Skeletons of the Eye: An Evolutionary and Developmental Perspective.

The ocular skeleton, composed of the scleral cartilage and scleral ossicles, is present in many vertebrates. The morphology of the scleral cartilage and ossicles varies within different extant reptiles (including birds) and also varies dramatically from the morphology in extant teleosts. This incredible range of diverse morphologies is the result of millions of years of evolution. Both the position of these elements within the eye and the timing of development vary amongst different vertebrates. While the development of both the scleral cartilage and scleral ossicles is somewhat understood in reptiles and in teleosts, the functional advantage of these elements is still debated. Most reptiles have a multi-component scleral ossicle ring composed of a series of flat bone plates and a scleral cartilage cup lining the retina, some sharks have calcified cartilage plates, and some teleosts have two bones while most others only have a ring of scleral cartilage. The data presented shows that different vertebrates have adapted to similar selective pressures in different ways. However, the reason why sarcopterygians have a series of overlapping bones in the sclera remains unclear. A better understanding of the ocular skeletal diversity in Reptilia as well as a better understanding of the mechanisms of vision within different environments (i.e., air vs. water) and that used by secondarily aquatic organisms is needed. This review discusses the observed variation in morphology and development of the ocular skeleton in the context of evolution and highlights our knowledge gaps in these areas. Anat Rec, 2018. © 2018 American Association for Anatomy.

Genetic linkage between altered tooth and eye development in lens-ablated Astyanax mexicanus.

The phenotype of lens-ablated Mexican tetra (Astyanax mexicanus) compared to wild-type surface fish has been described and includes, among other effects, eye degeneration, changes in tooth number and cranial bone changes. Here, we investigate the spatiotemporal expression patterns of several key genes involved in the development of these structures. Specifically, we show that the expression of pitx2, bmp4 and shh is altered in the eye, oral jaw, nasal pit and forebrain in these lens-ablated fish. Furthermore, for the first time, we show altered pitx2 expression in the cavefish, which also has altered eye and tooth phenotypes. We thus provide evidence for a genetic linkage between the eye and tooth modules in this fish species. Furthermore, the altered pitx2 expression pattern, together with the described morphological features of the lens-ablated fish suggests that Astyanax mexicanus could be considered as an alternative teleost model organism in which to study Axenfeld-Rieger syndrome (ARS), a rare autosomal dominant developmental disorder that is associated with PITX2 and which has both ocular and non-ocular abnormalities.

A wide temporal window for conjunctival papillae development ensures the formation of a complete sclerotic ring.

BACKGROUND: The conjunctival papillae are epithelial thickenings of the conjunctiva that are required for the induction of underlying bones (the scleral ossicles). These transient papillae develop and become inductively active over an extended temporal period (HH 30-36, 6.5-10 dpf). While their inductive capacity was discovered in the mid-1900s, little is known about their development. RESULTS: Through a series of timed surgical ablations followed by in situ hybridization for Bmp2, we show that the ring of conjunctival papillae is not altered if the conjunctival epithelium is ablated either prior to or shortly after papillae induction (i.e., HH 29-30, 6.5-7 dpf). A conjunctival papilla ablated at or prior to HH 34 (8 dpf), when the complete ring is present, regenerates and quickly becomes inductively active, inducing an underlying scleral condensation with only a slight delay. This regenerative capacity extends until HH 35.5, a full 36 hours beyond the normal timeline of papillae induction. As such, the period of epithelial competency for papilla induction is longer than previously identified. CONCLUSIONS: Papilla regeneration is a mechanism that ensures the formation of a complete sclerotic ring and provides another level of redundancy for the induction of a complete sclerotic ring during the normal inductive period. Developmental Dynamics 246:381-391, 2017. © 2017 Wiley Periodicals, Inc.

Gene expression analysis during the induction and patterning of the conjunctival papillae in the chick embryonic eye.

The induction and patterning of the conjunctival papillae (i.e. epithelial thickenings of the conjunctiva required for the induction of the underlying, neural crest-derived scleral ossicles) is complex. It takes place over a period of two days and follows a defined spatiotemporal sequence. In this study, we investigated the spatial and temporal expression pattern of four genes over seven morphological stages of development of these papillae. We show that ß-catenin is expressed during the pre-patterning of the epithelium prior to papilla induction and second that ß-catenin, Ednrb and Inhba are expressed during the induction and patterning of the conjunctival papillae. Furthermore, we identified two genes, ß-catenin and Prox1, that may be involved in the induction of the underlying scleral bones. These data provide an excellent baseline for future studies, setting the stage for functional studies aimed at examining the role of these genes in the patterning of the scleral ossicle system. This study also outlines the similarities between the conjunctival papillae and other placodes and may provide insights into the evolution and development of the conjunctival papillae.

Optimized ex-ovo culturing of chick embryos to advanced stages of development.

Research in anatomy, embryology, and developmental biology has largely relied on the use of model organisms. In order to study development in live embryos model organisms, such as the chicken, are often used. The chicken is an excellent model organism due to its low cost and minimal maintenance, however they present observational challenges because they are enclosed in an opaque eggshell. In order to properly view the embryo as it develops, the shell must be windowed or removed. Both windowing and ex ovo techniques have been developed to assist researchers in the study of embryonic development. However, each of the methods has limitations and challenges. Here, we present a simple, optimized ex ovo culture technique for chicken embryos that enables the observation of embryonic development from stage HH 19 into late stages of development (HH 40), when many organs have developed. This technique is easy to adopt in both undergraduate classes and more advanced research laboratories where embryo manipulations are conducted.

Perturbing the developing skull: using laser ablation to investigate the robustness of the infraorbital bones in zebrafish (Danio rerio).

BACKGROUND: The development of the craniofacial skeleton from embryonic mesenchyme is a complex process that is not yet completely understood, particularly for intramembranous bones. This study investigates the development of the neural crest derived infraorbital (IO) bones of the zebrafish (Danio rerio) skull. Located under the orbit, the IO bones ossify in a set sequence and are closely associated with the lateral line system. We conducted skeletogenic condensation and neuromast laser ablation experiments followed by shape analyses in order to investigate the relationship between a developing IO bone and the formation of the IO series as well as to investigate the highly debated inductive potential of neuromasts for IO ossification. RESULTS: We demonstrate that when skeletogenic condensations recover from laser ablation, the resulting bone differs in shape compared to controls. Interestingly, neighbouring IO bones in the bone series are unaffected. In addition, we show that the amount of canal wall mineralization is significantly decreased following neuromast laser ablation at juvenile and larval stages. CONCLUSIONS: These results highlight the developmental robustness of the IO bones and provide direct evidence that canal neuromasts play a role in canal wall development in the head. Furthermore, we provide evidence that the IO bones may be two distinct developmental modules. The mechanisms underlying developmental robustness are rarely investigated and are important to increase our understanding of evolutionary developmental biology of the vertebrate skull.

Spatial and temporal events in tooth development of Astyanax mexicanus.

The Mexican tetra (Astyanax mexicanus), a freshwater teleost fish, is an excellent vertebrate model organism to study tooth development, specifically the spatiotemporal events related to the development of the oral and pharyngeal dentitions. In contrast to the coordinated early tooth development in the premaxilla and mandible, the maxillary teeth develop much later in life at 60 dpf. By analysing a growth series of bone and cartilage stained tetra and histological sectioning of the tooth bearing bones, we track the developmental events of tooth development over ontogeny of this animal. Whole mount in situ hybridisation with bone morphogenetic proteins and their inhibitor Noggin was conducted to track the late tooth development events. Our data show that the first generation teeth are small and unicuspid irrespective of their location. Oral jaw teeth become multicuspid and large over ontogeny while the pharyngeal dentition remains unicuspid and disorganised. Tooth eruption occurs late in the maxillary bone. The distinct expression pattern of the BMP antagonist, Noggin, suggests that Noggin plays an inhibitory role by preventing early tooth development in the maxillary bone. These data further support and highlight the use of the Mexican tetra in understanding the spatio-temporal differences in tooth development in vertebrate jaws.

The zebrafish infraorbital bones: a descriptive study.

The infraorbital (IO) bone series, a component of the circumorbital series, makes up five of the eight dermal bones found in the orbital region of the zebrafish skull. Ossifying in a set sequence, the IOs are closely associated with the cranial lateral line system as they house neuromast sensory receptors in bony canals. We conducted a detailed analysis of the condensation to mineralization phases of development of these bones. Our analyses involved both bone and osteoblast staining of zebrafish at 20 different time points. IO bone condensations are shaped as templates for the final bone shape, and they mineralize at one or more centers of ossification. Initially, mineralization is closely associated with the lateral line canals and/or foramen, and the onset of mineralization is temporally variable. Canal wall mineralization is a process that continues into adulthood and completely mineralized canal roofs were not found. Our comprehensive growth series detailing the ossification of each IO bone provides important insight into the growth and development of this series of neural crest-derived flat bones in the zebrafish craniofacial skeleton.

Comparative developmental osteology of the seahorse skeleton reveals heterochrony amongst Hippocampus sp. and progressive caudal fin loss.

BACKGROUND: Seahorses are well known for their highly derived head shape, prehensile tail and armoured body. They belong to the family of teleosts known as Syngnathidae, which also includes the pipefishes, pipehorses and seadragons. Very few studies have investigated the development of the skeleton of seahorses because larvae are extremely difficult to obtain in the wild and breeding in captivity is rarely successful. Here we compare the developmental osteology of Hippocampus reidi over an ontogenetic series spanning the first 93 days after release from the brood pouch to that of a smaller series of Hippocampus; namely H. subelongatus. RESULTS: We compare the osteology in these two species over growth to the published description of the dwarf species, H. zosterae. We show that ossification onset in H. subelongatus is earlier than in H. reidi, despite similar sizes at parturition. Interestingly, the timing of development of the bony skeleton in H. zosterae is similar to that of the larger species, H. subelongatus. Furthermore, we show that the growth rate of all three species is similar up until about 30 days post pouch release. From this stage onwards in the life history, the size of the dwarf species H. zosterae remains relatively constant whilst the other two species continue growing with an accelerated growth phase. CONCLUSION: This data together with a phylogenetic assessment suggests that there has been a heterochronic shift (a delay) in the timing of ossification in H. reidi and accelerated bonedevelopment in H. zosterae. That is, H. zosterae is not a developmentally truncated dwarf species but rather a smaller version of its larger ancestor, "a proportioned dwarf" species. Furthermore, we show that caudal fin loss is incomplete in Hippocampus seahorses. This study shows that these three species of Hippocampus seahorses have evolved (either directly or indirectly) different osteogenic strategies over the last 20-30 million years of seahorse evolution.

Adaptive evolution of the lower jaw dentition in Mexican tetra (Astyanax mexicanus).

BACKGROUND: The Mexican tetra (Astyanax mexicanus) has emerged as a good animal model to study the constructive and regressive changes associated with living in cave environments, as both the ancestral sighted morph and the cave dwelling morph are extant. The cave dwelling morphs lack eyes and body pigmentation, but have well developed oral and sensory systems that are essential for survival in dark environments. The cave forms and surface forms are interfertile and give rise to F1 hybrids progeny known as intermediates. In cavefish, degeneration of the lens is one of the key events leading to eye regression. We have previously shown that surgical lens removal in surface fish embryos has an effect on the craniofacial skeleton. Surprisingly, lens removal was also found to have an effect on the caudal teeth in the lower jaw. In order to understand this result, we analyzed the lower jaw and upper jaw dentitions of surface, cavefish and F1 hybrids of surface and cavefish and compared our findings with surface fish that underwent lens removal. We also investigated the upper jaw (premaxillae and maxillae) dentition in these fish. RESULTS: Our tooth analyses shows that cavefish have the highest numbers of teeth in the mandible and maxillae, surface forms have the lowest numbers and F1 hybrids are between these groups. These differences are not observed in the premaxillae. A wide diversity of cuspal morphology can also be found in these fish. Jaw size also differs amongst the groups, with the mandible exhibiting the greatest differences. Interestingly, tooth number in surgery fish is different only in the caudal region of the mandible; this is the region that is constrained in size in all morphs. CONCLUSION: Our data provides the first detailed description of the jaw dentitions of two morphs of Astyanax mexicanus, as well as in F1 hybrids. Tooth number, patterning and cuspal morphology are enhanced in cavefish in all jaws. This is in contrast to the increase in tooth number previously observed on the lens ablated side of the surgery fish. These findings indicate that the mechanisms which govern the constructive traits in cavefish are different to the mechanisms causing an increase tooth number in surgery fish.

The ocular skeleton through the eye of evo-devo.

An evolutionary developmental (evo-devo) approach to understanding the evolution, homology, and development of structures has proved important for unraveling complex integrated skeletal systems through the use of modules, or modularity. An ocular skeleton, which consists of cartilage and sometimes bone, is present in many vertebrates; however, the origin of these two components remains elusive. Using both paleontological and developmental data, I propose that the vertebrate ocular skeleton is neural crest derived and that a single cranial neural crest module divided early in vertebrate evolution, possibly during the Ordovician, to give rise to an endoskeletal component and an exoskeletal component within the eye. These two components subsequently became uncoupled with respect to timing, placement within the sclera and inductive epithelia, enabling them to evolve independently and to diversify. In some extant groups, these two modules have become reassociated with one another. Furthermore, the data suggest that the endoskeletal component of the ocular skeleton was likely established and therefore evolved before the exoskeletal component. This study provides important insights into the evolution of the ocular skeleton, a region with a long evolutionary history among vertebrates.