Genetic Control of Skeletogenesis in the Zebrafish

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DESCRIPTION: (Applicant's Abstract) Human skeletal dysplasias, although a significant cause of morbidity, have also proven a rich source of information about the biology of skeletal development and function. The mutated genes encode a wide range of proteins, including transcription factors, signaling molecules, and structural proteins. However, the genes responsible for many diseases affecting the skeleton remain unidentified. To identify similar mutations in a tractable model organism would not only allow detailed study into the underlying disease mechanisms, but could also aid in the cloning of human disease genes. Recent studies have proven the zebrafish, Danio rerio, to be the most practical vertebrate organism for performing large-scale mutational screens. However, previous screens have focused on identifying mutations affecting early development. We propose to screen for mutations affecting the structure and morphology of the adult zebrafish skeleton, using radiography as an efficient method to examine the skeleton in live animals. We also propose to take advantage of the easily visible and accessible zebrafish embryo, to screen for mutations specifically affecting osteoblasts. We will make lines of transgenic fish expressing green fluorescent protein (GFP) under control of the promoter of cbfal, a transcription factor expressed in early osteoblasts. Using this line as a background, we will screen for mutations affecting the pattern of GFP expression, at stages before the osteoblasts can be distinguished morphologically. Using these two screening approaches, we aim to identify genes controlling all stages of skeletogenesis, from patterning to morphogenesis. Additionally, we are focusing on identifying classes of mutants that were unlikely to have been isolated in previous screens, thus further expanding the usefulness of the zebrafish as a model genetic system. The knowledge gained by this work should aid in the identification of human mutations causing skeletal abnormalities, and also lead to better understanding of common problems affecting the skeleton, such as osteoporosis, arthritis, and regeneration after injury.