Ferritins in Chordata: Potential evolutionary trajectory marked by discrete selective pressures: History and reclassification of ferritins in chordates and geological events' influence on their evolution and radiation.

Ferritins (FTs) are iron storage proteins that are involved in managing iron-oxygen balance. In our work, we present a hypothesis on the putative effect of geological changes that have affected the evolution and radiation of ferritin proteins. Based on sequence analysis and phylogeny reconstruction, we hypothesize that two significant factors have been involved in the evolution of ferritin proteins: fluctuations of atmospheric oxygen concentrations, altering redox potential, and changing availability of water rich in bioavailable ferric ions. Fish, ancient amphibians, reptiles, and placental mammals developed the broadest repertoire of singular FTs, attributable to embryonic growth in aquatic environments containing low oxygen levels and abundant forms of soluble iron. In contrast, oviparous land vertebrates, like reptiles and birds, that have developed in high oxygen levels and limited levels of environmental Fe2+ exhibit a lower diversity of singular FTs, but display a broad repertoire of subfamilies, particularly notable in early reptiles.

A chordate species lacking Nodal utilizes calcium oscillation and Bmp for left-right patterning.

Larvaceans are chordates with a tadpole-like morphology. In contrast to most chordates of which early embryonic morphology is bilaterally symmetric and the left-right (L-R) axis is specified by the Nodal pathway later on, invariant L-R asymmetry emerges in four-cell embryos of larvaceans. The asymmetric cell arrangements exist through development of the tailbud. The tail thus twists 90° in a counterclockwise direction relative to the trunk, and the tail nerve cord localizes on the left side. Here, we demonstrate that larvacean embryos have nonconventional L-R asymmetries: 1) L- and R-cells of the two-cell embryo had remarkably asymmetric cell fates; 2) Ca2+ oscillation occurred through embryogenesis; 3) Nodal, an evolutionarily conserved left-determining gene, was absent in the genome; and 4) bone morphogenetic protein gene (Bmp) homolog Bmp.a showed right-sided expression in the tailbud and larvae. We also showed that Ca2+ oscillation is required for Bmp.a expression, and that BMP signaling suppresses ectopic expression of neural genes. These results indicate that there is a chordate species lacking Nodal that utilizes Ca2+ oscillation and Bmp.a for embryonic L-R patterning. The right-side Bmp.a expression may have arisen via cooption of conventional BMP signaling in order to restrict neural gene expression on the left side.

Evolutionary diversification of the canonical Wnt signaling effector TCF/LEF in chordates.

Wnt signaling is essential during animal development and regeneration, but also plays an important role in diseases such as cancer and diabetes. The canonical Wnt signaling pathway is one of the most conserved signaling cascades in the animal kingdom, with the T-cell factor/lymphoid enhancer factor (TCF/LEF) proteins being the major mediators of Wnt/ß-catenin-regulated gene expression. In comparison with invertebrates, vertebrates possess a high diversity of TCF/LEF family genes, implicating this as a possible key change to Wnt signaling at the evolutionary origin of vertebrates. However, the precise nature of this diversification is only poorly understood. The aim of this study is to clarify orthology, paralogy, and isoform relationships within the TCF/LEF gene family within chordates via in silico comparative study of TCF/LEF gene structure, molecular phylogeny, and gene synteny. Our results support the notion that the four TCF/LEF paralog subfamilies in jawed vertebrates (gnathostomes) evolved via the two rounds of whole-genome duplications that occurred during early vertebrate evolution. Importantly, gene structure comparisons and synteny analysis of jawless vertebrate (cyclostome) TCFs suggest that a TCF7L2-like form of gene structure is a close proxy for the ancestral vertebrate structure. In conclusion, we propose a detailed evolutionary path based on a new pre-whole-genome duplication vertebrate TCF gene model. This ancestor gene model highlights the chordate and vertebrate innovations of TCF/LEF gene structure, providing the foundation for understanding the role of Wnt/ß-catenin signaling in vertebrate evolution.

Massive cytoplasmic transport and microtubule organization in fertilized chordate eggs.

Eggs have developed their own strategies for early development. Amphibian, teleost fish, and ascidian eggs show cortical rotation and an accompanying structure, a cortical parallel microtubule (MT) array, during the one-cell embryonic stage. Cortical rotation is thought to relocate maternal deposits to a certain compartment of the egg and to polarize the embryo. The common features and differences among chordate eggs as well as localized maternal proteins and mRNAs that are related to the organization of MT structures are described in this review. Furthermore, recent studies report progress in elucidating the molecular nature and functions of the noncentrosomal MT organizing center (ncMTOC). The parallel array of MT bundles is presumably organized by ncMTOCs; therefore, the mechanism of ncMTOC control is likely inevitable for these species. Thus, the molecules related to the ncMTOC provide clues for understanding the mechanisms of early developmental systems, which ultimately determine the embryonic axis.

Enhancer evolution in chordates: Lessons from functional analyses of cephalochordate cis-regulatory modules.

Chordates comprise three major groups, cephalochordates (amphioxus), tunicates (urochordates), and vertebrates. Since cephalochordates were the early branching group, comparisons between amphioxus and other chordates help us to speculate about ancestral chordates. Here, I summarize accumulating data from functional studies analyzing amphioxus cis-regulatory modules (CRMs) in model systems of other chordate groups, such as mice, chickens, clawed frogs, fish, and ascidians. Conservatism and variability of CRM functions illustrate how gene regulatory networks have evolved in chordates. Amphioxus CRMs, which correspond to CRMs deeply conserved among animal phyla, govern reporter gene expression in conserved expression domains of the putative target gene in host animals. In addition, some CRMs located in similar genomic regions (intron, upstream, or downstream) also possess conserved activity, even though their sequences are divergent. These conservative CRM functions imply ancestral genomic structures and gene regulatory networks in chordates. However, interestingly, if expression patterns of amphioxus genes do not correspond to those of orthologs of experimental models, some amphioxus CRMs recapitulate expression patterns of amphioxus genes, but not those of endogenous genes, suggesting that these amphioxus CRMs are close to the ancestral states of chordate CRMs, while vertebrates/tunicates innovated new CRMs to reconstruct gene regulatory networks subsequent to the divergence of the cephalochordates. Alternatively, amphioxus CRMs may have secondarily lost ancestral CRM activity and evolved independently. These data help to solve fundamental questions of chordate evolution, such as neural crest cells, placodes, a forebrain/midbrain, and genome duplication. Experimental validation is crucial to verify CRM functions and evolution.

Sex and dose-dependent responses of urinary and faecal glucocorticoid metabolite concentrations following an ACTH challenge in eastern rock sengis (Elephantulus myurus).

Non-invasive methods for measuring glucocorticoids and their metabolites are frequently used in ecological, behavioural and physiological studies of mammals. Using faeces, urine and other matrices for such a measurement has considerable advantages in comparison to more traditional methods, but also requires thorough validation of the methods used. Eastern rock sengis (Elephantulus myurus) are fascinating African mammals and the non-invasive monitoring of the adrenocortical activity opens up new opportunities to study their biology. We were able to validate two assays for measuring urinary (uGCM) and faecal glucocorticoid metabolite (fGCM) concentrations in this species using a dose-dependent challenge with adrenocorticotropic hormone (ACTH). A higher concentration of ACTH elicited higher uGCM and fGCM concentrations in both males and females. Interestingly, uGCM and fGCM concentrations and the responses to ACTH were higher in females than in males and small changes in faecal glucocorticoid metabolites could not be reliably detected in males. In contrast to ACTH, a saline injection did not result in an increase in uGCM or fGCM concentrations. The study also provided insight into when responses to a stressor are likely to be detected in the urine and faeces of sengis and opens up new opportunities to study the stress physiology of this and other sengi species. It further emphasises the importance of thoroughly validating non-invasive methods for measuring hormones in both sexes of a species and for incorporating dose-dependent approaches.

Immunomodulatory Activity of Low Molecular-Weight Peptides from Nibea japonica in RAW264.7 Cells via NF-κB Pathway.

In this study, a low molecular-weight (Mw) peptide named NJP (<1 kDa), was purified from a protein hydrolysate of Nibea japonica by ultrafiltration, and its immunomodulatory effect on RAW264.7 cells was evaluated. The lactate dehydrogenase (LDH) and MTT assays were performed to explore the cytotoxicity of NJP. The results showed that NJP promoted cell proliferation and had no significant toxic effects on RAW264.7 cells. Moreover, the cells formed multiple pseudopodia indicating that they were in activated state. Further tests showed that NJP significantly promoted phagocytic capacity, and the secretion of proinflammatory cytokines tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-1ß (IL-1ß). It also increased the synthesis of nitric oxide (NO) by upregulating inducible nitric oxide synthase (iNOS) protein level. Flow cytometry revealed that NJP promoted cell cycle progression and increased the percentage of cells in G0/G1 phase. NJP promoted IκBα degradation, p65 and nuclear factor (NF)-κB activation and translocation by up-regulating IKKα/ß protein expression. In conclusion, these results indicated that NJP exerts immunomodulatory effects on RAW264.7 cells through the NF-κB signaling pathway. Therefore, NJP can be incorporated in the production of functional foods or nutraceuticals.

Distinct core promoter codes drive transcription initiation at key developmental transitions in a marine chordate.

BACKGROUND: Development is largely driven by transitions between transcriptional programs. The initiation of transcription at appropriate sites in the genome is a key component of this and yet few rules governing selection are known. Here, we used cap analysis of gene expression (CAGE) to generate bp-resolution maps of transcription start sites (TSSs) across the genome of Oikopleura dioica, a member of the closest living relatives to vertebrates. RESULTS: Our TSS maps revealed promoter features in common with vertebrates, as well as striking differences, and uncovered key roles for core promoter elements in the regulation of development. During spermatogenesis there is a genome-wide shift in mode of transcription initiation characterized by a novel core promoter element. This element was associated with > 70% of male-specific transcription, including the use of cryptic internal promoters within operons. In many cases this led to the exclusion of trans-splice sites, revealing a novel mechanism for regulating which mRNAs receive the spliced leader. Binding of the cell cycle regulator, E2F1, is enriched at the TSS of maternal genes in endocycling nurse nuclei. In addition, maternal promoters lack the TATA-like element found in zebrafish and have broad, rather than sharp, architectures with ordered nucleosomes. Promoters of ribosomal protein genes lack the highly conserved TCT initiator. We also report an association between DNA methylation on transcribed gene bodies and the TATA-box. CONCLUSIONS: Our results reveal that distinct functional promoter classes and overlapping promoter codes are present in protochordates like in vertebrates, but show extraordinary lineage-specific innovations. Furthermore, we uncover a genome-wide, developmental stage-specific shift in the mode of TSS selection. Our results provide a rich resource for the study of promoter structure and evolution in Metazoa.

The vertebrate Hox gene regulatory network for hindbrain segmentation: Evolution and diversification: Coupling of a Hox gene regulatory network to hindbrain segmentation is an ancient trait originating at the base of vertebrates.

Hindbrain development is orchestrated by a vertebrate gene regulatory network that generates segmental patterning along the anterior-posterior axis via Hox genes. Here, we review analyses of vertebrate and invertebrate chordate models that inform upon the evolutionary origin and diversification of this network. Evidence from the sea lamprey reveals that the hindbrain regulatory network generates rhombomeric compartments with segmental Hox expression and an underlying Hox code. We infer that this basal feature was present in ancestral vertebrates and, as an evolutionarily constrained developmental state, is fundamentally important for patterning of the vertebrate hindbrain across diverse lineages. Despite the common ground plan, vertebrates exhibit neuroanatomical diversity in lineage-specific patterns, with different vertebrates revealing variations of Hox expression in the hindbrain that could underlie this diversification. Invertebrate chordates lack hindbrain segmentation but exhibit some conserved aspects of this network, with retinoic acid signaling playing a role in establishing nested domains of Hox expression.

Processing Optimization and Characterization of Angiotensin-Ι-Converting Enzyme Inhibitory Peptides from Lizardfish (Synodus macrops) Scale Gelatin.

Hypertension can cause coronary heart disease. Synthetic angiotensin-converting enzyme (ACE) inhibitors are effective antihypertensive drugs but often cause side effects. The aim of this study was to prepare potential ACE inhibitors from scales. Gelatin was extracted from lizardfish scales. Then, scale gelatin was enzymolyzed to prepare ACE inhibitory peptides using response surface methodology. Proteolytic conditions after optimization were as follows: pH 7.0, enzyme substrate ratio 3.2%, temperature 47 °C, and proteolysis lasting 2 h and 50 min. The experimental ACE inhibitory activity under optimal conditions was 86.0 ± 0.4%. Among the 118 peptides identified from gelatin hydrolysates, 87.3% were hydrophilic and 93.22% had a molecular weight <2000 Da. Gelatin peptides had high stability upon exposure to high temperature and pH as well as gastrointestinal tract enzymes. Gelatin peptides showed an antihypertensive effect in spontaneously hypertensive rats at a dosage of 2 g/kg in the long-term experiments. A new ACE inhibitory peptide was isolated from gelatin hydrolysates, and was identified as AGPPGSDGQPGAK with an IC50 value of 420 ± 20 μM. In this way, ACE inhibitory peptides derived from scale gelatin have the potential to be used as healthy ACE-inhibiting drug raw materials.

The structure, splicing, synteny and expression of lamprey COE genes and the evolution of the COE gene family in chordates.

COE genes encode transcription factors that have been found in all metazoans examined to date. They possess a distinctive domain structure that includes a DNA-binding domain (DBD), an IPT/TIG domain and a helix-loop-helix (HLH) domain. An intriguing feature of the COE HLH domain is that in jawed vertebrates it is composed of three helices, compared to two in invertebrates. We report the isolation and expression of two COE genes from the brook lamprey Lampetra planeri and compare these to COE genes from the lampreys Lethenteron japonicum and Petromyzon marinus. Molecular phylogenetic analyses do not resolve the relationship of lamprey COE genes to jawed vertebrate paralogues, though synteny mapping shows that they all derive from duplication of a common ancestral genomic region. All lamprey genes encode conserved DBD, IPT/TIG and HLH domains; however, the HLH domain of lamprey COE-A genes encodes only two helices while COE-B encodes three helices. We also identified COE-B splice variants encoding either two or three helices in the HLH domain, along with other COE-A and COE-B splice variants affecting the DBD and C-terminal transactivation regions. In situ hybridisation revealed expression in the lamprey nervous system including the brain, spinal cord and cranial sensory ganglia. We also detected expression of both genes in mesenchyme in the pharyngeal arches and underlying the notochord. This allows us to establish the primitive vertebrate expression pattern for COE genes and compare this to that of invertebrate chordates and other animals to develop a model for COE gene evolution in chordates.

TCF/Lef regulates the Gsx ParaHox gene in central nervous system development in chordates.

BACKGROUND: The ParaHox genes play an integral role in the anterior-posterior (A-P) patterning of the nervous system and gut of most animals. The ParaHox cluster is an ideal system in which to study the evolution and regulation of developmental genes and gene clusters, as it displays similar regulatory phenomena to its sister cluster, the Hox cluster, but offers a much simpler system with only three genes. RESULTS: Using Ciona intestinalis transgenics, we isolated a regulatory element upstream of Branchiostoma floridae Gsx that drives expression within the central nervous system of Ciona embryos. The minimal amphioxus enhancer region required to drive CNS expression has been identified, along with surrounding sequence that increases the efficiency of reporter expression throughout the Ciona CNS. TCF/Lef binding sites were identified and mutagenized and found to be required to drive the CNS expression. Also, individual contributions of TCF/Lef sites varied across the regulatory region, revealing a partial division of function across the Bf-Gsx-Up regulatory element. Finally, when all TCF/Lef binding sites are mutated CNS expression is not only abolished, but a latent repressive function is also unmasked. CONCLUSIONS: We have identified a B. floridae Gsx upstream regulatory element that drives CNS expression within transgenic Ciona intestinalis, and have shown that this CNS expression is dependent upon TCF/Lef binding sites. We examine the evolutionary and developmental implications of these results, and discuss the possibility of TCF/Lef not only as a regulator of chordate Gsx, but as a deeply conserved regulatory factor controlling all three ParaHox genes across the Metazoa.

FGF signaling induces mesoderm in the hemichordate Saccoglossus kowalevskii.

FGFs act in vertebrate mesoderm induction and also play key roles in early mesoderm formation in ascidians and amphioxus. However, in sea urchins initial characterizations of FGF function do not support a role in early mesoderm induction, making the ancestral roles of FGF signaling and mechanisms of mesoderm specification in deuterostomes unclear. In order to better characterize the evolution of mesoderm formation, we have examined the role of FGF signaling during mesoderm development in Saccoglossus kowalevskii, an experimentally tractable representative of hemichordates. We report the expression of an FGF ligand, fgf8/17/18, in ectoderm overlying sites of mesoderm specification within the archenteron endomesoderm. Embryological experiments demonstrate that mesoderm induction in the archenteron requires contact with ectoderm, and loss-of-function experiments indicate that both FGF ligand and receptor are necessary for mesoderm specification. fgf8/17/18 gain-of-function experiments establish that FGF8/17/18 is sufficient to induce mesoderm in adjacent endomesoderm. These experiments suggest that FGF signaling is necessary from the earliest stages of mesoderm specification and is required for all mesoderm development. Furthermore, they suggest that the archenteron is competent to form mesoderm or endoderm, and that FGF signaling from the ectoderm defines the location and amount of mesoderm. When considered in a comparative context, these data support a phylogenetically broad requirement for FGF8/17/18 signaling in mesoderm specification and suggest that FGF signaling played an ancestral role in deuterostome mesoderm formation.

Matrix adhesion polarizes heart progenitor induction in the invertebrate chordate Ciona intestinalis.

Cell-matrix adhesion strongly influences developmental signaling. Resulting impacts on cell migration and tissue morphogenesis are well characterized. However, the in vivo impact of adhesion on fate induction remains ambiguous. Here, we employ the invertebrate chordate Ciona intestinalis to delineate an essential in vivo role for matrix adhesion in heart progenitor induction. In Ciona pre-cardiac founder cells, invasion of the underlying epidermis promotes localized induction of the heart progenitor lineage. We found that these epidermal invasions are associated with matrix adhesion along the pre-cardiac cell/epidermal boundary. Through targeted manipulations of RAP GTPase activity, we were able to manipulate pre-cardiac cell-matrix adhesion. Targeted disruption of pre-cardiac cell-matrix adhesion blocked heart progenitor induction. Conversely, increased matrix adhesion generated expanded induction. We were also able to selectively restore cell-matrix adhesion and heart progenitor induction through targeted expression of Ci-Integrin ß2. These results indicate that matrix adhesion functions as a necessary and sufficient extrinsic cue for regional heart progenitor induction. Furthermore, time-lapse imaging suggests that cytokinesis acts as an intrinsic temporal regulator of heart progenitor adhesion and induction. Our findings highlight a potentially conserved role for matrix adhesion in early steps of vertebrate heart progenitor specification.

A bright monomeric green fluorescent protein derived from Branchiostoma lanceolatum.

We report a monomeric yellow-green fluorescent protein, mNeonGreen, derived from a tetrameric fluorescent protein from the cephalochordate Branchiostoma lanceolatum. mNeonGreen is the brightest monomeric green or yellow fluorescent protein yet described to our knowledge, performs exceptionally well as a fusion tag for traditional imaging as well as stochastic single-molecule superresolution imaging and is an excellent fluorescence resonance energy transfer (FRET) acceptor for the newest cyan fluorescent proteins.

Dact genes are chordate specific regulators at the intersection of Wnt and Tgf-ß signaling pathways.

BACKGROUND: Dacts are multi-domain adaptor proteins. They have been implicated in Wnt and Tgfß signaling and serve as a nodal point in regulating many cellular activities. Dact genes have so far only been identified in bony vertebrates. Also, the number of Dact genes in a given species, the number and roles of protein motifs and functional domains, and the overlap of gene expression domains are all not clear. To address these problems, we have taken an evolutionary approach, screening for Dact genes in the animal kingdom and establishing their phylogeny and the synteny of Dact loci. Furthermore, we performed a deep analysis of the various Dact protein motifs and compared the expression patterns of different Dacts. RESULTS: Our study identified previously not recognized dact genes and showed that they evolved late in the deuterostome lineage. In gnathostomes, four Dact genes were generated by the two rounds of whole genome duplication in the vertebrate ancestor, with Dact1/3 and Dact2/4, respectively, arising from the two genes generated during the first genome duplication. In actinopterygians, a further dact4r gene arose from retrotranscription. The third genome duplication in the teleost ancestor, and subsequent gene loss in most gnathostome lineages left extant species with a subset of Dact genes. The distribution of functional domains suggests that the ancestral Dact function lied with Wnt signaling, and a role in Tgfß signaling may have emerged with the Dact2/4 ancestor. Motif reduction, in particular in Dact4, suggests that this protein may counteract the function of the other Dacts. Dact genes were expressed in both distinct and overlapping domains, suggesting possible combinatorial function. CONCLUSIONS: The gnathostome Dact gene family comprises four members, derived from a chordate-specific ancestor. The ability to control Wnt signaling seems to be part of the ancestral repertoire of Dact functions, while the ability to inhibit Tgfß signaling and to carry out specialized, ortholog-specific roles may have evolved later. The complement of Dact genes coexpressed in a tissue provides a complex way to fine-tune Wnt and Tgfß signaling. Our work provides the basis for future structural and functional studies aimed at unraveling intracellular regulatory networks.

Coordination of mitosis and morphogenesis: role of a prolonged G2 phase during chordate neurulation.

Chordates undergo a characteristic morphogenetic process during neurulation to form a dorsal hollow neural tube. Neurulation begins with the formation of the neural plate and ends when the left epidermis and right epidermis overlying the neural tube fuse to close the neural fold. During these processes, mitosis and the various morphogenetic movements need to be coordinated. In this study, we investigated the epidermal cell cycle in Ciona intestinalis embryos in vivo using a fluorescent ubiquitination-based cell cycle indicator (Fucci). Epidermal cells of Ciona undergo 11 divisions as the embryos progress from fertilization to the tadpole larval stage. We detected a long G2 phase between the tenth and eleventh cell divisions, during which fusion of the left and right epidermis occurred. Characteristic cell shape change and actin filament regulation were observed during the G2 phase. CDC25 is probably a key regulator of the cell cycle progression of epidermal cells. Artificially shortening this G2 phase by overexpressing CDC25 caused precocious cell division before or during neural tube closure, thereby disrupting the characteristic morphogenetic movement. Delaying the precocious cell division by prolonging the S phase with aphidicolin ameliorated the effects of CDC25. These results suggest that the long interphase during the eleventh epidermal cell cycle is required for neurulation.

Ubiquity of insect-derived nitrogen transfer to plants by endophytic insect-pathogenic fungi: an additional branch of the soil nitrogen cycle.

The study of symbiotic nitrogen transfer in soil has largely focused on nitrogen-fixing bacteria. Vascular plants can lose a substantial amount of their nitrogen through insect herbivory. Previously, we showed that plants were able to reacquire nitrogen from insects through a partnership with the endophytic, insect-pathogenic fungus Metarhizium robertsii. That is, the endophytic capability and insect pathogenicity of M. robertsii are coupled so that the fungus acts as a conduit to provide insect-derived nitrogen to plant hosts. Here, we assess the ubiquity of this nitrogen transfer in five Metarhizium species representing those with broad (M. robertsii, M. brunneum, and M. guizhouense) and narrower insect host ranges (M. acridum and M. flavoviride), as well as the insect-pathogenic fungi Beauveria bassiana and Lecanicillium lecanii. Insects were injected with (15)N-labeled nitrogen, and we tracked the incorporation of (15)N into two dicots, haricot bean (Phaseolus vulgaris) and soybean (Glycine max), and two monocots, switchgrass (Panicum virgatum) and wheat (Triticum aestivum), in the presence of these fungi in soil microcosms. All Metarhizium species and B. bassiana but not L. lecanii showed the capacity to transfer nitrogen to plants, although to various degrees. Endophytic association by these fungi increased overall plant productivity. We also showed that in the field, where microbial competition is potentially high, M. robertsii was able to transfer insect-derived nitrogen to plants. Metarhizium spp. and B. bassiana have a worldwide distribution with high soil abundance and may play an important role in the ecological cycling of insect nitrogen back to plant communities.

Amphioxus FGF signaling predicts the acquisition of vertebrate morphological traits.

FGF signaling is one of the few cell-cell signaling pathways conserved among all metazoans. The diversity of FGF gene content among different phyla suggests that evolution of FGF signaling may have participated in generating the current variety of animal forms. Vertebrates possess the greatest number of FGF genes, the functional evolution of which may have been implicated in the acquisition of vertebrate-specific morphological traits. In this study, we have investigated the roles of the FGF signal during embryogenesis of the cephalochordate amphioxus, the best proxy for the chordate ancestor. We first isolate the full FGF gene complement and determine the evolutionary relationships between amphioxus and vertebrate FGFs via phylogenetic and synteny conservation analysis. Using pharmacological treatments, we inhibit the FGF signaling pathway in amphioxus embryos in different time windows. Our results show that the requirement for FGF signaling during gastrulation is a conserved character among chordates, whereas this signal is not necessary for neural induction in amphioxus, in contrast to what is known in vertebrates. We also show that FGF signal, acting through the MAPK pathway, is necessary for the formation of the most anterior somites in amphioxus, whereas more posterior somite formation is not FGF-dependent. This result leads us to propose that modification of the FGF signal function in the anterior paraxial mesoderm in an amphioxus-like vertebrate ancestor might have contributed to the loss of segmentation in the preotic paraxial mesoderm of the vertebrate head.

A dynamic history of gene duplications and losses characterizes the evolution of the SPARC family in eumetazoans.

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 to SPARC and SPARCB described 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, SPARC and SPARCB paralogues 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.