Retinoic acid (RA) is known to act as a signaling molecule during embryonic development, but little is known about the regulation of RA synthesis from retinol. The rate-limiting step in RA synthesis is the oxidation of retinol, a reaction that can be catalyzed by alcohol dehydrogenase (ADH). Ethanol is also a substrate for ADH, and high levels of ethanol inhibit ADH-catalyzed retinol oxidation. This has prompted us to hypothesize that ethanol-induced defects observed in fetal alcohol syndrome involve ethanol inhibition of ADH-catalyzed RA synthesis. Here, we have examined the effect of ethanol on RA levels in cultured mouse embryos by using a bioassay. Treatment with 100 mM ethanol, but no 10 mM, led to a significant decrease in RA detection in 7.5-day-old embryos. Using whole-mount in situ hybridization, we detected mRNA for class IV ADH, but not ethanol-active cytochrome P450 2E1, in 7.5- and 8.5-day-old embryos, indicating that an ADH-linked pathway exists at these stages for metabolizing retinol and ethanol. Thus, the observed ethanol-induced reduction in RA may be caused by ethanol inhibition of retinol oxidation catalyzed by class IV ADH. In our postulated mechanism for fetal alcohol syndrome, this enzyme may well play a crucial role.

Endogenous retinoic acid (RA) has been observed in vertebrate embryos as early as gastrulation, but the mechanism controlling spatiotemporal synthesis of this important regulatory molecule remains unknown. Some members of the alcohol dehydrogenase (ADH) family catalyze retinol oxidation, the rate-limiting step in RA synthesis. Here we have examined mouse embryos for the presence of endogenous RA and expression of ADH genes. RA was not detected in egg cylinder stage embryos but was detected in late primitive streak stage embryos. Detection of class IV ADH mRNA, but not class I or class III, coincided with the onset of RA synthesis, being absent in egg cylinder embryos but present in the posterior mesoderm of late primitive streak embryos. During neurulation, RA and class IV ADH mRNA were colocalized in the craniofacial region, trunk, and forelimb bud. Class IV ADH mRNA was detected in cranial neural crest cells and craniofacial mesenchyme as well as trunk and forelimb bud mesenchyme. The spatiotemporal expression pattern and enzymatic properties of class IV ADH are thus consistent with a crucial function in RA synthesis during embryogenesis. In addition, the finding of endogenous RA and class IV ADH mRNA in the craniofacial region has implications for the mechanism of fetal alcohol syndrome.

Vitamin A (retinol) regulates embryonic development and adult epithelial function via metabolism to retinoic acid, a pleiotrophic regulator of gene expression. Retinoic acid is synthesized locally and functions in an autocrine or paracrine fashion, but the enzymes involved remain obscure. Alcohol dehydrogenase (ADH) isozymes capable of metabolizing retinol include class I and class IV ADHs, with class III ADH unable to perform this function. ADHs also metabolize ethanol, and high levels of ethanol inhibit retinol metabolism, suggesting a possible mode of action for some of the medical complications of alcoholism. To explore whether any ADH isozymes are linked to retinoic acid synthesis, herein we have examined the expression patterns of all known classes of ADH in mouse embryonic and adult tissues, and also measured retinoic acid levels. Using in situ hybridization, class I ADH mRNA was localized in the embryo to the epithelia of the genitourinary tract, intestinal tract, adrenal gland, liver, conjunctival sac, epidermis, nasal epithelium, and lung, plus in the adult to epithelia within the testis, epididymis, uterus, kidney, intestine, adrenal cortex, and liver. Class IV ADH mRNA was localized in the embryo to the adrenal gland and nasal epithelium, plus in the adult to the epithelia of the esophagus, stomach, testis, epididymis, epidermis, and adrenal cortex. Class III ADH mRNA, in contrast, was present at low levels and not highly localized in the embryonic and adult tissues examined. We detected significant retinoic acid levels in the fetal kidney, fetal/adult intestine and adrenal gland, as well as the adult liver, lung, testis, epididymis, and uterus--all sites of class I and/or class IV ADH gene expression. These findings indicate that the expression patterns of class I ADH and class IV ADH, but not class III ADH, are consistent with a function in local retinoic acid synthesis needed for the development and maintenance of many specialized epithelial tissues.

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In order for nuclear retinoic acid receptors to mediate retinoid signaling, the ligand retinoic acid must first be produced from its vitamin A precursor retinal. Biochemical studies have shown that retinal can be metabolized in vitro to retinoic acid by members of the aldehyde dehydrogenase enzyme family, including ALDH1. Here we describe the first direct evidence that ALDH1 plays a physiological role in retinoic acid synthesis by analysis of retinoid signaling in Xenopus embryos, which have plentiful stores of maternally derived retinal. The Xenopus ALDH1 gene was cloned and shown to be highly conserved with chick and mammalian homologs. Xenopus ALDH1 was not expressed at blastula and gastrula stages, but was expressed at the neurula stage. We used a retinoic acid bioassay to demonstrate that retinoic acid is normally undetectable in embryos from fertilization to the initial gastrula stage, but that a tremendous increase in retinoic acid occurs during neurulation when ALDH1 is first expressed. Overexpression of ALDH1 by injection of Xenopus embryos with mRNAs encoding the mouse, chick or Xenopus ALDH1 homologs induced high levels of retinoic acid detection during the blastula stage. Thus, premature expression of ALDH1 stimulates premature synthesis of retinoic acid. These findings reveal an important conserved role for ALDH1 in retinoic acid synthesis in vivo, and demonstrate that conversion of retinoids from the aldehyde form to the carboxylic acid form is a crucial regulatory step in retinoid signaling.

Humans possess five classes of alcohol dehydrogenase (ADH), including forms able to oxidize ethanol or formaldehyde as part of a defense mechanism, as well as forms acting as retinol dehydrogenases in the synthesis of the regulatory ligand retinoic acid. However, the mouse has previously been shown to possess only three forms of ADH. Hybridization analysis of mouse genomic DNA using cDNA probes specific for each of the five classes of human ADH has now indicated that mouse DNA cross-hybridizes to only classes I, III, and IV. With human class II or class V ADH cDNA probes, hybridization to mouse genomic DNA was very weak or undetectable, suggesting either a lack of these genes in the mouse or a high degree of mutational divergence relative to the human genes. cDNAs for murine ADH classes I and III have previously been cloned, and we now report the cloning of a full-length mouse class IV ADH cDNA. In Northern blot analyses, mouse class IV ADH mRNA was abundant in the stomach, eye, skin, and ovary, thus correlating with the expression pattern for the mouse Adh-3 gene previously determined by enzyme analysis. In situ hybridization studies on mouse stomach indicated that class IV ADH transcripts were abundant in the mucosal epithelium but absent from the muscular layer. Comparison of the expression patterns for all three mouse ADH genes indicated that class III was expressed ubiquitously, whereas classes I and IV were differentially expressed in an overlapping set of tissues that all contain a large component of epithelial cells. This expression pattern is consistent with the ability of classes I and IV to oxidize retinol for the synthesis of retinoic acid known to regulate epithelial cell differentiation. The results presented here indicate that the mouse has a simpler ADH gene family than the human but has conserved class IV ADH previously shown to be a very active retinol dehydrogenase in humans.

The vitamin A metabolite retinoic acid plays an essential signaling role in spermatogenesis by acting as a ligand for nuclear retinoic acid receptors. However, little is known about the regulation of retinoic acid synthesis from vitamin A (retinol). Here we have examined mouse testis and epididymis for the presence of endogenous retinoic acid and for the expression of genes encoding class I and class IV alcohol dehydrogenases (ADH), both of which catalyze retinol oxidation, the rate-limiting step in the conversion of retinol to retinoic acid. Using a bioassay we found that mouse testis and epididymis both have significant levels of retinoic acid ranging from 7 to 8 pmol/g, an amount known to be sufficient to optimally activate retinoic acid receptors. In situ hybridization analysis of mouse testis revealed that class I ADH mRNA was localized in Sertoli cells and Leydig cells, while class IV ADH mRNA was confined to late spermatids. In the epididymis, class I ADH mRNA was detected in both principal and basal cells, whereas class IV ADH mRNA was limited to basal cells. Immunohistochemical analyses of testis indicated that class I ADH protein was localized in Sertoli and Leydig cells, whereas class IV ADH protein was observed only in late spermatids. Class I ADH protein was localized in principal and basal cells of the cauda epididymidis but only in basal cells of the caput epididymidis. Class IV ADH protein was limited to basal cells along the entire length of the epididymis. These results support a role for ADHs during spermatogenesis, potentially as retinol dehydrogenases catalyzing local retinoic acid synthesis in the testis and epididymis.

Retinoid signaling requires the conversion of retinol to retinoic acid by a two-step process, the first of which can be catalyzed in vitro by class I and class IV alcohol dehydrogenases (ADH). These enzymes may participate in local retinoic acid synthesis in some target tissues, although other studies suggest retinoic acid may also be supplied to tissues via the bloodstream, much like an endocrine hormone. Here we have analyzed the expression of these two ADHs as well as retinoic acid production in the adrenal gland, an organ known to be an endocrine source of other hormones. In situ hybridization revealed high levels of both class I and class IV ADH messenger RNAs in adrenal glands of 16.5-day mouse embryos and adults. Class I ADH protein was immunohistochemically detected in embryonic and adult adrenal glands, the latter primarily in the zona fasiculata of the cortex. Abundant class IV ADH protein was detected in the embryonic adrenal as well as in the zona glomerulosa and zona fasiculata of the adult adrenal cortex. Interestingly, class IV ADH protein was found in only a subset of adult cortical cells arranged in radial columns, thus providing further evidence for centripetal cell migration during adrenocortical differentiation. Using a retinoic acid bioassay, adrenal glands from 16.5 day embryos were found to have significantly higher levels of retinoic acid than embryonic liver. The adult adrenal was found to have approximately 15.5 pmol/g of retinoic acid, whereas the adult liver had 24.8 pmol/g, and brain, heart, and spleen each had less than 1.0 pmol/g. Because previous findings indicate that the adrenal gland is not a retinoid target tissue, our detection of both alcohol/retinol dehydrogenases and significant amounts of retinoic acid in this organ suggests that it functions as a potential endocrine source of this hormone during mouse development.