Endogenous retinoids are potential regulators of vertebrate embryogenesis that have been implicated in early anterior-posterior patterning and limb-bud development. We have characterized the temporal and spatial distribution of 9-cis-retinoic acid in the Xenopus embryo and compared it to two other retinoids, afl-tmns-retinoic acid and alI-frns-retinoyl-3glucuronide. 9-cis-Retinoic acid is first detected after the midblastula transition and by the end of gastruation is localz primarily within the anterior and posterior dorsal regions ofthe embryo. Since 9-cis-retinoic acid is a 6-fold more potent dysmorphogen than trans-retinoic acid, we suggest that it is involved in the early specification of the Xenopus anterior-posterior aids.The organization of the vertebrate body plan is dependent upon communication between groups of cells in the early embryo. It has been proposed that all-trans-retinoic acid (trans-RA), a metabolite of retinol (vitamin A) and a member of a class of compounds known as retinoids, functions as one of the molecular signals in early vertebrate development (1-9). In support of this proposal, experiments have shown that trans-RA affects cellular differentiation (10) and that embryonic exposure to trans-RA causes severe malformations in a number ofvertebrate species (11-13). Furthermore, retinoid deficiency during embryonic development also produces malformations (14,15). The cellular effects of RA isomers appear to be mediated by two distinct classes of receptors within the steroid-thyroid nuclear receptor superfamily, the RA receptors (RARs) and the retinoid X receptors (RXRs), which presumably function by altering the transcription of specific target genes (16). In addition, cellular RA and retinol binding proteins (CRABPs and CRBPs) appear to modulate the localization, availability, and metabolism of these retinoids within the organism (17).Despite the ability of exogenous all-trans-RA to cause major alterations to the anterior-posterior (A-P) axis of mice (2, 3) and frogs (1, 4-6), evidence that endogenous retinoids are important modulators of early development has been difficult to obtain. Indirect evidence that retinoids are present in restricted regions of the embryo has been obtained from in vivo studies with transgenic mice (18-21) and in vitro studies with rat (22) and chicken (23) tissue explants. Chicken embryos also exhibit regional differences in the capacity to convert retinol to trans-RA (24), with the maximum activity found in Hensen's node. Since these studies have not directly identified the specific retinoids in the early embryo or measured their spatial distribution during early embryonic stages, we have addressed these questions using the amphibian embryo. The results from these studies indicate that a number of retinoids were present in the early amphibian embryo and that the levels of three of these retinoids vary both spatially and temporally in early development. Notably, 9-cis-RA was first detected after the midblastula transition and at the end of gastrulation ...

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Sodium nitroprusside (SNP), a chemical that is readily converted to nitric oxide (NO) in biological systems, was microinjected into the amniotic fluids of cultured whole rat conceptuses on day 10.5 of gestation and dysmorphogenic/embryotoxic effects were evaluated after a 24 hr incubation period. Injections of 217 ng/embryo (approximately 800 microM) resulted in whitened zones of dead cells in a discretely circumscribed region within the mesencephalon closely associated with the neural tube. These zones were observed with a high incidence after SNP microinjections and were referred to as "white caps" because of their microscopic appearance. At higher concentrations, the whitened zone extended into the rhombencephalon and occasionally appeared to extend the full length of the dorsal midline. The whitened zones of tissue separated readily from the apparently normal underlying tissues upon removal or disturbance of the amniotic membrane. Coinjection of ferrous hemoglobin with SNP selectively prevented the appearance of "white caps" but not other embryotoxic manifestations. Microinjections of the breakdown products of light-exposed SNP elicited generalized embryotoxicity but "white caps" were not observed. In separate experiments, we found that embryonic enzymes catalyzed significant conversion of arginine to citrulline, indicating expression of NO-synthase during organogenesis. NG-monomethyl-L-arginine (L-NMMA), a specific inhibitor of NO-synthase, was microinjected (50-150 ng/embryo; approximately 200-600 microM) on day 10.5 of gestation and produced malformations that differed markedly from those elicited by SNP. Failure of anterior and posterior neural tube closure and profound underdevelopment of the hyoid arch and optic cup were observed at concentrations that produced no apparent growth deficit. These studies with SNP and L-NMMA indicated that both an excess and a deficiency of NO can be embryotoxic/dysmorphogenic and suggest important roles for optimal levels of NO and NO synthases in normal embryonic development.

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