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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., This work was supported by the Australian Research Council (ARC) Centre of Excellence programme in Plant Energy Biology (grant no. CE140100008). R.L. was supported by a Sylvia and Charles Viertel Senior Medical Research Fellowship, ARC Future Fellowship (no. FT120100862) and Howard Hughes Medical Institute International Research Scholarship. A.d.M. was funded by an EMBO long-term fellowship (no. ALTF 144-2014). J.L.G.-S. was supported by the Spanish government (grant no. BFU2016- 74961-P) and the institutional grant Unidad de Excelencia María de Maeztu (no. MDM-2016-0687). B.V. was supported by the Biomedical Research Council of the Agency for Science, Technology and Research of Singapore. F.G. was supported by an ARC Future Fellowship (no. FT160100267). C.W.R. was supported by an NSF grant (no. IOS-1354898). J.R.E. is an investigator of the Howard Hughes Medical Institute. Genomic data was generated at the Australian Cancer Research Foundation Centre for Advanced Cancer Genomics.

International audience; 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.

RNA interference (RNAi) requires RNA-dependent RNA polymerases (RdRPs) in many eukaryotes, and RNAi amplification constitutes the only known function for eukaryotic RdRPs. Yet in animals, classical model organisms can elicit RNAi without possessing RdRPs, and only nematode RNAi was shown to require RdRPs. Here we show that RdRP genes are much more common in animals than previously thought, even in insects, where they had been assumed not to exist. RdRP genes were present in the ancestors of numerous clades, and they were subsequently lost at a high frequency. In order to probe the function of RdRPs in a deuterostome (the cephalochordate Branchiostoma lanceolatum), we performed high-throughput analyses of small RNAs from various Branchiostoma developmental stages. Our results show that Branchiostoma RdRPs do not appear to participate in RNAi: we did not detect any candidate small RNA population exhibiting classical siRNA length or sequence features. Our results show that RdRPs have been independently lost in dozens of animal clades, and even in a clade where they have been conserved (cephalochordates) their function in RNAi amplification is not preserved. Such a dramatic functional variability reveals an unexpected plasticity in RNA silencing pathways., Author summary RNA interference (RNAi) is a conserved gene regulation system in eukaryotes. In non-animal eukaryotes, it necessitates RNA-dependent RNA polymerases (“RdRPs”). Among animals, only nematodes appear to require RdRPs for RNAi. Yet additional animal clades have RdRPs and it is assumed that they participate in RNAi. Here, we find that RdRPs are much more common in animals than previously thought, but their genes were independently lost in many lineages. Focusing on a species with RdRP genes (a cephalochordate), we found that it does not use them for RNAi. While RNAi is the only known function for eukaryotic RdRPs, our results suggest additional roles. Eukaryotic RdRPs thus have a complex evolutionary history in animals, with frequent independent losses and apparent functional diversification.

International audience; 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. floridaedthat 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.

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.

T-box transcription factors are found in all metazoans and play diverse roles during embryogenesis. In the cephalochordate amphioxus, nine T-box genes were previously identified. In this work we undertook the analysis of the embryonic expression pattern of Tbx6/16 and Tbx20, the last two T-box genes for which no such data are available. We found that Tbx6/16 is expressed in the unsegmented paraxial mesoderm, in a subpopulation of neurons, and in the tail epidermis. Comparison with the expression patterns of the different vertebrate orthologues indicates a conserved role of those genes in posterior mesoderm formation in chordates. Tbx20 expression is detected in the ventral mesoderm of amphioxus embryos, in cells that are proposed to be precursors of the amphioxus myocardium, in some neurons of the neural tube, and in the pre-oral pit which is thought to be the homologue of the vertebrate adenohypophysis. In vertebrates, Tbx20 is also one of the first genes expressed in the embryonic heart field, suggesting that the function of this gene in heart development has been conserved during chordate evolution.

The vertebrate glycoprotein hormones (GpHs), gonadotropins and thyrotropin, are heterodimers composed of a common alpha- and specific beta-subunit. The recombinant heterodimer of two additional, structurally related proteins identified in vertebrate and protostome genomes, the glycoproteins-alpha2 (GPA2) and-beta5 (GPB5), was shown to activate the thyrotropin receptor and was therefore named thyrostimulin. However, differences in tissue distribution and expression levels of these proteins suggested that they might act as nonassociated factors, prompting further investigation on these proteins. In this study we show that GPA2 and GPB5 appeared with the emergence of bilateria and were maintained in most groups. These genes are tightly associated at the genomic level, an association, however, lost in tetrapods. Our structural and genomic environment comparison reinforces the hypothesis of their phylogenetic relationships with GpH-alpha and -beta. In contrast, the glycosylation status of GPA2 and GPB5 is highly variable further questioning heterodimer secretory efficiency and activity. As a first step toward understanding their function, we investigated the spatiotemporal expression of GPA2 and GPB5 genes at different developmental stages in a basal chordate, the amphioxus. Expression of GPB5 was essentially ubiquitous with an anteroposterior gradient in embryos. GPA2 embryonic and larvae expression was restricted to specific areas and, interestingly, partially overlapped that of a GpH receptor-related gene. In conclusion, we speculate that GPA2 and GPB5 have nondispensable and coordinated functions related to a novelty appeared with bilateria. These proteins would be active during embryonic development in a manner that does not require their heterodimerization.

Tyrosine kinase (TK) proteins play a central role in cellular behavior and development of animals. The expansion of this superfamily is regarded as a key event in the evolution of the complex signaling pathways and gene networks of metazoans and is a prominent example of how shuffling of protein modules may generate molecular novelties. Using the intron/exon structure within the TK domain (TK intron code) as a complementary tool for the assignment of orthology and paralogy, we identified and studied the 118 TK proteins of the amphioxus Branchiostoma floridae genome to elucidate TK gene family evolution in metazoans and chordates in particular. Unlike all characterized metazoans to date, amphioxus has members of all known widespread TK families, with not a single loss. Putting amphioxus TKs in an evolutionary context, including new data from the cnidarian Nematostella vectensis, the echinoderm Strongylocentrotus purpuratus, and the ascidian Ciona intestinalis, we suggest new evolutionary histories for different TK families and draw a new global picture of gene loss/gain in the different phyla. Surprisingly, our survey also detected an unprecedented expansion of a group of closely related TK families, including TIE, FGFR, PDGFR, and RET, due most probably to massive gene duplication and exon shuffling. Based on their highly similar intron/exon structure at the TK domain, we suggest that this group of TK families constitute a superfamily of TK proteins, which we termed EXpanding TK, after their seemingly unique propensity to gene duplication and exon shuffling, not only in amphioxus but also across all metazoan groups. Due to this extreme tendency to both retention and expansion of TK genes, amphioxus harbors the richest and most diverse TK repertoire among all metazoans studied so far, retaining most of the gene complement of its ancestors, but having evolved its own repertoire of genetic novelties.

Acemel, Rafael D. et al., The HoxA and HoxD gene clusters of jawed vertebrates are organized into bipartite three-dimensional chromatin structures that separate long-range regulatory inputs coming from the anterior and posterior Hox-neighboring regions. This architecture is instrumental in allowing vertebrate Hox genes to pattern disparate parts of the body, including limbs. Almost nothing is known about how these three-dimensional topologies originated. Here we perform extensive 4C-seq profiling of the Hox cluster in embryos of amphioxus, an invertebrate chordate. We find that, in contrast to the architecture in vertebrates, the amphioxus Hox cluster is organized into a single chromatin interaction domain that includes long-range contacts mostly from the anterior side, bringing distant cis-regulatory elements into contact with Hox genes. We infer that the vertebrate Hox bipartite regulatory system is an evolutionary novelty generated by combining ancient long-range regulatory contacts from DNA in the anterior Hox neighborhood with new regulatory inputs from the posterior side., Work was funded by grants from the Ministerio de Economía y Competitividad (BFU2013-41322-P to J.L.G.-S.; Juan de la Cierva postdoctoral contract to I.M.; BFU2014-58449-JIN to J.J.T.); the Andalusian government (BIO-396 to J.L.G.-S.; C2A (EE: 2013/2506) to D.P.D. and I.I.-A.); the European Research Council (ERC; grant 268513) to P.W.H.H. and F.M.; a European Molecular Biology Organization (EMBO) short fellowship to I.M.; the Universidad Pablo de Olavide to J.J.T.; and Conicyt 'Becas Chile' to D.A.

The cephalochordate amphioxus (Branchiostoma sp.) is an important animal model for studying the evolution of chordate developmental mechanisms. Obtaining amphioxus embryos is a key step for these studies. It has been shown that an increase of 3-4 degrees C in water temperature triggers spawning of the European amphioxus (Branchiostoma lanceolatum) in captivity, however, very little is known about the natural spawning behavior of this species in the field. In this work, we have followed the spawning behavior of the European amphioxus during two spawning seasons (2004 and 2005), both in the field and in captivity. We show that animals in the field spawn approximately from mid-May through early July, but depending on the year, they show different patterns of spawning. Thus, even if temperature has a critical role in the induction of the spawning in captivity, it is not the major factor in the field. Moreover, we report some improvements on the methodology for inducing spawning in captivity (e.g. in maintenance, light cycle control and induction of spawning in a laboratory without running sea water system). These studies have important implications for amphioxus animal husbandry and for improving laboratory techniques to develop amphioxus as an experimental animal model. J. Exp. Zool. (Mol. Dev. Evol.) 308B, 2007. (c) 2007 Wiley-Liss, Inc.

Guidance of cells and tissue sheets is an essential function in developing and differentiating animal tissues. In Hydra, where cells and tissue move dynamically due to constant cell proliferation towards the termini or into lateral, vegetative buds, factors essential for guidance are still unknown. Good candidates to take over this function are fibroblast growth factors (FGFs). We present the phylogeny of several Hydra FGFs and analysis of their expression patterns. One of the FGFs is expressed in all terminal regions targeted by tissue movement and at boundaries crossed by moving tissue and cells with an expression pattern slightly differing in two Hydra strains. A model addressing an involvement of this FGF in cell movement and morphogenesis is proposed: Hydra FGFf-expressing cells might serve as sources to attract tissue and cells towards the termini of the body column and across morphological boundaries. Moreover, a function in morphogenesis and/or differentiation of cells and tissue is suggested.

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.

Association pour la Recherche contre le Cancer, Centre National de la Recherche Scientifique, the European Molecular Biology Organization, and the Cascade network of excellence in the 6th Framework Programme for financial support. The laboratory of HE is supported by the Cascade EU Network of Excellence and Agence Nationale de la Recherche. The laboratory of JGF is supported by BMC2008-03776 (Ministerio de Educación y Ciencia). IS' postdoctoral position was supported by a CNRS fellowship. SB's postdoctoral position was supported by an ARC fellowship. 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. Publisher PDF

International audience; Comparative endocrinology considers the evolution of bioregulatory systems and the anatomical structures and molecules that constitute the neuroendocrine and endocrine systems. One aim of comparative endocrinology is to trace the origins of the main endocrine systems. The understanding of the evolution of the ligand/receptor couple is central to this objective. One classical approach to tackle this question is the characterization of receptors and ligands in various types of non-model organisms using as a starting point the knowledge accumulated on classical models such as mammals (mainly human and mouse) and arthropods (with Drosophila among other insects). In this review we discuss the potential caveats associated to this two-by-two comparison between a classical model and non-model organisms. We suggest that the use of an evolutionary approach involving comparisons of several organisms in a coherent framework permits reconstruction of the most probable scenarios. The use of the vast amount of genomic data now available, coupled to functional experiments, offers unprecedented possibilities to trace back the origins of the main ligand/receptor couples

Cephalochordates, the most basal extant group in the phylum Chordata, are represented chiefly by about 20 species of the genus Branchiostoma, commonly called amphioxus or lancelets. In recent years, insights into the evolutionary origin of the vertebrates have been gained from molecular genetic studies during the development of three of these amphioxus species (Branchiostoma floridae in North America, Branchiostoma lanceolatum in Europe, and Branchiostoma belcheri in East Asia). In spite of an estimated divergence time of 100-200 Myr among these species, all three are remarkably similar morphologically, and students of amphioxus have tacitly assumed that such resemblances arise during ontogeny from nearly identical networks of developmental genes. We felt that this assumption needed to be reexamined because instances are known--even in comparisons of closely related species--where characters seeming homologous on the basis of morphology actually develop under the control of conspicuously divergent genetic programs (a phenomenon termed "genetic piracy"). In the present work, we tested the hypothesis that morphological similarities reflect strict conservation of developmentally important genes' expression patterns in order to assess whether the developmental genetics of different amphioxus species show evidence of genetic piracy. To these ends, we cloned 18 genes implicated in different developmental functions in B. lanceolatum and compared their gene expression patterns with the known expression patterns of their orthologous genes in B. floridae. We show that, for the most part, conservation of gene expression parallels that of morphology in these two species. We also identified some differences in gene expression, likely reflecting experimental sensitivity, with the exception of Pax1/9, which may result from true developmental specificities in each amphioxus species. Our results demonstrate that morphological conservation reflects stasis in developmental gene expression patterns and find no evidence for genetic piracy. Thus, different species of amphioxus appear to be very similar, not only morphologically, but also in the genetic programs directing the development of their structural features. Moreover, we provide the first catalogue of gene expression data for the European species, B. lanceolatum.

The online version of this article (doi:10.1007/s00427-008-0251-y) contains supplementary material, which is available to authorized users.; International audience; The nuclear hormone receptors (NRs) form a superfamily of transcription factors unified by conserved protein structure and mode of function. While most members of this superfamily are activated by ligands, such as thyroid hormones, steroids, vitamin D or retinoic acid, other NRs are called orphan receptors because they have no known ligand. NR-dependent signaling is crucial for vertebrate development with the majority of receptors being expressed in the developing embryo. Due to massive gene duplications during vertebrate diversification, there are usually more NRs in vertebrates than in invertebrates. In this study, we examine the evolutionary diversification of the NR superfamily and of NR-dependent signaling in chordates (vertebrates, tunicates, and amphioxus). We take advantage of the unique features of the genome of the invertebrate amphioxus, which is characterized by a vertebrate-like gene content without having undergone massive duplications, to assess the NR signaling complement (NRs and NR coregulators) of the ancestral chordate. We find 33 NRs in amphioxus, which are more NRs than originally anticipated. This increase is mainly due to an amphioxus-specific duplication of genes encoding receptors of the NR1H group. In addition, there are three heterologous NRs in amphioxus that could not be placed within the framework of the NR superfamily. Apart from these exceptions, there is usually one amphioxus NR or NR signaling coregulator for each paralogous group of two, three, or four human receptors suggesting that the ancestral chordate had a set of 22 different NRs plus one copy of each NR coregulator.

Nuclear receptors (NRs) are transcription factors that are implicated in several biological processes such as embryonic development, homeostasis, and metabolic diseases. To study the role of NRs in development, it is critically important to know when and where individual genes are expressed. Although systematic expression studies using reverse transcriptase PCR and/or DNA microarrays have been performed in classical model systems such as Drosophila and mouse, no systematic atlas describing NR involvement during embryonic development on a global scale has been assembled. Adopting a systems biology approach, we conducted a systematic analysis of the dynamic spatiotemporal expression of all NR genes as well as their main transcriptional coregulators during zebrafish development (101 genes) using whole-mount in situ hybridization. This extensive dataset establishes overlapping expression patterns among NRs and coregulators, indicating hierarchical transcriptional networks. This complete developmental profiling provides an unprecedented examination of expression of NRs during embryogenesis, uncovering their potential function during central nervous system and retina formation. Moreover, our study reveals that tissue specificity of hormone action is conferred more by the receptors than by their coregulators. Finally, further evolutionary analyses of this global resource led us to propose that neofunctionalization of duplicated genes occurs at the levels of both protein sequence and RNA expression patterns. Altogether, this expression database of NRs provides novel routes for leading investigation into the biological function of each individual NR as well as for the study of their combinatorial regulatory circuitry within the superfamily., Author Summary NRs are key molecules controlling development, metabolism, and reproduction in metazoans. Since NRs are implicated in many human diseases such as cancer, metabolic syndrome, and hormone resistance, they are important pharmaceutical targets and are under intense scrutiny to better understand their biological functions. In the present study, we determined the expression patterns of all NR genes as well as their main transcriptional coregulators during zebrafish development. We used zebrafish because the transparency of its embryo allows us to perform whole-mount in situ hybridization from early development to late organogenesis. This complete developmental profiling offers an unprecedented view of NR expression during embryogenesis, uncovering their potential function during central nervous system and retina formation. We observed that in contrast to NR genes, only a few coregulators exhibit a restricted expression pattern, suggesting that tissue specificity of hormone action is conferred more by the receptors than by their coregulators. Lastly, by evolutionary analysis of expression pattern divergence of duplicated genes, we observed that neofunctionalization occurs at the levels of both protein sequence and mRNA expression patterns. Taken together, our data provide the starting point for functional analysis of an entire gene family during development and call for the study of the intersection between metabolism and development.

The orphan nuclear receptor Rev-erbα (NR1D1) plays an important role in the regulation of the circadian pacemaker and its expression has been shown to be regulated with a robust circadian rhythm in zebrafish and mammals. In addition, in zebrafish its expression has been shown to be developmentally regulated. In order to analyze the mechanisms of the zfRev-erbα gene regulation, we have isolated its 5′-upstream region. We found that two promoters control the zfRev-erbα expression. The first one (ZfP1) is characterized by a very high degree of sequence identity with the mammalian P1 promoter and contains, as the mammalian P1, a functional Rev-erbα-binding site (RevDR2). Inhibition of zfRev-erbα activity in zebrafish embryos using antisense-morpholino knockdown results in an increase of zfRev-erbα gene expression suggesting that zfRev-erbα is repressing its own transcription in vivo. In addition, we show that ROR orphan receptors also regulate in vitro and in vivo zfRev-erbα gene expression through the same RevDR2 element. In contrast, the second promoter ZfP2 is strikingly different from the mammalian P2: its sequence is not conserved between zebrafish and mammals and is not regulated by the same transcription factors. Together, these data suggest that ZfP1 is orthologous to the mammalian P1 promoter, whereas zebrafish ZfP2 has no mammalian ortholog and does not function like ZfP1 to control Rev-erbα expression.

Hypermethylated in Cancer 1 (HIC1) is a human tumor suppressor gene located at chromosome 17p13.3 which is frequently hypermethylated and transcriptionally silent in many types of tumors. In addition, its location in the Miller-Dieker syndrome's (MDS) deletion region, its embryonic expression pattern in mice and the phenotype of the HIC1-deficient mice have provided strong evidence for its implication in this contiguous-gene syndrome. HIC1 encodes a five C2H2-type zinc finger transcriptional repressor belonging to the BTB/POZ family. We have isolated the true zebrafish orthologue of human HIC1 since it has a comparable intron-exon structure and since its predicted gene product, ZfHIC1 displays much higher sequence similarities in its overall sequence (737 residues) with human HIC1 (714 residues) than the 454 residues encoded by the only zebrafish HIC1 sequence (AF111712) described so far, which has been renamed ZfHIC1alpha. Notably, the C-terminal end and one zinc finger in the DNA-binding domain are missing in ZfHIC1alpha. As a consequence, ZfHIC1 proteins bind the human HIC1 consensus DNA-binding sequence in vitro, whereas ZfHIC1alpha cannot. Analyses of the expression pattern of ZfHIC1 and of its paralogue ZfHRG22 (HIC1 related gene on chromosome 22) show that they share expression domains with their respective orthologous vertebrate genes. ZfHRG22 is prominently expressed in the brain and in neural tissues. Interestingly, the predominant expression of ZfHIC1 in the mesenchyme of the head, around the nose and the eye and in the branchial arches is possibly consistent with some of the abnormalities seen in the HIC1-deficient mice and provides another clue for the implication of HIC1 in MDS.

Bilaterian animals are notably characterized by complex endocrine systems. The receptors for many steroids, retinoids, and other hormones belong to the superfamily of nuclear receptors, which are transcription factors regulating many aspects of development and homeostasis. Despite a diversity of regulatory mechanisms and physiological roles, nuclear receptors share a common protein organization. To obtain the broad picture of bilaterian nuclear hormone receptor evolution, we have characterized the complete set of nuclear receptor genes from nine animal genome sequences and analyzed it in a phylogenetic framework. In addition, expressed sequence tags from key lineages with no available genome sequence were also searched. This allows us to date the evolutionary events that led from an ancestral nuclear receptor gene, in an early metazoan, to present day diversity. We show that there were approximately 25 nuclear receptor genes in Urbilateria, the ancestor of bilaterians, at which point the fundamental diversity of the subfamily was already established. Surprisingly, differential gene loss played an important role in the evolution of different nuclear receptor sets in bilaterian lineages. The nuclear receptor distribution was also shaped by periods of gene duplication, essentially in vertebrates, as well as a lineage-specific duplication burst in nematodes. Our results imply that the genes for major receptors such as steroid receptors or thyroid hormone receptors were present in Urbilateria.

The vertebrates share the ability to produce a skeleton made of mineralized extracellular matrix. However, our understanding of the molecular changes that accompanied their emergence remains scarce. Here, we describe the evolutionary history of the SPARC (secreted protein acidic and rich in cysteine) family, because its vertebrate orthologues are expressed in cartilage, bones and teeth where they have been proposed to bind calcium and act as extracellular collagen chaperones, and because further duplications of specific SPARC members produced the small calcium-binding phosphoproteins (SCPP) family that is crucial for skeletal mineralization to occur. Both phylogeny and synteny conservation analyses reveal that, in the eumetazoan ancestor, a unique ancestral gene duplicated to give rise toSPARCandSPARCBdescribed here for the first time. Independent losses have eliminated one of the two paralogues in cnidarians, protostomes and tetrapods. Hence, only non-tetrapod deuterostomes have conserved both genes. Remarkably,SPARCandSPARCBparalogues are still linked in the amphioxus genome. To shed light on the evolution of the SPARC family members in chordates, we performed a comprehensive analysis of their embryonic expression patterns in amphioxus, tunicates, teleosts, amphibians and mammals. Our results show that in the chordate lineage SPARC and SPARCB family members were recurrently recruited in a variety of unrelated tissues expressing collagen genes. We propose that one of the earliest steps of skeletal evolution involved the co-expression of SPARC paralogues with collagenous proteins.

Background The basally divergent phylogenetic position of amphioxus (Cephalochordata), as well as its conserved morphology, development and genetics, make it the best proxy for the chordate ancestor. Particularly, studies using the amphioxus model help our understanding of vertebrate evolution and development. Thus, interest for the amphioxus model led to the characterization of both the transcriptome and complete genome sequence of the American species, Branchiostoma floridae. However, recent technical improvements allowing induction of spawning in the laboratory during the breeding season on a daily basis with the Mediterranean species Branchiostoma lanceolatum have encouraged European Evo-Devo researchers to adopt this species as a model even though no genomic or transcriptomic data have been available. To fill this need we used the pyrosequencing method to characterize the B. lanceolatum transcriptome and then compared our results with the published transcriptome of B. floridae. Results Starting with total RNA from nine different developmental stages of B. lanceolatum, a normalized cDNA library was constructed and sequenced on Roche GS FLX (Titanium mode). Around 1.4 million of reads were produced and assembled into 70,530 contigs (average length of 490 bp). Overall 37% of the assembled sequences were annotated by BlastX and their Gene Ontology terms were determined. These results were then compared to genomic and transcriptomic data of B. floridae to assess similarities and specificities of each species. Conclusion We obtained a high-quality amphioxus (B. lanceolatum) reference transcriptome using a high throughput sequencing approach. We found that 83% of the predicted genes in the B. floridae complete genome sequence are also found in the B. lanceolatum transcriptome, while only 41% were found in the B. floridae transcriptome obtained with traditional Sanger based sequencing. Therefore, given the high degree of sequence conservation between different amphioxus species, this set of ESTs may now be used as the reference transcriptome for the Branchiostoma genus.

The proprotein convertases (PCs) comprise a family of subtilisin-like endoproteases that activate precursor proteins (including, prohormones, growth factors, and adhesion molecules) during their transit through secretory pathways or at the cell surface. To explore the evolution of the PC gene family in chordates, we made a phylogenetic analysis of PC genes found in databases, with special attention to three PC genes of the cephalochordate amphioxus, the closest living invertebrate relative to the vertebrates. Since some vertebrate PC genes are essential for early development, we investigated the expression pattern of the C isoform of the amphioxus PC6 gene (aPC6C). In amphioxus embryos and larvae, aPC6C is expressed at places where epithelia fuse. Several kinds of fusions occur: ectoderm-to-ectoderm during neurulation; mesoderm-to-ectoderm during formation of the preoral ciliated pit; and endoderm-to-ectoderm during formation of the mouth, pharyngeal slits, anus, and external opening of the club-shaped gland. Presumably, at all these sites, aPC6C is activating proteins favoring association between previously disjunct cell populations.

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.