Vertebrate Lineage Research Articles

A Ball Python Colour Morph Implicates MC1R in Melanophore-Xanthophore Distribution and Pattern Formation.

Reptiles showcase an extensive array of skin colours and patterns, yet little is known about the genetics of reptile colouration. Here, we investigate the genetic basis of the Clown colour morph found in captive-bred ball pythons (Python regius) to study skin pigmentation and patterning in snakes. We obtained samples by crowdsourcing shed skin from commercial breeders and hobbyists. We applied a case-control design, whole-genome pool sequencing, variant annotation, histological analyses, and electron microscopy imaging. We identified a missense mutation in a transmembrane region of the melanocortin-1 receptor (MC1R) associated with the Clown phenotype. In classic avian and mammalian model species, MC1R is known for controlling the type and amount of melanin produced. In contrast, our results suggest that MC1R signalling might play a key role in pattern formation in ball pythons, affecting xanthophore-melanophore distribution. This work highlights the varied functions of MC1R across different vertebrate lineages and promotes a novel model system to study reptile colouration.

ARPP19 phosphorylation site evolution and the switch in cAMP control of oocyte maturation in vertebrates.

cAMP-PKA signaling initiates the critical process of oocyte meiotic maturation in many animals, but inhibits it in vertebrates. To address this "cAMP paradox", we exchanged the key PKA substrate, ARPP19, between representative species, the vertebrate Xenopus and the cnidarian Clytia, comparing its phosphorylation and function. We found that as in Xenopus, Clytia maturing oocytes undergo ARPP19 phosphorylation on a highly conserved Gwl site, which inhibits PP2A and promotes M-phase entry. In contrast, despite a PKA phosphorylation signature motif recognisable across most animals, Clytia ARPP19 was only poorly phosphorylated by PKA in vitro and in vivo. Furthermore, unlike Xenopus ARPP19, exogenous Clytia ARPP19 did not delay Xenopus oocyte maturation. We conclude that in Clytia ARPP19 does not intervene in oocyte maturation initiation because of both poor recognition by PKA and the absence of effectors that mediate vertebrate oocyte prophase arrest. We propose that ancestral ARPP19 phosphorylated by Gwl has retained a key role in M-phase across eukaryotes and it has acquired new functions during animal evolution mediated by enhanced PKA phosphorylation, allowing co-option into oocyte maturation regulation in the vertebrate lineage.

Retinoblastoma protein activity revealed by CRISPRi study of divergent Rbf1 and Rbf2 paralogs.

Retinoblastoma tumor suppressor proteins (Rb) are highly conserved metazoan transcriptional corepressors involved in regulating the expression of thousands of genes. The vertebrate lineage and the Drosophila genus independently experienced an Rb gene duplication event, leading to the expression of several Rb paralogs whose unique and redundant roles in gene regulation remain to be fully explored. Here, we used a novel CRISPRi system in Drosophila to identify the significance of paralogy in the Rb family. We engineered dCas9 fusions to the fly Rbf1 and Rbf2 paralogs and deployed them to gene promoters in vivo, studying them in their native chromatin context. By directly querying the in vivo response of dozens of genes to Rbf1 and Rbf2 targeting, using both transcriptional as well as sensitive developmental readouts, we find that Rb paralogs function as "soft repressors" and have highly context-specific activities. Our comparison of targeting endogenous genes to reporter genes in cell culture identified striking differences in activity, underlining the importance of using CRISPRi effectors in a physiologically relevant context to identify paralog-specific activities. Our study uncovers the complexity of Rb-mediated transcriptional regulation in a living organism, and serves as a stepping stone for future CRISPRi development in Drosophila.

Adaptive evolution and functional significance of the PPARGC1A gene across diverse animal species.

Codon-based analyses of the PPARGC1A gene across 38 vertebrate species were deployed to elucidate patterns of evolutionary change. Employing maximum likelihood assessments through MEGA, we scrutinized 447 codon positions addressing the entire coding region, excluding positions mired by gaps or missing data. Distinct codons manifested variance in selection pressures, particularly codons 4, 11, 66, and 123, which exhibited positive dN-dS values suggestive of positive selection. Codon 137 displayed the most pronounced dN-dS value, signifying intensified selective advantage. Meanwhile, codons 30 and 90 portrayed near-neutral scores, indicative of purifying selection. Complementary computational methods (IFEL, REL, FUBAR, and SLAC) confirmed positive selection at specific codon sites, with varying degrees of corroboration. The integration of mixed-effect modeling (MEME) identified episodic diversifying selection, pinpointing codons that underwent selection episodes in certain lineages. Refined codon model selection lent insight into substitution rates, revealing nuanced degrees of evolutionary conservation among different codons. Supporting these genetic insights, the phylogenetic analysis highlighted relationships among the PPARGC1A sequences and domain analysis confirmed conserved features across species, while protein-protein interaction networks suggested a complex web of functional interdependencies. These findings advance our understanding of the PPARGC1A gene's evolutionary trajectory and underscore the gene's potential adaptive significance within diverse vertebrate lineages.

Bactericidal ability of target acidic phospholipids and phagocytosis of CDC42 GTPase-mediated cytoskeletal rearrangement underlie functional conservation of CXCL12 in vertebrates.

Chemokine CXCL12 plays a crucial role in both direct bactericidal activity and phagocytosis in humans. However, the mechanisms and evolutionary functions of these processes in vertebrates remain largely unknown. In this study, we found that the direct bactericidal activity of CXCL12 is highly conserved across various vertebrate lineages, including Arctic lamprey (Lampetra japonica), Basking shark (Cetorhinus maximus), grass carp (Ctenopharyngodon idella), Western clawed frog (Xenopus tropicalis), Green anole (Anolis carolinensis), chicken (Gallus gallus), and human (Homo sapiens). CXCL12 also has been shown to promote phagocytosis in lower and higher vertebrates. We then employed C. idella CXCL12a (CiCXCL12a) as a model to further investigate its immune functions and underlying mechanisms. CiCXCL12a exerts direct broad-spectrum antibacterial activity by targeting bacterial acidic phospholipids, resulting in bacterial cell membrane perforation, and eventual lysis. Monocytes/macrophages are attracted to the infection sites for phagocytosis through the rapid production of CiCXCL12a during bacterial infection. CiCXCL12a induces CDC42 and CDC42 GTPase activation, which in turn mediates F-actin polymerization and cytoskeletal rearrangement. The interaction between F-actin and Aeromonas hydrophila facilitates bacterial internalization into monocytes/macrophages. Additionally, A. hydrophila is colocalized within early endosomes, late endosomes and lysosomes, ultimately degrading within phagolysosomes. CiCXCL12a also activates PI3K-AKT, JAK-STAT5 and MAPK-ERK signaling pathways. Notably, only the PI3K-AKT signaling pathway inhibits LPS-induced monocyte/macrophage apoptosis. Thus, CiCXCL12a plays key roles in reducing tissue bacterial loads, attenuating organ injury, and decreasing mortality rates. Altogether, our findings elucidate the conserved mechanisms underlying CXCL12-mediated bactericidal activity and phagocytosis, providing novel perspectives into the immune functions of CXCL12 in vertebrates.

Evolution of Key Oxygen-Sensing Genes Is Associated with Hypoxia Tolerance in Fishes.

Low dissolved oxygen (hypoxia) is recognized as a major threat to aquatic ecosystems worldwide. Because oxygen is paramount for the energy metabolism of animals, understanding the functional and genetic drivers of whole-animal hypoxia tolerance is critical to predicting the impacts of aquatic hypoxia. In this study, we investigate the molecular evolution of key genes involved in the detection of and response to hypoxia in ray-finned fishes: the prolyl hydroxylase domain (PHD)-hypoxia-inducible factor (HIF) oxygen-sensing system, also known as the EGLN (egg-laying nine)-HIF oxygen-sensing system. We searched fish genomes for HIFA and EGLN genes, discovered new paralogs from both gene families, and analyzed protein-coding sites under positive selection. The physicochemical properties of these positively selected amino acid sites were summarized using linear discriminants for each gene. We employed phylogenetic generalized least squares to assess the relationship between these linear discriminants for each HIFA and EGLN and hypoxia tolerance as reflected by the critical oxygen tension (Pcrit) of the corresponding species. Our results demonstrate that Pcrit in ray-finned fishes correlates with the physicochemical variation of positively selected sites in specific HIFA and EGLN genes. For HIF2A, two linear discriminants captured more than 90% of the physicochemical variation of these sites and explained between 20% and 39% of the variation in Pcrit. Thus, variation in HIF2A among fishes may contribute to their capacity to cope with aquatic hypoxia, similar to its proposed role in conferring tolerance to high-altitude hypoxia in certain lineages of terrestrial vertebrates.

Evolutionary and functional analyses reveal a role for the RHIM in tuning RIPK3 activity across vertebrates.

Receptor interacting protein kinases (RIPK) RIPK1 and RIPK3 play important roles in diverse innate immune pathways. Despite this, some RIPK1/3-associated proteins are absent in specific vertebrate lineages, suggesting that some RIPK1/3 functions are conserved while others are more evolutionarily labile. Here, we perform comparative evolutionary analyses of RIPK1-5 and associated proteins in vertebrates to identify lineage-specific rapid evolution of RIPK3 and RIPK1 and recurrent loss of RIPK3-associated proteins. Despite this, diverse vertebrate RIPK3 proteins are able to activate NF-κB and cell death in human cells. Additional analyses revealed a striking conservation of the RIP homotypic interaction motif (RHIM) in RIPK3, as well as other human RHIM-containing proteins. Interestingly, diversity in the RIPK3 RHIM can tune activation of NF-κB while retaining the ability to activate cell death. Altogether, these data suggest that NF-κB activation is a core, conserved function of RIPK3, and the RHIM can tailor RIPK3 function to specific needs within and between species.

Conditions for establishing fin primary cell cultures in a wide range of ray-finned fishes.

Ray-finned fishes (Actinopterygii) represent the most diverse vertebrate lineage that show extensive variations in physiology, ways of life, and adaptations to marine and freshwater environments, and several species have been established as biological research models. The in vitro culture of cells is fundamental for several fields of biological research, being an alternative for studies that use animals. Hundreds of fish cell lines have been established using specific methods for each cell type and species. Here is described a protocol which can be used commonly for obtaining cell cultures from the caudal fin of a wide range of ray-finned fishes including marine and freshwater species. Conditions for sample collection, microbial disinfection, tissue dissociation, plating and incubation, cryopreservation and thawing, and karyotyping are described in detail. Primary cell cultures were developed for 20 species grouped into 12 different orders. Eleven of these species have been cultivated in vitro for the first time. In the beginning, the fish cell cultures showed different capacities of proliferation among them; however throughout the passages, most cultures began to have a similar proliferation rate. Throughout the passages, it was noticed that cells similar to fibroblasts began to predominate. The great proliferative ability of these cultures reveals their potential to become cell lines. The culture of A. mexicanus, for example, has been proliferating for months and is already in its 65th passage. Moreover, these cell cultures showed conserved diploid chromosome numbers in comparison with in vivo descriptions which suggest these cultures have stable karyotypes. Therefore, these cultures have potential to be used in several fields, such as toxicology, cytogenetics, genomics, pathology, immunology, cellular agriculture, and conservation, and this method has the potential to be expanded to species not yet tested, as well as to other organs.

Functional characterization of follicle-stimulating hormone and luteinizing hormone receptors in cloudy catshark, Scyliorhinus torazame

The follicle-stimulating hormone receptor (FSHR) and luteinizing hormone receptor (LHR) in cloudy catshark were cloned, and recombinant FSHR and LHR were expressed for characterization. Ventral lobe extract (VLE) from the pituitary contains homologous FSH and LH, and it stimulated the cAMP signaling of FSHR and LHR dose-dependently. Two transcript variants of LHR (LHR-L with exon 10 and LHR-S without) were identified, and LHR-S was the dominant form with higher basal cAMP activity without VLE stimulation. Among various developmental stages of follicles, FSHR expression was mainly associated with the pre-vitellogenic and early white follicles. When follicles were recruited into vitellogenesis, the expression of FSHR decreased while of LHR was upregulated reciprocally, suggesting that LHR may also be responsible for the control of vitellogenesis in chondrichthyans. The expression of LHR-L was upregulated among maturing follicles before ovulation, indicating LHR-L could have a specific role in receiving the LH surge signal for final maturation. Plasma LH-like activity was transiently increased prior to the progesterone (P4)-surge and testosterone-drop at the beginning of P4-phase, supporting a pituitary control of follicle-maturation via LH signaling in chondrichthyans. The expression of follicular LHR was downregulated during the P4-phase when LH-like activity was high, indicating that the LH-dependent downregulation of LHR is conserved in chondrichthyans as it is in other vertebrate lineages. (213 words)

Adult sex change leads to extensive forebrain reorganization in clownfish

BackgroundSexual differentiation of the brain occurs in all major vertebrate lineages but is not well understood at a molecular and cellular level. Unlike most vertebrates, sex-changing fishes have the remarkable ability to change reproductive sex during adulthood in response to social stimuli, offering a unique opportunity to understand mechanisms by which the nervous system can initiate and coordinate sexual differentiation.MethodsThis study explores sexual differentiation of the forebrain using single nucleus RNA-sequencing in the anemonefish Amphiprion ocellaris, producing the first cellular atlas of a sex-changing brain.ResultsWe uncover extensive sex differences in cell type-specific gene expression, relative proportions of cells, baseline neuronal excitation, and predicted inter-neuronal communication. Additionally, we identify the cholecystokinin, galanin, and estrogen systems as central molecular axes of sexual differentiation. Supported by these findings, we propose a model of sexual differentiation in the conserved vertebrate social decision-making network spanning multiple subtypes of neurons and glia, including neuronal subpopulations within the preoptic area that are positioned to regulate gonadal differentiation.ConclusionsThis work deepens our understanding of sexual differentiation in the vertebrate brain and defines a rich suite of molecular and cellular pathways that differentiate during adult sex change in anemonefish.

Multiple mechanisms of aminoglycoside ototoxicity are distinguished by subcellular localization of action.

Mechanosensory hair cells of the inner ears and lateral line of vertebrates display heightened vulnerability to environmental insult, with damage resulting in hearing and balance disorders. An important example is hair cell loss due to exposure to toxic agents including therapeutic drugs such as the aminoglycoside antibiotics such as neomycin and gentamicin and antineoplastic agents. We describe two distinct cellular pathways for aminoglycoside-induced hair cell death in zebrafish lateral line hair cells. Neomycin exposure results in death from acute exposure with most cells dying within 1 hour of exposure. By contrast, exposure to gentamicin results primarily in delayed hair cell death, taking up to 24 hours for maximal effect. Washout experiments demonstrate that delayed death does not require continuous exposure, demonstrating two mechanisms where downstream responses differ in their timing. Acute damage is associated with mitochondrial calcium fluxes and can be alleviated by the mitochondrially-targeted antioxidant mitoTEMPO, while delayed death is independent of these factors. Conversely delayed death is associated with lysosomal accumulation and is reduced by altering endolysosomal function, while acute death is not sensitive to lysosomal manipulations. These experiments reveal the complexity of responses of hair cells to closely related compounds, suggesting that intervention focusing on early events rather than specific death pathways may be a successful therapeutic strategy.

Atypical Course of the Habenulo-Interpeduncular Tract in Chick Embryos.

Classical studies of the avian diencephalon hardly mention the habenulo-interpeduncular tract (a.k.a. retroflex tract), although both the habenula (HB) (its origin) and the interpeduncular nuclear complex (its target) are present. Retroflex tract fibers were described at early embryonic stages but seem absent in the adult in routine stains. However, this tract is a salient diencephalic landmark in all other vertebrate lineages. It typically emerges out of the caudal HB, courses dorsoventrally across thalamic alar and basal plates just in front of the thalamo-pretectal boundary, and then sharply bends 90° caudalwards at paramedian basal plate levels (this is the "retroflexion"), to approach longitudinally via paramedian pretectum and midbrain the rostralmost hindbrain, specifically the prepontine median interpeduncular complex across isthmus and rhombomere 1. We systematize this habenulo-interpeduncular course into four parts named subhabenular, retrothalamic, tegmental, and interpeduncular. We reexamined the chicken habenulo-interpeduncular fibers at stages HH30 and HH35 (6.5- and 9-day incubation) by mapping them specifically with immunoreaction for BEN protein, a well-known marker. We found that only a small fraction of the stained retroflex tract fibers approaches the basal plate by coursing along the standard dorsoventral pathway in front of the thalamo-pretectal boundary. Many other habenular fibers instead diverge into atypical dispersed courses across the thalamic cell mass (implying alteration of the first subhabenular part of the standard course) before reaching the basal plate; this dispersion explains their invisibility. A significant number of such transthalamic habenular fibers cross orthogonally the zona limitans (ZLI) (the rostral thalamic boundary) and invade the caudal alar prethalamus. Here, they immediately descend dorsoventrally, just rostrally to the ZLI, until reaching the prethalamic basal plate, where they bend (retroflex) caudalwards, entering the thalamic basal paramedian area. These atypical fibers gradually fasciculate with the other groups of habenular efferent fibers in their final longitudinal approach to the hindbrain interpeduncular complex. We conclude that the poor visibility of this tract in birds is due to its dispersion into a diversity of atypical alternative routes, though all components eventually reach the interpeduncular complex. This case merits further analysis of the diverse permissive versus nonpermissive guidance mechanisms called into action, which partially correlate distinctly with successive diencephalic, mesencephalic, and hindbrain neuromeric fields and their boundaries.

Pseudogenization of NK3 homeobox 2 (Nkx3.2) in monotremes provides insight into unique gastric anatomy and physiology.

The enzymatic breakdown and regulation of food passage through the vertebrate antral stomach and pyloric sphincter (antropyloric region) is a trait conserved over 450 million years. Development of the structures involved is underpinned by a highly conserved signalling pathway involving the hedgehog, bone morphogenetic protein and Wingless/Int-1 (Wnt) protein families. Monotremes are one of the few vertebrate lineages where acid-based digestion has been lost, and this is consistent with the lack of genes for hydrochloric acid secretion and gastric enzymes in the genomes of the platypus (Ornithorhynchus anatinus) and short-beaked echidna (Tachyglossus aculeatus) . Furthermore, these species feature unique gastric phenotypes, both with truncated and aglandular antral stomachs and the platypus with no pylorus. Here, we explore the genetic underpinning of monotreme gastric phenotypes, investigating genes important in antropyloric development using the newest monotreme genomes (mOrnAna1.pri.v4 and mTacAcu1) together with RNA-seq data. We found that the pathway constituents are generally conserved, but surprisingly, NK3 homeobox 2 (Nkx3.2) was pseudogenized in both platypus and echidna. We speculate that the unique sequence evolution of Grem1 and Bmp4 sequences in the echidna lineage may correlate with their pyloric-like restriction and that the convergent loss of gastric acid and stomach size genotypes and phenotypes in teleost and monotreme lineages may be a result of eco-evolutionary dynamics. These findings reflect the effects of gene loss on phenotypic evolution and further elucidate the genetic control of monotreme stomach anatomy and physiology.

Decoupled evolution of ventral and dorsal scales in agamid lizards: ventral keels are associated with arboreality.

Arboreality has evolved in all major vertebrate lineages and is often associated with morphological adaptations and increased diversification concomitant with accessing novel niche space. In squamate reptiles, foot, claw, and tail morphology are well-studied adaptations shown to be associated with transitions to arboreality. Here, we examined a less well understood trait-the keeled scale-in relation to microhabitat, climate, and diversification dynamics across a diverse lizard radiation, Agamidae. We found that the ancestral agamid had keeled dorsal but not ventral scales; further, dorsal and ventral keels are evolutionarily decoupled. Ventral keeled scales evolved repeatedly in association with arboreality and may be advantageous in reducing wear or by promoting interlocking when climbing. We did not find an association between keeled scales and diversification, suggesting keels do not allow finer-scale microhabitat partitioning observed in other arboreal-associated traits. We additionally found a relationship between keeled ventral scales and precipitation in terrestrial species where we posit that the keels may function to reduce scale degradation. Our results suggest that keeled ventral scales facilitated transitions to arboreality across agamid lizards, and highlight a need for future studies that explore their biomechanical function in relation to microhabitat and climate.

A new type of spermiogenesis in teleost fish: Formation of the aflagellate sperm in Campylomormyrus compressirostris (Osteoglossomorpha: Mormyridae)

Osteoglossomorpha, the bony tongue fishes, show great variation in morphology, behavioural strategies, reproductive biology and gamete ultrastructure. The order Osteoglossiformes is the only vertebrate taxon, in which four types of sperm (monoflagellate, biflagellate and aflagellate aquasperm and the complex introsperm) have been described. It is also the only vertebrate lineage in which aflagellate spermatozoa exist. The aim of this study was to analyse the structure of the testis and the process of spermiogenesis in the mormyrid Campylomormyrus compressirostris during the breeding season using light and electron microscopy (transmission and scanning). Males of this species have a single testis of the anastomosing tubular type. The tubules of the anterior part of the testis contain cysts with developing germ cells, and this region is much wider than the posterior part, which consists of efferent ducts filled with sperm cells. The cysts are filled with single or mitotic spermatogonia, primary and secondary spermatocytes and early spermatids. At the stage of spermatids with fine granular chromatin, the cysts rupture and successive stages of spermatid differentiation take place in the testicular lumen; we therefore characterise this process as ‘extracystic spermiogenesis’. Sperm development in C. compressirostris is extremely simple and involves chromatin condensation in the central region of the nucleus, a slight decrease in nuclear volume, the appearance of numerous vesicles in the cytoplasm that form a tubular-vesicular system at the base of the nucleus. Both centrioles and mitochondria are translocated to the peripheral region of the midpiece, which forms the opposite pole to the nucleus. There are many differences between the types of spermiogenesis described so far in teleosts and that found in C. compressirostris, including the loss of flagellum formation. This unique type of spermiogenesis is restricted to species of the families Mormyridae and Gymnarchidae, all of which possess aflagellate spermatozoa. Our data demonstrate that the spermatid differentiation and existence of the aflagellate spermatozoon are a unique phenomena not only among teleosts but also in the whole vertebrate lineage.

Gap-junction-mediated bioelectric signaling required for slow muscle development and function in zebrafish

Bioelectric signaling, intercellular communication facilitated by membrane potential and electrochemical coupling, is emerging as a key regulator of animal development. Gap junction (GJ) channels can mediate bioelectric signaling by creating a fast, direct pathway between cells for the movement of ions and other small molecules. In vertebrates, GJ channels are formed by a highly conserved transmembrane protein family called the connexins. The connexin gene family is large and complex, creating challenges in identifying specific connexins that create channels within developing and mature tissues. Using the embryonic zebrafish neuromuscular system as a model, we identify a connexin conserved across vertebrate lineages, gjd4, which encodes the Cx46.8 protein, that mediates bioelectric signaling required for slow muscle development and function. Through mutant analysis and invivo imaging, we show that gjd4/Cx46.8 creates GJ channels specifically in developing slow muscle cells. Using genetics, pharmacology, and calcium imaging, we find that spinal-cord-generated neural activity is transmitted to developing slow muscle cells, and synchronized activity spreads via gjd4/Cx46.8 GJ channels. Finally, we show that bioelectrical signal propagation within the developing neuromuscular system is required for appropriate myofiber organization and that disruption leads to defects in behavior. Our work reveals a molecular basis for GJ communication among developing muscle cells and reveals how perturbations to bioelectric signaling in the neuromuscular system may contribute to developmental myopathies. Moreover, this work underscores a critical motif of signal propagation between organ systems and highlights the pivotal role of GJ communication in coordinating bioelectric signaling during development.

Colonization of the ocean floor by jawless vertebrates across three mass extinctions

BackgroundThe deep (> 200 m) ocean floor is often considered to be a refugium of biodiversity; many benthic marine animals appear to share ancient common ancestry with nearshore and terrestrial relatives. Whether this pattern holds for vertebrates is obscured by a poor understanding of the evolutionary history of the oldest marine vertebrate clades. Hagfishes are jawless vertebrates that are either the living sister to all vertebrates or form a clade with lampreys, the only other surviving jawless fishes.ResultsWe use the hagfish fossil record and molecular data for all recognized genera to construct a novel hypothesis for hagfish relationships and diversification. We find that crown hagfishes persisted through three mass extinctions after appearing in the Permian ~ 275 Ma, making them one of the oldest living vertebrate lineages. In contrast to most other deep marine vertebrates, we consistently infer a deep origin of continental slope occupation by hagfishes that dates to the Paleozoic.ConclusionOur results establish hagfishes as ancient members of demersal continental slope faunas and suggest a prolonged accumulation of deep sea jawless vertebrate biodiversity.

Slc26a12-A novel member of the slc26 family, is located in tandem with slc26a2 in coelacanths, amphibians, reptiles, and birds.

Solute carrier family 26 (Slc26) is a family of anion exchangers with 11 members in mammals (named Slc26a1-a11). Here, we identified a novel member of the slc26 family, slc26a12, located in tandem with slc26a2 in the genomes of several vertebrate lineages. BLAST and synteny analyses of various jawed vertebrate genome databases revealed that slc26a12 is present in coelacanths, amphibians, reptiles, and birds but not in cartilaginous fishes, lungfish, mammals, or ray-finned fishes. In some avian and reptilian lineages such as owls, penguins, egrets, and ducks, and most turtles examined, slc26a12 was lost or pseudogenized. Phylogenetic analysis showed that Slc26a12 formed an independent branch with the other Slc26 members and Slc26a12, Slc26a1 and Slc26a2 formed a single branch, suggesting that these three members formed a subfamily in Slc26. In jawless fish, hagfish have two genes homologous to slc26a2 and slc26a12, whereas lamprey has a single gene homologous to slc26a2. African clawed frogs express slc26a12 in larval gills, skin, and fins. These results show that slc26a12 was present at least before the separation of lobe-finned fish and tetrapods; the name slc26a12 is appropriate because the gene duplication occurred in the distant past.

DNA methylation reprogramming in teleosts.

Early embryonic development is crucially important but also remarkably diverse among animal taxa. Axis formation and cell lineage specification occur due to both spatial and temporal control of gene expression. This complex system involves various signaling pathways and developmental genes such as transcription factors as well as other molecular interactants that maintain cellular states, including several types of epigenetic marks. 5mC DNA methylation, the chemical modification of cytosines in eukaryotes, represents one such mark. By influencing the compaction of chromatin (a high-order DNA structure), DNA methylation can either repress or induce transcriptional activity. Mammals exhibit a reprogramming of DNA methylation from the parental genomes in the zygote following fertilization, and later in primordial germ cells (PGCs). Whether these periods of methylation reprogramming are evolutionarily conserved, or an innovation in mammals, is an emerging question. Looking into these processes in other vertebrate lineages is thus important, and teleost fish, with their extensive species richness, phenotypic diversity, and multiple rounds of whole genome duplication, provide the perfect research playground for answering such a question. This review aims to present a concise state of the art of DNA methylation reprogramming in early development in fish by summarizing findings from different research groups investigating methylation reprogramming patterns in teleosts, while keeping in mind the ramifications of the methodology used, then comparing those patterns to reprogramming patterns in mammals.

Xenopus tropicalis osteoblast-specific open chromatin regions reveal promoters and enhancers involved in human skeletal phenotypes and shed light on early vertebrate evolution

While understanding the genetic underpinnings of osteogenesis has far-reaching implications for skeletal diseases and evolution, a comprehensive characterization of the osteoblastic regulatory landscape in non-mammalian vertebrates is still lacking. Here, we compared the ATAC-Seq profile of Xenopus tropicalis (Xt) osteoblasts to a variety of non mineralizing control tissues, and identified osteoblast-specific nucleosome free regions (NFRs) at 527 promoters and 6747 distal regions. Sequence analyses, Gene Ontology, RNA-Seq and ChIP-Seq against four key histone marks confirmed that the distal regions correspond to bona fide osteogenic transcriptional enhancers exhibiting a shared regulatory logic with mammals. We report 425 regulatory regions conserved with human and globally associated to skeletogenic genes. Of these, 35 regions have been shown to impact human skeletal phenotypes by GWAS, including one trps1 enhancer and the runx2 promoter, two genes which are respectively involved in trichorhinophalangeal syndrome type I and cleidocranial dysplasia. Intriguingly, 60 osteoblastic NFRs also align to the genome of the elephant shark, a species lacking osteoblasts and bone tissue. To tackle this paradox, we chose to focus on dlx5 because its conserved promoter, known to integrate regulatory inputs during mammalian osteogenesis, harbours an osteoblast-specific NFR in both frog and human. Hence, we show that dlx5 is expressed in Xt and elephant shark odontoblasts, supporting a common cellular and genetic origin of bone and dentine. Taken together, our work (i) unravels the Xt osteogenic regulatory landscape, (ii) illustrates how cross-species comparisons harvest data relevant to human biology and (iii) reveals that a set of genes including bnc2, dlx5, ebf3, mir199a, nfia, runx2 and zfhx4 drove the development of a primitive form of mineralized skeletal tissue deep in the vertebrate lineage.