Squamates as a model to understand key dental features of vertebrates.

Thanks to their exceptional diversity, teeth are among the most distinctive features of vertebrates. Parameters such as tooth size, shape, number, identity, and implantation can have substantial implications for the ecology and certain social behaviors of toothed species. Despite decades of research primarily focused on mammalian dentition, particularly using the laboratory mouse model, squamate reptiles ("lizards" and snakes) offer a wide array of tooth types and dentition variations. This diversity, which includes differences in size, shape, function, and replacement capacity, provides invaluable opportunities for investigating these fundamental properties. The central bearded dragon (Pogona vitticeps), a popular pet species with well-established husbandry practices, is of particular interest. It features a broad spectrum of morphs and spontaneous mutants and exhibits a wide range of heterodont phenotypes, including variation in the size, shape, number, implantation, and renewal of teeth at both posterior and anterior positions. These characteristics position the species as a crucial model organism for developmental studies in tooth research and for gaining deeper insights into evolutionary patterns of vertebrate dentitions. In this article, we provide an overview of the current understanding of squamate dentition, its diversity, development, and replacement. Furthermore, we discuss the significant advantages offered by squamate species as model organisms for investigating the evolutionary and developmental aspects of vertebrate dentition.

Toward a universal measure of robustness across model organs and systems.

The development of an individual must be capable of resisting the harmful effects of internal and external perturbations. This capacity, called robustness, can make the difference between normal variation and disease. Some systems and organs are more resilient in their capacity to correct the effects of internal disturbances such as mutations. Similarly, organs and organisms differ in their capacity to be resilient against external disturbances, such as changes in temperature. Furthermore, all developmental systems must be somewhat flexible to permit evolutionary change, and understanding robustness requires a comparative framework. Over the last decades, most research on developmental robustness has been focusing on specific model systems and organs. Hence, we lack tools that would allow cross-species and cross-organ comparisons. Here, we emphasize the need for a uniform framework to experimentally test and quantify robustness across study systems and suggest that the analysis of fluctuating asymmetry might be a powerful proxy to do so. Such a comparative framework will ultimately help to resolve why and how organs of the same and different species differ in their sensitivity to internal (e.g., mutations) and external (e.g., temperature) perturbations and at what level of biological organization buffering capacities exist and therefore create robustness of the developmental system.

Embryonic development of skull bones in the Sahara horned viper (Cerastes cerastes), with new insights into structures related to the basicranium and braincase roof.

Ontogenetic studies are crucial for understanding functional morphology, origin and adaptation of skulls in vertebrates. However, very few studies have so far released complete embryonic series focusing on skull embryonic development in species showing diverse and extreme cranial morphologies such as snakes. The wide distribution and unique reproductive and ecological behaviors of venomous vipers, including the heterogeneity in breeding and egg incubation periods in oviparous species, make this group an excellent new model for studying the diversity of skull developmental processes in snakes. Here we present the first complete description of osteocranium development in a viperine snake, Cerastes cerastes, using detailed analysis of the ossification pattern of individual bones across different embryonic stages based on high-resolution micro-computed tomography data. Particularly, we describe in detail the development of the laterosphenoid from its dorsal and ventral components, dividing the trigeminal foramen into maxillary and mandibular foramina. Furthermore, our data help clarify some controversy concerning the presence and/or origin of structures related to the snake basicranium and braincase roof. For example, our detailed description of supraoccipital development suggests that this bone derived, at least in part, from the tectum posterius, although the involvement of the tectum synoticum cannot be totally excluded. Similarly, the epiotic centers of supraoccipital ossification are confirmed during braincase development, and the ancestral lacrimal bone primordium is observed as a ventral element at the early stages of prefrontal development. Finally, our embryonic C. cerastes data highlight a plausible asymmetry in snake skull development, mostly occurring in the basicranium region, but further investigations of embryonic samples and viper species would be required to confirm such phenomenon.

Changes in Hox genes' structure and function during the evolution of the squamate body plan.

Hox genes are central to the specification of structures along the anterior-posterior body axis, and modifications in their expression have paralleled the emergence of diversity in vertebrate body plans. Here we describe the genomic organization of Hox clusters in different reptiles and show that squamates have accumulated unusually large numbers of transposable elements at these loci, reflecting extensive genomic rearrangements of coding and non-coding regulatory regions. Comparative expression analyses between two species showing different axial skeletons, the corn snake and the whiptail lizard, revealed major alterations in Hox13 and Hox10 expression features during snake somitogenesis, in line with the expansion of both caudal and thoracic regions. Variations in both protein sequences and regulatory modalities of posterior Hox genes suggest how this genetic system has dealt with its intrinsic collinear constraint to accompany the substantial morphological radiation observed in this group.

Dynamic evolutionary interplay between ontogenetic skull patterning and whole-head integration.

The arrangement and morphology of the vertebrate skull reflect functional and ecological demands, making it a highly adaptable structure. However, the fundamental developmental and macroevolutionary mechanisms leading to different vertebrate skull phenotypes remain unclear. Here we exploit the morphological diversity of squamate reptiles to assess the developmental and evolutionary patterns of skull variation and covariation in the whole head. Our geometric morphometric analysis of a complex squamate ontogenetic dataset (209 specimens, 169 embryos, 44 species), covering stages from craniofacial primordia to fully ossified bones, reveals that morphological differences between snake and lizard skulls arose gradually through changes in spatial relationships (heterotopy) followed by alterations in developmental timing or rate (heterochrony). Along with dynamic spatiotemporal changes in the integration pattern of skull bone shape and topology with surrounding brain tissues and sensory organs, we identify a relatively higher phenotypic integration of the developing snake head compared with lizards. The eye, nasal cavity and Jacobson's organ are pivotal in skull morphogenesis, highlighting the importance of sensory rearrangements in snake evolution. Furthermore, our findings demonstrate the importance of early embryonic, ontogenetic and tissue interactions in shaping craniofacial evolution and ecological diversification in squamates, with implications for the nature of cranio-cerebral relations across vertebrates.

The macroevolutionary singularity of snakes.

Snakes and lizards (Squamata) represent a third of terrestrial vertebrates and exhibit spectacular innovations in locomotion, feeding, and sensory processing. However, the evolutionary drivers of this radiation remain poorly known. We infer potential causes and ultimate consequences of squamate macroevolution by combining individual-based natural history observations (>60,000 animals) with a comprehensive time-calibrated phylogeny that we anchored with genomic data (5400 loci) from 1018 species. Due to shifts in the dynamics of speciation and phenotypic evolution, snakes have transformed the trophic structure of animal communities through the recurrent origin and diversification of specialized predatory strategies. Squamate biodiversity reflects a legacy of singular events that occurred during the early history of snakes and reveals the impact of historical contingency on vertebrate biodiversity.

Reconstructing the origin and early evolution of the snake brain.

Snakes represent one-eighth of terrestrial vertebrate diversity, encompassing various lifestyles, ecologies, and morphologies. However, the ecological origins and early evolution of snakes are controversial topics in biology. To address the paucity of well-preserved fossils and the caveats of osteological traits for reconstructing snake evolution, we applied a different ecomorphological hypothesis based on high-definition brain reconstructions of extant Squamata. Our predictive models revealed a burrowing lifestyle with opportunistic behavior at the origin of crown snakes, reflecting a complex ancestral mosaic brain pattern. These findings emphasize the importance of quantitatively tracking the phenotypic diversification of soft tissues-including the accurate definition of intact brain morphological traits such as the cerebellum-in understanding snake evolution and vertebrate paleobiology. Furthermore, our study highlights the power of combining extant and extinct species, soft tissue reconstructions, and osteological traits in tracing the deep evolution of not only snakes but also other groups where fossil data are scarce.

Endocast, brain, and bones: Correspondences and spatial relationships in squamates.

Vertebrate endocasts are widely used in the fields of paleoneurology and comparative neuroanatomy. The validity of endocranial studies is dependent upon the extent to which an endocast reflects brain morphology. Due to the variable neuroanatomical resolution of vertebrate endocasts, direct information about the brain morphology can be sometimes difficult to assess and needs to be investigated across lineages. Here, we employ X-ray computed tomography (CT), including diffusible iodine-based contrast-enhanced CT, to qualitatively compare brains and endocasts in different species of squamates. The relative position of the squamate brain within the skull, as well as the variability that may exist in such spatial relationships, was examined to help clarify the neurological regions evidence on their endocasts. Our results indicate that squamate endocasts provide variable representation of the brain, depending on species and neuroanatomical regions. The olfactory bulbs and peduncles, cerebral hemispheres, as well as the medulla oblongata represent the most easily discernable brain regions from squamate endocasts. In contrast, the position of the optic lobes, the ventral diencephalon and the pituitary may be difficult to determine depending on species. Finally, squamate endocasts provide very limited or no information about the cerebellum. The spatial relationships revealed here between the brain and the surrounding bones may help to identify each of the endocranial region. However, as one-to-one correspondences between a bone and a specific region appear limited, the exact delimitation of these regions may remain challenging according to species. This study provides a basis for further examination and interpretation of squamate endocast disparity.

A landmarking protocol for geometric morphometric analysis of squamate endocasts.

Landmark-based geometric morphometrics is widely used to study the morphology of the endocast, or internal mold of the braincase, and the diversity associated with this structure across vertebrates. Landmarks, as the basic unit of such methods, are intended to be points of correspondence, selected depending on the question at hand, whose proper definition is essential to guarantee robustness and reproducibility of results. In this study, 20 landmarks are defined to provide a framework to analyze the morphological variability in squamate endocasts. Ten species representing a cross-section of the diversity of Squamata from both phylogenetic and ecological (i.e., habitat) perspectives were considered, to select landmarks replicable throughout the entire clade, regardless of the degree of neuroanatomical resolution of the endocast. To assess the precision, accuracy, and repeatability of these newly defined landmarks, both intraobserver and interobserver error were investigated. Estimates of measurement error show that most of the landmarks established here are highly replicable, and preliminary results suggest that they capture aspects of endocast shape related to both phylogenetic and ecologic signals. This study provides a basis for further examinations of squamate endocast disparity using landmark-based geometric morphometrics.

Additive and global functions of HoxA cluster genes in mesoderm derivatives.

Hox genes encode transcription factors that play a central role in the specification of regional identities along the anterior to posterior body axis. In the developing mouse embryo, Hox genes from all four genomic clusters are involved in range of developmental processes, including the patterning of skeletal structures and the formation of several organs. However, the functional redundancy observed either between paralogous genes, or among neighboring genes from the same cluster, has hampered functional analyses, in particular when synergistic, cluster-specific functions are considered. Here, we report that mutant mice lacking the entire HoxA cluster in mesodermal lineages display the expected spectrum of postnatal respiratory, cardiac and urogenital defects, previously reported for single gene mutations. Likewise, mild phenotypes are observed in both appendicular and axial skeleton. However, a striking effect was uncovered in the hematopoietic system, much stronger than that seen for Hoxa9 inactivation alone, which involves stem cells (HSCs) as well as the erythroid lineage, indicating that several Hoxa genes are necessary for normal hematopoiesis to occur. Finally, the combined deletions of Hoxa and Hoxd genes reveal abnormalities in axial elongation as well as skin morphogenesis that are likely the results of defects in epithelial-mesenchymal interactions.

The developmental origins of heterodonty and acrodonty as revealed by reptile dentitions.

Despite the exceptional diversity and central role of dentitions in vertebrate evolution, many aspects of tooth characters remain unknown. Here, we exploit the large array of dental phenotypes in acrodontan lizards, including EDA mutants showing the first vertebrate example of positional transformation in tooth identity, to assess the developmental origins and evolutionary patterning of tooth types and heterodonty. We reveal that pleurodont versus acrodont dentition can be determined by a simple mechanism, where modulation of tooth size through EDA signaling has major consequences on dental formula, thereby providing a new flexible tooth patterning model. Furthermore, such implication of morphoregulation in tooth evolution allows predicting the dental patterns characterizing extant and fossil lepidosaurian taxa at large scale. Together, the origins and diversification of tooth types, long a focus of multiple research fields, can now be approached through evo-devo approaches, highlighting the importance of underexplored dental features for illuminating major evolutionary patterns.

Multiple evolutionary origins and losses of tooth complexity in squamates.

Teeth act as tools for acquiring and processing food, thus holding a prominent role in vertebrate evolution. In mammals, dental-dietary adaptations rely on tooth complexity variations controlled by cusp number and pattern. Complexity increase through cusp addition has dominated the diversification of mammals. However, studies of Mammalia alone cannot reveal patterns of tooth complexity conserved throughout vertebrate evolution. Here, we use morphometric and phylogenetic comparative methods across fossil and extant squamates to show they also repeatedly evolved increasingly complex teeth, but with more flexibility than mammals. Since the Late Jurassic, multiple-cusped teeth evolved over 20 times independently from a single-cusped common ancestor. Squamates frequently lost cusps and evolved varied multiple-cusped morphologies at heterogeneous rates. Tooth complexity evolved in correlation with changes in plant consumption, resulting in several major increases in speciation. Complex teeth played a critical role in vertebrate evolution outside Mammalia, with squamates exemplifying a more labile system of dental-dietary evolution.

Distinct roles and regulations for HoxD genes in metanephric kidney development.

Hox genes encode homeodomain-containing proteins that control embryonic development in multiple contexts. Up to 30 Hox genes, distributed among all four clusters, are expressed during mammalian kidney morphogenesis, but functional redundancy between them has made a detailed functional account difficult to achieve. We have investigated the role of the HoxD cluster through comparative molecular embryological analysis of a set of mouse strains carrying targeted genomic rearrangements such as deletions, duplications, and inversions. This analysis allowed us to uncover and genetically dissect the complex role of the HoxD cluster. Regulation of metanephric mesenchyme-ureteric bud interactions and maintenance of structural integrity of tubular epithelia are differentially controlled by some Hoxd genes during renal development, consistent with their specific expression profiles. We also provide evidence for a kidney-specific form of colinearity that underlies the differential expression of two distinct sets of genes located on both sides and overlapping at the Hoxd9 locus. These insights further our knowledge of the genetic control of kidney morphogenesis and may contribute to understanding certain congenital kidney malformations, including polycystic kidney disease and renal hypoplasia.

Glia-Mediated Regenerative Response Following Acute Excitotoxic Damage in the Postnatal Squamate Retina.

The retina is a complex tissue responsible for both detection and primary processing of visual stimuli. Although all vertebrate retinas share a similar, multi-layered organization, the ability to regenerate individual retinal cells varies tremendously, being extremely limited in mammals and birds when compared to anamniotes such as fish and amphibians. However, little is yet known about damage response and regeneration of retinal tissues in "non-classical" squamate reptiles (lizards, snakes), which occupy a key phylogenetic position within amniotes and exhibit unique regenerative features in many tissues. Here, we address this gap by establishing and characterizing a model of excitotoxic retinal damage in bearded dragon lizard (Pogona vitticeps). We particularly focus on identifying, at the cellular and molecular level, a putative endogenous cellular source for retinal regeneration, as diverse self-repair strategies have been characterized in vertebrates using a variety of retinal injury and transgenic models. Our findings reveal for the first time that squamates hold the potential for postnatal retinal regeneration following acute injury. Although no changes occur in the activity of physiologically active progenitors recently identified at the peripheral retinal margin of bearded dragon, two distinct successive populations of proliferating cells at central retina respond to neurotoxin treatment. Following an initial microglia response, a second source of proliferating cells exhibit common hallmarks of vertebrate Müller glia (MG) activation, including cell cycle re-entry, dedifferentiation into a progenitor-like phenotype, and re-expression of proneural markers. The observed lizard glial responses, although not as substantial as in anamniotes, appear more robust than the absent or neonatal-limited regeneration reported without exogenous stimulation in other amniotes. Altogether, these results help to complete our evolutionary understanding of regenerative potential of the vertebrate retina, and further highlight the major importance of glial cells in retinal regeneration. Furthermore, our work offers a new powerful vertebrate model to elucidate the developmental and evolutionary bases of retinal regeneration within amniotes. Such new understanding of self-repair mechanisms in non-classical species endowed with regenerative properties may help designing therapeutic strategies for vertebrate retinal diseases.

Heterochronic Developmental Shifts Underlying Squamate Cerebellar Diversity Unveil the Key Features of Amniote Cerebellogenesis.

Despite a remarkable conservation of architecture and function, the cerebellum of vertebrates shows extensive variation in morphology, size, and foliation pattern. These features make this brain subdivision a powerful model to investigate the evolutionary developmental mechanisms underlying neuroanatomical complexity both within and between anamniote and amniote species. Here, we fill a major evolutionary gap by characterizing the developing cerebellum in two non-avian reptile species-bearded dragon lizard and African house snake-representative of extreme cerebellar morphologies and neuronal arrangement patterns found in squamates. Our data suggest that developmental strategies regarded as exclusive hallmark of birds and mammals, including transit amplification in an external granule layer (EGL) and Sonic hedgehog expression by underlying Purkinje cells (PCs), contribute to squamate cerebellogenesis independently from foliation pattern. Furthermore, direct comparison of our models suggests the key importance of spatiotemporal patterning and dynamic interaction between granule cells and PCs in defining cortical organization. Especially, the observed heterochronic shifts in early cerebellogenesis events, including upper rhombic lip progenitor activity and EGL maintenance, are strongly expected to affect the dynamics of molecular interaction between neuronal cell types in snakes. Altogether, these findings help clarifying some of the morphogenetic and molecular underpinnings of amniote cerebellar corticogenesis, but also suggest new potential molecular mechanisms underlying cerebellar complexity in squamates. Furthermore, squamate models analyzed here are revealed as key animal models to further understand mechanisms of brain organization.

Variations in the proliferative activity of the peripheral retina correlate with postnatal ocular growth in squamate reptiles.

The retina is a complex, multilayered tissue responsible for the perception of visual stimuli from the environment. Contrary to mammals, the capacity for postnatal eye growth in fish and amphibians, and to a lower extent in birds, is coordinated with a progenitor population residing in the ciliary marginal zone (CMZ) at the retinal peripheral margin. However, little is known about embryonic retinogenesis and postnatal retinal growth in squamates (lizards, snakes), despite their exceptional array of ecologies and ocular morphologies. Here, we address this gap by performing the first large-scale study assessing both ontogenetic and adult changes in the stem/progenitor activity of the squamate peripheral retina. Our study reveals for the first time that squamates exhibit a source of proliferating progenitors persisting post embryogenesis in a newly identified retinociliary junction anteriorly adjacent to the retina. This region is strikingly similar to the vertebrate CMZ by its peripheral location and pseudostratified nature, and shares a common pattern of slow-cycling cells, spatial differentiation gradient, and response to postnatal ocular growth. Additionally, its proliferative activity varies considerably among squamate species, in correlation with embryonic and postnatal differences in eye size and growth. Together our data indicate that squamates possess a proliferative peripheral retina that acts as a source of progenitors to compensate, at least in part, for postnatal ocular growth. Our findings also highlight the remarkable variation in activity and location of vertebrate retinal progenitors, indicating that the currently accepted scenario of reduced CMZ activity over the course of evolution is too simplistic.

Comparative analysis of squamate brains unveils multi-level variation in cerebellar architecture associated with locomotor specialization.

Ecomorphological studies evaluating the impact of environmental and biological factors on the brain have so far focused on morphology or size measurements, and the ecological relevance of potential multi-level variations in brain architecture remains unclear in vertebrates. Here, we exploit the extraordinary ecomorphological diversity of squamates to assess brain phenotypic diversification with respect to locomotor specialization, by integrating single-cell distribution and transcriptomic data along with geometric morphometric, phylogenetic, and volumetric analysis of high-definition 3D models. We reveal significant changes in cerebellar shape and size as well as alternative spatial layouts of cortical neurons and dynamic gene expression that all correlate with locomotor behaviours. These findings show that locomotor mode is a strong predictor of cerebellar structure and pattern, suggesting that major behavioural transitions in squamates are evolutionarily correlated with mosaic brain changes. Furthermore, our study amplifies the concept of 'cerebrotype', initially proposed for vertebrate brain proportions, towards additional shape characters.

The alternative regenerative strategy of bearded dragon unveils the key processes underlying vertebrate tooth renewal.

Deep understanding of tooth regeneration is hampered by the lack of lifelong replacing oral dentition in most conventional models. Here, we show that the bearded dragon, one of the rare vertebrate species with both polyphyodont and monophyodont teeth, constitutes a key model for filling this gap, allowing direct comparison of extreme dentition types. Our developmental and high-throughput transcriptomic data of microdissected dental cells unveils the critical importance of successional dental lamina patterning, in addition to maintenance, for vertebrate tooth renewal. This patterning process happens at various levels, including directional growth but also gene expression levels, dynamics, and regionalization, and involves a large number of yet uncharacterized dental genes. Furthermore, the alternative renewal mechanism of bearded dragon dentition, with dual location of slow-cycling cells, demonstrates the importance of cell migration and functional specialization of putative epithelial stem/progenitor niches in tissue regeneration, while expanding the diversity of dental replacement strategies in vertebrates.

Shared and differential features of Robo3 expression pattern in amniotes.

In Bilaterians, commissural neurons project their axons across the midline of the nervous system to target neurons on the opposite side. In mammals, midline crossing at the level of the hindbrain and spinal cord requires the Robo3 receptor which is transiently expressed by all commissural neurons. Unlike other Robo receptors, mammalian Robo3 receptors do not bind Slit ligands and promote midline crossing. Surprisingly, not much is known about Robo3 distribution and mechanism of action in other vertebrate species. Here, we have used whole-mount immunostaining, tissue clearing and light-sheet fluorescent microscopy to study Robo3 expression pattern in embryonic tissue from diverse representatives of amniotes at distinct stages, including squamate (African house snake), birds (chicken, duck, pigeon, ostrich, emu and zebra finch), early postnatal marsupial mammals (fat-tailed dunnart), and eutherian mammals (mouse and human). The analysis of this rich and unique repertoire of amniote specimens reveals conserved features of Robo3 expression in midbrain, hindbrain and spinal cord commissural circuits, which together with subtle but meaningful modifications could account for species-specific evolution of sensory-motor and cognitive capacities. Our results also highlight important differences of precerebellar nuclei development across amniotes.

Conserved gene signalling and a derived patterning mechanism underlie the development of avian footpad scales.

BACKGROUND: Vertebrates possess a diverse range of integumentary epithelial appendages, including scales, feathers and hair. These structures share extensive early developmental homology, as they mostly originate from a conserved anatomical placode. In the context of avian epithelial appendages, feathers and scutate scales are known to develop from an anatomical placode. However, our understanding of avian reticulate (footpad) scale development remains unclear. RESULTS: Here, we demonstrate that reticulate scales develop from restricted circular domains of thickened epithelium, with localised conserved gene expression in both the epithelium and underlying mesenchyme. These domains constitute either anatomical placodes, or circular initiatory fields (comparable to the avian feather tract). Subsequent patterning of reticulate scales is consistent with reaction-diffusion (RD) simulation, whereby this primary domain subdivides into smaller secondary units, which produce individual scales. In contrast, the footpad scales of a squamate model (the bearded dragon, Pogona vitticeps) develop synchronously across the ventral footpad surface. CONCLUSIONS: Widely conserved gene signalling underlies the initial development of avian reticulate scales. However, their subsequent patterning is distinct from the footpad scale patterning of a squamate model, and the feather and scutate scale patterning of birds. Therefore, we suggest reticulate scales are a comparatively derived epithelial appendage, patterned through a modified RD system.