Dr Athanasia Tzika

Skin coloration is crucial for the survival of animals and ranges from spectacular colorful displays used to attract a mate to cryptic camouflage used to avoid predators. Among the 3 main types of chromatophores, melanophores are the most widespread in vertebrates and can set the skin tone by the amount of melanin they produce and store in dedicated vesicles, the melanosomes. Mutations associated with melanophore differentiation and maturation result in hypomelanistic and amelanistic phenotypes, both extensively studied in mammals but less so in snakes and lizards. Here, we characterize at the genomic, transcriptomic, and histological level, the Hypomelanistic corn snake morph and 3 hypomelanistic leopard gecko morphs. To minimize bias in studying leopard gecko color morphs, we first assembled a chromosome-level genome from a wild-type individual in terms of coloration. We propose that candidate mutations in 3 melanogenesis factors generate these phenotypes: (i) tyrosinase (TYR), an essential enzyme for melanin synthesis, (ii) NCKX5 (SLC24A5), an ion exchanger involved in melanosome maturation, and (iii) the P protein (OCA2), a transmembrane transporter for tyrosine. Our extended bulk RNA sequencing analyses show that additional pigmentation-related genes, affecting melanin production, melanosome motility, and melanophore migration, are dysregulated in the embryonic skin of the mutated animals. This observation highlights the likely associations among the corresponding pathways and is in line with our electron microscopy imaging results. Indeed, the subcellular structure of melanophores is uniquely altered at each of the 4 morphs and likely reflects a multigenic effect. These findings demonstrate that conserved pigmentation genes can produce species-specific effects, underscoring the modular nature of skin coloration in vertebrates. Our work establishes reptiles as comparative models for studying pigment cell biology and reveals evolutionary flexibility in the genetic regulation of melanogenesis.

Vertebrate reproduction is controlled by 2 pituitary gonadotropin hormones (GtHs), FSH and LH, binding to gonadotropin hormone receptors (GtHRs) in gonadal tissues. All gnathostome vertebrates have been confirmed to possess at least 1 receptor for each GtH [LH receptor (LHR) and FSH receptor (FSHR)], except for species of the reptilian (nonavian sauropsidan) orders, such as lepidosauria, testudines, and crocodylia, which showed inexplicable reactions to heterologous amphibian, avian, and mammalian GtHs in early endocrinological studies. This study investigated the number and function of reptilian GtHRs. Genomic and transcriptomic analyses of selected tetrapod species now strongly suggest the inactivation of the LHR in all nonavian sauropsidans. This gene inactivation likely occurred independently in 3 branches of the sauropdisan clade, sparing only the avian class. Bioassays served to investigate the binding specificity of squamate, chelonian, crocodilian, avian, and mammalian GtHRs with their homologous and heterologous GtHs. The FSHR of a squamate lizard proved completely promiscuous to both its homologous GtHs, while the chelonian FSHR responded slightly stronger to the homologous LH than FSH, and the crocodylian FSHR was only stimulated by the homologous LH but not FSH. We therefore propose a modified paradigm with a neuroendocrine control of nonavian reptilian reproduction by a single GtHR and either 1 GtH in crocodylians or 2 GtHs in chelonians and squamate reptiles. Finally, we discuss hypotheses of tightly regulated temporal and spatial expression of the remaining FSHR in different gonadal somatic cells and temperature-dependent functions of the single nonavian reptilian GtHR.

Snakes exhibit a broad variety of adaptive colors and color patterns, generated by the spatial arrangement of chromatophores, but little is known of the mechanisms responsible for these spectacular traits. Here, we investigate a mono-locus trait with two recessive alleles, motley and stripe, that both cause pattern aberrations in the corn snake.

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.

Corn snakes are emerging models for animal colouration studies. Here, we focus on the Terrazzo morph, whose skin pattern is characterized by stripes rather than blotches. Using genome mapping, we discover a disruptive mutation in the coding region of the Premelanosome protein (PMEL) gene. Our transcriptomic analyses reveal that PMEL expression is significantly downregulated in Terrazzo embryonic tissues. We produce corn snake PMEL knockouts, which present a comparable colouration phenotype to Terrazzo and the subcellular structure of their melanosomes and xanthosomes is also similarly impacted. Our single-cell expression analyses of wild-type embryonic dorsal skin demonstrate that all chromatophore progenitors express PMEL at varying levels. Finally, we show that in wild-type embryos PMEL-expressing cells are initially uniformly spread before forming aggregates and eventually blotches, as seen in the adults. In Terrazzo embryos, the aggregates fail to form. Our results provide insights into the mechanisms governing colouration patterning in reptiles.

Reptilian skin coloration is spectacular and diverse, yet little is known about the ontogenetic processes that govern its establishment and the molecular signaling pathways that determine it. Here, we focus on the development of the banded pattern of leopard gecko hatchlings and the transition to black spots in the adult. With our histological analyses, we show that iridophores are present in the white and yellow bands of the hatchling and they gradually perish in the adult skin. Furthermore, we demonstrate that melanophores can autonomously form spots in the absence of the other chromatophores both on the regenerated skin of the tail and on the dorsal skin of the Mack Super Snow (MSS) leopard geckos. This color morph is characterized by uniform black coloration in hatchlings and black spots in adulthood; we establish that their skin is devoid of xanthophores and iridophores at both stages. Our genetic analyses identified a 13-nucleotide deletion in the PAX7 transcription factor of MSS geckos, affecting its protein coding sequence. With our single-cell transcriptomics analysis of embryonic skin, we confirm that PAX7 is expressed in iridophores and xanthophores, suggesting that it plays a key role in the differentiation of both chromatophores. Our in situ hybridizations on whole-mount embryos document the dynamics of the skin pattern formation and how it is impacted in the PAX7 mutants. We hypothesize that the melanophores-iridophores interactions give rise to the banded pattern of the hatchlings and black spot formation is an intrinsic capacity of melanophores in the postembryonic skin.

In some mammals, notably humans, recombination occurs almost exclusively where the protein PRDM9 binds, whereas in vertebrates lacking an intact , such as birds and canids, recombination rates are elevated near promoter-like features. To determine whether PRDM9 directs recombination in nonmammalian vertebrates, we focused on an exemplar species with a single, intact ortholog, the corn snake (). Analyzing historical recombination rates along the genome and crossovers in pedigrees, we found evidence that PRDM9 specifies the location of recombination events, but we also detected a separable effect of promoter-like features. These findings reveal that the uses of PRDM9 and promoter-like features need not be mutually exclusive and instead reflect a tug-of-war that is more even in some species than others.

Reptilian species, particularly snakes and lizards, are emerging models of animal coloration. Here, I focus on the role of the TFEC transcription factor in snake and lizard coloration based on a study on wild-type and piebald ball pythons. Genomic mapping previously identified a TFEC mutation linked to the piebald ball python phenotype. The association of TFEC with skin coloration was further supported by gene-editing experiments in the brown anole lizard. However, novel histological analyses presented here reveal discrepancies between the ball python and the anole TFEC mutants phenotype, cautioning against broad generalizations. Indeed, both wild-type and piebald ball pythons completely lack iridophores, whereas the TFEC anole lizard mutants lose their iridophores compared to the wild-type anole. Based on these findings, I discuss the potential role of the MiT/TFE family in skin pigmentation across vertebrate lineages and advocate the need for developmental analyses and additional gene-editing experiments to explore the reptilian coloration diversity.

Changes in gene expression represent an important source of phenotypic innovation. Yet how such changes emerge and impact the evolution of traits remains elusive. Here, we explore the molecular mechanisms associated with the development of masculinizing ovotestes in female moles. By performing integrative analyses of epigenetic and transcriptional data in mole and mouse, we identified the co-option of SALL1 expression in mole ovotestes formation. Chromosome conformation capture analyses highlight a striking conservation of the 3D organization at the SALL1 locus, but an evolutionary divergence of enhancer activity. Interspecies reporter assays support the capability of mole-specific enhancers to activate transcription in urogenital tissues. Through overexpression experiments in transgenic mice, we further demonstrate the capability of SALL1 to induce kidney-related gene programs, which are a signature of mole ovotestes. Our results highlight the co-option of gene expression, through changes in enhancer activity, as a plausible mechanism for the evolution of traits.

In vertebrates, there are two known mechanisms by which meiotic recombination is directed to the genome: in humans, mice, and other mammals, recombination occurs almost exclusively where the protein PRDM9 binds, while in species lacking an intact , such as birds and canids, recombination rates are elevated near promoter-like features. To test if PRDM9 also directs recombination in non-mammalian vertebrates, we focused on an exemplar species, the corn snake (). Unlike birds, this species possesses a single, intact ortholog. By inferring historical recombination rates along the genome from patterns of linkage disequilibrium and identifying crossovers in pedigrees, we found that PRDM9 specifies the location of recombination events outside of mammals. However, we also detected an independent effect of promoter-like features on recombination, which is more pronounced on macro- than microchromosomes. Thus, our findings reveal that the uses of PRDM9 and promoter-like features are not mutually-exclusive, and instead reflect a tug of war, which varies in strength along the genome and is more lopsided in some species than others.