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Developmental complexity stemming from the dynamic interplay between genetic and biomechanic factors canalizes the ways genotypes and phenotypes can change in evolution. As a paradigmatic system, we explore how changes in developmental factors generate typical tooth shape transitions. Since tooth development has mainly been researched in mammals, we contribute to a more general understanding by studying the development of tooth diversity in sharks. To this end, we build a general, but realistic, mathematical model of odontogenesis. We show that it reproduces key shark-specific features of tooth development as well as real tooth shape variation in small-spotted catsharks Scyliorhinus canicula . We validate our model by comparison with experiments in vivo. Strikingly, we observe that developmental transitions between tooth shapes tend to be highly degenerate, even for complex phenotypes. We also discover that the sets of developmental parameters involved in tooth shape transitions tend to depend asymmetrically on the direction of that transition. Together, our findings provide a valuable base for furthering our understanding of how developmental changes can lead to both adaptive phenotypic change and trait convergence in complex, phenotypically highly diverse, structures.

While cartilage is an ancient tissue found both in protostomes and deuterostomes, its mineralization evolved more recently, within the vertebrate lineage. SPARC, SPARC-L, and the SCPP members (Secretory Calcium-binding PhosphoProtein genes which evolved from SPARC-L) are major players of dentine and bone mineralization, but their involvement in the emergence of the vertebrate mineralized cartilage remains unclear. We performed in situ hybridization on mineralizing cartilaginous skeletal elements of the frog Xenopus tropicalis (Xt) and the shark Scyliorhinus canicula (Sc) to examine the expression of SPARC (present in both species), SPARC-L (present in Sc only) and the SCPP members (present in Xt only). We show that while mineralizing cartilage expresses SPARC (but not SPARC-L) in Sc, it expresses the SCPP genes (but not SPARC) in Xt, and propose two possible evolutionary scenarios to explain these opposite expression patterns. In spite of these genetic divergences, our data draw the attention on an overlooked and evolutionarily conserved peripheral cartilage subdomain expressing SPARC or the SCPP genes and exhibiting a high propensity to mineralize.

International audience; Teeth in sharks are shed and replaced throughout their lifetime. Morphological dental changes through ontogeny have been identified in several species and have been correlated with shifts in diet and the acquisition of sexual maturity. However, these changes were rarely quantified in detail along multiple ontogenetic stages, which makes it difficult to infer the developmental processes responsible for the observed plasticity. In this work, we use micro‐computed tomography and 3D geometric morphometrics to describe and analyze the tooth size and shape diversity across three ontogenetic stages (hatchling, juvenile, and sexually mature) in the large‐spotted catshark Scyliorhinus stellaris (Linnaeus, 1758). We first describe the intra‐individual variation of tooth form for each sex at each ontogenetic stage. We provide a tooth morphospace for palatoquadrate and Meckelian teeth and identify dental features, such as relative size and number of cusps, involved in the range of variation of the observed morphologies. We then use these shape data to draw developmental trajectories between ontogenetic stages and for each tooth position within the jaw to characterize ontogenetic patterns of sexual dimorphism. We highlight the emergence of gynandric heterodonty between the juvenile and mature ontogenetic stages, with mature females having tooth morphologies more similar to juveniles’ than mature males that display regression in the number of accessory cusps. From these data, we speculate on the developmental processes that could account for such developmental plasticity in S. stellaris.

Most extant vertebrates display a high variety of tooth and tooth-like organs (odontodes) that vary in shape, position over the body and nature of composing tissues. The development of these structures is known to involve similar genetic cascades and teeth and odontodes are believed to share a common evolutionary history. Gene expression patterns have previously been compared between mammalian and teleost tooth development but we highlight how the comparative framework was not always properly defined to deal with different tooth types or tooth developmental stages. Larger-scale comparative analyses also included cartilaginous fishes: sharks display oral teeth and dermal scales for which the gene expression during development started to be investigated in the small-spotted catshark Scyliorhinus canicula during the past decade. We report several descriptive approaches to analyse the embryonic tooth and caudal scale gene expressions in S. canicula. We compare these expressions wih the ones reported in mouse molars and teleost oral and pharyngeal teeth and highlight contributions and biases that arise from these interspecific comparisons. We finally discuss the evolutionary processes that can explain the observed intra and interspecific similarities and divergences in the genetic cascades involved in tooth and odontode development in jawed vertebrates.

In order to characterize the molecular bases of mineralizing cell evolution, we targeted type X collagen, a nonfibrillar network forming collagen encoded by the Col10a1 gene. It is involved in the process of endochondral ossification in ray-finned fishes and tetrapods (Osteichthyes), but until now unknown in cartilaginous fishes (Chondrichthyes). We show that holocephalans and elasmobranchs have respectively five and six tandemly duplicated Col10a1 gene copies that display conserved genomic synteny with osteichthyan Col10a1 genes. All Col10a1 genes in the catshark Scyliorhinus canicula are expressed in ameloblasts and/or odontoblasts of teeth and scales, during the stages of extracellular matrix protein secretion and mineralization. Only one duplicate is expressed in the endoskeletal (vertebral) mineralizing tissues. We also show that the expression of type X collagen is present in teeth of two osteichthyans, the zebrafish Danio rerio and the western clawed frog Xenopus tropicalis, indicating an ancestral jawed vertebrate involvement of type X collagen in odontode formation. Our findings push the origin of Col10a1 gene prior to the divergence of osteichthyans and chondrichthyans, and demonstrate its ancestral association with mineralization of both the odontode skeleton and the endoskeleton.

International audience; The skeleton of cartilaginous fishes is notoriously difficult to prepare, and the literature dealing with the subject is extremely rare compared to other vertebrate groups. As a result, chondrichthyan skeletal specimens held in museums and other scientific institutions are often limited to isolated sets of jaws, taxidermised specimens and small individuals preserved whole in fluid. However, skeletal characters are heavily relied upon in several taxonomical and phylogenetical analyses, highlighting the importance of properly prepared skeletal specimens in research collections. The relevant literature is briefly reviewed herein, with a proposal for a new and simple protocol involving limited and easily obtainable chemicals , for the quick preparation of clean and durable chon-drichthyan skeletal specimens. Various features of the chondrichthyan skeleton are also discussed, along with the microstructural effects of the chemicals involved in the protocol .

Background The molecular bases explaining the diversity of dental tissue mineralization across gnathostomes are still poorly understood. Odontodes, such as teeth and body denticles, are serial structures that develop through deployment of a gene regulatory network shared between all gnathostomes. Dentin, the inner odontode mineralized tissue, is produced by odontoblasts and appears well-conserved through evolution. In contrast, the odontode hypermineralized external layer (enamel or enameloid) produced by ameloblasts of epithelial origin, shows extensive structural variations. As EMP (Enamel Matrix Protein) genes are as yet only found in osteichthyans where they play a major role in the mineralization of teeth and others skeletal organs, our understanding of the molecular mechanisms leading to the mineralized odontode matrices in chondrichthyans remains virtually unknown. Results We undertook a phylogenetic analysis of the SPARC/SPARC-L gene family, from which the EMPs are supposed to have arisen, and examined the expression patterns of its members and of major fibrillar collagens in the spotted catshark Scyliorhinus canicula, the thornback ray Raja clavata, and the clawed frog Xenopus tropicalis. Our phylogenetic analyses reveal that the single chondrichthyan SPARC-L gene is co-orthologous to the osteichthyan SPARC-L1 and SPARC-L2 paralogues. In all three species, odontoblasts co-express SPARC and collagens. In contrast, ameloblasts do not strongly express collagen genes but exhibit strikingly similar SPARC-L and EMP expression patterns at their maturation stage, in the examined chondrichthyan and osteichthyan species, respectively. Conclusions A well-conserved odontoblastic collagen/SPARC module across gnathostomes further confirms dentin homology. Members of the SPARC-L clade evolved faster than their SPARC paralogues, both in terms of protein sequence and gene duplication. We uncover an osteichthyan-specific duplication that produced SPARC-L1 (subsequently lost in pipidae frogs) and SPARC-L2 (independently lost in teleosts and tetrapods).Our results suggest the ameloblastic expression of the single chondrichthyan SPARC-L gene at the maturation stage reflects the ancestral gnathostome situation, and provide new evidence in favor of the homology of enamel and enameloids in all gnathostomes. Electronic supplementary material The online version of this article (10.1186/s12862-018-1241-y) contains supplementary material, which is available to authorized users.

International audience; The mitogenome is an inescapable tool in conservation biology studies. Yet, its routine sequencing may remain tricky despite next-generation sequencing technologies. An enrichment step is often necessary but not always straightforward depending on the initial DNA quality or quantity. Furthermore, the availability of close mitochondrial DNA reference sequences for non-model species limits the primer design for long-range PCR or bait synthesis. Here we propose an easy and cost-effective protocol without enrichment step for building and sequencing multiplexed Illumina libraries from small quantities of either high-quality or degraded genomic DNA. We validated the approach through the successful assembly of the complete mitogenome of 60 bats and 7 tunicates. Our protocol allows the sequencing and assembly of mitochondrial genomes from non-model species with sufficient coverage for applications in conservation genetics.

Evolution of Developmental Control Mechanisms.-- et al., The acquisition of jaws constitutes a landmark event in vertebrate evolution, one that in large part potentiated their success and diversification. Jaw development and patterning involves an intricate spatiotemporal series of reciprocal inductive and responsive interactions between the cephalic epithelia and the cranial neural crest (CNC) and cephalic mesodermal mesenchyme. The coordinated regulation of these interactions is critical for both the ontogenetic registration of the jaws and the evolutionary elaboration of variable jaw morphologies and designs. Current models of jaw development and evolution have been built on molecular and cellular evidence gathered mostly in amniotes such as mice, chicks and humans, and augmented by a much smaller body of work on the zebrafish. These have been partnered by essential work attempting to understand the origins of jaws that has focused on the jawless lamprey. Chondrichthyans (cartilaginous fish) are the most distant group to amniotes within extant gnathostomes, and comprise the crucial clade uniting amniotes and agnathans; yet despite their critical phylogenetic position, evidence of the molecular and cellular underpinnings of jaw development in chondrichthyans is still lacking. Recent advances in genome and molecular developmental biology of the lesser spotted dogfish shark, Scyliorhinus canicula, make it ideal for the molecular study of chondrichthyan jaw development. Here, following the 'Hinge and Caps' model of jaw development, we have investigated evidence of heterotopic (relative changes in position) and heterochronic (relative changes in timing) shifts in gene expression, relative to amniotes, in the jaw primordia of S. canicula embryos. We demonstrate the presence of clear proximo-distal polarity in gene expression patterns in the shark embryo, thus establishing a baseline molecular baüplan for branchial arch-derived jaw development and further validating the utility of the 'Hinge and Caps' model in comparative studies of jaw development and evolution. Moreover, we correlate gene expression patterns with the absence of a lambdoidal junction (formed where the maxillary first arch meets the frontonasal processes) in chondrichthyans, further highlighting the importance of this region for the development and evolution of jaw structure in advanced gnathostomes., This work was supported by funding to M.J.D. from the Royal Society, the Dental Institute of King’s College London, and Friends of Guy’s Hospital. C.C. and J.G. were funded by Marie Curie Early Training Fellowships (MEST-CT-2004-504025). J.L.F. was assisted by a HFSP Long Term Fellowship (LT 01061/2007-L). S.M. was funded by Région Centre, Région Bretagne (EVOVERT grant number 049755), National Research Agency (grant ANR-09-BLAN-026201), CNRS, Université d’Orléans and the Université Pierre et Marie Curie. M.C. benefited from a doctoral fellowship from CNRS.

International audience; Current knowledge on the skeletogenesis of Chondrichthyes is scarce compared with their extant sister group, the bony fishes. Most of the previously described developmental tables in Chondrichthyes have focused on embryonic external morphology only. Due to its small body size and relative simplicity to raise eggs in laboratory conditions, the small-spotted catshark Scyliorhinus canicula has emerged as a reference species to describe developmental mechanisms in the Chondrichthyes lineage. Here we investigate the dynamic of mineralization in a set of six embryonic specimens using X-ray microtomography and describe the developing units of both the dermal skeleton (teeth and dermal scales) and endoskeleton (vertebral axis). This preliminary data on skeletogenesis in the catshark sets the first bases to a more complete investigation of the skeletal developmental in Chondrichthyes. It should provide comparison points with data known in osteichthyans and could thus be used in the broader context of gnathostome skeletal evolution.

During vertebrate forebrain formation, Dlx homeobox genes play essential roles in the differentiation, migration and survival of subpallial precursor cells that will later give rise to diverse subtypes of γ-aminobutyric acid (GABA)-expressing neurons, including inhibitory cortical interneurons in mammals. They also participate in the regulation of the Gad genes encoding the enzymes necessary for GABA synthesis. In mice, at least four cis-regulatory elements (CREs) control Dlx expression in the telencephalon and diencephalon: URE2 and I12b in the Dlx1/Dlx2 bigene cluster, and I56i and I56ii in the Dlx5/Dlx6 bigene cluster. However, little is known so far with respect to the function of orthologous dlx genes and their regulatory elements during zebrafish GABAergic neuron development. To investigate whether similar dlx-mediated pathways exist in the early developing zebrafish forebrain, we generated independent lines of transgenic zebrafish carrying two distinct GFP reporter constructs driven by a β-globin minimal promoter: one containing a ∼1.4kb dlx5a/dlx6a intergenic sequence (encompassing I56i and I56ii) and one with a ∼1.1kb fragment containing only the I56i CRE, respectively. The expression patterns of these two transgenes were compared with that obtained with another construct containing the ∼1.4kb dlx5a/dlx6a intergenic sequence and driven by a ∼3.5kb dlx6a 5'-flanking fragment. Our comparative analysis showed that GFP expression of the three transgene is largely overlapping throughout the ventral forebrain. Intriguingly, the dlx6a 5'-flanking fragment has a major impact on transgene expression in the mesencephalic tectum. Furthermore, comparison of transgene expression between the ∼1.4kb and ∼1.1kb intergenic fragments did not show any specific spatial expression conferred by I56ii. Almost all GFP-expressing cells in the transgenic zebrafish are GABA-positive and also express various GABAergic interneuron markers. Together, our data suggest that zebrafish dlx5a/dlx6a intergenic CREs may be involved in a conserved genetic pathway necessary for proper dlx expression during zebrafish GABAergic neuron development.

SUMMARY The Hox gene family encodes homeodomain-containing transcription factors involved in the patterning of structures composed of repeated elements along the antero-posterior axis of Bilateralia embryos. In vertebrate, Hox genes are thought to control the segmental identity of the rhombomeres, the branchial arches, and the somites. They are therefore thought to have played a key role in the morphological evolution of structures like the jaw, girdles, and vertebrae in gnathostomes. Thus far, our knowledge about the expression patterns of the Hox genes, the Hox code, has been mainly restricted to osteichthyans species and little is known about chondrichthyans. Recently, we identified 34 Hox genes clustered in three complexes (HoxA, HoxB, and HoxD) in the dogfish (Scyliorhinus canicula) genome suggesting that in sharks most, if not all, genes belonging to the HoxC complex are lost. To gain insights into the evolution of gnathostome Hox transcription, we present here expression patterns along the anteroposterior axis for all Hox genes known in the dogfish. A comparison of these patterns with those of osteichthyans shows that the expression patterns of the Hox genes in serially homologous compartments such as the branchial arches, the hindbrain, and the somites underwent only subtle changes during the evolution of gnathostomes. Therefore, the nested expression of Hox genes in these structures, the Hox code, is a ground plan, which predates the morphological diversification of serially homologous structures along the body axis.

Summary The Dlx genes play an important role in the development of the pharyngeal arches and the structures derived from these tissues, including the craniofacial skeleton. They are typically expressed in a nested pattern along the proximo-distal axis of the branchial arches in mice. This pattern is known as the “Dlx code” and has been proposed to be responsible for an early regional patterning of branchial arches in mammals. A number of cis- regulatory elements (CREs) have been identified within the Dlx loci, which target reporter expression to the developing pharyngeal arches of the mouse. Most of these CREs are located in the intergenic regions between the two genes constituting a Dlx bigene cluster. Therefore, the regionalized dlx expression in the branchial arches could be the result of the shared activities of these regulatory regions. Here we analyze previously published and new results showing these CREs are highly conserved in both their sequence and their activity in the pharyngeal arches of two distantly related vertebrates, the zebrafish and the mouse. A better understanding of Dlx gene regulation of the Dlx genes and of the genetic cascades in which they are involved can lead to clues explaining variations in morphology of the craniofacial regions of vertebrates.

Zaprionus vittiger Coquillett is the type species of the genus Zaprionus Coquillett. However, the species is only known from five old museum specimens collected from South Africa and Malawi. It has often been confused with many other Zaprionus species, especially with Z. spinipilus Chassagnard & McEvey, a widespread species in Africa known from Madagascar, Malawi, Ethiopia and Cameroon. We have recently collected flies from the type localities of both species (South Africa and Madagascar, respectively). This has prompted us to test the taxonomic boundaries of these two nominal species using molecular (the mitochondrial COII and the nuclear Amyrel genes), chromosomal, morphological (internal and external genitalia), and reproductive isolation analyses. The results suggest Z. spinipilus to be a junior synonym to Z. vittiger.

BackgroundThe gene regulatory network involved in tooth morphogenesis has been extremely well described in mammals and its modeling has allowed predictions of variations in regulatory pathway that may have led to evolution of tooth shapes. However, very little is known outside of mammals to understand how this regulatory framework may also account for tooth shape evolution at the level of gnathostomes. In this work, we describe expression patterns and proliferation/apoptosis assays to uncover homologous regulatory pathways in the catsharkScyliorhinus canicula.ResultsBecause of their similar structural and developmental features, gene expression patterns were described over the four developmental stages of both tooth and scale buds in the catshark. These gene expression patterns differ from mouse tooth development, and discrepancies are also observed between tooth and scale development within the catshark. However, a similar nested expression of Shh and Fgf suggests similar signaling involved in morphogenesis of all structures, although apoptosis assays do not support a strictly equivalent enamel knot system in sharks. Similarities in the topology of gene expression pattern, including Bmp signaling pathway, suggest that mouse molar development is more similar to scale bud development in the catshark.ConclusionsThese results support the fact that no enamel knot, as described in mammalian teeth, can be described in the morphogenesis of shark teeth or scales. However, homologous signaling pathways are involved in growth and morphogenesis with variations in their respective expression patterns. We speculate that variations in this topology of expression are also a substrate for tooth shape evolution, notably in regulating the growth axis and symmetry of the developing structure.

Understanding the evolutionary emergence and subsequent diversification of the vertebrate skeleton requires a comprehensive view of the diverse skeletal cell types found in distinct developmental contexts, tissues, and species. To date, our knowledge of the molecular nature of the shark calcified extracellular matrix, and its relationships with osteichthyan skeletal tissues, remain scarce. Here, based on specific combinations of expression patterns of the Col1a1, Col1a2, and Col2a1 fibrillar collagen genes, we compare the molecular footprint of endoskeletal elements from the chondrichthyan Scyliorhinus canicula and the tetrapod Xenopus tropicalis. We find that, depending on the anatomical location, Scyliorhinus skeletal calcification is associated to cell types expressing different subsets of fibrillar collagen genes, such as high levels of Col1a1 and Col1a2 in the neural arches, high levels of Col2a1 in the tesserae, or associated to a drastic Col2a1 downregulation in the centrum. We detect low Col2a1 levels in Xenopus osteoblasts, thereby revealing that the osteoblastic expression of this gene was significantly reduced in the tetrapod lineage. Finally, we uncover a striking parallel, from a molecular and histological perspective, between the vertebral cartilage calcification of both species and discuss the evolutionary origin of endochondral ossification.

International audience; Using X-ray microtomography, we describe the ossification events during the larval development of a non-teleost actinopterygian species: the Cuban gar Atractosteus tristoechus from the order Lepisosteiformes. We provide a detailed developmental series for each anatomical structure, covering a large sequence of mineralization events going from an early stage (13 days post-hatching, 21mm total length) to an almost fully ossified larval stage (118dph or 87mm in standard length). With this work, we expect to bring new developmental data to be used in further comparative studies with other lineages of bony vertebrates. We also hope that the online publication of these twelve successive 3D reconstructions, fully flagged, will be an educational tool for all students in comparative anatomy.

International audience; During development of the mouse forebrain interneurons, the Dlx genes play a key role in a gene regulatory network (GRN) that leads to the GABAergic phenotype. Here, we have examined the regulatory relationships between the ascl1a, dlx, and gad1b genes in the zebrafish forebrain. Expression of ascl1a overlaps with dlx1a in the telencephalon and diencephalon during early forebrain development. The loss of Ascl1a function results in a loss of dlx expression, and subsequent losses of dlx5a and gad1b expression in the diencephalic prethalamus and hypothalamus. Loss of Dlx1a and Dlx2a function, and, to a lesser extent, of Dlx5a and Dlx6a, impairs gad1b expression in the prethalamus and hypothalamus. We conclude that dlx1a/2a act downstream of ascl1a but upstream of dlx5a/dlx6a and gad1b to activate GABAergic specification. This pathway is conserved in the diencephalon, but has diverged between mammals and teleosts in the telencephalon.

The Dlx genes encode a family of transcription factors important for the development of the vertebrate forebrain. These genes have very similar expression domains during the development of the telencephalon in mice and play a role in gamma-aminobutyric acid (GABAergic) interneuron differentiation. We have used triple fluorescent in situ hybridization to study the relative expression domains of the dlx and gad1 genes in the zebrafish telencephalon and diencephalon. We also generated transgenic zebrafish with regulatory elements from the zebrafish dlx1a/2a locus. The zebrafish dlx regulatory elements recapitulated dlx expression in the forebrain and mimicked the relationship between the expression of the dlx genes and gad1. Finally, we show that a putative enhancer located downstream of dlx2b can also activate reporter gene expression in a tissue-specific manner similar to endogenous dlx2b expression. Our results indicate the dlx genes are regulated by an evolutionarily conserved genetic pathway and may play a role in GABAergic interneuron differentiation in the zebrafish forebrain.

International audience; It is now well established that there were four Hox gene clusters in the genome of the last common ancestor of extant gnathostomes. To better understand the evolution of the organization and expression of these genomic regions, we have studied the Hox gene clusters of a shark (Scyliorhinus canicula). We sequenced 225,580 expressed sequence tags from several embryonic cDNA libraries. Blast searches identified corresponding transcripts to almost all the HoxA, HoxB, and HoxD cluster genes. No HoxC transcript was identified, suggesting that this cluster is absent or highly degenerate. Using Hox gene sequences as probes, we selected and sequenced seven clones from a bacterial artificial chromosome library covering the complete region of the three gene clusters. Mapping of cDNAs to these genomic sequences showed extensive alternative splicing and untranslated exon sharing between neighboring Hox genes. Homologous noncoding exons could not be identified in transcripts from other species using sequence similarity. However, by comparing conserved noncoding sequences upstream of these exons in different species, we were able to identify homology between some exons. Some alternative splicing variants are probably very ancient and were already coded for by the ancestral Hox gene cluster. We also identified several transcripts that do not code for Hox proteins, are probably not translated, and all but one are in the reverse orientation to the Hox genes. This survey of the transcriptome of the Hox gene clusters of a shark shows that the high complexity observed in mammals is a gnathostome ancestral feature.

Background The phylogenetic position of the elephant shark (Callorhinchus milii ) is particularly relevant to study the evolution of genes and gene regulation in vertebrates. Here we examine the evolution of Dlx homeobox gene regulation during vertebrate embryonic development with a particular focus on the forebrain. We first identified the elephant shark sequence orthologous to the URE2 cis -regulatory element of the mouse Dlx1/Dlx2 locus (herein named CmURE2). We then conducted a comparative study of the sequence and enhancer activity of CmURE2 with that of orthologous regulatory sequences from zebrafish and mouse. Results The CmURE2 sequence shows a high percentage of identity with its mouse and zebrafish counterparts but is overall more similar to mouse URE2 (MmURE2) than to zebrafish URE2 (DrURE2). In transgenic zebrafish and mouse embryos, CmURE2 displayed enhancer activity in the forebrain that overlapped with that of DrURE2 and MmURE2. However, we detected notable differences in the activity of the three sequences in the diencephalon. Outside of the forebrain, CmURE2 shows enhancer activity in areas such as the pharyngeal arches and dorsal root ganglia where its' counterparts are also active. Conclusions Our transgenic assays show that part of the URE2 enhancer activity is conserved throughout jawed vertebrates but also that new characteristics have evolved in the different groups. Our study demonstrates that the elephant shark is a useful outgroup to study the evolution of regulatory mechanisms in vertebrates and to address how changes in the sequence of cis -regulatory elements translate into changes in their regulatory activity.

International audience; One of the goals of evolutionary developmental biology is to link specific adaptations to changes in developmental pathways. The dentition of cypriniform fishes, which in contrast to many other teleost fish species possess pharyngeal teeth but lack oral teeth, provides a suitable model to study the development of feeding adaptations. Here, we have examined the involvement of retinoic acid (RA) in tooth development and show that RA is specifically required to induce the pharyngeal tooth developmental program in zebrafish. Perturbation of RA signaling at this stage abolished tooth induction without affecting the development of tooth-associated ceratobranchial bones. We show that this inductive event is dependent on RA synthesis from aldh1a2 in the ventral posterior pharynx. Fibroblast growth factor (FGF) signaling has been shown to be critical for tooth induction in zebrafish, and its loss has been associated with oral tooth loss in cypriniform fishes. Pharmacological treatments targeting the RA and FGF pathways revealed that both pathways act independently during tooth induction. In contrast, we find that in Mexican tetra and medaka, species that also possess oral teeth, both oral and pharyngeal teeth are induced independently of RA. Our analyses suggest an evolutionary scenario in which the gene network controlling tooth development obtained RA dependency in the lineage leading to the cypriniforms. The loss of pharyngeal teeth in this group was cancelled out through a shift in aldh1a2 expression, while oral teeth might have been lost ultimately due to deficient RA signaling in the oral cavity.-Gibert, Y., Bernard, L., Debiais-Thibaud, M., Bourrat, F., Joly, J.-S., Pottin, K., Meyer, A., Retaux, S., Stock, D. W., Jackman, W. R., Seritrakul, P., Begemann, G., Laudet, V. Formation of oral and pharyngeal dentition in teleosts depends on differential recruitment of retinoic acid signaling.

SUMMARY Serially homologous structures are believed to originate from the redeployment of a genetic cascade in different locations of the body. Serial homologs may diverge at the genetic and morphological level and acquire developmental independency (individualization). Teeth are repeated units that form dentitions found on different bones of the oral–pharyngeal cavity in gnathostomes and provide a good model to study such processes. Previous comparisons of dlx gene expression patterns between mouse oral teeth and zebrafish pharyngeal teeth showed a high level of divergence. Furthermore, these genes are differentially expressed in different teeth of the zebrafish, and in the mouse they are responsible for tooth identity (incisors vs. molars). We examined the potential divergence of dlx gene expression between oral and pharyngeal teeth by examining the expression pattern in the development of the first generation teeth of the medaka and comparing it with data from the zebrafish and the mouse. Out of the seven medaka dlx genes, five are expressed during odontogenesis compared with six in both the zebrafish and the mouse. The only difference observed between oral and pharyngeal teeth in the medaka is an earlier expression of dlx5a in the oral dental epithelium. The subset of dlx genes expressed in the medaka, zebrafish, and mouse is slightly different but their detailed expression patterns are highly divergent. Our results demonstrate a low constraint on dlx gene expression shuffling in the odontogenic cascade within osteichtyans but the non-individualization of oral and pharyngeal dentitions in the medaka.

International audience; Gnathostome teeth are one of the most promising models for developmental evolutionary studies, they are the most abundant organ in the fossil record and an excellent example of organogenesis. Teeth have a complex morphology and are restricted to the mouth in mammals, whereas actinopterygian teeth have a simple morphology and are found in several locations, notably on pharyngeal bones. Morphological and developmental similarities support the hypothesis that oral and pharyngeal teeth are serially homologous. Gene expression data from the mouse and some teleosts have shown that the gene families involved in pharyngeal odontogenesis are also involved in oral tooth formation, with the notable exception of the evx gene family. Here, we present a complete description of early odontogenesis in the medaka (Oryzias latipes), which has both oral and pharyngeal dentition. We show that oral and pharyngeal teeth share deep developmental similarities. In the medaka, like in the zebrafish, eve1 is the only evx gene expressed during odontogenesis. In each forming tooth, regardless of its location, eve1 transcription is activated in the placode, then becomes restricted to the inner dental epithelium and is activated in the dental mesenchyme during early differentiation, and finally ceases at late differentiation. Thus eve1 expression is not specific to pharyngeal teeth development as was previously suggested. Because it permits direct comparisons between oral and pharyngeal teeth by molecular, development and functional studies, the medaka is an excellent model to develop further insights into the evolution of odontogenesis in gnathostomes.. 2007. Development of oral and pharyngeal teeth in the medaka (Oryzias latipes): comparison of morphology and expression of eve1 gene. J. Exp. Zool. (Mol. Dev. Evol.) 308B:693-708. Teeth are regarded by both palaeontologists and developmental geneticists as an excellent model for studying evolutionary mechanisms for several reasons. They are the most abundant organ in the vertebrate fossil record, chiefly because they are mainly composed of hard tissues and are partially hyper mineralised. They have undergone extensive morphological variation during the course of evolution, are therefore a very informative character, and have been used in a wide range of palaeontological studies. Finally, although the tooth is one of the simplest organs that form during gnathostomes development, it represents a model of organ ontogeny.

With the availability of an increasing number of whole genome sequences in chordates, exhaustive comparisons of multigene families become feasible. Relationships of orthology/paralogy can not only be inferred from sequence similarity but also by comparing synteny conservation on chromosomes. More accurate scenarios for gene and expression domain gain or loss can now be proposed. Here, we take benefit from the recent release of the medaka (Oryzias latipes) genome to analyse the orthology relationships and expression patterns of the three different sub-families of the pitx homeobox genes belonging to the paired class. They are involved in a wide variety of developmental processes and have pleiotropic expression patterns, especially in the case of the pitx2 sub-family. The emerging picture is a strong conservation of expression domains, suggesting that most functions have been present in the common ancestor of actinopterygians and sarcopterygians. Almost all pitx genes are expressed in anterior placodes in all species studied so far, including medaka. It has previously been shown that in mammals, pitx1 and 2 are expressed in the pituitary. Interestingly we demonstrate here that only pitx3 is expressed in medaka pituitary. It will be interesting to analyze what are the corresponding changes in the regulatory elements of pitx genes.

We have used dlx genes to test the hypothesis of a separate developmental program for dermal and cartilage bones within the neuro- and splanchnocranium by comparing expression patterns of all eight dlx genes during cranial bone formation in zebrafish from 1 day postfertilization (dPF) to 15 dPF. dlx genes are expressed in the visceral skeleton but not during the formation of dermal or cartilage bones of the braincase. The spatiotemporal expression pattern of all the members of the dlx gene family, support the view that dlx genes impart cellular identity to the different arches, required to make arch-specific dermal bones. Expression patterns seemingly associated with cartilage (perichondral) bones of the arches, in contrast, are probably related to ongoing differentiation of the underlying cartilage rather than with differentiation of perichondral bones themselves. Whether dlx genes originally functioned in the visceral skeleton only, and whether their involvement in the formation of neurocranial bones (as in mammals) is secondary, awaits clarification.

In order to investigate similarities and differences in genetic control of development among teeth within and between species, we determined the expression pattern of all eight Dlx genes of the zebrafish during development of the pharyngeal dentition and compared these data with that reported for mouse molar tooth development. We found that (i) dlx1a and dlx6a are not expressed in teeth, in contrast to their murine orthologs, Dlx1 and Dlx6; (ii) the expression of the six other zebrafish Dlx genes overlaps in time and space, particularly during early morphogenesis; (iii) teeth in different locations and generations within the zebrafish dentition differ in the number of genes expressed; (iv) expression similarities and differences between zebrafish Dlx genes do not clearly follow phylogenetic and linkage relationships; and (v) similarities and differences exist in the expression of zebrafish and mouse Dlx orthologs. Taken together, these results indicate that the Dlx gene family, despite having been involved in vertebrate tooth development for over 400 million years, has undergone extensive diversification of expression of individual genes both within and between dentitions. The latter type of difference may reflect the highly specialized dentition of the mouse relative to that of the zebrafish, and/or genome duplication in the zebrafish lineage facilitating a redistribution of Dlx gene function during odontogenesis.

International audience; Background: The Dlx gene family encodes transcription factors involved in the development of a wide variety of morphological innovations that first evolved at the origins of vertebrates or of the jawed vertebrates. This gene family expanded with the two rounds of genome duplications that occurred before jawed vertebrates diversified. It includes at least three bigene pairs sharing conserved regulatory sequences in tetrapods and teleost fish, but has been only partially characterized in chondrichthyans, the third major group of jawed vertebrates. Here we take advantage of developmental and molecular tools applied to the shark Scyliorhinus canicula to fill in the gap and provide an overview of the evolution of the Dlx family in the jawed vertebrates. These results are analyzed in the theoretical framework of the DDC (Duplication-Degeneration-Complementation) model.Results: The genomic organisation of the catshark Dlx genes is similar to that previously described for tetrapods. Conserved non-coding elements identified in bony fish were also identified in catshark Dlx clusters and showed regulatory activity in transgenic zebrafish. Gene expression patterns in the catshark showed that there are some expression sites with high conservation of the expressed paralog(s) and other expression sites with events of paralog sub-functionalization during jawed vertebrate diversification, resulting in a wide variety of evolutionary scenarios within this gene family.Conclusion: Dlx gene expression patterns in the catshark show that there has been little neo-functionalization in Dlx genes over gnathostome evolution. In most cases, one tandem duplication and two rounds of vertebrate genome duplication have led to at least six Dlx coding sequences with redundant expression patterns followed by some instances of paralog sub-functionalization. Regulatory constraints such as shared enhancers, and functional constraints including gene pleiotropy, may have contributed to the evolutionary inertia leading to high redundancy between gene expression patterns.

Bony fish display a skeleton that develops mainly as cartilage, locally replaced by endochondral bone tissue. Cartilaginous fish, however, have a cartilaginous skeleton, locally mineralized in various ways, some of which are evolutionary innovations specific to this lineage (prismatic cartilage, for instance). The cells associated with these tissues are thus capable of biomineralization by precipitation of calcium phosphate, a property that seems to be limited to the vertebrate clade. In this thesis, I focus on the genetics of skeletal mineralization development in a cartilaginous fish: the small-spotted catshark (Scyliorhinus canicula). I describe in detail the putative genetic factors underlying the mineralization process, and I do so through two approaches: through candidate gene studies and through transcriptomic studies. This allows me to better characterize the set of genetic mineralization tools in chondrichthyans, so that through comparative analysis, an ancestral state for jawed vertebrates can be inferred, at least partially. First, through the candidate gene approach, I test the level of conservation regarding genes known to be associated with mineralization in bony fish. Second, I describe how I used recently generated genomic and transcriptomic data in the small-spotted catshark to study intrinsic genes specifically expressed in mineralizing tissues. These two approaches allow me to detect interesting genes that are then studied through phylogenetic analyses, quantification/localization of expression in tissues, as well as comparative analyses with bony fish (when possible). In the last part, I present two papers in preparation that result from the work we have done with the help of two master students. These papers stem from a candidate gene approach but involve large gene families and in an effort to include all paralogs, the papers move towards a molecular evolutionary framework rather than an Evo-Devo framework.; Les poissons osseux présentent un squelette se développant principalement sous la forme de cartilage, localement remplacé par du tissu osseux endochondral. Les poissons cartilagineux, eux, présentent un squelette de nature cartilagineuse, localement minéralisé selon diverses modalités dont certaines sont des innovations évolutives propres à cette lignée (le cartilage prismatique, par exemple). Les cellules associées à ces tissus sont donc capables de biominéralisation par précipitation de phosphate de calcium, une propriété qui semble limitée au groupe des vertébrés. Au cours de cette thèse, je me concentre sur la génétique du développement de la minéralisation du squelette chez un poisson cartilagineux : la petite roussette (Scyliorhinus canicula). Je décris en détail les facteurs génétiques putatifs qui sous-tendent le processus de minéralisation, et je le fais par deux approches : par l'étude de gènes candidats et par la transcriptomique. Cela me permet de mieux caractériser l'ensemble des outils de minéralisation chez les chondrichtyens, de sorte que par une analyse comparative, un état ancestral pour les vertébrés à mâchoire peut être déduit, au moins partiellement. Tout d'abord, à travers l'approche par gènes candidats, je teste le niveau de conservation concernant des gènes connus par leur association à la minéralisation chez les poissons osseux. Ensuite, je décris comment j'ai utilisé les données génomiques et transcriptomiques récemment générées chez la petite roussette pour étudier les gènes intrinsèques exprimés spécifiquement dans les tissus minéralisants. Ces deux approches me permettent de détecter des gènes intéressants qui sont ensuite étudiés à travers des analyses phylogénétiques, de quantification/localisation de l'expression dans les tissues, ainsi que des analyses comparatives avec les poissons osseux (lorsque cela est possible). Dans la dernière partie, je présente deux articles en préparation qui résultent du travail que nous avons réalisé avec l'aide de deux étudiants de master. Ces articles sont issus d'une approche gène candidat mais concernent des familles de gènes étendues et dans un effort d'inclure tous les paralogues, les articles s'orientent vers un cadre d'évolution moléculaire plutôt qu'un cadre Evo-Devo.

Teeth are serial structures whose evolutionary and developmental history is intricately linked with the emergence of mineralised tissues in vertebrates. Teeth display a broad range of forms and differ in developmental patterns in extant vertebrates, making them remarkable elements to study species diversification. Selachian teeth renew permanently and display morphologies that are correlated with mating and trophic behaviours.This work first assesses the variation of tooth forms in two scyliorhinids by using 3D geometric morphometrics and machine learning. The emergence of gynandric heterodonty is detailed for the first time along the ontogeny of sharks and it is demonstrated that this natural variation should be first assessed before performing species discrimination.This work also questions the role of specific proteins on the acquisition of a shark tooth form over development. Functional tests suggest an impact of Shh and Fgf3 in the cusp morphogenesis and in the mineralisation process. These proteins are promising explanatory variables to the inter- and intraspecific tooth differences observed, leading to hypotheses on their role in the evolution of structures with speciation and trophic and mating behaviours.Histological data on extant chondrichthyan vertebrae finally highlight the unsuspected proportion of extant elasmobranchs exhibiting fibrous mineralisation in the neural arches, a bone-like tissue which occurrence had long been refuted in this group. Evolutionary considerations are discussed in the light of the evolution of jawed vertebrates and question on the ecological factors that led particular tissues to be restricted to specific shark and batoid groups., Les dents sont des structures répétées dont l’histoire évolutive est étroitement liée à celle de l’émergence des tissus minéralisés des vertébrés. Les dents des vertébrés actuels présentent une importante diversité de tissus, de formes et donc de développement, ce qui permet de les étudier dans un contexte de diversification des espèces. Les dents des sélaciens se renouvellent en permanence et présentent des morphologies variées en lien avec les comportements reproducteur et alimentaire.Ce travail aborde dans un premier temps la variation des formes dentaires de deux scyliorhinidés en utilisant de la morphométrie géométrique en 3D et un algorithme d’apprentissage automatique. Il décrit pour la première fois en détail l’émergence de l’hétérodontie gynandrique au cours de l’ontogénie chez des requins et discute des implications de cette variation pour la taxonomie.Les tests fonctionnels réalisés au cours de cette thèse suggèrent par ailleurs que les facteurs de signalisation Shh et Fgf influencent la morphogenèse des cuspides et la minéralisation dentaire. Ces résultats sont discutés dans le cadre de la divergence entre espèces et de l’acquisition de certains comportements reproducteur et alimentaire.Les données histologiques de vertèbres révèlent enfin une proportion inattendue d’élasmobranches actuels dont les arcs neuraux présentent de la minéralisation fibreuse, un tissu similaire à l’os dont la présence au sein de ce clade a longtemps été supposée perdue. Ces résultats permettent de discuter de l’évolution des tissus squelettiques et de questionner quels pourraient être les facteurs écologiques expliquant la restriction de tissus minéralisés particuliers à certains groupes de requins et de raies.

Chondrichthyans exhibit a number of interesting features which make them valuable organisms to investigate from an evo-devo perspective. However, due to their cartilaginous skeleton, isolated teeth are usually the only available fossil material to reconstruct their evolutionary history. Their teeth are covered by enameloid, a hypermineralized tissue whose microstructure has proven a useful taxonomic tool to differentiate between modern forms (neoselachians) and their extinct relatives. However it is poorly known in several groups, thus casting doubts on the validity of such characters.In this work, I first describe the enameloid microstructure of batoids based on an extensive sampling of both their extant and extinct diversity. I highlight unexpected diversity in the microstructural organization of enameloid which was thought to be very stable in neoselachians. The developmental basis for this diversity was then investigated in two extant chondrichthyan models through classic histological techniques and in situ hybridization. The results highlight differences in their enameloid organic matrix, as well as important differences with enameloid formation in osteichthyans, casting doubts on the homology of the two tissues. Finally I investigate skeletogenesis in the lesser spotted catshark from both a morphological and molecular perspective. Using X-Ray microtomography and in situ hybridization, I highlight both the mineralization sequence of the cartilaginous skeleton and the molecular context in which it calcifies over the course of embryonic development. I find that skeletal calcification in chondrichthyans appears to be much more versatile than in osteichthyans., Les chondrichthyens sont caractérisés par des particularités anatomiques uniques, ce qui, combiné à leur position phylogénétique, en fait un groupe particulièrement attrayant en biologie de l’évolution. Compte tenu de leur squelette cartilagineux, leur registre fossile est principalement constitué de dents isolées, dont la microstructure des tissus est utilisée depuis longtemps dans un contexte taxonomique. Celle-ci permet en effet de différencier les chondrichthyens modernes (néosélaciens) des groupes éteints dans le registre fossile. La microstructure dentaire de nombreux d’entre eux demeure toutefois peu connue, notamment celle des batoïdes, pourtant le groupe de chondrichthyen le plus diversifié à l’heure actuelle.La première partie de ce travail porte sur une description approfondie de la microstructure dentaire des batoïdes, basée sur un large échantillonnage de formes fossiles et actuelles. Ces observations ont permis de décrire une diversité microstructurale importante et inattendue compte tenu de la stabilité évolutive de ces tissus chez les requins. Cette variation est ensuite abordée à travers une approche histologique et transcriptionelle du développement dentaire de formes actuelles. Les résultats obtenus suggèrent la convergence de ces tissus avec l’émailloïde présent chez certains ostéichthyens. La troisième partie de ce travail porte sur la calcification du squelette cartilagineux au cours du développement embryonnaire, dans un contexte morphologique et moléculaire. Les modalités de calcification des pièces squelettiques apparaissent ainsi beaucoup plus versatiles chez les chondrichthyens que chez les ostéichthyens.