Comparative Proteome Analysis of Four Stages of Spermatogenesis in the Small-Spotted Catshark (Scyliorhinus canicula), Using High-Resolution NanoLC-ESI-MS/MS.

Spermatogenesis is a highly specialized process of cell proliferation and differentiation leading to the production of spermatozoa from spermatogonial stem cells. Due to its testicular anatomy, Scyliorhinus canicula is an interesting model to explore stage-based changes in proteins during spermatogenesis. The proteomes of four testicular zones corresponding to the germinative niche and to spermatocysts (cysts) with spermatogonia (zone A), cysts with spermatocytes (zone B), cysts with young spermatids (zone C), and cysts with late spermatids (zone D) have been analyzed by nanoLC-ESI-MS/MS. Gene ontology and KEGG annotations were also performed. A total of 3346 multiple protein groups were identified. Zone-specific protein analyses highlighted RNA-processing, chromosome-related processes, cilium organization, and cilium activity in zones A, D, C, and D, respectively. Analyses of proteins with zone-dependent abundance revealed processes related to cellular stress, ubiquitin-dependent degradation by the proteasome, post-transcriptional regulation, and regulation of cellular homeostasis. Our results also suggest that the roles of some proteins, such as ceruloplasmin, optineurin, the pregnancy zone protein, PA28ß or the Culling-RING ligase 5 complex, as well as some uncharacterized proteins, during spermatogenesis could be further explored. Finally, the study of this shark species allows one to integrate these data in an evolutionary context of the regulation of spermatogenesis. Mass spectrometry data are freely accessible via iProX-integrated Proteome resources (https://www.iprox.cn/) for reuse purposes.

K092A and K092B, Two Peptides Isolated from the Dogfish (Scyliorhinus canicula L.), with Potential Antineoplastic Activity Against Human Prostate and Breast Cancer Cells.

Cancer therapy is currently a major challenge within the research community, especially in reducing the side effects of treatments and to develop new specific strategies against cancers that still have a poor prognosis. In this context, alternative strategies using biotechnologies, such as marine peptides, have been developed based on their promise of effectivity associated with a low toxicity for healthy cells. The purpose of the present paper is to investigate the active mechanism of two peptides that were isolated from the epigonal tissue of the lesser spotted dogfish Scyliorhinus canicula L., identified NFDTDEQALEDVFSKYG (K092A) and EAPPEAAEEDEW (K092B) on the in vitro growth inhibition of ZR-75-1 mammary carcinoma cells and MDA-Pca-2b prostate cancer cells. The effects of the peptides on cell proliferation and cell death mechanisms were studied by the flow cytometry and immunofluorescence microscopy approaches. The results have shown the onset of both K092A- and K092B-induced early cytoskeleton changes, and then cell cycle perturbations followed by non-apoptotic cell death. Moreover, impedance perturbation and plasma membrane perforation in ZR-75-1 K092A-treated cell cultures and autophagy inhibition in MDA-Pca-2b K092B-treated cells have been observed. In conclusion, these two bioactive peptides from dogfish exhibit antineoplastic activity on the human prostate and breast cancer cells in vitro.

A Potential Antineoplastic Peptide of Human Prostate Cancer Cells Derived from the Lesser Spotted Dogfish (Scyliorhinus canicula L.).

The purpose of the present paper is to investigate the mechanism of action of a pyroglutamate-modified peptide (pE-K092D) on in vitro growth inhibition of MDA-Pca-2b prostate cancer cells. This peptide was derived from a peptide previously isolated from the testis of the lesser spotted dogfish and identified as QLTPEALADEEEMNALAAR (K092D). The effect of the peptide on cell proliferation and cell death mechanisms was studied by flow cytometry. Cellular morphology and cytoskeleton integrity of peptide-treated cells were observed by immunofluorescence microscopy. Results showed the onset of peptide induced early cytoskeleton perturbation, inhibition of autophagy, inhibition of cell proliferation and, at the end, non-apoptotic cell death mechanisms (membrane destabilization and necrosis). All those mechanisms seem to contribute to MDA-Pca-2b growth inhibition by a main cytostatic fate.

The nanos1 gene was duplicated in early Vertebrates and the two paralogs show different gonadal expression profiles in a shark.

Nanos are RNA-binding proteins playing crucial roles in germ cell development and maintenance. Based on phylogenetic and synteny analyses, this study reveals that nanos1 gene has undergone multiple duplications and gene copies losses in Vertebrates. Chondrichthyan species display two nanos1 genes (named nanos1A/1B), which were both retrieved in some Osteichthyes at basal positions in Sarcopterygii and Actinopterygii lineages. In contrast, Teleosts have lost nanos1A but duplicated nanos1B leading to the emergence of two ohnologs (nanos1Ba/1Bb), whereas Tetrapods have lost nanos1B gene. The two successive nanos gene duplications may result from the second and third whole genome duplication events at the basis of Vertebrates and Teleosts respectively. The expression profiles of nanos1A and nanos1B paralogs were characterized in the dogfish, Scyliorhinus canicula. Nanos1A was strongly expressed in brain and also localized in all germ cell types in the polarized testis. In contrast, nanos1B was detected in testis with the highest expression in the germinative zone. In addition, Nanos1B protein was predominantly located in the nuclei of male germinal cells. In the ovary, both paralogs were detected in germinal and somatic cells. Our study opens new perspectives concerning the complex evolution of nanos1 paralogs and their potential distinct roles in Vertebrates gonads.

Dynamics of DNA methylomes underlie oyster development.

DNA methylation is a critical epigenetic regulator of development in mammals and social insects, but its significance in development outside these groups is not understood. Here we investigated the genome-wide dynamics of DNA methylation in a mollusc model, the oyster Crassostrea gigas, from the egg to the completion of organogenesis. Large-scale methylation maps reveal that the oyster genome displays a succession of methylated and non methylated regions, which persist throughout development. Differentially methylated regions (DMRs) are strongly regulated during cleavage and metamorphosis. The distribution and levels of methylated DNA within genomic features (exons, introns, promoters, repeats and transposons) show different developmental lansdscapes marked by a strong increase in the methylation of exons against introns after metamorphosis. Kinetics of methylation in gene-bodies correlate to their transcription regulation and to distinct functional gene clusters, and DMRs at cleavage and metamorphosis bear the genes functionally related to these steps, respectively. This study shows that DNA methylome dynamics underlie development through transcription regulation in the oyster, a lophotrochozoan species. To our knowledge, this is the first demonstration of such epigenetic regulation outside vertebrates and ecdysozoan models, bringing new insights into the evolution and the epigenetic regulation of developmental processes.

Transcriptome analysis reveals strong and complex antiviral response in a mollusc.

Viruses are highly abundant in the oceans, and how filter-feeding molluscs without adaptive immunity defend themselves against viruses is not well understood. We studied the response of a mollusc Crassostrea gigas to Ostreid herpesvirus 1 µVar (OsHV-1µVar) infections using transcriptome sequencing. OsHV-1µVar can replicate extremely rapidly after challenge of C. gigas as evidenced by explosive viral transcription and DNA synthesis, which peaked at 24 and 48 h post-inoculation, respectively, accompanied by heavy oyster mortalities. At 120 h post-injection, however, viral gene transcription and DNA load, and oyster mortality, were greatly reduced indicating an end of active infections and effective control of viral replication in surviving oysters. Transcriptome analysis of the host revealed strong and complex responses involving the activation of all major innate immune pathways that are equipped with expanded and often novel receptors and adaptors. Novel Toll-like receptor (TLR) and MyD88-like genes lacking essential domains were highly up-regulated in the oyster, possibly interfering with TLR signal transduction. RIG-1/MDA5 receptors for viral RNA, interferon-regulatory factors, tissue necrosis factors and interleukin-17 were highly activated and likely central to the oyster's antiviral response. Genes related to anti-apoptosis, oxidation, RNA and protein destruction were also highly up-regulated, while genes related to anti-oxidation were down-regulated. The oxidative burst induced by the up-regulation of oxidases and severe down-regulation of anti-oxidant genes may be important for the destruction of viral components, but may also exacerbate oyster mortality. This study provides unprecedented insights into antiviral response in a mollusc. The mobilization and complex regulation of expanded innate immune-gene families highlights the oyster genome's adaptation to a virus-rich marine environment.

Maintenance of potential spermatogonial stem cells in vitro by GDNF treatment in a chondrichthyan model (Scyliorhinus canicula L.).

Previous work in dogfish, Scyliorhinus canicula, has identified the testicular germinative area as the spermatogonial stem cell niche. In the present study, an in vitro co-culture system of spermatogonia and somatic cells from the germinative area was developed. Long-term maintenance of spermatogonia has been successful, and addition of GDNF has promoted the development of clones of spermatogonia expressing stem cell characteristics such as alkaline phosphatase activity and has allowed maintenance of self-renewal in spermatogonia for at least 5 mo under culture conditions, notably by decreasing cell apoptosis. Furthermore, clones of spermatogonia expressed the receptor of GDNF, GFRalpha1, which is consistent with the effect of GDNF on cells despite the lack of identification of a GDNF sequence in the dogfish's transcriptome. However, a sequence homologous to artemin has been identified, and in silico analysis supports the hypothesis that artemin could replace GDNF in the germinative area in dogfish. This study, as the first report on long-term in vitro maintenance of spermatogonia in a chondrichthyan species, suggests that the GFRalpha1 signaling function in self-renewal of spermatogonial stem cells is probably conserved in gnathostomes.

Cg-SoxE and Cg-ß-catenin, two new potential actors of the sex-determining pathway in a hermaphrodite lophotrochozoan, the Pacific oyster Crassostrea gigas.

Sex determination is poorly known in mollusks, lophotrochozoa and most hermaphrodites. In the oyster Crassostrea gigas, an irregular successive hermaphrodite, sex determination may occur at the end of a gametogenetic cycle to prepare the next cycle. To investigate further into these aspects we have focused on orthologs of SoxE and ß-catenin, key players of the male and female gonadic differentiation respectively in mammals. Based on phylogenetic analyses, Cg-SoxE, the oyster's SoxE ortholog, is closely related to vertebrate SoxE genes while Cg-ß-catenin, the oyster's ß-catenin ortholog, is classified amongst lophotrochozoa. The mRNA expression measured by qPCR in the gonadic area during a gametogenetic cycle is maximal for Cg-SoxE when sex is indiscernible and for Cg-ß-catenin in mature females. Both expressions are localized from early germ cells to spermatocytes and pre-vitellogenic oocytes, and potentially in somatic cells. Cg-ß-catenin is also expressed in vitellogenic oocytes. These actors may be involved in early oyster gonadic differentiation, which includes sex determination. Our results enhance the understanding of sex determination in C. gigas and in mollusks and they provide additional knowledge in compared genomics of reproduction and in molecular phylogeny.

Characterization of spermatogonial markers in the mature testis of the dogfish (Scyliorhinus canicula L.).

In dogfish, spermatogenesis progresses from a restricted germinative zone, which lines the dorsal testicular vessel. Single spermatogonia (A(s)), including the spermatogonial stem cells (SSCs), produce successively paired (A(p)), undifferentiated (A(u4) to A(u512)), and differentiated (A(d1) to A(d8)) spermatogonia and preleptotene (PL) spermatocytes through 13 mitoses. Dogfish spermatogonial subpopulations present classical morphological characteristics but cannot be distinguished on the basis of molecular markers. This characterization has been initiated in mammals despite the difficulty to separate each spermatogonial subpopulation. For instance, both glial cell-derived neurotrophic factor family receptor alpha 1 (GFRα1) and promyelocytic leukemia zinc finger protein (PLZF) are markers of undifferentiated spermatogonia, whereas receptor tyrosine kinase C-kit is a marker of differentiated spermatogonia. The aim of this study is to characterize spermatogonial markers and to differentiate several spermatogonial subpopulations. Dogfish cDNA sequences have been identified and validated by phylogenetic analyses for gfrα1, plzf, pou2, as well as for high-mobility group box proteins 2 and 3 (hmgb2 and 3) and for mini-chromosome maintenance protein 6 (mcm6). We have used the anatomical advantage of the polarized dogfish testis to analyze the expression of those markers by RT-PCR and in situ hybridization. gfrα1, pou2, and plzf have been detected in the testicular germinative zone, suggesting that spermatogonial markers are relatively well conserved among vertebrates but with a less restricted expression for plzf. Moreover, hmgb3 and mcm6 have been identified as new markers of differentiated spermatogonia. Finally, this first molecular characterization of spermatogonial subpopulations in a chondrichthyan model will be useful for further studies on the SSC niche evolution.

DNA methylation is crucial for the early development in the Oyster C. gigas.

In vertebrates, epigenetic modifications influence gene transcription, and an appropriate DNA methylation is critical in development. Indeed, a precise temporal and spatial pattern of early gene expression is mandatory for a normal embryogenesis. However, such a regulation and its underlying mechanisms remain poorly understood in more distant organisms such as Lophotrochozoa. Thus, despite DNA in the oyster genome being methylated, the role of DNA methylation in development is unknown. To clarify this point, oyster genomic DNA was examined during early embryogenesis and found differentially methylated. Reverse transcriptase quantitative polymerase chain reaction indicated stage-specific levels of transcripts encoding DNA-methyltransferase (DNMT) and methyl-binding domain proteins. In addition, as highlighted by electronic microscopy and immunohistochemistry, the DNMT inhibitor 5-aza-cytidine induced alterations in the quantity and the localisation of methylated DNA and severe dose-dependent development alterations and was lethal after zygotic genome reinitiation. Furthermore, methyl-DNA-immunoprecipitation-quantitative polymerase chain reaction revealed that the transcription level of most of the homeobox gene orthologues examined, but not of the other early genes investigated, was inversely correlated with their specific DNA methylation. Altogether, our results demonstrate that DNA methylation influences gene expression in Crassostrea gigas and is critical for oyster development, possibly by specifically controlling the transcription level of homeobox orthologues. These findings provide evidence for the importance of epigenetic regulation of development in Lophotrochozoans and bring new insights into the early life of C. gigas, one of the most important aquaculture resources worldwide.

Proteomic identification of protein associated to mature spermatozoa in the Pacific oyster Crassostrea gigas.

Knowledge of sperm maturation process is limited in the Pacific oyster Crassostrea gigas and major factors of fertilization success of this free spawning animal are unknown. We investigated proteins associated to spermatozoa by analyzing two cellular fractions obtained from a 40-80% Percoll gradient fractioning of germ cell of mature male gonads. Mature spermatozoa were enriched in the lower Percoll fraction while the upper fraction contained less mature or earlier germ cells. A 2-DE proteomic approach was used to identify differentially expressed proteins in both fractions. We screened out 31 differential proteins (P<0.05) which included 14 up-regulated and 17 down-regulated proteins. Using MALDI-TOF/TOF MS and bioinformatics search against a C. gigas database, 13 and 8 proteins were identified for the up-regulated and down-regulated groups, respectively. In the spermatozoa enriched fraction, proteins regarding flagellum formation and control, energy production and Proteosome subunit beta were increased. In less mature germ cell fraction, proteins regarding developmental processes and chaperon molecules were mainly increased. Our results improve current knowledge of proteins associated with spermatozoa maturation related to zootechnical practices used in mollusk hatcheries. BIOLOGICAL SIGNIFICANCE: This is the revised version of the manuscript "Proteomic identification of protein associated to mature spermatozoa in the Pacific oyster Crassostrea gigas" by Kingtong et al. to the Journal of Proteomics. The corrections have been done by the team carefully. This work highlight the enrichment method of spermatozoa of Pacific oyster from stripped complex sample using Percoll gradient. The results reflexed developmental stages of germ cells in gonadal tubules of this species. We have used proteomic approach to identify differentially expressed proteins in mature spermatozoa fraction compared to less mature spermatozoa fraction which provided candidates of protein associated to mature spermatozoa in the Pacific oyster. This work improves the current knowledge in marine bivalve hatchery.

Oyster sex determination is influenced by temperature - first clues in spat during first gonadic differentiation and gametogenesis.

The sex-determining system of Crassostrea gigas is still poorly known, especially regarding the potential influence of temperature. In order to address this question, mRNA expressions of actors of the molecular cascade (Cg-DMl, Cg-SoxE, Cg-ß-catenin, Cg-Foxl2/Cg-Foxl2os) and of Oyvlg, a germ cell marker, were investigated by real-time PCR in spat grown at different temperatures (18, 22, 25 and 28°C). In parallel, gonadic differentiation, gametogenesis and sex ratios were assessed by histology at each of these temperatures. Whatever the temperature, Cg-DMl, Cg-SoxE, Cg-ß-catenin and Oyvlg expressions peaked at the same developmental stage, always after Cg-Foxl2/Cg-Foxl2os (around 40-44dpf for spat grown at 18°C). Temperatures increased the kinetics of first gonadic differentiation and gametogenesis. At 25°C a significant switch occurred in sex ratio towards males and in the balance of expression between male and female genes, in favor of males. A slight gametogenesis disturbance was also observed. These results strengthen the hypotheses about the sex-determining time window and molecular cascade governing the development of C. gigas, with notably the involvement of Cg-Foxl2/Cg-Foxl2os in the very early steps. They also suggest an influence of temperature on the oyster's sex determination which, associated to genetic control, would induce a mixed sex determination system (GSD+TSD).

Proteomic analysis of the spermatogonial stem cell compartment in dogfish Scyliorhinus canicula L.

In the dogfish (Scyliorhinus canicula L.) the testicular germinative zone (GZ), composed of large isolated spermatogonia surrounded by elongating pre-Sertoli cells, is located between the albuginea and the ventrolateral intratesticular vessel. During the spermatogenic wave, cysts radiate in maturational order forming distinct testicular zones. In this study, soluble proteins of the GZ and of the zone containing cysts with spermatocytes were separated by two-dimensional electrophoresis. Gel images were matched and then evaluated for GZ-specific proteins. From the1400 protein spots identified, 680 were found to be apparently specific to this zone. Using MALDI-TOF/TOF mass spectrometry, de novo sequences were obtained for 33 proteins out of the 169 selected for identification by mass spectrometry, but only 16 of these 169 proteins were identified. One of them, proteasome subunit alpha-6, was analyzed further by immunohistochemistry. This study demonstrates the utility of the dogfish as a model for proteome analysis of the spermatogonial stem cell niche, even if it remains restricted by the lack of genomic data available on Elasmobranchs.

Stage-specific gene expression during spermatogenesis in the dogfish (Scyliorhinus canicula).

In the dogfish testis, the cystic arrangement and polarization of germ cell stages make it possible to observe all stages of spermatogenesis in a single transverse section. By taking advantage of the zonation of this organ, we have used suppressive subtractive libraries construction, real-time PCR, and in situ hybridization to identify 32 dogfish genes showing differential expressions during spermatogenesis. These include homologs of genes already known to be expressed in the vertebrate testis, but found here to be specifically expressed either in pre-meiotic and/or meiotic zones (ribosomal protein S8, high-mobility group box 3, ubiquitin carboxyl-terminal esterase L3, 20beta-hydroxysteroid dehydrogenase, or cyclophilin B) or in post-meiotic zone (speriolin, Soggy, zinc finger protein 474, calreticulin, or phospholipase c-zeta). We also report, for the first time, testis-specific expression patterns for dogfish genes coding for A-kinase anchor protein 5, ring finger protein 152, or F-box only protein 7. Finally, the study highlights the differential expression of new sequences whose identity remains to be assessed. This study provides the first molecular characterization of spermatogenesis in a chondrichthyan, a key species to gain insight into the evolution of this process in gnathostomes.

Proliferating cell nuclear antigen in gonad and associated storage tissue of the Pacific oyster Crassostrea gigas: seasonal immunodetection and expression in laser microdissected tissues.

To understand the processes involved in tissue remodeling associated with the seasonal reproductive cycle of the oyster Crassostrea gigas, we used immunodetection and expression measurements of proliferating cell nuclear antigen (PCNA). The expression of the PCNA gene was measured by real-time polymerase chain reaction in the whole gonadal area compared with laser microdissected gonad and storage tissue. Results underlined the advantage of the laser microdissection approach to detect expression, mainly for early stages of spermatogenesis. In the storage tissue, PCNA expression was reduced in the gonadal tubules, but immunolabeled hemocytes and vesicular cells were detected when the storage tissue was being restored. In the gonadal tubules, the PCNA gene was more highly expressed in males than in females. As soon as spermatogenesis was initiated, PCNA expression showed a high and constant level. In females, the expression level increased gradually until the ripe stage. The immunological approach established the involvement of peritubular cells in gonadal tubule expansion during early gametogenesis. In both sexes, gonial mitosis was immunodetected throughout the reproductive cycle. In males, the occurrence of two types of spermatogonia was ascertained by differential immunolabeling, and intragonadal somatic cell proliferation was noted. As expected, immunolabeling was never observed from stage II spermatocytes to spermatozoa. In females, positively stained cells were detected from oogonia to growing oocytes with various labeled intracellular locations.

Molecular cloning and gene expression of Cg-Foxl2 during the development and the adult gametogenetic cycle in the oyster Crassostrea gigas.

A Foxl2 ortholog has been identified in a lophotrochozoa, the pacific oyster, which is a successive irregular hermaphrodite mollusc. Its cDNA has been called Cg-Foxl2 (Crassostrea gigas Foxl2) and the deduced protein sequence is 367aa long. This sequence contains the conserved domain Forkhead box and its gene is devoid of intron at least in the first 926 bp of the cDNA, as found for Foxl2 factors. Real time PCR and in situ hybridization have shown a gonadic male and female Cg-Foxl2 expression which increases during the adult gametogenetic cycle for both sexes, but with a significant increase occurring earlier in females than in males. In females this increase corresponds to the vitellogenetic stage. During development, a peak of Cg-DMl (a potential factor of the male gonadic differentiation) and Oyvlg (a germ cell marker) expression and a significant decrease of Cg-Foxl2 expression were observed after metamorphosis in 1-1.5-month-old spats, a period of development when primordial germ cells may differentiate into germinal stem cells during the first gonadic establishment.

Identification and expression of a factor of the DM family in the oyster Crassostrea gigas.

The Pacific oyster Crassostrea gigas is a successive not systematic protandric hermaphrodite. Searching for an ortholog to Dmrt1, a conserved sex determinism factor, we have identified the first complete cDNA of a DM factor in Lophotrochozoa which we have called Cg-DMl (Crassostrea gigas DMRT-like). It is 359aa long, with the DM domain common to all the family factors, and one DMA domain specific to members such as Dmrt4 and Dmrt5. Its gene presents one intron of 598 bp. Real time PCR and in situ hybridization have shown that Cg-DMl was expressed in both sexes, with a significantly higher expression in male than in female gonads at the end of the adult gametogenetic cycle and that a significant peak of expression was observed in spat between 1 and 2 months of age. These results suggest that Cg-DMl may be involved in the development of the gonad and may constitute preliminary clues for future work in order to better understand DM protein evolution.

Study of the potential spermatogonial stem cell compartment in dogfish testis, Scyliorhinus canicula L.

In the lesser-spotted dogfish (Scyliorhinus canicula), spermatogenesis takes place within spermatocysts made up of Sertoli cells associated with stage-synchronized germ cells. As shown in testicular cross sections, cysts radiate in maturational order from the germinative area, where they are formed, to the opposite margin of the testis, where spermiation occurs. In the germinative zone, which is located in a specific area between the tunica albuginea of the testis and the dorsal testicular vessel, individual large spermatogonia are surrounded by elongated somatic cells. The aim of this study has been to define whether these spermatogonia share characteristics with spermatogonial stem cells described in vertebrate and non-vertebrate species. We have studied their ultrastructure and their mitotic activity by 5'-bromo-2'-deoxyuridine (BrdU) incorporation and proliferating cell nuclear antigen (PCNA) immunodetection. Additionally, immunodetection of c-Kit receptor, a marker of differentiating spermatogonia in rodents, and of alpha- and beta-spectrins, as constituents of the spectrosome and the fusome, has been performed. Ultrastructurally, nuclei of stage I spermatogonia present the same mottled aspect in dogfish as undifferentiated spermatogonia nuclei in rodents. Moreover, intercellular bridges are not observed in dogfish spermatogonia, although they are present in stage II spermatogonia. BrdU and PCNA immunodetection underlines their low mitotic activity. The presence of a spectrosome-like structure, a cytological marker of the germline stem cells in Drosophila, has been observed. Our results constitute the first step in the study of spermatogonial stem cells and their niche in the dogfish.

The Dogfish Scyliorhinus canicula: A Reference in Jawed Vertebrates.

INTRODUCTIONDue to their large size and long generation times, chondrichthyans have been largely ignored by geneticists. However, their key phylogenetic position makes them ideal subjects to study the molecular bases of the important morphological and physiological innovations that characterize jawed vertebrates. Such analyses are crucial to understanding the origin of the complex genetic mechanisms unraveled in osteichthyans. The small spotted dogfish Scyliorhinus canicula, a representative of the largest order of extant sharks, presents a number of advantages in this context. Due to its relatively small size among sharks, its abundance, and easy maintenance, the dogfish has been an important model in comparative anatomy and physiology for more than a century. Recently, revived interest has occurred with the development of large-scale transcriptomic and genomic resources, together with the establishment of facilities allowing massive egg and embryo production. These new tools open the way to molecular analyses of the elaborate physiological and sensory systems used by sharks. They also make it possible to take advantage of unique characteristics of these species, such as organ zonation, in analyses of cell proliferation and differentiation. Finally, they offer important perspectives to evolutionary developmental biology that will provide a better understanding of the origin and diversifications of jawed vertebrates. The dogfish whole-genome sequence, which may shortly become accessible, should establish this species as an essential shark reference, complementary to other chondrichthyan models. These analyses are likely to reveal an organism of an underestimated complexity, far from the primitive prototypical gnathostome anticipated in gradistic views.