoderm. Finally, our work provides evidence that the appearance of the specific mesodermal structures of the vertebrate head was associated to both segregation of pre-existing cell populations, and co-option of new genes for the control of myogenesis., (© 2024. The Author(s).)

Cephalochordates and tunicates represent the only two groups of invertebrate chordates, and extant cephalochordates - commonly known as amphioxus or lancelets - are considered the best proxy for the chordate ancestor, from which they split around 520 million years ago. Amphioxus has been an important organism in the fields of zoology and embryology since the 18 th century, and the morphological and genomic simplicity of cephalochordates (compared to vertebrates) makes amphioxus an attractive model for studying chordate biology at the cellular and molecular levels. Here we describe the life cycle of amphioxus, and discuss the natural histories and habitats of the different species of amphioxus. We also describe their use as laboratory animal models, and discuss the techniques that have been developed to study different aspects of amphioxus., Competing Interests: SD, SB, HE No competing interests declared, (© 2023, D'Aniello et al.)

Oceanic plastic pollution is of major concern to marine organisms, especially filter feeders. However, limited is known about the toxic effects of the weathered microplastics instead of the pristine ones. This study evaluates the effects of weathered polystyrene microplastic on a filter-feeder amphioxus under starvation conditions via its exposure to the microplastics previously deployed in the natural seawater allowing for the development of a mature biofilm (so-called plastisphere). The study focused on the integration of physiological, histological, biochemical, molecular, and microbiota impacts on amphioxus. Overall, specific alterations in gene expression of marker genes were observed to be associated with oxidative stresses and immune systems. Negligible impacts were observed on antioxidant biochemical activities and gut microbiota of amphioxus, while we highlighted the potential transfer of 12 bacterial taxa from the plastisphere to the amphioxus gut microbiota. Moreover, the classical perturbation of body shape detected in control animals under starvation conditions (a slim and curved body) but not for amphioxus exposed to microplastic, indicates that the microorganisms colonizing plastics could serve as a nutrient source for this filter-feeder, commitment with the elevated proportions of goblet cell-like structures after the microplastic exposure. The multidisciplinary approach developed in this study underlined the trait of microplastics that acted as vectors for transporting microorganisms from the plastisphere to amphioxus., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

DNA methylation [5-methylcytosine (5mC)] is a repressive gene-regulatory mark required for vertebrate embryogenesis. Genomic 5mC is tightly regulated through the action of DNA methyltransferases, which deposit 5mC, and ten-eleven translocation (TET) enzymes, which participate in its active removal through the formation of 5-hydroxymethylcytosine (5hmC). TET enzymes are essential for mammalian gastrulation and activation of vertebrate developmental enhancers; however, to date, a clear picture of 5hmC function, abundance, and genomic distribution in nonvertebrate lineages is lacking. By using base-resolution 5mC and 5hmC quantification during sea urchin and lancelet embryogenesis, we shed light on the roles of nonvertebrate 5hmC and TET enzymes. We find that these invertebrate deuterostomes use TET enzymes for targeted demethylation of regulatory regions associated with developmental genes and show that the complement of identified 5hmC-regulated genes is conserved to vertebrates. This work demonstrates that active 5mC removal from regulatory regions is a common feature of deuterostome embryogenesis suggestive of an unexpected deep conservation of a major gene-regulatory module.

Background: Amphioxus are non-vertebrate chordates characterized by a slow morphological and molecular evolution. They share the basic chordate body-plan and genome organization with vertebrates but lack their 2R whole-genome duplications and their developmental complexity. For these reasons, amphioxus are frequently used as an outgroup to study vertebrate genome evolution and Evo-Devo. Aside from whole-genome duplications, genes continuously duplicate on a smaller scale. Small-scale duplicated genes can be found in both amphioxus and vertebrate genomes, while only the vertebrate genomes have duplicated genes product of their 2R whole-genome duplications. Here, we explore the history of small-scale gene duplications in the amphioxus lineage and compare it to small- and large-scale gene duplication history in vertebrates., Results: We present a study of the European amphioxus (Branchiostoma lanceolatum) gene duplications thanks to a new, high-quality genome reference. We find that, despite its overall slow molecular evolution, the amphioxus lineage has had a history of small-scale duplications similar to the one observed in vertebrates. We find parallel gene duplication profiles between amphioxus and vertebrates and conserved functional constraints in gene duplication. Moreover, amphioxus gene duplicates show levels of expression and patterns of functional specialization similar to the ones observed in vertebrate duplicated genes. We also find strong conservation of gene synteny between two distant amphioxus species, B. lanceolatum and B. floridae, with two major chromosomal rearrangements., Conclusions: In contrast to their slower molecular and morphological evolution, amphioxus' small-scale gene duplication history resembles that of the vertebrate lineage both in quantitative and in functional terms., (© 2022. The Author(s).)

Photoconvertible proteins are powerful tools widely used in cellular biology to study cell dynamics and organelles. Over the past decade, photoconvertible proteins have also been used for developmental biology applications to analyze cell lineage and cell fate during embryonic development. One of these photoconvertible proteins called Kaede, from the stony coral Trachyphyllia geoffroyi, undergoes irreversible photoconversion from green to red fluorescence when illuminated with UV light. Undertaking a cell tracing approach using photoconvertible proteins can be challenging when using unconventional animal models. In this protocol, we describe the use of Kaede to track specific cells during embryogenesis of the cephalochordate Branchiostoma lanceolatum. This protocol can be adapted to other unconventional models, especially marine animals., Competing Interests: The authors have declared that no competing interests exist.

The fibroblast growth factor (FGF) signalling pathway plays various roles during vertebrate embryogenesis, from mesoderm formation to brain patterning. This diversity of functions relies on the fact that vertebrates possess the largest FGF gene complement among metazoans. In the cephalochordate amphioxus, which belongs to the chordate clade together with vertebrates and tunicates, we have previously shown that the main role of FGF during early development is the control of rostral somite formation. Inhibition of this signalling pathway induces the loss of these structures, resulting in an embryo without anterior segmented mesoderm, as in the vertebrate head. Here, by combining several approaches, we show that the anterior presumptive paraxial mesoderm cells acquire an anterior axial fate when FGF signal is inhibited and that they are later incorporated in the anterior notochord. Our analysis of notochord formation in wild type and in embryos in which FGF signalling is inhibited also reveals that amphioxus anterior notochord presents transient prechordal plate features. Altogether, our results give insight into how changes in FGF functions during chordate evolution might have participated to the emergence of the complex vertebrate head., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2022. Published by The Company of Biologists Ltd.)

Neurons are a highly specialized cell type only found in metazoans. They can be scattered throughout the body or grouped together, forming ganglia or nerve cords. During embryogenesis, centralized nervous systems develop from the ectoderm, which also forms the epidermis. How pluripotent ectodermal cells are directed toward neural or epidermal fates, and to which extent this process is shared among different animal lineages, are still open questions. Here, by using micromere explants, we were able to define in silico the putative gene regulatory networks (GRNs) underlying the first steps of the epidermis and the central nervous system formation in the cephalochordate amphioxus. We propose that although the signal triggering neural induction in amphioxus (i.e., Nodal) is different from vertebrates, the main transcription factors implicated in this process are conserved. Moreover, our data reveal that transcription factors of the neural program seem to not only activate neural genes but also to potentially have direct inputs into the epidermal GRN, suggesting that the Nodal signal might also contribute to neural fate commitment by repressing the epidermal program. Our functional data on whole embryos support this result and highlight the complex interactions among the transcription factors activated by the signaling pathways that drive ectodermal cell fate choice in chordates., (© The Author(s) 2022. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

During animal ontogenesis, body axis patterning is finely regulated by complex interactions among several signaling pathways. Nitric oxide (NO) and retinoic acid (RA) are potent morphogens that play a pivotal role in vertebrate development. Their involvement in axial patterning of the head and pharynx shows conserved features in the chordate phylum. Indeed, in the cephalochordate amphioxus, NO and RA are crucial for the correct development of pharyngeal structures. Here, we demonstrate the functional cooperation between NO and RA that occurs during amphioxus embryogenesis. During neurulation, NO modulates RA production through the transcriptional regulation of Aldh1a.2 that irreversibly converts retinaldehyde into RA. On the other hand, RA directly or indirectly regulates the transcription of Nos genes. This reciprocal regulation of NO and RA pathways is essential for the normal pharyngeal development in amphioxus and it could be conserved in vertebrates., Competing Interests: FC, GA, LS, HE, SB, SD No competing interests declared, (© 2021, Caccavale et al.)

About eight million animal species are estimated to live on Earth, and all except those belonging to one subphylum are invertebrates. Invertebrates are incredibly diverse in their morphologies, life histories, and in the range of the ecological niches that they occupy. A great variety of modes of reproduction and sex determination systems is also observed among them, and their mosaic-distribution across the phylogeny shows that transitions between them occur frequently and rapidly. Genetic conflict in its various forms is a long-standing theory to explain what drives those evolutionary transitions. Here, we review (1) the different modes of reproduction among invertebrate species, highlighting sexual reproduction as the probable ancestral state; (2) the paradoxical diversity of sex determination systems; (3) the different types of genetic conflicts that could drive the evolution of such different systems.

Mammalian brains feature exceptionally high levels of non-CpG DNA methylation alongside the canonical form of CpG methylation. Non-CpG methylation plays a critical regulatory role in cognitive function, which is mediated by the binding of MeCP2, the transcriptional regulator that when mutated causes Rett syndrome. However, it is unclear whether the non-CpG neural methylation system is restricted to mammalian species with complex cognitive abilities or has deeper evolutionary origins. To test this, we investigated brain DNA methylation across 12 distantly related animal lineages, revealing that non-CpG methylation is restricted to vertebrates. We discovered that in vertebrates, non-CpG methylation is enriched within a highly conserved set of developmental genes transcriptionally repressed in adult brains, indicating that it demarcates a deeply conserved regulatory program. We also found that the writer of non-CpG methylation, DNMT3A, and the reader, MeCP2, originated at the onset of vertebrates as a result of the ancestral vertebrate whole-genome duplication. Together, we demonstrate how this novel layer of epigenetic information assembled at the root of vertebrates and gained new regulatory roles independent of the ancestral form of the canonical CpG methylation. This suggests that the emergence of non-CpG methylation may have fostered the evolution of sophisticated cognitive abilities found in the vertebrate lineage.

The cephalochordates amphioxus or lancelets are benthic marine animals representing the earliest divergent evolutionary lineage within chordates. Although amphioxus are present in most of the world's tropical and temperate oceans, only about thirty different species grouped into three different genera, Branchiostoma, Epigonichthys and Asymmetron have been described. In the genus Asymmetron, only two species have been characterized, although for one of them, A. lucayanum, several cryptic lineages exist. In this work we have sequenced and analyzed the mitogenome of an A. lucayanum population previously described in the Red Sea. The phylogenetic study using this complete mitogenome as well as the analysis of COI gene sequences of several individuals of this Red Sea population show that the Red Sea population is a new cryptic species. We propose to call this new species Asymmetron rubrum., Competing Interests: The authors have declared that no competing interests exist

The evolution of vertebrates from an ancestral chordate was accompanied by the acquisition of a predatory lifestyle closely associated to the origin of a novel anterior structure, the highly specialized head. While the vertebrate head mesoderm is unsegmented, the paraxial mesoderm of the earliest divergent chordate clade, the cephalochordates (amphioxus), is fully segmented in somites. We have previously shown that fibroblast growth factor signalling controls the formation of the most anterior somites in amphioxus; therefore, unravelling the fibroblast growth factor signalling downstream effectors is of crucial importance to shed light on the evolutionary origin of vertebrate head muscles. By using a comparative RNA sequencing approach and genetic functional analyses, we show that several transcription factors, such as Six1/2, Pax3/7 and Zic, act in combination to ensure the formation of three different somite populations. Interestingly, these proteins are orthologous to key regulators of trunk, and not head, muscle formation in vertebrates. Contrary to prevailing thinking, our results suggest that the vertebrate head mesoderm is of visceral and not paraxial origin and support a multistep evolutionary scenario for the appearance of the unsegmented mesoderm of the vertebrates new 'head'.

Amphioxus represents the most basally divergent group in chordates and probably the best extant proxy to the ancestor of all chordates including vertebrates. The amphioxus, or lancelets, are benthic filter feeding marine animals and their interest as a model in research is due to their phylogenetic position and their anatomical and genetic stasis throughout their evolutionary history. From the first works in the 19th century to the present day, enormous progress is made mainly favored by technical development at different levels, from spawning induction and husbandry techniques, through techniques for studies of gene function or of the role of different signalling pathways through embryonic development, to functional genomics techniques. Together, these advances foretell a plethora of interesting developments in the world of research with the amphioxus model. Here, the discovery and development of amphioxus as a superb model organism in evolutionary and evolutionary-developmental biology are reviewed., (© 2018 WILEY Periodicals, Inc.)

Vertebrates have greatly elaborated the basic chordate body plan and evolved highly distinctive genomes that have been sculpted by two whole-genome duplications. Here we sequence the genome of the Mediterranean amphioxus (Branchiostoma lanceolatum) and characterize DNA methylation, chromatin accessibility, histone modifications and transcriptomes across multiple developmental stages and adult tissues to investigate the evolution of the regulation of the chordate genome. Comparisons with vertebrates identify an intermediate stage in the evolution of differentially methylated enhancers, and a high conservation of gene expression and its cis-regulatory logic between amphioxus and vertebrates that occurs maximally at an earlier mid-embryonic phylotypic period. We analyse regulatory evolution after whole-genome duplications, and find that-in vertebrates-over 80% of broadly expressed gene families with multiple paralogues derived from whole-genome duplications have members that restricted their ancestral expression, and underwent specialization rather than subfunctionalization. Counter-intuitively, paralogues that restricted their expression increased the complexity of their regulatory landscapes. These data pave the way for a better understanding of the regulatory principles that underlie key vertebrate innovations.

Glutamate receptors are divided in two unrelated families: ionotropic (iGluR), driving synaptic transmission, and metabotropic (mGluR), which modulate synaptic strength. The present classification of GluRs is based on vertebrate proteins and has remained unchanged for over two decades. Here we report an exhaustive phylogenetic study of GluRs in metazoans. Importantly, we demonstrate that GluRs have followed different evolutionary histories in separated animal lineages. Our analysis reveals that the present organization of iGluRs into six classes does not capture the full complexity of their evolution. Instead, we propose an organization into four subfamilies and ten classes, four of which have never been previously described. Furthermore, we report a sister class to mGluR classes I-III, class IV. We show that many unreported proteins are expressed in the nervous system, and that new Epsilon receptors form functional ligand-gated ion channels. We propose an updated classification of glutamate receptors that includes our findings., Competing Interests: DR, JJ, EG, GG, RR, JL, DB, EN, JG, PF, HE, NR, DS, ÀB No competing interests declared, (© 2018, Ramos-Vicente et al.)

In 2016, Kaji et al. concluded that the amphioxus mouth has the quality of a coelomoduct and is, therefore, not homologous to the oral opening of any other animal. They studied a Japanese population of Branchiostoma japonicum and based their conclusion, in part, on the larval expression of BMP2/4 in cells that reportedly joined the rim of the forming mouth. They did not detect transcription of that gene in any other tissues in the anterior region of the larva. Their results were almost the inverse of findings for B. floridae by Panopoulou et al. (1998), who detected BMP2/4 expression in several anterior tissues, but not in cells intimately associated with the nascent mouth. To resolve this discrepancy, we have studied BMP2/4 in a Chinese population of B. japonicum as well as in an additional species, the European B. lanceolatum. In both species, larval expression of BMP2/4 closely resembles the pattern previously reported for B. floridae-that is, transcription is undetectable in tissues juxtaposed to the forming mouth, but is seen in several other anterior structures (most conspicuously in the lining of the rostral coelom and the club-shaped gland). In sum, we could not repeat the BMP2/4 expression pattern of Kaji et al. (2016) even in the same species, and their findings for this gene, at least, cannot be counted as a support for their hypothesis for a coelomoduct mouth., (Copyright © 2017. Published by Elsevier B.V.)

Neural induction is the process through which pluripotent cells are committed to a neural fate. This first step of Central Nervous System formation is triggered by the "Spemann organizer" in amphibians and by homologous embryonic regions in other vertebrates. Studies in classical vertebrate models have produced contrasting views about the molecular nature of neural inducers and no unifying scheme could be drawn. Moreover, how this process evolved in the chordate lineage remains an unresolved issue. In this work, by using graft and micromanipulation experiments, we definitively establish that the dorsal blastopore lip of the cephalochordate amphioxus is homologous to the vertebrate organizer and is able to trigger the formation of neural tissues in a host embryo. In addition, we demonstrate that Nodal/Activin is the main signal eliciting neural induction in amphioxus, and that it also functions as a bona fide neural inducer in the classical vertebrate model Xenopus . Altogether, our results allow us to propose that Nodal/Activin was a major player of neural induction in the ancestor of chordates. This study further reveals the diversity of neural inducers deployed during chordate evolution and advocates against a universally conserved molecular explanation for this process., Competing Interests: Competing Financial Interests statement The authors declare no competing financial interests.

During embryonic development, cells of metazoan embryos need to communicate in order to construct the correct bodyplan. To do so, they use several signals that usually act through interactions between ligands and receptors. Interestingly, only a few pathways are known to be fundamental during animal development, and they are usually found in all the major metazoan clades, raising the following question: how have evolution of the actors and of the functions of these pathways participated in the appearance of the current diversity of animal morphologies? The chordate lineage comprises vertebrates, their sister group the urochordates, and the cephalochordates (i.e. amphioxus). Urochordates are quite derived relative to the chordate ancestor, whereas cephalochordates and vertebrates share many morphological traits. Thus, comparing embryonic development between vertebrates and cephalochordates should give us some insight into the ancestral characters present in chordates and into the morphological evolution in this clade. However, while much is known about the function of different signalling pathways in vertebrates, data are still scarce in the literature for cephalochordates. In this review, we summarize the current state of the field concerning the expression of actors and the function of the major cell-cell communication pathways, including Hedgehog (Hh), Notch, Nuclear Receptor (NR), Receptor Tyrosine Kinase (RTK), Transforming Growth Factor-β (TGF-β) and Wingless/Int (Wnt), in amphioxus.

Co-option of RAG1 and RAG2 for antigen receptor gene assembly by V(D)J recombination was a crucial event in the evolution of jawed vertebrate adaptive immunity. RAG1/2 are proposed to have arisen from a transposable element, but definitive evidence for this is lacking. Here, we report the discovery of ProtoRAG, a DNA transposon family from lancelets, the most basal extant chordates. A typical ProtoRAG is flanked by 5-bp target site duplications and a pair of terminal inverted repeats (TIRs) resembling V(D)J recombination signal sequences. Between the TIRs reside tail-to-tail-oriented, intron-containing RAG1-like and RAG2-like genes. We demonstrate that ProtoRAG was recently active in the lancelet germline and that the lancelet RAG1/2-like proteins can mediate TIR-dependent transposon excision, host DNA recombination, transposition, and low-efficiency TIR rejoining using reaction mechanisms similar to those used by vertebrate RAGs. We propose that ProtoRAG represents a molecular "living fossil" of the long-sought RAG transposon., (Copyright © 2016 Elsevier Inc. All rights reserved.)

During vertebrate development, the paraxial mesoderm becomes segmented, forming somites that will give rise to dermis, axial skeleton and skeletal muscles. Although recently challenged, the "clock and wavefront" model for somitogenesis explains how interactions between several cell-cell communication pathways, including the FGF, RA, Wnt and Notch signals, control the formation of these bilateral symmetric blocks. In the cephalochordate amphioxus, which belongs to the chordate phylum together with tunicates and vertebrates, the dorsal paraxial mesendoderm also periodically forms somites, although this process is asymmetric and extends along the whole body. It has been previously shown that the formation of the most anterior somites in amphioxus is dependent upon FGF signalling. However, the signals controlling somitogenesis during posterior elongation in amphioxus are still unknown. Here we show that, contrary to vertebrates, RA and FGF signals act independently during posterior elongation and that they are not mandatory for posterior somites to form. Moreover, we show that RA is not able to buffer the left/right asymmetry machinery that is controlled through the asymmetric expression of Nodal pathway actors. Our results give new insights into the evolution of the somitogenesis process in chordates. They suggest that RA and FGF pathways have acquired specific functions in the control of somitogenesis in vertebrates. We propose 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.

Insulin is one of the most studied proteins since it is central to the regulation of carbohydrate and fat metabolism in vertebrates and its expression and release are disturbed in diabetes, the most frequent human metabolic disease worldwide. However, the evolution of the function of the insulin protein family is still unclear. In this study, we present a phylogenetic and developmental analysis of the Insulin Like Peptide (ILP) in the cephalochordate amphioxus. We identified an ILP in the European amphioxus Branchiostoma lanceolatum that displays structural characteristics of both vertebrate insulin and Insulin-like Growth Factors (IGFs). Our phylogenetic analysis revealed that amphioxus ILP represents the sister group of both vertebrate insulin and IGF proteins. We also characterized both temporal and spatial expression of ILP in amphioxus. We show that ilp is highly expressed in endoderm and paraxial mesoderm during development, and mainly expressed in the gut of both the developing embryo and adult. We hypothesize that ILP has critical implications in both developmental processes and metabolism and could display IGF- and insulin-like functions in amphioxus supporting the idea of a common ancestral protein.

Vertebrates are equipped with so-called camera eyes, which provide them with image-forming vision. Vertebrate image-forming vision evolved independently from that of other animals and is regarded as a key innovation for enhancing predatory ability and ecological success. Evolutionary changes in the neural circuits, particularly the visual center, were central for the acquisition of image-forming vision. However, the evolutionary steps, from protochordates to jaw-less primitive vertebrates and then to jawed vertebrates, remain largely unknown. To bridge this gap, we present the detailed development of retinofugal projections in the lamprey, the neuroarchitecture in amphioxus, and the brain patterning in both animals. Both the lateral eye in larval lamprey and the frontal eye in amphioxus project to a light-detecting visual center in the caudal prosencephalic region marked by Pax6, which possibly represents the ancestral state of the chordate visual system. Our results indicate that the visual system of the larval lamprey represents an evolutionarily primitive state, forming a link from protochordates to vertebrates and providing a new perspective of brain evolution based on developmental mechanisms and neural functions., (© 2014 Wiley Periodicals, Inc.)

Complex metazoan bodies require cell-to-cell communication for development, a process often mediated by signaling molecules binding to specific receptors. Relatively few signaling pathways have been recruited during evolution to build multicellular animals from unicellular zygotes. Of these few signaling pathways, one of particular importance is the receptor tyrosine kinase (RTK) pathway. In metazoans, fibroblast growth factors (FGFs) bind to receptors in the RTK family, but the origin of the FGF gene family has so far remained a mystery. Here we show that extant bona fide FGFs most likely originated from proteins bearing an FGF-like domain that arose in a choanoflagellate/metazoan ancestor. We found orthologous genes closely related to FGF in choanoflagellates as well as in many metazoans such as sponges, acoels, protostomes, or nonvertebrate deuterostomes. We also show that these genes have a common evolutionary history with Retinitis Pigmentosa 1 (RP1). Even if some metazoan signaling pathways emerged long before multicellularity, we show that FGFs, like their receptors, originated in a eumetazoan ancestor.

Bone morphogenetic proteins (BMPs) are members of the Transforming Growth Factor-β (TGF-β) family implicated in many developmental processes in metazoans such as embryo axes specification. Their wide variety of actions is in part controlled by inhibitors that impede the interaction of BMPs with their specific receptors. Here, we focused our attention on the Differential screening-selected gene Aberrative in Neuroblastoma (DAN) family of inhibitors. Although they are well-characterized in vertebrates, few data are available for this family in other metazoan species. In order to understand the evolution of potential developmental roles of these inhibitors in chordates, we identified the members of this family in the cephalochordate amphioxus, and characterized their expression patterns during embryonic development. Our data suggest that the function of Cerberus/Dand5 subfamily genes is conserved among chordates, whereas Gremlin1/2 and NBL1 subfamily genes seem to have acquired divergent expression patterns in each chordate lineage. On the other hand, the expression of Gremlin in the amphioxus neural plate border during early neurulation strengthens the hypothesis of a conserved neural plate border gene network in chordates., (Copyright © 2013 Elsevier B.V. All rights reserved.)

The question of whether the ancestral bilaterian had a central nervous system (CNS) or a diffuse ectodermal nervous system has been hotly debated. Considerable evidence supports the theory that a CNS evolved just once. However, an alternative view proposes that the chordate CNS evolved from the ectodermal nerve net of a hemichordate-like ancestral deuterostome, implying independent evolution of the CNS in chordates and protostomes. To specify morphological divisions along the anterior/posterior axis, this ancestor used gene networks homologous to those patterning three organizing centers in the vertebrate brain: the anterior neural ridge, the zona limitans intrathalamica and the isthmic organizer, and subsequent evolution of the vertebrate brain involved elaboration of these ancestral signaling centers; however, all or part of these signaling centers were lost from the CNS of invertebrate chordates. The present review analyzes the evidence for and against these theories. The bulk of the evidence indicates that a CNS evolved just once - in the ancestral bilaterian. Importantly, in both protostomes and deuterostomes, the CNS represents a portion of a generally neurogenic ectoderm that is internalized and receives and integrates inputs from sensory cells in the remainder of the ectoderm. The expression patterns of genes involved in medio/lateral (dorso/ventral) patterning of the CNS are similar in protostomes and chordates; however, these genes are not similarly expressed in the ectoderm outside the CNS. Thus, their expression is a better criterion for CNS homologs than the expression of anterior/posterior patterning genes, many of which (for example, Hox genes) are similarly expressed both in the CNS and in the remainder of the ectoderm in many bilaterians. The evidence leaves hemichordates in an ambiguous position - either CNS centralization was lost to some extent at the base of the hemichordates, or even earlier, at the base of the hemichordates + echinoderms, or one of the two hemichordate nerve cords is homologous to the CNS of protostomes and chordates. In any event, the presence of part of the genetic machinery for the anterior neural ridge, the zona limitans intrathalamica and the isthmic organizer in invertebrate chordates together with similar morphology indicates that these organizers were present, at least in part, at the base of the chordates and were probably elaborated upon in the vertebrate lineage.