The corn snake (Pantherophis guttatus) is a new model species particularly appropriate for investigating the processes generating colours in reptiles because numerous colour and pattern mutants have been isolated in the last five decades. Using our captive-bred colony of corn snakes, transcriptomic and genomic next-generation sequencing, exome assembly, and genotyping of SNPs in multiple families, we delimit the genomic interval bearing the causal mutation of amelanism, the oldest colour variant observed in that species. Proceeding with sequencing the candidate gene OCA2 in the uncovered genomic interval, we identify that the insertion of an LTR-retrotransposon in its 11(th) intron results in a considerable truncation of the p protein and likely constitutes the causal mutation of amelanism in corn snakes. As amelanistic snakes exhibit white, instead of black, borders around an otherwise normal pattern of dorsal orange saddles and lateral blotches, our results indicate that melanocytes lacking melanin are able to participate to the normal patterning of other colours in the skin. In combination with research in the zebrafish, this work opens the perspective of using corn snake colour and pattern variants to investigate the generative processes of skin colour patterning shared among major vertebrate lineages.
Despite the availability of deep-sequencing techniques, genomic and transcriptomic data remain unevenly distributed across phylogenetic groups. For example, reptiles are poorly represented in sequence databases, hindering functional evolutionary and developmental studies in these lineages substantially more diverse than mammals. In addition, different studies use different assembly and annotation protocols, inhibiting meaningful comparisons. Here, we present the "Reptilian Transcriptomes Database 2.0," which provides extensive annotation of transcriptomes and genomes from species covering the major reptilian lineages. To this end, we sequenced normalized complementary DNA libraries of multiple adult tissues and various embryonic stages of the leopard gecko and the corn snake and gathered published reptilian sequence data sets from representatives of the four extant orders of reptiles: Squamata (snakes and lizards), the tuatara, crocodiles, and turtles. The LANE runner 2.0 software was implemented to annotate all assemblies within a single integrated pipeline. We show that this approach increases the annotation completeness of the assembled transcriptomes/genomes. We then built large concatenated protein alignments of single-copy genes and inferred phylogenetic trees that support the positions of turtles and the tuatara as sister groups of Archosauria and Squamata, respectively. The Reptilian Transcriptomes Database 2.0 resource will be updated to include selected new data sets as they become available, thus making it a reference for differential expression studies, comparative genomics and transcriptomics, linkage mapping, molecular ecology, and phylogenomic analyses involving reptiles. The database is available at www.reptilian-transcriptomes.org and can be enquired using a wwwblast server installed at the University of Geneva.
Lizards and snakes exhibit colour variation of adaptive value for thermoregulation, camouflage, predator avoidance, sexual selection and speciation. Furcifer pardalis, the panther chameleon, is one of the most spectacular reptilian endemic species in Madagascar, with pronounced sexual dimorphism and exceptionally large intraspecific variation in male coloration. We perform here an integrative analysis of molecular phylogeography and colour variation after collecting high-resolution colour photographs and blood samples from 324 F. pardalis individuals in locations spanning the whole species distribution. First, mitochondrial and nuclear DNA sequence analyses uncover strong genetic structure among geographically restricted haplogroups, revealing limited gene flow among populations. Bayesian coalescent modelling suggests that most of the mitochondrial haplogroups could be considered as separate species. Second, using a supervised multiclass support vector machine approach on five anatomical components, we identify patterns in 3D colour space that efficiently predict assignment of male individuals to mitochondrial haplogroups. We converted the results of this analysis into a simple visual classification key that can assist trade managers to avoid local population overharvesting.
Many chameleons, and panther chameleons in particular, have the remarkable ability to exhibit complex and rapid colour changes during social interactions such as male contests or courtship. It is generally interpreted that these changes are due to dispersion/aggregation of pigment-containing organelles within dermal chromatophores. Here, combining microscopy, photometric videography and photonic band-gap modelling, we show that chameleons shift colour through active tuning of a lattice of guanine nanocrystals within a superficial thick layer of dermal iridophores. In addition, we show that a deeper population of iridophores with larger crystals reflects a substantial proportion of sunlight especially in the near-infrared range. The organization of iridophores into two superposed layers constitutes an evolutionary novelty for chameleons, which allows some species to combine efficient camouflage with spectacular display, while potentially providing passive thermal protection.
While recent imaging techniques provide insights into biological processes from the molecular to the cellular scale, phenotypes at larger scales remain poorly amenable to quantitative analyses. For example, investigations of the biophysical mechanisms generating skin morphological complexity and diversity would greatly benefit from 3D geometry and colour-texture reconstructions. Here, we report on R2OBBIE-3D, an integrated system that combines a robotic arm, a high-resolution digital colour camera, an illumination basket of high-intensity light-emitting diodes and state-of-the-art 3D-reconstruction approaches. We demonstrate that R2OBBIE generates accurate 3D models of biological objects between 1 and 100 cm, makes multiview photometric stereo scanning possible in practical processing times, and enables the capture of colour-texture and geometric resolutions better than 15 mum without the use of magnifying lenses. R2OBBIE has the potential to greatly improve quantitative analyses of phenotypes in addition to providing multiple new applications in, e.g., biomedical science.
Syncytins are fusogenic envelope (env) genes of retroviral origin that have been captured for a function in placentation. Syncytins have been identified in Euarchontoglires (primates, rodents, Leporidae) and Laurasiatheria (Carnivora, ruminants) placental mammals. Here, we searched for similar genes in species that retained characteristic features of primitive mammals, namely the Malagasy and mainland African Tenrecidae. They belong to the superorder Afrotheria, an early lineage that diverged from Euarchotonglires and Laurasiatheria 100 Mya, during the Cretaceous terrestrial revolution. An in silico search for env genes with full coding capacity within a Tenrecidae genome identified several candidates, with one displaying placenta-specific expression as revealed by RT-PCR analysis of a large panel of Setifer setosus tissues. Cloning of this endogenous retroviral env gene demonstrated fusogenicity in an ex vivo cell-cell fusion assay on a panel of mammalian cells. Refined analysis of placental architecture and ultrastructure combined with in situ hybridization demonstrated specific expression of the gene in multinucleate cellular masses and layers at the materno-fetal interface, consistent with a role in syncytium formation. This gene, which we named "syncytin-Ten1," is conserved among Tenrecidae, with evidence of purifying selection and conservation of fusogenic activity. To our knowledge, it is the first syncytin identified to date within the ancestrally diverged Afrotheria superorder.
Background Mammals exhibit a remarkable variety of phenotypes and comparative studies using novel model species are needed to uncover the evolutionary developmental mechanisms generating this diversity. Here, we undertake a developmental biology and numerical modeling approach to investigate the development of skin appendages in the spiny mouse, Acomys dimidiatus. Results We demonstrate that Acomys spines, possibly involved in display and protection, are enlarged awl hairs with a concave morphology. The Acomys spines originate from enlarged placodes that are characterized by a rapid downwards growth which results in voluminous follicles. The dermal condensation (dermal papilla) at the core of the follicle is very large and exhibits a curved geometry. Given its off-centered position, the dermal papilla generates two waves of anisotropic proliferation, first of the posterior matrix, then of the anterior inner root sheath (IRS). Higher in the follicle, the posterior and anterior cortex cross-section areas substantially decrease due to cortex cell elongation and accumulation of keratin intermediate filaments. Milder keratinization in the medulla gives rise to a foamy material that eventually collapses under the combined compression of the anterior IRS and elongation of the cortex cells. Simulations, using linear elasticity theory and the finite-element method, indicate that these processes are sufficient to replicate the time evolution of the Acomys spine layers and the final shape of the emerging spine shaft. Conclusions Our analyses reveal how hair follicle morphogenesis has been altered during the evolution of the Acomys lineage, resulting in a shift from ancestral awl follicles to enlarged asymmetrical spines. This study contributes to a better understanding of the evolutionary developmental mechanisms that generated the great diversity of skin appendage phenotypes observed in mammals.
BACKGROUND: Colour traits in animals play critical roles in thermoregulation, photoprotection, camouflage, and visual communication and are amenable to objective quantification and modelling. However, the extensive variation in non-melanic pigments and structural colours in squamate reptiles has been largely disregarded. Here, we use an integrated approach to investigate the morphological basis and physical mechanisms generating variation in colour traits in tropical day geckos of the genus Phelsuma. RESULTS: Combining histology, optics, mass spectrometry, as well as UV and Raman spectroscopy, we show that the extensive variation in colour patterns observed within and among Phelsuma species is generated by complex interactions between, on the one hand, chromatophores containing yellow/red pteridine pigments and, on the other hand, iridophores producing structural colour by constructive interference of light with guanine nanocrystals. More specifically, we show that (i) the hue of the vivid dorso-lateral skin is modulated both by variation in geometry of structural highly-ordered narrowband reflectors and by the presence of yellow pigments, and (ii) the reflectivity of the white belly, as well as of dorso-lateral pigmentary red marks, is increased by underlying structural disorganised broadband reflectors. Most importantly, these interactions require precise co-localisation of yellow and red chromatophores with different types of iridophores, characterised by ordered and disordered nanocrystals, respectively. We validated these results through numerical simulations combining pigmentary components with a multilayer interferential optical model. Finally, we show that melanophores form dark lateral patterns but do not significantly contribute to variation in blue/green or red coloration, and that changes in the pH- or redox-state of pigments provide yet an additional source of colour variation in squamates. CONCLUSIONS: Precisely co-localised interacting pigmentary and structural elements generate extensive variation in lizard colour patterns. Our results indicate the need to identify the developmental mechanisms responsible for the control of nanocrystals size, shape and orientation, as well as the superposition of specific chromatophore types. This study opens up new perspectives for Phelsuma lizards as models in evolutionary developmental biology.
Hox genes are required for the development of the intestinal cecum, a major organ of plant-eating species. We have analyzed the transcriptional regulation of Hoxd genes in cecal buds and show that they are controlled by a series of enhancers located in a gene desert flanking the HoxD cluster. The start site of two opposite long noncoding RNAs (lncRNAs), Hotdog and Twin of Hotdog, selectively contacts the expressed Hoxd genes in the framework of a topological domain, coinciding with robust transcription of these genes during cecum budding. Both lncRNAs are specifically transcribed in the cecum, albeit bearing no detectable function in trans. Hedgehogs have kept this regulatory potential despite the absence of the cecum, suggesting that these mechanisms are used in other developmental situations. In this context, we discuss the implementation of a common "budding toolkit" between the cecum and the limbs.
BACKGROUND: During their evolution towards a complete life cycle on land, stem reptiles developed both an impermeable multi-layered keratinized epidermis and skin appendages (scales) providing mechanical, thermal, and chemical protection. Previous studies have demonstrated that, despite the presence of a particularly armored skin, crocodylians have exquisite mechanosensory abilities thanks to the presence of small integumentary sensory organs (ISOs) distributed on postcranial and/or cranial scales. RESULTS: Here, we analyze and compare the structure, innervation, embryonic morphogenesis and sensory functions of postcranial, cranial, and lingual sensory organs of the Nile crocodile (Crocodylus niloticus) and the spectacled caiman (Caiman crocodilus). Our molecular analyses indicate that sensory neurons of crocodylian ISOs express a large repertoire of transduction channels involved in mechano-, thermo-, and chemosensory functions, and our electrophysiological analyses confirm that each ISO exhibits a combined sensitivity to mechanical, thermal and pH stimuli (but not hyper-osmotic salinity), making them remarkable multi-sensorial micro-organs with no equivalent in the sensory systems of other vertebrate lineages. We also show that ISOs all exhibit similar morphologies and modes of development, despite forming at different stages of scale morphogenesis across the body. CONCLUSIONS: The ancestral vertebrate diffused sensory system of the skin was transformed in the crocodylian lineages into an array of scattered discrete multi-sensory micro-organs innervated by multiple pools of sensory neurons. This discretization of skin sensory expression sites is unique among vertebrates and allowed crocodylians to develop a highly-armored, but very sensitive, skin.