Targeted disruption of Aldh1a1 (Raldh1) provides evidence for a complex mechanism of retinoic acid synthesis in the developing retina.

Genetic studies have shown that retinoic acid (RA) signaling is required for mouse retina development, controlled in part by an RA-generating aldehyde dehydrogenase encoded by Aldh1a2 (Raldh2) expressed transiently in the optic vesicles. We examined the function of a related gene, Aldh1a1 (Raldh1), expressed throughout development in the dorsal retina. Raldh1(-/-) mice are viable and exhibit apparently normal retinal morphology despite a complete absence of Raldh1 protein in the dorsal neural retina. RA signaling in the optic cup, detected by using a RARE-lacZ transgene, is not significantly altered in Raldh1(-/-) embryos at embryonic day 10.5, possibly due to normal expression of Aldh1a3 (Raldh3) in dorsal retinal pigment epithelium and ventral neural retina. However, at E16.5 when Raldh3 is expressed ventrally but not dorsally, Raldh1(-/-) embryos lack RARE-lacZ expression in the dorsal retina and its retinocollicular axonal projections, whereas normal RARE-lacZ expression is detected in the ventral retina and its axonal projections. Retrograde labeling of adult Raldh1(-/-) retinal ganglion cells indicated that dorsal retinal axons project to the superior colliculus, and electroretinography revealed no defect of adult visual function, suggesting that dorsal RA signaling is unnecessary for retinal ganglion cell axonal outgrowth. We observed that RA synthesis in liver of Raldh1(-/-) mice was greatly reduced, thus showing that Raldh1 indeed participates in RA synthesis in vivo. Our findings suggest that RA signaling may be necessary only during early stages of retina development and that if RA synthesis is needed in dorsal retina, it is catalyzed by multiple enzymes, including Raldh1.

Novel retinoic acid generating activities in the neural tube and heart identified by conditional rescue of Raldh2 null mutant mice.

Retinoid control of vertebrate development depends upon tissue-specific metabolism of retinol to retinoic acid (RA). The RA biosynthetic enzyme RALDH2 catalyzes much, but not all, RA production in mouse embryos, as revealed here with Raldh2 null mutants carrying an RA-responsive transgene. Targeted disruption of Raldh2 arrests development at midgestation and eliminates all RA synthesis except that associated with Raldh3 expression in the surface ectoderm of the eye field. Conditional rescue of Raldh2(-/-) embryos by limited maternal RA administration allows development to proceed and results in the establishment of additional sites of RA synthesis linked to Raldh1 expression in the dorsal retina and to Raldh3 expression in the ventral retina, olfactory pit and urinary tract. Unexpectedly, conditionally rescued Raldh2(-/-) embryos also possess novel sites of RA synthesis in the neural tube and heart that do not correspond to expression of Raldh1-3. RA synthesis in the mutant neural tube was localized in the spinal cord, posterior hindbrain and portions of the midbrain and forebrain, whereas activity in the mutant heart was localized in the conotruncus and sinus venosa. In the posterior hindbrain, this novel RA-generating activity was expressed during establishment of rhombomeric boundaries. In the spinal cord, the novel activity was localized in the floorplate plus in the intermediate region where retinoid-dependent interneurons develop. These novel RA-generating activities in the neural tube and heart fill gaps in our knowledge of how RA is generated spatiotemporally and may, along with Raldh1 and Raldh3, contribute to rescue of Raldh2(-/-) embryos by producing RA locally.

Distinct retinoid metabolic functions for alcohol dehydrogenase genes Adh1 and Adh4 in protection against vitamin A toxicity or deficiency revealed in double null mutant mice.

The ability of class I alcohol dehydrogenase (ADH1) and class IV alcohol dehydrogenase (ADH4) to metabolize retinol to retinoic acid is supported by genetic studies in mice carrying Adh1 or Adh4 gene disruptions. To differentiate the physiological roles of ADH1 and ADH4 in retinoid metabolism we report here the generation of an Adh1/4 double null mutant mouse and its comparison to single null mutants. We demonstrate that loss of both ADH1 and ADH4 does not have additive effects, either for production of retinoic acid needed for development or for retinol turnover to minimize toxicity. During gestational vitamin A deficiency Adh4 and Adh1/4 mutants exhibit completely penetrant postnatal lethality by day 15 and day 24, respectively, while 60% of Adh1 mutants survive to adulthood similar to wild-type. Following administration of a 50-mg/kg dose of retinol to examine retinol turnover, Adh1 and Adh1/4 mutants exhibit similar 10-fold decreases in retinoic acid production, whereas Adh4 mutants have only a slight decrease. LD(50) studies indicate a large increase in acute retinol toxicity for Adh1 mutants, a small increase for Adh4 mutants, and an intermediate increase for Adh1/4 mutants. Chronic retinol supplementation during gestation resulted in 65% postnatal lethality in Adh1 mutants, whereas only approximately 5% for Adh1/4 and Adh4 mutants. These studies indicate that ADH1 provides considerable protection against vitamin A toxicity, whereas ADH4 promotes survival during vitamin A deficiency, thus demonstrating largely non-overlapping functions for these enzymes in retinoid metabolism.