Hydrocortisone treatment as a tool to study conjunctival placode induction.

BACKGROUND: Conjunctival placodes are a series of placodes that develop into the conjunctival (scleral) papillae and ultimately induce a series of scleral ossicles in the eyes of many vertebrates. This study establishes a hydrocortisone injection procedure (incl. dosage) that consistently inhibits all conjunctival papillae in the embryonic chicken eye. The effects of this hydrocortisone treatment on apoptosis, vasculature, and placode-related gene expression were assessed. RESULTS: Hydrocortisone treatment does not increase apoptotic cell death or have a major effect on the ciliary artery or vascular plexus in the eye. ß-catenin and Eda expression levels were not significantly altered following hydrocortisone treatment, despite the absence of conjunctival papillae. Notably, Fgf20 expression was significantly reduced following hydrocortisone treatment, and the distribution of ß-catenin was altered. CONCLUSIONS: Our study showed that conjunctival papillae induction begins as early as HH27.5 (E5.5). Hydrocortisone treatment reduces Fgf20 expression independently of ß-catenin and Eda and may instead affect other members of the Wnt/ß-catenin or Eda/Edar pathways, or it may affect the ability of morphogens to diffuse through the extracellular matrix. This study contributes to a growing profile of gene expression data during placode development and enhances our understanding of how some vertebrate eyes develop these fascinating bones.

Intravitreal injection of FGF and TGF-ß inhibitors disrupts cranial cartilage development.

Cartilage development is a tightly regulated process that requires the interaction of epithelial and mesenchymal tissues layers to initiate the aggregation of mesenchyme in a condensation. Several signaling molecules have been implicated in cartilage formation including FGFs, WNTs, and members of the TGF-ß super family. However, little is known about the earliest signals involved in these initial phases of development. Here we aimed to investigate whether direct intravitreal injection of pharmaceutical inhibitors for FGF and TGF-ß signaling would perturb cranial cartilages in zebrafish. Via wholemount bone and cartilage staining, we found effects on multiple cranial cartilage elements. We found no effect on scleral cartilage development, however, the epiphyseal bar, basihyal, and basicapsular cartilages were disrupted. Interestingly, the epiphyseal bar arises from the same progenitor pool as the scleral cartilage, namely, the periocular ectomesenchyme. This study adds to the foundational knowledge about condensation induction of cranial cartilage development and provides insight into the timing and signaling involved in the early development of several craniofacial cartilage elements in zebrafish.

Vibration exposure uncovers a critical early developmental window for zebrafish caudal fin development.

Mechanical influencers have long been shown to affect mature bone. Bone mechanosensation is a key feature that allows the skeleton to adapt to environmental constraints. In this study, we describe the response of immature, developing bones to a mechanical stimulus. To do so, zebrafish larvae at different stages of development were exposed to whole-body vibration (WBV) at a low frequency of 20 Hz, for up to 4 days. Whole mount Alizarin red and Alcian blue staining revealed age-related and bone type-specific defects. Specifically, the parhypural and hypural 1 caudal fin endoskeletal elements were affected when the exposure to WBV started early during their development. We show that these WBV-induced parhypural and hypural 1 patterning defects are triggered by a Sox9-independent pathway, potentially by reducing the distance separating adjacent chondrogenic condensations in the developing tail skeleton. The remaining hypurals were unaffected by the WBV treatment. Altogether, our results indicate that, upon exposure to vibration, chondrogenic cell progenitors can react to mechanical stimuli early during their development, which ultimately affects the skeletal patterning of the growing zebrafish larvae. These findings open a new research avenue to better understand the cellular processes involved in developing, patterning, and maintaining skeletal tissue.

Quantification and comparison of teleost scleral cartilage development and growth.

The ocular skeleton is composed of the scleral cartilage and the scleral ossicles. Teleost scleral cartilage is composed of a single layer of chondrocytes embedded in the sclera of the eye. The teleost scleral cartilage ring can vary in depth across teleost families and species, from a narrow ring a few cells wide to a deeper ring that resembles a cup and surrounds the entire sclera. However, very little research has been conducted on the development and morphology of teleost scleral cartilage. Thus, this study aims to characterize the development of the scleral cartilage in the zebrafish and Mexican tetra, with respect to the timing of emergence, depth throughout development, and positioning within the eye. We hypothesized that the scleral cartilage would first emerge in the scleral tissue closely abutting the ora serrata, and that growth would proceed in an anterior-to-posterior direction, resulting in differences in scleral cartilage depth between different fish species. We found that the scleral cartilage ring does not develop uniformly along its circumference, and that its relationship to the ora serrata varies between the rostral and caudal regions. Furthermore, distinct differences in the growth trajectory of the scleral cartilage indicate that the deep scleral cartilage of the Pachón cavefish is the result of both decreased eye size and prolonged cartilage growth. A significant difference in the size of the scleral chondrocytes was also noted. Overall, this study provides the first characterization of early scleral cartilage development in teleost fish and indicates that some aspects of scleral cartilage development and morphology are highly conserved while others are not.

Elucidating the early signaling cues involved in zebrafish chondrogenesis and cartilage morphology.

Across the teleost skeleton, cartilages are diverse in their composition suggesting subtle differences in their developmental mechanisms. This study aims to elucidate the regulatory role of bone morphogenetic protein (BMPs) during the morphogenesis of two cartilage elements in zebrafish: the scleral cartilage in the eye and the caudal fin endoskeleton. Zebrafish larvae were exposed to a BMP inhibitor (LDN193189) at a series of timepoints preceding the initial appearance of the scleral cartilage and caudal fin endoskeleton. Morphological assessments of the cartilages in later stages, revealed that BMP-inhibited fish harbored striking disruptions in caudal fin endoskeletal morphology, regardless of the age at which the inhibitor treatment was performed. In contrast, scleral cartilage morphology was unaffected in all age groups. Morphometric and principal component analysis, performed on the caudal fin endoskeleton, revealed differential clustering of principal components one and two in BMP-inhibited and control fish. Additionally, the expression of sox9a and sox9b were reduced in BMP-inhibited fish when compared to controls, indicating that LDN193189 acts via a Sox9-dependent pathway. Further examination of notochord flexion also revealed a disruptive effect of BMP inhibition on this process. This study provides a detailed characterization of the effects of BMP inhibition via LDN193189 on zebrafish cartilage morphogenesis and development. It highlights the specific, localized role of the BMP-signaling pathways during the development of different cartilage elements and sheds some light on the morphological characteristics of fossil teleosts that together suggest an uncoupling of the developmental processes between the upper and lower lobes of the caudal fin.

An overlooked placode: Recharacterizing the papillae in the embryonic eye of reptilia.

The papillae in the chicken embryonic eye, described as scleral papillae in the well-known Hamburger and Hamilton (1951) staging table, are one of the key anatomical features used to stage reptilian (including bird) embryos from HH30-36. These papillae are epithelial thickenings of the conjunctiva and are situated above the mesenchymal sclera. Here, we present evidence that the conjunctival papillae, which are required for the induction and patterning of the underlying scleral ossicles, require epithelial pre-patterning and have a placodal stage similar to other placode systems. We also suggest modifications to the Hamburger Hamilton staging criteria that incorporate this change in terminology (from "scleral" to "conjunctival" papillae) and provide a more detailed description of this anatomical feature that includes its placode stage. This enables a more complete and accurate description of chick embryo staging. The acknowledgment of a placode phase, which shares molecular and morphological features with other cutaneous placodes, will direct future research into the early inductive events leading to scleral ossicle formation.

Dark world rises: The emergence of cavefish as a model for the study of evolution, development, behavior, and disease.

A central question in biology is how naturally occurring genetic variation accounts for morphological and behavioral diversity within a species. The Mexican tetra, Astyanax mexicanus, has been studied for nearly a century as a model for investigating trait evolution. In March of 2019, researchers representing laboratories from around the world met at the Sixth Astyanax International Meeting in Santiago de Querétaro, Mexico. The meeting highlighted the expanding applications of cavefish to investigations of diverse aspects of basic biology, including development, evolution, and disease-based applications. A broad range of integrative approaches are being applied in this system, including the application of state-of-the-art functional genetic assays, brain imaging, and genome sequencing. These advances position cavefish as a model organism for addressing fundamental questions about the genetics and evolution underlying the impressive trait diversity among individual populations within this species.

A heterochronic shift in skeletal development in the barn owl (Tyto furcata): A description of the ocular skeleton and tubular eye shape formation.

BACKGROUND: The eyes of some birds of prey (Strigiformes and some eagles) are tubular in shape, which contrasts strongly with those in others, which are more globose (e.g., Galliformes) or flat (most diurnal birds). Regardless, all birds have an ocular skeleton composed of a ring of ossicles (annulus ossicularis sclerae) and a cartilage cup within the sclera. RESULTS: We show that the tubular eye of the barn owl, Tyto furcata, grows substantially in length to achieve its long axial length several weeks after hatching, well after the period when the visual input adjusts the optical system and when the scleral ossicles mineralize. This is in contrast to the chicken. The conjunctival papillae are morphologically different in each species, however, they are present for about 3 days in both birds before they degenerate. CONCLUSIONS: Our data shows a heterochronic shift in the timing of scleral cartilage development and ossicle mineralization (but not induction) to later in development compared to in the chicken. These shifts likely relate to the altricial vs precocial nature of these birds and suggests that the scleral ossicles are likely functionally important bones for vision in owls and possibly other altricial species.

Towards understanding the dose and timing effect of hydrocortisone treatment on the scleral ossicle system within the chicken eye.

Previous work, almost four decades ago, showed that hydrocortisone (HC) treatment reduces the number of skeletogenic condensations that give rise to the scleral ossicles in the chicken eye. The scleral ossicles are a ring of overlapping intramembranous bones, the sclerotic ring, and are present in most reptiles, including birds. The scleral condensations that give rise to the scleral ossicles are induced by a series of overlying thickenings (or papillae) of the conjunctival epithelium. Here, we further explore the effects of altering the dosage and timing of HC treatment on the morphology and number of skeletogenic condensations and conjunctival papillae. We show that high doses can completely obliterate the entire sclerotic ring. Significantly, the reduction in papillae number we observed was less extreme than that of the scleral condensations, indicating that additional factors contribute to the observed skeletogenic condensation loss. Via immunohistochemical analyses, we show that HC treatment alters the spatial expression pattern of several extracellular matrix components (tenascin-C, decorin and procollagen I) and also alters the vasculature network within the sclera. This research provides important insights into understanding the role of the scleral tissue components in ossicle development within the vertebrate eye.

Formation of the inner ear during embryonic and larval development of the cichlid fish (Oreochromis mossambicus).

BACKGROUND: The vertebrate inner ear comprises mineralized elements, namely the otoliths (fishes) or the otoconia (mammals). These elements serve vestibular and auditory functions. The formation of otoconia and otoliths is described as a stepwise process, and in fish, it is generally divided into an aggregation of the otolith primordia from precursor particles and then a growth process that continues throughout life. RESULTS: This study was undertaken to investigate the complex transition between these two steps. Therefore, we investigated the developmental profiles of several inner ear structural and calcium-binding proteins during the complete embryonic and larval development of the cichlid fish Oreochromis mossambicus in parallel with the morphology of inner ear and especially otoliths. We show that the formation of otoliths is a highly regulated temporal and spatial process which takes place throughout embryonic and larval development. CONCLUSIONS: Based on our data we defined eight phases of otolith differentiation from the primordia to the mature otolith.

Vascular endothelial growth factor signaling affects both angiogenesis and osteogenesis during the development of scleral ossicles.

Intramembranous ossification is a complex multi-step process which relies on extensive interactions among bone cells and surrounding tissues. The embryonic vasculature is essential in regulating endochondral ossification; however, its role during intramembranous ossification remains poorly understood, and in vivo studies are lacking. Previous research from our lab on the development of the intramembranous scleral ossicles has demonstrated an intriguing pattern of vascular development in which the areas of future osteogenesis remain avascular until after bone induction has occurred. Such avascular zones are located directly beneath each of the conjunctival papillae, epithelial structures which provide osteogenic signals to the underlying mesenchyme. Here we provide a high-resolution map of the developing vasculature from the time of ossicle induction to mineralization using a novel technique. We show that vegfa is expressed by the papillae and nearby mesenchymal tissue throughout HH 34-37, when vascular growth is taking place, and is down-regulated thereafter. Localized inhibition of Vegf results in expansion of the avascular zone surrounding the implanted papilla and mispatterning of the scleral ossicles. These results demonstrate that Vegf signaling could provide important insights into the complex relationship between bone and vasculature during intramembranous bone development.

Functional bone histology of zebrafish reveals two types of endochondral ossification, different types of osteoblast clusters and a new bone type.

The zebrafish is as an important vertebrate animal model system for studying developmental processes, gene functions and signalling pathways. It is also used as a model system for the understanding of human developmental diseases including those related to the skeleton. However, surprisingly little is known about normal zebrafish skeletogenesis and osteogenesis. As in most vertebrates, it is commonly known that the bones of adult zebrafish are cellular unlike that of some other teleosts. After careful histological analyses of each zebrafish adult bone, we identified several acellular bones, with no entrapped osteocytes in addition to several cellular bones. We show that both cellular and acellular bones can even occur within the same skeletal element and transitions between these two cell types can be found. Furthermore, we describe two types of osteoblast clusters during skeletogenesis and two different types of endochondral ossification. The epiphyseal plate, for example, lacks a zone of calcification and a degradation zone with osteoblasts. A new bone type that we term tubular bone was also identified. This bone is completely filled with adipose tissue, unlike spongy bones. This study provides important insight on how osteogenesis takes place in zebrafish, and especially on the transition from cellular to acellular bones. Overall, this study leads to a deeper understanding of the functional histological composition of adult zebrafish bones.

The sclerotic ring of squamates: an evo-devo-eco perspective.

The sclerotic ring consists of several bones that form in the sclera of many reptiles. This element has not been well studied in squamates, a diverse order of reptiles with a rich fossil record but debated phylogeny. Squamates inhabit many environments, display a range of behaviours, and have evolved several different body plans. Most importantly, many species have secondarily lost their sclerotic rings. This research investigates the presence of sclerotic rings in squamates and traces the lineage of these bones across evolutionary time. We compiled a database on the presence/absence of the sclerotic ring in extinct and extant squamates and investigated the evolutionary history of the sclerotic ring and how its presence/absence and morphology is correlated with environment and behaviour within this clade. Of the 400 extant species examined (59 families, 214 genera), 69% have a sclerotic ring. Those species that do not are within Serpentes, Amphisbaenia, and Dibamidae. We find that three independent losses of the sclerotic ring in squamates are supported when considering both evolutionary and developmental evidence. We also show that squamate species that lack, or have a reduced, sclerotic ring, are fossorial and headfirst burrowers. Our dataset is the largest squamate dataset with measurements of sclerotic rings, and supports previous findings that size of the ring is related to both environment occupied and behaviour. Specifically, scotopic species tend to have both larger inner and outer sclerotic ring apertures, resulting in a narrower ring of bone than those found in photopic species. Non-fossorial species also have a larger sclerotic ring than fossorial species. This research expands our knowledge of these fascinating bones; with further phylogenetic analyses scleral ossicles could become an extremely useful character trait for inferring the behaviour of fossil squamates.

Morphological diversity in the orbital bones of two teleosts with experimental and natural variation in eye size.

BACKGROUND: Understanding differences in tissue morphology has not been well researched, yet provides crucial insight into evolution. We investigate the effect of eye reduction on the shape of surrounding bones by examining two morphs of the Mexican tetra (Tinaja cavefish and sighted fish), F1 intermediates, zebrafish, a sighted tetra after lens removal and a zebrafish mutant, bum-/- , which has a degenerating lens. RESULTS: Significantly, by comparing the skulls, we show that there are broadly similar effects on bone shape after eye reduction with bones posterior and dorsal to the eye consistently most affected in both species. We conclude that there are conserved mechanisms underlying bone shape changes in response to a reduced or lost eye. Of interest, when we compare the shapes of individual bones and the mode of eye reduction, differences suggest that the finer details of these underlying mechanisms may indeed vary. We also show that cavefish occupy a unique morphospace with respect to skull morphology and that F1 intermediates are most similar to sighted fish than their cavefish parent. CONCLUSIONS: This study highlights the dynamic nature of the vertebrate skull and its ability to respond to tissue changes within the head, a topic which has been largely overlooked in the literature. Developmental Dynamics 244:1109-1120, 2015. © 2015 Wiley Periodicals, Inc.

Expression of SPARC and the osteopontin-like protein during skeletal development in the cichlid fish Oreochromis mossambicus.

BACKGROUND: Bones are mainly composed of calcium hydroxyapatite and a proteinous matrix. In this study, we focus on the bone matrix proteins, the fish osteopontin orthologous protein (osteopontin-like protein; OP-L) and SPARC, because the current knowledge regarding their expression is fragmentary or contradictory. RESULTS: We first provide a comprehensive and detailed description of skeletal development in the cichlid fish Oreochromis mossambicus. Following this, we analyzed the expression pattern of OP-L and SPARC in detail during development. OP-L expression was only found in tissues that undergo ossification (i.e., developing bones and teeth). Furthermore, we show that there is a fundamental difference in cartilage formation of the splanchnocranium and all other cartilages, concerning SPARC expression. Significantly, we show that the initial calcification of cranial bones occurs simultaneously with the expression of OP-L and SPARC in the osteoblast-like cells, which appear early in development. CONCLUSIONS: The difference in SPARC expression during chondrogenesis of the splanchnocranium is likely based on its different evolutionary history compared with the dermatocranium and chondrocranium. Moreover, our results suggest a co-occurrence of the initial calcium deposition and bone matrix protein expression during osteogenesis. Overall, this study enhances our understanding of fish skeletal development and evolution.

Reduced ossification caused by 3D simulated microgravity exposure is short-term in larval zebrafish.

Understanding how skeletal tissues respond to microgravity is ever more important with the increased interest in human space travel. Here, we exposed larval Danio rerio at 3.5 dpf to simulated microgravity (SMG) using a 3D mode of rotation in a ground-based experiment and then studied different cellular, molecular, and morphological bone responses both immediately after exposure and one week later. Our results indicate an overall decrease in ossification in several developing skeletal elements immediately after SMG exposure with the exception of the otoliths, however ossification returns to normal levels seven days after exposure. Coincident with the reduction in overall ossification tnfsf11 (RANKL) expression is highly elevated after 24 h of SMG exposure and also returns to normal levels seven days after exposure. We also show that genes associated with osteoblasts are unaffected immediately after SMG exposure. Thus, the observed reduction in ossification is primarily the result of a high level of bone resorption. This study sheds insight into the nuances of how osteoblasts and osteoclasts in the skeleton of a vertebrate organism respond to an external environmental disturbance, in this case simulated microgravity.