Regulation of retinoic acid in early zebrafish development.

摘要

Retinoic acid (RA) is an important developmental signaling molecule responsible for the patterning of multiple vertebrate tissues. RA is also a potent teratogen, causing multi-organ birth defects in humans. Endogenous RA levels must therefore be tightly controlled in the developing embryo. Based on a microarray approach to identify genes that function as negative feedback regulators of retinoic acid signaling, I screened for genes expressed in early somite-stage embryos that respond oppositely to treatment with RA versus RA antagonists, and validated them by RNA in situ hybridization. To study the in vivo RA related phenotype at early developmental stages, I designed and tested assays on two zebrafish mutants, cyp26a1 mutant giraffe and aldh1a2 mutant neckless, both having aberrant endogenous RA level. By using these assays and the specific marker for RA dependent developmental processes, I characterize the cyp26a1 mutant in detail to study the function of Cyp26a1 on zebrafish early development. It turned out that cyp26a1 mutant only has limited developmental defects in vivo, which suggesting other regulating mechanisms must play important roles. Focusing on genes known to be involved in RA metabolism, I determined that a dehydrogenase and reducatse short-chain protein family member, dhrs3a, is both RA dependent and strongly RA inducible. Dhrs3a is known to catalyze the dehydrogenation of the RA precursor retinaldehyde to vitamin A, however its developmental function has not been demonstrated. Using morpholino knock down and mRNA over-expression, I demonstrated that Dhrs3a functions as an RA feedback inhibitor with primary effects on retinoic acid-dependent events in the central nervous system. Based on the finding of dhrs3a function and our validated microarray results, as well as results from other labs, I proposed a feedback regulation model on how the accuracy of RA function is achieved. In this model, the RA was regulated by both the positive and negative feedback circuits through multiple RA responsive genes, and forming a regulatory network from multiple levels.