Teaching Zebrafish Development in a STEM-Based Science Camp for Middle School Students.

Zebrafish have been used as an education tool for students of all ages and can be used in many learning environments to teach different fields of science. In this study, we focus on the biology of zebrafish. We describe an educational program within a weeklong science camp for students between 12 and 14 years old. The methodology described is based on running annual science camps over an 11-year period. In these camps, students learnt about the developmental stages of zebrafish, as well as general zebrafish biology, husbandry, ecology, behavior, and reproduction. This article describes how to provide students and educators with an educational program to explore, discover, and contribute to the ever-evolving landscape of biological understanding through active and visual learning. We describe the methodology, the evaluation, revisions to our program over time, and future directions for expansion.

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.

The elusive scleral cartilages: Comparative anatomy and development in teleosts and avians.

The sclera of all vertebrate eyes is comprised of connective tissue, with some organisms developing cartilage within this tissue. A review of the cartilages that have been described in the vertebrate sclera and their anatomical relationships is discussed together with their potential homology. Incorrect terminology erroneously implies similarity in location, development, morphology, and evolution, which may lead some scientists to assume all cartilages in orbit are the same elements when reading the literature. Therefore, new terminology to distinguish the different types of cartilage associated with the vertebrate eye is proposed. The scleral cartilages that are likely homologous to one another and which are situated in the sclera, should be termed scleral cartilages sensu stricto, while other cartilages in the sclera should be termed ocular cartilages. Some of the cartilages also ossify, and these bones should be distinguished from the scleral ossicles. The plasticity of the scleral tissue layer and its range of morphologies from fibrous to cartilaginous connective tissue across different vertebrate lineages are also described. This review also highlights several gaps in our understanding of the vertebrate scleral cartilages, in particular.

Clomiphene Citrate and Enclomiphene Hydrochloride Exposure Is Associated With Interfrontal Suture Fusion in Zebrafish.

Background: The last several decades have witnessed an increase in metopic craniosynostosis incidence. Population-based studies suggest that pharmacological exposure in utero may be responsible. This study examined effects of the fertility drug clomiphene citrate (CC) on calvarial development in an established model for craniofacial development, the zebrafish Danio rerio. Results: Zebrafish larvae were exposed to clomiphene citrate or its isomer enclomiphene for five days at key points during calvarial development. Larvae were then raised to adulthood in normal rearing water. Zebrafish were analyzed using whole-mount skeletal staining. We observed differential effects on survivability, growth and suture formation depending on the treatment. Treatments with CC or enclomiphene at 5.5 mm SL led to increased fusion of the interfrontal suture (p < .01) compared to controls. Conclusions: Exposure to fertility drugs appears to affect development of the cranial vault, specifically the interfrontal suture, in zebrafish. Further research is required to identify the signaling mechanisms at play. This work suggests that fertility drug treatment may contribute to the increased incidence of metopic craniosynostosis observed globally.

Historique : L'incidence de craniosynostoses métopiques a augmenté ces dernières décennies. D'après les études en population, l'exposition aux médicaments in utero pourrait en être responsable. La présente étude traite des effets d'un agent ovulatoire, le citrate de clomifène (CC), sur le développement de la voûte crânienne d'un modèle établi de développement crâniofacial, le poisson-zèbre Danio rerio. Résultats : Les chercheurs ont exposé des larves de poisson-zèbre au citrate de clomifène ou à son isomère, l'enclomiphène, sur une période de cinq jours à des moments clés du développement de la voûte crânienne. Ils les ont ensuite élevés jusqu'à l'âge adulte dans de l'eau normale. Ils ont analysé les poissons-zèbres par coloration du squelette entier et observé des effets différentiels sur la capacité de survie, la croissance et la formation des sutures en fonction du traitement. Les traitements au CC ou à l'enclomiphène d'une longueur standard de 5,5 mm ont entraîné une fusion accrue de la suture métopique (p < 0,01) par rapport aux sujets témoins. Conclusions : L'exposition aux agents ovulatoires semble influer sur le développement de la voûte crânienne, notamment la suture métopique, chez le poisson-zèbre. D'autres recherches devront être réalisées pour déterminer les mécanismes de signalisation en jeu. Selon la présente étude, les agents ovulatoires peuvent contribuer à l'incidence accrue de craniosynostose métopique sur la scène mondiale.

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.

Effects of simulated microgravity and vibration on osteoblast and osteoclast activity in cultured zebrafish scales.

Zebrafish cultured scales have been used effectively to study cellular and molecular responses of bone cells. In order to expose zebrafish scales to simulated microgravity (SMG) and/or vibration, we first determined via apoptosis staining whether cells of the scale survive in culture for two days and hence, we restricted our analyses to two-day durations. Next, we measured the effects of SMG and vibration on cell death, osteoclast tartrate-resistant acid phosphatase, and osteoblast alkaline phosphatase activity and on the number of Runx2a positive cells. We found that during the SMG treatment, osteoclast tartrate-resistant acid phosphatase activity increased on average, while the number of Runx2a positive cells decreased significantly. In contrast, SMG exposure caused a decrease in osteoblast activity. The vibration treatment showed an increase, on average, in the osteoblast alkaline phosphatase activity. This study demonstrates the effect of SMG and vibration on zebrafish scales and the effects of SMG on bone cells. We also show that zebrafish scales can be used to examine the effects of SMG on bone maintenance.

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.

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.

Girls Get WISE-A programming model for engaging girls+ in STEM.

The majority of STEM disciplines in Canada are male-dominated and there is a significant lack of programming available to girls. The Girls Get WISE program is a university-based program that is funded by the federal government, the university, and corporate sponsorship. This program is delivered in person by educational professionals, science students, and past participants. By engaging girls in hands-on interactive STEM activities in a safe and fun space, this program provides an opportunity for young women to showcase their talents and excitement for science-based topics. The features of this program and its evaluation over a 10 year period are described here.

Accurate whole-mount bone and cartilage staining requires acid-free conditions.

Bone and cartilage staining has provided anatomists with the ability to generate detailed descriptions of the adult and developing skeleton. Typically, Alizarin red S and Alcian blue are used for the staining of bone and cartilage, respectively. The binding of Alizarin red S and calcium is most stable at basic conditions, however, Alcian blue exhibits specific binding to polyanionic substances such as mucopolysaccharides under acidic conditions. Typical bone and cartilage staining protocols are conducted under acidic conditions. Because of this discrepancy in optimal pH, issues can arise in the staining of small specimens such as larval fish. Specifically, staining embryonic or larval specimens under acidic conditions can cause decalcification of small bones. Decalcification can completely inhibit the uptake of Alizarin red S in small bones. In order to mitigate this issue, researchers have developed an acid-free staining protocol that utilizes the concept of critical electrolyte concentration. While many researchers have adopted acid-free bone and cartilage staining, some researchers continue to stain these small specimens with acidic staining protocols. To ensure the reliability and validity of our skeletal descriptions, we urge scientists to utilize acid-free staining protocols when analyzing the skeletons of larval or embryonic specimens.

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.

Quantitative gene expression dynamics of key placode signalling factors in the embryonic chicken scleral ossicle system.

Development of the scleral ossicles, a ring of bony elements within the sclera, is directed by a series of papillae that arise from placodes in the conjunctival epithelium over a 1.5-day induction period in the chicken embryo. The regular spacing of the papillae around the corneal-scleral limbus suggests that their induction may be regulated by a reaction-diffusion mechanism, similar to other epithelial appendages. Some key placode signalling molecules, including ß-catenin, are known to be expressed throughout the induction period. However, others have been studied only at certain stages or have not been successfully detected. Here we use qPCR to study the gene expression patterns of the wingless integration (WNT)/ß-catenin, bone morphogenetic protein (BMP), ectodysplasin (EDA), fibroblast growth factor (FGF) and hedgehog (HH) signalling families in discrete regions of the eye throughout the complete conjunctival placode and papillae induction period. This comprehensive analysis revealed a variable level of gene expression within specific eye regions, with some genes exhibiting high, moderate or low changes. Most genes exhibited an initial increase in gene expression, followed by a decrease or plateau as development proceeded, suggesting that some genes are important for a brief initial period whilst the sustained elevated expression level of other genes is needed for developmental progression. The timing or magnitude of these changes, and/or the overall gene expression trend differed in the temporal, nasal and/or dorsal eye regions for some, but not all genes, demonstrating that gene expression may vary across different eye regions. Temporal and nasal EDA receptor (EDAR) had the greatest number of strong correlations (r > 0.700) with other genes and ß-catenin had the greatest number of moderate correlations (r = 0.400-0.700), while EDA had the greatest range in correlation strengths. Among the strongly correlated genes, two distinct signalling modules were identified, connected by some intermediate genes. The dynamic gene expression patterns of the five signalling pathways studied here from conjunctival placode formation through to papillae development is consistent with other epithelial appendages and confirms the presence of a conserved induction and patterning signalling network. Two unique gene expression patterns and corresponding gene interaction modules suggest functionally distinct roles throughout placode development. Furthermore, spatial differences in gene expression patterns among the temporal, nasal and dorsal regions of the eye may indicate that the expression of certain genes is influenced by mechanical forces exerted throughout development. Therefore, this study identifies key placode signalling factors and their interactions, as well as some potential region-specific features of gene expression in the scleral ossicle system and provides a basis for further exploration of the spatial expression of these genes and the patterning mechanism(s) active throughout conjunctival placode and papillae 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.

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.

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.