Alcohol is a common addictive substance and prenatal alcohol exposure could cause fetal alcohol spectrum disorder (FASD) and can lead to various birth defects. The small teleost zebrafish (Danio rerio) has been identified as a fine animal model in developmental biology and toxicological research. Zebrafish models are widely used to study the harmful effects of alcohol and limited studies are available on the craniofacial and skin malformations associated with FASD. The present study attempts to investigate the effect of alcohol on early zebrafish embryonic development. The effects of prenatal alcohol exposure on neural crest cell-derived organ formation, including pharyngeal dentition, palatal bones and skin melanocytes were analysed. Whole-mount cartilage and bone staining and imaging techniques were applied to determine the effects of alcohol on the above-mentioned structures. The tooth size and shape were affected by alcohol exposure, but the number of teeth in the pharyngeal dentition was not affected. Only first-generation teeth showed size differences. The alcohol-exposed ethmoid bone, which is homologous to the human hard palate, was smaller and less dense in cell arrangement compared with the control medial ethmoid bone. The skin pigmentation defects included reduced melanocyte density, melanin contraction, smaller melanocyte surface area and aberrations in melanosome dispersion, revealing that alcohol significantly influenced and downregulated each and every step of the melanocyte developmental process. This descriptive study summarises the effects of alcohol on the development of neural crest cell-derived structures and highlights the importance of zebrafish in studying the phenotypic characteristics of fetal alcohol spectrum disorder.
Astyanax mexicanus is being recognized as an intriguing teleost model in developmental biological studies. A. mexicanus exists as two morphs eyed, pigmented surface dwelling morph known as surface fish and an unpigmented blind cave dwelling cavefish. The midline bones of anterior neurocranium of A. mexicanus consists of parasphenoid, ethmoid and vomer bone. The detailed anatomical development of neuorocranial skeleton of this fish is unexplored thus far. The present comparative study is designed to understand the palatal bones, parasphenoid, ethmoid and vomer bone development among surface fish and in two population of cavefish Pachon and Tinaja. Cavefish and surface fish (Astyanax mexicanus) were included in to this study. Fish were fixed at different time point ranging from 10 dpf, 20 dpf, 35dpf, 60 dpf and 5 year old adults. Alizarin red bone stained were used to stain the skeleton. Dissected samples were examined under the stereo microscope for the ossification sequence of the palatal bones. The micro‐CT images of adult fish were taken using SkyScan micro‐CT imager (Bruker) and CT vox software (Bruker) was used for image analysis. First ossification center appears at the parasphenoid bone at 5 dpf. The anterior neurocranial bones ossified via endochondral ossification. Both cavefish and surface fish ossification starts from the ossification center locate in the center of the parasphenoid cartilaginous palate. Ossification progresses towards anterior direction and merge with the ossifying maxillary bone. The ossification extend up to the notochord in posterior direction. The Micro‐CT images showed that the morphology of parasphenoid bone is different in each eye morph. In surface fish parasphenoid bone curved and it has 20° angle of elevation towards the anterior direction. In contrast, parasphenoid bone found to be straight in Pachon and Tinaja cavefish. Absence of eye structures have an influence on determining the shape of parasphenoid bone. Support or Funding Information We would like to thank the Natural Sciences and Engineering Research Council of Canada (#2019‐05364) for funding this research. We acknowledge the support from University of Manitoba.
Astyanax mexicanus is a teleost fish that belongs to the order Characiform. Ithas two distinct eye morphs as surface‐dwelling, pigmented eyed surface fish population, and an eyeless cavefish population. Accumulation of functional mutations in the genes which are important for eye development led to the loss of eyes in cavefish. Some cranial changes have occurred from the direct effect of eye loss in cavefish including size and position of the circumorbital bones, ossified sclera, and the shape of both suborbital 3 and the supraorbital bone. The maxillary teeth, the positions of suborbital 4 to suborbital 6, and the shape of the opercular bone were found to be unaffected by the eye loss. Unlike higher vertebrates, the teleost eyeball does not cover by a bony socket and it has direct communication with the inner neurocranium. However, the influence of eye development on the inner neurocranial bones has gained little attention in research. In A. mexicanus palate bones are mainly made of a series of bones such as parasphenoid bone, basioccipital, vomer, and ethmoid. Among them, parasphenoid bone makes the largest part of the palate. This study was designed to understand the effect of eye loss in cavefish on parasphenoid bone development, shape determination, and bone remodeling activity. A. mexicanus populations; Surface fish and Pachon cavefish at 5dpf, 10 dpf, 20 dpf, 35 dpf, and 60 dpf subjected to alizarin red bone staining. Adult surface fish and two cave populations Pachon and Tinaja cavefish were subjected to micro‐CT imaging and morphometric analysis was carried out using those images to compare the parasphenoid bone anatomy amongst these three morphs. TRAP and ALP staining were used to determine the bone remodeling activity in 60 dpf, A. mexicanus populations of surface and Pachon cavefish. A. mexicanus cavefish and surface are similar to each other in bone development, articulation, and ossification with certain exceptions such as late protrusion of lateral wings in cavefish. The straightened parasphenoid bone observed in cavefish and curved parasphenoid bone observed in surface fish beneath the eye orbit. Bone remodeling activity in cavefish was found to be increased compared to the surface fish. Cavefish have a direct or indirect effect on eye loss on the development of bones in the cranial region. Differential bone remodeling activity observed in cave and surface fish may result from the limited resources in the cave environment and as a long‐term adaptation into troglomorphic cave environment.
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