The view that the RDH first reaction occurs in a ubiquitous and unregulated fashion was challenged by the identification of Rdh10trex, a mutation that dramatically impairs RA synthesis via disruption of RDH10, a spatially and temporally regulated membrane-bound SDR. However, because the RDH reaction had previously been thought to be carried out by redundant or ubiquitous ADH family members, and because the Rdh10trex mutant embryos exhibited a milder phenotype and survived longer than embryos lacking Aldha2, the RDH first reaction continued to be thought of as relatively unimportant for the spatiotemporal control of RA synthesis. The residual production of RA and the less severe phenotype of Rdh10trex embryos compared to Aldh1a2 mutant embryos indicated that some RDH activity remained intact in the Rdh10trex embryos, mediated possibly by remaining activity of the hypomorphic Rdh10trex point mutant enzyme, or by other RDH enzymes, or by ADH enzymes. Given that genetic or environmental perturbations causing excessive or insufficient levels of RA cause embryonic malformations, it is important to resolve the question of which enzymes catalyze the RDH reaction during embryogenesis and, further, to understand whether these enzymes function redundantly and ubiquitously or if their regulated expression may serve as a control point for production of RA. To gain insight into the enzymes and regulation of RA synthesis, we have generated a knockout mutation of Rdh10 and demonstrate that complete loss of the RDH10 enzyme results in embryonic death ,E10.5. Rdh10delta/delta embryos have severe defects in growth, axial turning, and cardiac and pharyngeal development. The severe embryonic phenotypes of Rdh10delta/delta mutants unequivocally demonstrate that redundant or ubiquitous enzymes, including the members of the ADH family, do not contribute significantly to physiologically relevant production of RA during embryogenesis. Moreover, the severity of the phenotypes resulting from loss of an individual enzyme, RDH10, provides clear proof that the first RDH reaction of Vitamin A metabolism is as critical a control point in the synthesis of RA as the second RALDH reaction. Although the phenotype of Rdh10delta/delta mutant embryos is very severe, it is not identical to the extreme phenotype of loss of Aldha2. Loss of RALDH2 function results in embryos that apparently cease to grow at by E8.5, whereas loss of Rdh10 results in embryos that apparently cease to grow at around E9.0. The slightly milder phenotype of the Rdh10delta/delta embryos is likely due to the small amount of residual RA produced at E9.5 within the trunk region of affected embryos. The identity of the enzymes responsible for the residual trunk RA at E9.5 is unknown. It is possible the small amount of residual RA production in Rdh10delta/delta mutant embryos represents the contribution of other, as yet unidentified, SDR enzymes closely related to RDH10 or, alterna- tively, ADH enzymes. Recently, we demonstrated that a compound knockout of multiple Adh genes does not impair the residual RA synthesis in Rdh10trex embryos nor exacerbate the phenotype [35], a result which argues against a role for ADHs. Whichever enzymes are ultimately found to be responsible for the small amount of residual RA, it is clear that they are insufficient to produce physiologically relevant levels of RA and are unable to compensate for loss of Rdh10. Concurrent with the present study, a distinct knockout allele of Rdh10, a deletion of Rdh10 exon 4, was reported in the literature [36]. The phenotypes of the Rdh10 exon 4 deletion allele (Rdh102/2) are largely consistent with the Rdh10delta allele we describe here. In both cases, the majority of embryos survive until ,E10.5 and exhibit defects in growth and extension of the body axis, absence of posterior pharyngeal arches, compacted somites, and small or duplicated otic vesicles. RA signaling is also notably absent from the forebrain region in both knockout models at this RDH10 Control of RA Synthesis stage. The primary difference is that we do not observe embryo survival beyond E10.5 in our Rdh10delta mutants, whereas a small fraction (,10%) of exon 4 deletion mutants survive until E12.5. This difference may be related to variation in strain background or the relative importance of the individual exons for Rdh10 activity that were deleted. We also examined RA signaling in Rdh10delta/delta embryos in a broader range of embryos and observe that between the 1–6 somite stages (Fig. 5C–F), RA signaling is not detected in Rdh10delta/delta embryos compared to the robust detection of reporter activity in control embryos. Taken together, these data indicate the important role of Rdh10 in RA signaling during the early critical E8.0–8.5 window of development. Feedback regulation on RA metabolism In addition to showing that RDH10 is the predominant enzyme responsible for embryonic conversion of retinol to retinal, the data presented here reveal that Rdh10 gene expression is modulated by presence or absence of RA, being upregulated by RA deficiency in Aldha2gri/gri mutants. The upregulation of Rdh10 expression when RA synthesis is impaired demonstrates that Rdh10 is a point for feedback control in Vitamin A metabolism and RA synthesis in mammals (Fig. 6C). A similar mode of regulation has been observed in Xenopus and zebrafish, indicating that the mechanistic feedback control of Rdh10 to modulate the RA synthesis pathway is evolutionarily conserved [24,25]. The evolutionary conservation of feedback regulation of the Rdh10 gene by RA lends further support to the concept that RDH10-mediated oxidation of retinol to retinal plays an important role in regulating the key process of embryonic Vitamin A metabolism and RA synthesis. The conversion of retinol to retinal is a reversible reaction. Enzymes of the SDR family can mediate both the forward and the reverse directions. Transcriptional regulation of SDR enzymes to mediate RA homeostasis is observed in other contexts. In zebrafish embryos, excess RA induces expression of dhrs3a, an SDR that catalyzes the reduction of retinal into retinol [25]. In adult mouse liver, chronic exposure to ethanol causes complex changes in RA metabolism and is associated with transcriptional changes in RA metabolic genes including induction of the SDR Dhrs9 [37]. Together these data suggest that reversible interconversion of retinol to retinal by SDRs serve as a key control step in RA homeostasis. Consistent with this, biochemical analysis of Vitamin A metabolism in embryos has indicated that the first enzymatic reaction, the oxidation of retinol to retinal, is the rate-limiting step in the synthesis of retinoic acid [38]. Spatial localization of RA production in tissue and cell The finding that the RDH reaction, mediated primarily by RDH10, is a major control point in RA synthesis has implications for understanding the tissue specificity and sub-cellular localization of the sequential steps of RA production. With respect to tissue distribution of Vitamin A metabolism and RA synthesis, it is notable that the expression pattern of Rdh10 within an embryo is very similar to, but not identical to that of Aldh1a2 [23,32]. In chick, the expression pattern of Rdh10 has also been shown to overlap with that of the gene encoding the retinol transporter Stra6 [26]. Together, these data suggest that retinol is imported into cells, oxidized into retinal and subsequently into the product RA all largely within the same population of cells. We have previously shown that maternal administration of intermediate retinal, which exposes all cells of the embryo to the retinal intermediate, can rescue RDH10 deficient embryos to birth [39] and recently this has now been extended postnataly [36]. Although viable, these rescued embryos still exhibit some abnormalities including limb PLoS ONE | www.plosone.org 9 February 2012 | Volume 7 | Issue 2 | e30698