Reptile enamel matrix proteins: Selection, divergence, and functional constraint.

The three major enamel matrix proteins (EMPs): amelogenin (AMEL), ameloblastin (AMBN), and enamelin (ENAM), are intrinsically linked to tooth development in tetrapods. However, reptiles and mammals exhibit significant differences in dental patterning and development, potentially affecting how EMPs evolve in each group. In most reptiles, teeth are replaced continuously throughout life, while mammals have reduced replacement to only one or two generations. Reptiles also form structurally simple, aprismatic enamel while mammalian enamel is composed of highly organized hydroxyapatite prisms. These differences, combined with reported low sequence homology in reptiles, led us to predict that reptiles may experience lower selection pressure on their EMPs as compared with mammals. However, we found that like mammals, reptile EMPs are under moderate purifying selection, with some differences evident between AMEL, AMBN, and ENAM. We also demonstrate that sequence homology in reptile EMPs is closely associated with divergence times, with more recently diverged lineages exhibiting high homology, along with strong phylogenetic signal. Lastly, despite sequence divergence, none of the reptile species in our study exhibited mutations consistent with diseases that cause degeneration of enamel (e.g. amelogenesis imperfecta). Despite short tooth retention time and simplicity in enamel structure, reptile EMPs still exhibit purifying selection required to form durable enamel.

Semaphorin 5B is a repellent cue for sensory afferents projecting into the developing spinal cord.

During vertebrate development, centrally projecting sensory axons of the dorsal root ganglia neurons first reach the embryonic spinal cord at the dorsolateral margin. Instead of immediately projecting into the grey matter, they bifurcate and extend rostrally and caudally to establish the longitudinal dorsal funiculus during a stereotyped waiting period of approximately 48 h. Collateral fibres then extend concurrently across multiple spinal segments and project to their appropriate targets within the grey matter. This rostrocaudal extension of sensory afferents is crucial for the intersegmental processing of information throughout the spinal cord. However, the precise cues that prevent premature entry during the waiting period remain to be identified. Here, we show that semaphorin 5B (Sema5B), a member of the semaphorin family of guidance molecules, is expressed in the chick spinal cord during this waiting period and dorsal funiculus formation. Sema5B expression is dynamic, with a reduction of expression apparent in the spinal cord concomitant with collateral extension. We show that Sema5B inhibits the growth of NGF-dependent sensory axons and that this effect is mediated in part through the cell adhesion molecule TAG-1. Knockdown of Sema5B in the spinal cord using RNA interference leads to the premature extension of cutaneous nociceptive axons into the dorsal horn grey matter. These premature projections predominantly occur at the site of dorsal root entry. Our results suggest that Sema5B contributes to a repulsive barrier for centrally projecting primary sensory axons, forcing them to turn and establish the dorsal funiculus.

Pannexin 3 is required for late stage bone growth but not for initiation of ossification in avian embryos.

BACKGROUND: Pannexin 3 (PANX3) is a channel-forming protein capable of stimulating osteogenesis in vitro. Here, we studied the in vivo roles of PANX3 in the chicken embryo using the RCAS retroviral system to over-express and knockdown expression during endochondral bone formation. RESULTS: In the limbs, PANX3 RNA was first detected in the cartilage condensations and became restricted to the prehypertrophic cartilage of the epiphyses, diaphysis, and perichondrium. The increase in PANX3 was not sufficient to alter osteogenesis; however, knockdown with a virus containing an interference RNA construct caused a 20% reduction in bone volume. The control virus containing an shEGFP cassette did not affect development. Interestingly, the phenotype was restricted to later stages rather than to proliferation of the skeletogenic mesenchyme, formation of the cartilage condensation, or creation of the hypertrophic zones. In addition, there was also no change in readouts of Hedgehog, WNT, fibroblast growth factor, or bone morphogenetic protein signaling using either quantitative real-time polymerase chain reaction or radioactive in situ hybridization. CONCLUSIONS: Based on the normal expression domains of PANX3 and the relatively late manifestation of the phenotype, it is possible that PANX3 hemichannels may be required to facilitate the transition of hypertrophic chondrocytes to osteoblasts, thereby achieving final bone size. Developmental Dynamics 245:913-924, 2016. © 2016 Wiley Periodicals, Inc.

Recent insights into the morphological diversity in the amniote primary and secondary palates.

The assembly of the upper jaw is a pivotal moment in the embryonic development of amniotes. The upper jaw forms from the fusion of the maxillary, medial nasal, and lateral nasal prominences, resulting in an intact upper lip/beak and nasal cavities; together called the primary palate. This process of fusion requires a balance of proper facial prominence shape and positioning to avoid craniofacial clefting, whilst still accommodating the vast phenotypic diversity of adult amniotes. As such, variation in craniofacial ontogeny is not tolerated beyond certain bounds. For clarity, we discuss primary palatogenesis of amniotes into in two categories, according to whether the nasal and oral cavities remain connected throughout ontogeny or not. The transient separation of these cavities occurs in mammals and crocodilians, while remaining connected in birds, turtles and squamates. In the latter group, the craniofacial prominences fuse around a persistent choanal groove that connects the nasal and oral cavities. Subsequently, all lineages except for turtles, develop a secondary palate that ultimately completely or partially separates oral and nasal cavities. Here, we review the shared, early developmental events and highlight the points at which development diverges in both primary and secondary palate formation.

Diversity in primary palate ontogeny of amniotes revealed with 3D imaging.

The amniote primary palate encompasses the upper lip and the nasal cavities. During embryonic development, the primary palate forms from the fusion of the maxillary, medial nasal and lateral nasal prominences. In mammals, as the primary palate fuses, the nasal and oral cavities become completely separated. Subsequently, the tissue demarcating the future internal nares (choanae) thins and becomes the bucconasal membrane, which eventually ruptures and allows for the essential connection of the oral and nasal cavities to form. In reptiles (including birds), the other major amniote group, primary palate ontogeny is poorly studied with respect to prominence fusion, especially the formation of a bucconasal membrane. Using 3D optical projection tomography, we found that the prominences that initiate primary palate formation are similar between mammals and crocodilians but distinct from turtles and lizards, which are in turn similar to each other. Chickens are distinct from all non-avian lineages and instead resemble human embryos in this aspect. The majority of reptiles maintain a communication between the oral and nasal cavities via the choanae during primary palate formation. However, crocodiles appear to have a transient separation between the oral and nasal cavities. Furthermore, the three lizard species examined here, exhibit temporary closure of their external nares via fusion of the lateral nasal prominences with the frontonasal mass, subsequently reopening them just before hatching. The mechanism of the persistent choanal opening was examined in chicken embryos. The mesenchyme posterior/dorsal to the choana had a significant decline in proliferation index, whereas the mesenchyme of the facial processes remained high. This differential proliferation allows the choana to form a channel between the oral and nasal cavities as the facial prominences grow and fuse around it. Our data show that primary palate ontogeny has been modified extensively to support the array of morphological diversity that has evolved among amniotes.

Divergent palate morphology in turtles and birds correlates with differences in proliferation and BMP2 expression during embryonic development.

During embryonic development, amniotes typically form outgrowths from the medial sides of the maxillary prominences called palatal shelves or palatine processes. In mammals the shelves fuse in the midline and form a bony hard palate that completely separates the nasal and oral cavities. In birds and lizards, palatine processes develop but remain unfused, leaving a natural cleft. Adult turtles do not possess palatine processes and unlike other amniotes, the internal nares open into the oral cavity. Here we investigate craniofacial ontogeny in the turtle, Emydura subglobosa to determine whether vestigial palatine processes develop and subsequently regress, or whether development fails entirely. We found that the primary palate in turtles develops similarly to other amniotes, but secondary palate ontogeny diverges. Using histology, cellular dynamics and in situ hybridization we found no evidence of palatine process development at any time during ontogeny of the face in the turtle. Furthermore, detailed comparisons with chicken embryos (the model organism most closely related to turtles from a molecular phylogeny perspective), we identified differences in proliferation and gene expression patterns that correlate with the differences in palate morphology. We propose that, in turtles, palatine process outgrowth is never initiated due to a lack of mesenchymal bone morphogenetic protein 2 (BMP2) expression in the maxillary mesenchyme, which in turn fails to induce the relatively higher cellular proliferation required for medial tissue outgrowth. It is likely that these differences between turtles and birds arose after the divergence of the lineage leading to modern turtles.

Characterization of the developing axolotl nasal cavity supports multiple evolution of the vertebrate choana.

All tetrapods (mammals, birds, reptiles, and amphibians) share the ability to breathe with their mouths closed due to the formation of choanae, which are openings that allow communication between the nasal and oral cavities. In most fishes, the nasal cavities serve a strictly olfactory function, possessing incurrent and excurrent nares that lie outside of the mouth and therefore, never communicate with the respiratory system. It is not until the evolution of tetrapods, in which the nasal cavities consistently open into the mouth, that they are used both for olfaction and for respiration. However, this developmental transition is poorly understood, with no consensus on the evolutionary origin of the choana in various groups despite decades of debate. Here, we use high-contrast 3D imaging in conjunction with histology and apoptotic cell analysis in non-mineralized embryonic tissues to study the formation of the choana in the axolotl (Ambystoma mexicanum), an aquatic salamander species. We show that the axolotl choana forms from an extension of the embryonic nasal sac, which pushes through intervening mesenchyme and connects with the palate epithelium of the oral cavity, eventually breaking through. This mechanism differs from caecilians, mammals and reptiles, where fusion across a bucconasal groove plays an active role in choana formation. Nevertheless, caecilians, mammals and axolotls converge on the development of a transient epithelial tissue that has to break down in order to develop a patent choana, adding another twist to the intriguing arguments on the evolutionary history of the choana.

Novel insights into the development of the avian nasal cavity.

In embryonic amniotes, patterning of the oral and nasal cavities requires bilateral fusion between craniofacial prominences, ensuring an intact primary palate and upper jaw. After fusion has taken place, the embryonic nasal cavities open anteriorly through paired external nares positioned directly above the fusion zones and bordered by the medial nasal and lateral nasal prominences. In this study, we show that in the chicken embryo, the external nares initially form as patent openings but only remain so for a short period of time. Soon after the nasal cavities form, the medial nasal and lateral nasal prominences fuse together in stage 29 embryos, entirely closing off the external nares for a substantial portion of embryonic and fetal development. The epithelium between the fused prominences is then retained and eventually develops into a nasal plug that obstructs the nasal vestibule through the majority of the fetal period. At stage 40, the nasal plug begins to break down through a combination of cellular remodeling, apoptosis, as well as non-apoptotic necrosis, leading to completely patent nasal cavities at hatching. These findings place chickens in a category with several species of nonavian reptiles and mammals (including humans) that have been found to develop a transient embryonic nasal plug. Our findings are discussed in the context of previously reported cases of nasal plugs as part of normal embryonic development and provide novel insight into the craniofacial development of a key model organism in developmental biology.

Role of Cell Death in Cellular Processes During Odontogenesis.

The development of a tooth germ in a precise size, shape, and position in the jaw, involves meticulous regulation of cell proliferation and cell death. Apoptosis, as the most common type of programmed cell death during embryonic development, plays a number of key roles during odontogenesis, ranging from the budding of the oral epithelium during tooth initiation, to later tooth germ morphogenesis and removal of enamel knot signaling center. Here, we summarize recent knowledge about the distribution and function of apoptotic cells during odontogenesis in several vertebrate lineages, with a special focus on amniotes (mammals and reptiles). We discuss the regulatory roles that apoptosis plays on various cellular processes during odontogenesis. We also review apoptosis-associated molecular signaling during tooth development, including its relationship with the autophagic pathway. Lastly, we cover apoptotic pathway disruption, and alterations in apoptotic cell distribution in transgenic mouse models. These studies foster a deeper understanding how apoptotic cells affect cellular processes during normal odontogenesis, and how they contribute to dental disorders, which could lead to new avenues of treatment in the future.

Pseudogenized Amelogenin Reveals Early Tooth Loss in True Toads (Anura: Bufonidae).

Extant anurans (frogs and toads) exhibit reduced dentition, ranging from a lack of mandibular teeth to complete edentulation, as observed in the true toads of the family Bufonidae. The evolutionary time line of these reductions remains vague due to a poor fossil record. Previous studies have demonstrated an association between the lack of teeth in edentulous vertebrates and the pseudogenization of the major tooth enamel gene amelogenin (AMEL) through accumulation of deleterious mutations and the disruption of its coding sequence. In this study, we have harnessed the pseudogenization of AMEL as a molecular dating tool to correlate loss of dentition with genomic mutation patterns during the rise of the family Bufonidae. Specifically, we have utilized AMEL pseudogenes in three members of the family as a tool to estimate the putative date of edentulation in true toads. Comparison of AMEL sequences from Rhinella marina, Bufo gargarizans and Bufo bufo, with nine extant, dentulous frogs, revealed mutations confirming AMEL inactivation in Bufonidae. AMEL pseudogenes in modern bufonids also exhibited remarkably high 86-93% sequence identity among each other, with only a slight increase in substitution rate and relaxation of selective pressure, in comparison with functional copies in other anurans. Moreover, using selection intensity estimates and synonymous substitution rates, analysis of functional and pseudogenized AMEL resulted in an estimated inactivation window of 46-60 million years ago in the lineage leading to modern true toads, a time line that coincides with the rise of the family Bufonidae.

Molecular characterization of the Bidder's organ in the cane toad (Bufo marinus).

In toads, both males and females develop a unique gonadal structure called the Bidder's organ (BO), which resembles ovarian tissue and is attached to the anterior part of the gonad. It is not clear whether the BO is a vestigial organ, or has an endocrine function. In this study, we investigated the expression of the gonadal development genes Dmrt1, Sox9, Sf1, Dax1, and p450arom in the developing BO as compared with the gonads of male and female cane toads. We demonstrate that Sf1, Dax1, and p450arom, key genes involved in vertebrate steroidogenesis, are transcriptionally active in the BO during developmental milestones associated with sexual development and maturation. Furthermore, the pattern of transcriptional activity in the BO is completely independent of the corresponding gonads in both sexes, despite its ovary-like morphology. These results suggest that the BO likely has a steroidogenic role in the development of the cane toad, distinct from that of the gonads.

Z and W sex chromosomes in the cane toad (Bufo marinus).

The cane toad (Bufo marinus) is one of the most notorious animal pests encountered in Australia. Members of the genus Bufo historically have been regarded as having genotypic sex determination with male homogamety/female heterogamety. Nevertheless, as with many toads, karyotypic analyses of the cane toad have so far failed to identify heteromorphics sex chromosomes. In this study, we used comparative genomic hybridization, reverse fluorescence staining, C-banding, and morphometric analyses of chromosomes to characterize sex chromosome dimorphism in B. marinus. We found that females consistently had a length dimorphism associated with a nucleolus organizer region (NOR) on one of the chromosome 7 pair. A strong signal over the longer NOR in females, and the absence of a signal in males indicated sex-specific DNA sequences. All females were heterozygous and all males homozygous, indicating a ZZ/ZW sex chromosomal system. Our study confirms the existence of sex chromosomes in this species. The ability to reliably identify genotypic sex of cane toads will be of value in monitoring and control efforts in Australia and abroad.

Cloning and expression of candidate sexual development genes in the cane toad (Bufo marinus).

The development of the reproductive system in bufonids (true toads) is unique in several respects: sexual differentiation occurs later than in other anurans, and toads develop a Bidder's organ, a rudimentary ovary that can be manipulated in males to produce mature oocytes. To illuminate the genesis of this unusual reproductive system, we isolated from the cane toad (Bufo marinus) the orthologues of several known vertebrate sex-determining genes, determined their primary structure, and studied their expression by reverse transcriptase-polymerase chain reaction and in situ hybridization of tissue sections. We report here that cane toad Sox9, Dmrt1, and p450aromatase (Cyp19a1) are highly homologous to their counterparts in other vertebrates. They show profiles of expression that generally follow patterns observed in other taxa, but with some novel features. Our data suggest that these genes likely play key roles in sex determination and early gonad development in bufonids.

Disconnect between the developing eye and craniofacial prominences in the avian embryo.

In the amniote embryo, the upper jaw and nasal cavities form through coordinated outgrowth and fusion of craniofacial prominences. Adjacent to the embryonic prominences are the developing eyes, which abut the maxillary and lateral nasal prominences. The embryos of extant sauropsids (birds and nonavian reptiles) develop particularly large eyes in comparison to mammals, leading researchers to propose that the developing eye may facilitate outgrowth of prominences towards the midline in order to aid prominence fusion. To test this hypothesis, we performed unilateral and bilateral ablation of the developing eyes in chicken embryos, with the aim of evaluating subsequent prominence formation and fusion. Our analyses revealed minor interaction between the developing craniofacial prominences and the eyes, inconsequential to the fusion of the upper beak. At later developmental stages, the skull exhibited only localized effects from missing eyes, while geometric morphometrics revealed minimal effect on overall shape of the upper jaw when it develops without eyes. Our results indicate that the substantial size of the developing eyes in the chicken embryo exert little influence over the fusion of the craniofacial prominences, despite their effect on the size and shape of maxillary prominences and components of the skull.

Hedgehog Signaling and Embryonic Craniofacial Disorders.

Since its initial discovery in a Drosophila mutagenesis screen, the Hedgehog pathway has been revealed to be instrumental in the proper development of the vertebrate face. Vertebrates possess three hedgehog paralogs: Sonic hedgehog (Shh), Indian hedgehog (Ihh), and Desert hedgehog (Dhh). Of the three, Shh has the broadest range of functions both in the face and elsewhere in the embryo, while Ihh and Dhh play more limited roles. The Hedgehog pathway is instrumental from the period of prechordal plate formation early in the embryo, until the fusion of the lip and secondary palate, which complete the major patterning events of the face. Disruption of Hedgehog signaling results in an array of developmental disorders in the face, ranging from minor alterations in the distance between the eyes to more serious conditions such as severe clefting of the lip and palate. Despite its critical role, Hedgehog signaling seems to be disrupted through a number of mechanisms that may either be direct, as in mutation of a downstream target of the Hedgehog ligand, or indirect, such as mutation in a ciliary protein that is otherwise seemingly unrelated to the Hedgehog pathway. A number of teratogens such as alcohol, statins and steroidal alkaloids also disrupt key aspects of Hedgehog signal transduction, leading to developmental defects that are similar, if not identical, to those of Hedgehog pathway mutations. The aim of this review is to highlight the variety of roles that Hedgehog signaling plays in developmental disorders of the vertebrate face.

Craniofacial development: discoveries made in the chicken embryo.

The aim of this review is to highlight some of the key contributions to our understanding of craniofacial research from work carried out with the chicken and other avian embryos. From the very first observations of neural crest cell migration to the fusion of the primary palate, the chicken has proven indispensable in facilitating craniofacial research. In this review we will look back to the premolecular studies where "cut and paste" grafting experiments mapped the fate of cranial neural crest cells, the role of different tissue layers in patterning the face, and more recently the contribution of neural crest cells to jaw size and identity. In the late 80's the focus shifted to the molecular underpinnings of facial development and, in addition to grafting experiments, various chemicals and growth factors were being applied to the face. The chicken is above all else an experimental model, inviting hands-on manipulations. We describe the elegant discoveries made by directly controlling signaling either in the brain, in the pharyngeal arches or in the face itself. We cover how sonic hedgehog (Shh) signals to the face and how various growth factors regulate facial prominence identity, growth and fusion. We also review abnormal craniofacial development and how several type of spontaneous chicken mutants shed new light on diseases affecting the primary cilium in humans. Finally, we bring out the very important role that the bird beak has played in understanding amniote evolution. The chicken, duck and quail have been and will continue to be used as experimental models to explore the evolution of jaw diversity and the morphological constraints of the vertebrate face.

Lineage-specific loss of FGF17 within the avian orders Galliformes and Passeriformes.

The genomic and developmental complexity of vertebrates is commonly attributed to two rounds of whole genome duplications which occurred at the base of the vertebrate radiation. These duplications led to the rise of several, multi-gene families of developmental proteins like the fibroblast growth factors (FGFs); a signaling protein family which functions at various stages of embryonic development. One of the major FGF assemblages arising from these duplications is the FGF8 subfamily, which includes FGF8, FGF17, and FGF18 in tetrapods. While FGF8 and FGF18 are found in all tetrapods and are critical for embryonic survival, genomic analyses suggest putative loss of FGF17 in various lineages ranging from frogs and fish, to the chicken. This study utilizes 27 avian genomes in conjunction with molecular analyses of chicken embryos to confirm the loss of FGF17 in chicken as a true, biological occurrence. FGF17 is also missing in the turkey, black grouse, Japanese quail and northern bobwhite genomes. These species, along with chicken, form a monophyletic clade in the order Galliformes. Four additional species, members of the clade Passeroidea, within the order Passeriformes, are also missing FGF17. Additionally, analysis of intact FGF17 in other avian lineages reveals that it is still under strong purifying selection, despite being seemingly dispensable. Thus, FGF17 likely represents a molecular spandrel arising from a genome duplication event and due to its high connectivity with FGF8/FGF18, and potential for interference with their function, is retained under strong purifying selection, despite itself not having a strong selective advantage.

The western painted turtle genome, a model for the evolution of extreme physiological adaptations in a slowly evolving lineage.

BACKGROUND: We describe the genome of the western painted turtle, Chrysemys picta bellii, one of the most widespread, abundant, and well-studied turtles. We place the genome into a comparative evolutionary context, and focus on genomic features associated with tooth loss, immune function, longevity, sex differentiation and determination, and the species' physiological capacities to withstand extreme anoxia and tissue freezing. RESULTS: Our phylogenetic analyses confirm that turtles are the sister group to living archosaurs, and demonstrate an extraordinarily slow rate of sequence evolution in the painted turtle. The ability of the painted turtle to withstand complete anoxia and partial freezing appears to be associated with common vertebrate gene networks, and we identify candidate genes for future functional analyses. Tooth loss shares a common pattern of pseudogenization and degradation of tooth-specific genes with birds, although the rate of accumulation of mutations is much slower in the painted turtle. Genes associated with sex differentiation generally reflect phylogeny rather than convergence in sex determination functionality. Among gene families that demonstrate exceptional expansions or show signatures of strong natural selection, immune function and musculoskeletal patterning genes are consistently over-represented. CONCLUSIONS: Our comparative genomic analyses indicate that common vertebrate regulatory networks, some of which have analogs in human diseases, are often involved in the western painted turtle's extraordinary physiological capacities. As these regulatory pathways are analyzed at the functional level, the painted turtle may offer important insights into the management of a number of human health disorders.

Species-specific chitin-binding module 18 expansion in the amphibian pathogen Batrachochytrium dendrobatidis.

UNLABELLED: Batrachochytrium dendrobatidis is the causative agent of chytridiomycosis, which is considered one of the driving forces behind the worldwide decline in populations of amphibians. As a member of the phylum Chytridiomycota, B. dendrobatidis has diverged significantly to emerge as the only pathogen of adult vertebrates. Such shifts in lifestyle are generally accompanied by various degrees of genomic modifications, yet neither its mode of pathogenicity nor any factors associated with it have ever been identified. Presented here is the identification and characterization of a unique expansion of the carbohydrate-binding module family 18 (CBM18), specific to B. dendrobatidis. CBM (chitin-binding module) expansions have been likened to the evolution of pathogenicity in a variety of fungus species, making this expanded group a prime candidate for the identification of potential pathogenicity factors. Furthermore, the CBM18 expansions are confined to three categories of genes, each having been previously implicated in host-pathogen interactions. These correlations highlight this specific domain expansion as a potential key player in the mode of pathogenicity in this unique fungus. The expansion of CBM18 in B. dendrobatidis is exceptional in its size and diversity compared to other pathogenic species of fungi, making this genomic feature unique in an evolutionary context as well as in pathogenicity. IMPORTANCE: Amphibian populations are declining worldwide at an unprecedented rate. Although various factors are thought to contribute to this phenomenon, chytridiomycosis has been identified as one of the leading causes. This deadly fungal disease is cause by Batrachochytrium dendrobatidis, a chytrid fungus species unique in its pathogenicity and, furthermore, its specificity to amphibians. Despite more than two decades of research, the biology of this fungus species and its deadly interaction with amphibians had been notoriously difficult to unravel. Due to the alarming rate of worldwide spread and associated decline in amphibian populations, it is imperative to incorporate novel genomic and genetic techniques into the study of this species. In this study, we present the first reported potential pathogenicity factors in B. dendrobatidis. In silico studies such as this allow us to identify putative targets for more specific molecular analyses, furthering our hope for the control of this pathogen.