Specific teratogenic effects of different retinoic acid isomers and analogs in the developing anterior central nervous system of zebrafish.

Vertebrate embryos are sensitive to retinoic acid, either in deficiency or in excess. Although all-trans retinoic acid (RA) and 9-cis RA are known to have distinct but overlapping activities in higher organisms, only the all-trans isomer has been investigated in detail as a teratogen in zebrafish. We have identified profound and specific effects of 9-cis RA when administered to zebrafish embryos, and have confirmed the results of prior studies on the teratogenic effects of exogenous all-trans RA. Moreover, we have identified a 1-hr period during gastrulation in which embryos are particularly sensitive to the teratogenic effects of RA. In the course of these investigations, we have also studied the effects of two synthetic retinoids-a 9-cis RA analog, SR11217, and an all-trans RA analog, TTAB. An application of all-trans RA to the early zebrafish gastrula leads to defects that are limited to the caudal midbrain and rostral hindbrain. Our experiments show that an application of exogenous 9-cis RA for a period as short as 1 hr and at a concentration as low as 0.1 mu M can block differentiation of the rostral CNS. We have observed abnormal phenotypes using DIC optics, and have demonstrated further abnormalities using whole-mount immunocytochemical staining with antibodies to HNK-1 and acetylated alpha-tubulin. Major axon tract formation in the anterior CNS is unambiguously disrupted by the administration of 9-cis RA but not all-trans RA. Furthermore, exogenous 9-cis RA produces a qualitative alteration in the multiple-site expression pattern of the hlx-1 gene within the rostral CNS, while treatment with all-trans RA leads only to a weakened expression signal. The administration of TTAB and SR11217 result in distinctive inhibitions of hlx-1 expression. Unlike all-trans RA, which causes premature par-2 expression in the posterior midbrains of a majority of embryos, 9-cis RA leads to a complete deletion of this domain throughout development. These results suggest that 9-cis RA is a more active teratogen than all-trans RA in rostral CNS structures of the zebrafish embryo.