A protocol for in ovo electroporation of chicken and snake embryos to study forebrain development.

In vivo electroporation has become a key technique to study genetic mechanisms of brain development. However, electroporation of the embryonic pallium in oviparous species, interesting for evolutionary studies but distinct from in utero electroporation, is quite infrequent. Here, we detail the in ovo electroporation of the developing pallium in chick and snake embryos. This protocol allows gene manipulation through introducing exogenous DNA into brain progenitor cells and can be adapted to any type of gene manipulation of the embryonic telencephalon. For complete information on the use and execution of this protocol, please refer to Cárdenas et al. (2018).

Ultrastructural studies of developing egg tooth in grass snake Natrix natrix (Squamata, Serpentes) embryos, supported by X-ray microtomography analysis.

The egg tooth development is similar to the development of all the other vertebrate teeth except earliest developmental stages because the egg tooth develops directly from the oral epithelium instead of the dental lamina similarly to null generation teeth. The developing egg tooth of Natrix natrix changes its curvature differently than the egg tooth of the other investigated unidentates due to the presence of the rostral groove. The developing grass snake egg tooth comprises dental pulp and the enamel organ. The fully differentiated enamel organ consists of outer enamel epithelium, stellate reticulum, and ameloblasts in its inner layer. The enamel organ directly in contact with the oral cavity is covered with periderm instead of outer enamel epithelium. Stellate reticulum cells in the grass snake egg tooth share intercellular spaces with the basal part of ameloblasts and are responsible for their nutrition. Ameloblasts during egg tooth differentiation pass through the following stages: presecretory, secretory, and mature. The ameloblasts from the grass snake egg tooth show the same cellular changes as reported during mammalian amelogenesis but are devoid of Tomes' processes. Odontoblasts of the developing grass snake egg tooth pass through the following classes: pre-odontoblasts, secretory odontoblasts, and ageing odontoblasts. They have highly differentiated secretory apparatus and in the course of their activity accumulate lipofuscin. Grass snake odontoblasts possess processes which are poor in organelles. In developing egg tooth cilia have been identified in odontoblasts, ameloblasts and cells of the stellate reticulum. Dental pulp cells remodel collagen matrix during growth of the grass snake egg tooth. They degenerate in a way previously not described in other teeth.

Intrauterine and post-ovipositional embryonic development of Amerotyphlops brongersmianus (Vanzolini, 1976) (Serpentes: Typhlopidae) from northeastern Argentina.

The reproductive biology and embryonic development of Typhlopidae have rarely been explored. This family of snakes includes mostly oviparous species with uterine egg retention, but the morphology and development of embryos remain unknown. This work aimed to describe the embryonic development of Amerotyphlops brongersmianus from the northeast of Argentina. For this purpose, embryos from intrauterine eggs of gravid females and eight post-ovipositional eggs incubated in the laboratory were analyzed. Embryonic stages, corresponding to the early, mid and advanced development, and a hatchling were described. The main organs and systems form during the period of intrauterine embryonic retention. Comparing to other snakes, differences in the development of cranial structures such as encephalic vesicles and mandibular and maxillary processes were identified. After oviposition the development and differentiation of the tissues and organs completes, the body scales develop, the characteristic pattern of pigmentation establishes and the embryo grows and consumes the yolk. On average, the incubation period lasts 55 days. Differences in the stage of development at oviposition among females of different populations were observed. Embryonic retention could extend up to advanced stages of development.

Getting out of an egg: Merging of tooth germs to create an egg tooth in the snake.

BACKGROUND: The egg tooth is a vital structure allowing hatchlings to escape from the egg. In squamates (snakes and lizards), the egg tooth is a real tooth that develops within the oral cavity at the top of the upper jaw. Most squamates have a single large midline egg tooth at hatching, but a few families, such as Gekkonidae, have two egg teeth. In snakes the egg tooth is significantly larger than the rest of the dentition and is one of the first teeth to develop. RESULTS: We follow the development of the egg tooth in four snake species and show that the single egg tooth is formed by two tooth germs. These two tooth germs are united at the midline and grow together to produce a single tooth. In culture, this merging can be perturbed to give rise to separate smaller teeth, confirming the potential of the developing egg tooth to form two teeth. CONCLUSIONS: Our data agrees with previous hypotheses that during evolution one potential mechanism to generate a large tooth is through congrescence of multiple tooth germs and suggests that the ancestors of snakes could have had two egg teeth.

Reflections on Model Organisms in Evolutionary Developmental Biology.

This chapter reflects on and makes explicit the distinctiveness of reasoning practices associated with model organisms in the context of evolutionary developmental research. Model organisms in evo-devo instantiate a unique synthesis of model systems strategies from developmental biology and comparative strategies from evolutionary biology that negotiate a tension between developmental conservation and evolutionary change to address scientific questions about the evolution of development and the developmental basis of evolutionary change. We review different categories of model systems that have been advanced to understand practices found in the life sciences in order to comprehend how evo-devo model organisms instantiate this synthesis in the context of three examples: the starlet sea anemone and the evolution of bilateral symmetry, leeches and the origins of segmentation in bilaterians, and the corn snake to understand major evolutionary change in axial and appendicular morphology.

Prenatal development of the sound transmitting apparatus in different embryonic stages of Malpolon monsspesulanus (squamata-serpentes) / Desenvolvimento pré-natal do aparelho transmissor de som em diferentes estágios embrionários de Malpolon monsspesulanus (Squamata-serpentes)

Abstract The developmental investigation of sound transmitting apparatus is important in understanding the ontogenetic processes behind morphological diversity. The development of sound conducting apparatus was studied in Montpellier snake; Malpolon monspessulanus at 6.5, 7.2, 8.3 and 9.3 cm total body lengths using light microscopy study. The columella auris firstly appeared as undifferentiated rod shape mesenchymal cells. As the growth proceeded, it chondrified and differentiates into two main parts. In addition, the viscerocranium components which participate in formation of sound transmitting apparatus undergo critical organization. In more advanced stages, procartilagenous stylohyal chondrified and fuse with the well organized quadrate. These data considered as a base for functional and molecular mechanisms of sound transmitting apparatus studies and identification of diseases that may infect them.

Resumo A investigação do desenvolvimento de equipamentos de transmissão de som é importante na compreensão dos processos ontogenéticos atrás diversidade morfológica. O desenvolvimento de aparelhos de som realização foi estudada em Montpellier cobra; Monspessulanus Malpolon em 6.5, 7.2, 8.3 e 9.3 cm corporal total utilizando comprimentos de estudo de microscopia de luz. O auris columelar em primeiro lugar apareceu como células mesenquimais forma haste indiferenciada. Como o crescimento passou, ele chondrified e diferencia em duas partes principais. Além disso, os componentes viscerocrânio que participam na formação do aparelho de transmissão de som submeter a organização crítico. Em estágios mais avançados, stylohyal procartilagenous chondrified e se fundem com o quadrado bem organizado. Estes dados considerados como uma base para os mecanismos funcionais e estudos moleculares do aparelho de transmissão de som e identificação de doenças que podem infectar-los.

Evolution of Cortical Neurogenesis in Amniotes Controlled by Robo Signaling Levels.

Cerebral cortex size differs dramatically between reptiles, birds, and mammals, owing to developmental differences in neuron production. In mammals, signaling pathways regulating neurogenesis have been identified, but genetic differences behind their evolution across amniotes remain unknown. We show that direct neurogenesis from radial glia cells, with limited neuron production, dominates the avian, reptilian, and mammalian paleocortex, whereas in the evolutionarily recent mammalian neocortex, most neurogenesis is indirect via basal progenitors. Gain- and loss-of-function experiments in mouse, chick, and snake embryos and in human cerebral organoids demonstrate that high Slit/Robo and low Dll1 signaling, via Jag1 and Jag2, are necessary and sufficient to drive direct neurogenesis. Attenuating Robo signaling and enhancing Dll1 in snakes and birds recapitulates the formation of basal progenitors and promotes indirect neurogenesis. Our study identifies modulation in activity levels of conserved signaling pathways as a primary mechanism driving the expansion and increased complexity of the mammalian neocortex during amniote evolution.

Prenatal development of the sound transmitting apparatus in different embryonic stages of Malpolon monsspesulanus (squamata-serpentes).

The developmental investigation of sound transmitting apparatus is important in understanding the ontogenetic processes behind morphological diversity. The development of sound conducting apparatus was studied in Montpellier snake; Malpolon monspessulanus at 6.5, 7.2, 8.3 and 9.3 cm total body lengths using light microscopy study. The columella auris firstly appeared as undifferentiated rod shape mesenchymal cells. As the growth proceeded, it chondrified and differentiates into two main parts. In addition, the viscerocranium components which participate in formation of sound transmitting apparatus undergo critical organization. In more advanced stages, procartilagenous stylohyal chondrified and fuse with the well organized quadrate. These data considered as a base for functional and molecular mechanisms of sound transmitting apparatus studies and identification of diseases that may infect them.

Reduced egg shell permeability affects embryonic development and hatchling traits in Lycodon rufozonatum and Pelodiscus sinensis.

The response of embryos to unpredictable hypoxia is critical for successful embryonic development, yet there remain significant gaps in our understanding of such responses in reptiles with different types of egg shell. We experimentally generated external regional hypoxia by sealing either the upper half or bottom half of the surface area of eggs in 2 species of reptiles (snake [Lycodon rufozonatum] with parchment egg shell and Chinese soft-shelled turtle [Pelodiscus sinensis] with rigid egg shell), then monitored the growth pattern of the opaque white patch in turtle eggs (a membrane that attaches the embryo to the egg shell and plays an important role in gas exchange), the embryonic heart rate, the developmental rate and the hatchling traits in turtle and snake eggs in response to external regional hypoxia. The snake embryos from the hypoxia treatments facultatively increased their heart rate during incubation, and turtle embryos from the upper-half hypoxia treatment enhanced their growth of the opaque white patch. Furthermore, the incubation period and hatching success of embryos were not affected by the hypoxia treatment in these 2 species. External regional hypoxia significantly affected embryonic yolk utilization and offspring size in the snake and turtle. Compared to sham controls, embryos from the upper-half hypoxia treatment used less energy from yolk and, therefore, developed into smaller hatchlings, but embryos from the bottom-half hypoxia treatment did not.

Does the oviparity-viviparity transition alter the partitioning of yolk in embryonic snakes?

BACKGROUND: The oviparity-viviparity transition is a major evolutionary event, likely altering the reproductive process of the organisms involved. Residual yolk, a portion of yolk remaining unutilized at hatching or birth as parental investment in care, has been investigated in many oviparous amniotes but remained largely unknown in viviparous species. Here, we used data from 20 (12 oviparous and 8 viviparous) species of snakes to see if the oviparity-viviparity transition alters the partitioning of yolk in embryonic snakes. We used ANCOVA to test whether offspring size, mass and components at hatching or birth differed between the sexes in each species. We used both ordinary least squares and phylogenetic generalized least squares regressions to test whether relationships between selected pairs of offspring components were significant. We used phylogenetic ANOVA to test whether offspring components differed between oviparous and viviparous species and, more specifically, the hypothesis that viviparous snakes invest more in the yolk as parental investment in embryogenesis to produce more well developed offspring that are larger in linear size. RESULTS: In none of the 20 species was sex a significant source of variation in any offspring component examined. Newborn viviparous snakes on average contained proportionally more water and, after accounting for body dry mass, had larger carcasses but smaller residual yolks than did newly hatched oviparous snakes. The rates at which carcass dry mass (CDM) and fat body dry mass (FDM) increased with residual yolk dry mass (YDM) did not differ between newborn oviparous and viviparous snakes. Neither CDM nor FDM differed between newborn oviparous and viviparous snakes after accounting for YDM. CONCLUSIONS: Our results are not consistent with the hypothesis that the partitioning of yolk between embryonic and post-embryonic stages differs between snakes that differ in parity mode, but instead show that the partitioning of yolk in embryonic snakes is species-specific or phylogenetically related. We conclude that the oviparity-viviparity transition does not alter yolk partitioning in embryonic snakes.

Three-dimensional reconstruction of the embryonic pancreas in the grass snake Natrix natrix L. (Lepidosauria, Serpentes) based on histological studies.

The aim of this study was to evaluate two research hypotheses: H0-the embryonic pancreas in grass snakes develops in the same manner as in all previously investigated amniotes (from three buds) and its topographical localization within the adult body has no relation to its development; H1-the pancreas develops in a different manner and is related to the different topography of internal organs in snakes. For the evaluation of these hypotheses we used histological methods and three-dimensional (3D) reconstructions of the position of the pancreatic buds and surrounding organs at particular developmental stages and of the final position and shape of the pancreatic gland. Our results indicate that the pancreas primordium in the grass snake is formed by only two buds - a dorsal and a ventral one - that are not connected until the end of stage II. This differs from the majority of vertebrates investigated so far. The gall bladder of the grass snake embryos is connected with the liver only by a thin cystic duct, which also differs from many other vertebrates. Our histological study also indicates a different distribution of the endocrine cells in the embryonic pancreas of the grass snake because the first endocrine cells appeared in the dorsal part of the pancreas in a region located close to the spleen. During the entire developmental period no evidence of these cells was found in the ventral part of the pancreas. The endocrine cells form elongated, large and irregular-shaped islets. They can also form structures resembling "inverted acini". Such an arrangement is characteristic of snakes only. The differentiating pancreas penetrates the ventral part of the developing spleen and divides it into three separate parts at developmental stage IX. This is unique among vertebrates. At the end of the embryonic development (stage XI), the pancreas, the spleen and the gall bladder are located in close proximity and form the so-called triad. Our results suggest that the untypical topography of the organ systems in snakes may determine the unique development of the pancreas in these animals.

Reorganisation of Hoxd regulatory landscapes during the evolution of a snake-like body plan.

Within land vertebrate species, snakes display extreme variations in their body plan, characterized by the absence of limbs and an elongated morphology. Such a particular interpretation of the basic vertebrate body architecture has often been associated with changes in the function or regulation of Hox genes. Here, we use an interspecies comparative approach to investigate different regulatory aspects at the snake HoxD locus. We report that, unlike in other vertebrates, snake mesoderm-specific enhancers are mostly located within the HoxD cluster itself rather than outside. In addition, despite both the absence of limbs and an altered Hoxd gene regulation in external genitalia, the limb-associated bimodal HoxD chromatin structure is maintained at the snake locus. Finally, we show that snake and mouse orthologous enhancer sequences can display distinct expression specificities. These results show that vertebrate morphological evolution likely involved extensive reorganisation at Hox loci, yet within a generally conserved regulatory framework.

Oct4 Is a Key Regulator of Vertebrate Trunk Length Diversity.

Vertebrates exhibit a remarkably broad variation in trunk and tail lengths. However, the evolutionary and developmental origins of this diversity remain largely unknown. Posterior Hox genes were proposed to be major players in trunk length diversification in vertebrates, but functional studies have so far failed to support this view. Here we identify the pluripotency factor Oct4 as a key regulator of trunk length in vertebrate embryos. Maintaining high Oct4 levels in axial progenitors throughout development was sufficient to extend trunk length in mouse embryos. Oct4 also shifted posterior Hox gene-expression boundaries in the extended trunks, thus providing a link between activation of these genes and the transition to tail development. Furthermore, we show that the exceptionally long trunks of snakes are likely to result from heterochronic changes in Oct4 activity during body axis extension, which may have derived from differential genomic rearrangements at the Oct4 locus during vertebrate evolution.

Efficient harvesting methods for early-stage snake and turtle embryos.

Reptile development is an intriguing research target for understating the unique morphogenesis of reptiles as well as the evolution of vertebrates. However, there are numerous difficulties associated with studying development in reptiles. The number of available reptile eggs is usually quite limited. In addition, the reptile embryo is tightly adhered to the eggshell, making it a challenge to isolate reptile embryos intact. Furthermore, there have been few reports describing efficient procedures for isolating intact embryos especially prior to pharyngula stage. Thus, the aim of this review is to present efficient procedures for obtaining early-stage reptilian embryos intact. We first describe the method for isolating early-stage embryos of the Japanese striped snake. This is the first detailed method for obtaining embryos prior to oviposition in oviparous snake species. Second, we describe an efficient strategy for isolating early-stage embryos of the soft-shelled turtle.

Heartbeat, embryo communication and hatching synchrony in snake eggs.

Communication is central to life at all levels of complexity, from cells to organs, through to organisms and communities. Turtle eggs were recently shown to communicate with each other in order to synchronise their development and generate beneficial hatching synchrony. Yet the mechanism underlying embryo to embryo communication remains unknown. Here we show that within a clutch, developing snake embryos use heart beats emanating from neighbouring eggs as a clue for their metabolic level, in order to synchronise development and ultimately hatching. Eggs of the water snake Natrix maura increased heart rates and hatched earlier than control eggs in response to being incubated in physical contact with more advanced eggs. The former produced shorter and slower swimming young than their control siblings. Our results suggest potential fitness consequences of embryo to embryo communication and describe a novel driver for the evolution of egg-clustering behaviour in animals.

Viviparous placentotrophy in reptiles and the parent-offspring conflict.

In placentotrophic viviparous reptiles, pregnant females deliver nutrients to their developing fetuses by diverse morphological specializations that reflect independent evolutionary origins. A survey of these specializations reveals a major emphasis on histotrophy (uterine secretion and fetal absorption) rather than hemotrophy (transfer between maternal and fetal blood streams). Of available hypotheses for the prevalence of histotrophic transfer, the most promising derives insights from the theoretical parent-offspring conflict over nutrient investment. I suggest that histotrophy gives pregnant females greater control over nutrient synthesis, storage, and delivery than hemotrophic transfer, reflecting maternal preeminence in any potential parent-offspring competition over nutrient investment. One lizard species shows invasive ovo-implantation and direct contact between fetal tissues and maternal blood vessels, potentially conferring control over nutrient transfer to the embryo. Future research on squamates will benefit from application of parent-offspring conflict theory to the transition from incipient to substantial matrotrophy, as well as by testing theory-derived predictions on both facultatively and highly placentotrophic forms.

Placental specializations in lecithotrophic viviparous squamate reptiles.

Squamate reptiles have been thought to be predisposed to evolution of viviparity because embryos of most oviparous species undergo considerable development in the uterus prior to oviposition. A related hypothesis proposes that prolonged intrauterine gestation, an intermediate condition leading to viviparity, requires little or no physiological adjustment, other than reduction in thickness of the eggshell. This logical framework is often accompanied by an assumption that mode of parity (oviparity, viviparity) and pattern of embryonic nutrition (lecithotrophy, placentotrophy) are independent traits that evolve in sequence. Thus, specializations for viviparity should be absent in some lecithotrophic viviparous species. Studies of species of lizards with geographic variation in mode of parity challenge this scenario by demonstrating that placental specializations are correlated with viviparity. Uterine specializations for placental transport of calcium to viviparous embryos alter uterine physiology compared to oviparous females. In addition, comparative studies of oviparous and viviparous species, i.e., in which gene flow is disrupted, reveal that both uterine and embryonic structural modifications are commonly associated with viviparity, suggesting relatively rapid evolution of placental specializations. Studies of squamate reproductive biology support two hypotheses: 1) evolution of viviparity requires physiological adjustments of the uterine environment, and 2) evolution of viviparity promotes relatively rapid adaptations for placentation. Models for the evolution of viviparity from oviparity, or for reversals from viviparity to oviparity, should reflect current understanding of squamate reproductive biology and future studies should be designed to challenge these models.

Physiological effects of polybrominated diphenyl ether (PBDE-47) on pregnant gartersnakes and resulting offspring.

Polybrominated diphenyl ethers (PBDEs) are used as flame retardants and are persistent contaminants found in virtually every environment and organism sampled to date, including humans. There is growing evidence that PBDEs are the source of thyroid, neurodevelopmental, and reproductive toxicity. Yet little work has focused on how this pervasive contaminant may influence the reproduction and physiology of non-traditional model species. This is especially critical because in many cases non-model species, such as reptiles, are most likely to come into contact with PBDEs in nature. We tested how short-term, repeated exposure to the PBDE congener BDE-47 during pregnancy affected physiological processes in pregnant female gartersnakes (thyroid follicular height, bactericidal ability, stress responsiveness, reproductive output, and tendency to terminate pregnancy) and their resulting offspring (levels of corticosterone, bactericidal ability, and size differences). We found potential effects of BDE-47 on both the mother, such as increased size and higher thyroid follicular height, and her offspring (increased size), suggesting the effects on physiological function of PBDEs do indeed extend beyond the traditional rodent models.

Heterochrony and early left-right asymmetry in the development of the cardiorespiratory system of snakes.

Snake lungs show a remarkable diversity of organ asymmetries. The right lung is always fully developed, while the left lung is either absent, vestigial, or well-developed (but smaller than the right). A 'tracheal lung' is present in some taxa. These asymmetries are reflected in the pulmonary arteries. Lung asymmetry is known to appear at early stages of development in Thamnophis radix and Natrix natrix. Unfortunately, there is no developmental data on snakes with a well-developed or absent left lung. We examine the adult and developmental morphology of the lung and pulmonary arteries in the snakes Python curtus breitensteini, Pantherophis guttata guttata, Elaphe obsoleta spiloides, Calloselasma rhodostoma and Causus rhombeatus using gross dissection, MicroCT scanning and 3D reconstruction. We find that the right and tracheal lung develop similarly in these species. By contrast, the left lung either: (1) fails to develop; (2) elongates more slowly and aborts early without (2a) or with (2b) subsequent development of faveoli; (3) or develops normally. A right pulmonary artery always develops, but the left develops only if the left lung develops. No pulmonary artery develops in relation to the tracheal lung. We conclude that heterochrony in lung bud development contributes to lung asymmetry in several snake taxa. Secondly, the development of the pulmonary arteries is asymmetric at early stages, possibly because the splanchnic plexus fails to develop when the left lung is reduced. Finally, some changes in the topography of the pulmonary arteries are consequent on ontogenetic displacement of the heart down the body. Our findings show that the left-right asymmetry in the cardiorespiratory system of snakes is expressed early in development and may become phenotypically expressed through heterochronic shifts in growth, and changes in axial relations of organs and vessels. We propose a step-wise model for reduction of the left lung during snake evolution.

A relative shift in cloacal location repositions external genitalia in amniote evolution.

The move of vertebrates to a terrestrial lifestyle required major adaptations in their locomotory apparatus and reproductive organs. While the fin-to-limb transition has received considerable attention, little is known about the developmental and evolutionary origins of external genitalia. Similarities in gene expression have been interpreted as a potential evolutionary link between the limb and genitals; however, no underlying developmental mechanism has been identified. We re-examined this question using micro-computed tomography, lineage tracing in three amniote clades, and RNA-sequencing-based transcriptional profiling. Here we show that the developmental origin of external genitalia has shifted through evolution, and in some taxa limbs and genitals share a common primordium. In squamates, the genitalia develop directly from the budding hindlimbs, or the remnants thereof, whereas in mice the genital tubercle originates from the ventral and tail bud mesenchyme. The recruitment of different cell populations for genital outgrowth follows a change in the relative position of the cloaca, the genitalia organizing centre. Ectopic grafting of the cloaca demonstrates the conserved ability of different mesenchymal cells to respond to these genitalia-inducing signals. Our results support a limb-like developmental origin of external genitalia as the ancestral condition. Moreover, they suggest that a change in the relative position of the cloacal signalling centre during evolution has led to an altered developmental route for external genitalia in mammals, while preserving parts of the ancestral limb molecular circuitry owing to a common evolutionary origin.