The severe phenotypes of embryos lacking RALDH2 or RALDH3 clearly demonstrate the critical nature of the RALDH reaction in embryonic RA synthesis. In contrast to the well-established importance of the RALDH second reaction, the RDH first reaction of RA synthesis has received less attention due to the misperception that it occurs ubiquitously throughout embryogenesis. Two types of molecule are capable of RDH activity in vitro: members of the alcohol dehydrogenase (ADH) family, which are cytosolic medium chain reductases (MDR), and members of the RDH family, which are membrane-bound short chain dehydrogenase/reductases (SDR) (reviewed in [17]). ADH enzymes have the ability to catalyze the conversion of retinol to retinal in vitro [18]. Owing to this biochemical activity of ADH enzymes, together with an overlapping or ubiquitous pattern of Adh gene expression during embryogenesis [19], it was initially proposed that the embryonic oxidation of retinol to retinal was carried out by ADH enzymes within the cytosol [20,21,22]. However, despite their biochemical activity and their widespread in vivo expression pattern, disruption of single and compound Adh genes produced no embryonic defects [21], a lack of effect that was speculatively attributed to potential redundancy within the gene family. The failure to identify an Adh mutant embryonic phenotype led to the prevailing view that the first step of embryonic Vitamin A metabolism is mediated by ubiquitous or redundant enzymes and plays a minimal role in the spatiotemporal regulation of RA synthesis. The notion that the initial oxidative conversion of retinol occurs in an unregulated fashion mediated by ADHs has been challenged by recent discoveries highlighting a critical role for the SDR RDH10 in tissue specific synthesis of RA within the vertebrate embryo. The first indication that RDH10 is important for embryonic synthesis of RA came from characterization of mice with a point mutation termed Rdh10trex [23]. Embryos homozygous for this mutation exhibit a dramatic reduction in RA synthesis and severe defects in embryonic development including limb, cranio- facial and organ defects that result in lethality between E10.5– 14.5. Further evidence of a role for RDH10 came from Xenopus, in which XRDH10 was shown to be critical for RA synthesis and to be controlled by RA negative feedback inhibition [24]. In zebrafish, the rdh10a gene was found to be subject to positive and negative feedback regulation by RA, as was, inversely, dhrs3a, whose product catalyzes the reverse reaction converting retinal back into retinol [25]. In chick embryos, expression of Rdh10 is regulated spatially and temporally in a pattern overlapping with the retinol transporter Stra6, but does not appear to be regulated by feedback from excessive or reduced levels of RA [26]. These recent findings raise questions of the specific contribution of RDH10 versus ADHs and of the true mechanistic importance of the first oxidation step of Vitamin A metabolism in controlling embryonic RA synthesis. The embryonic abnormalities and RA reduction observed in Rdh10trex mutant mice are dramatic, but generally not as severe as those caused by loss of RALDH2. Embryos lacking RALDH2 do not survive past E10.5 [8,27], while most homozyogous Rdh10trex embryos remain viable through E10.5–E11.5 and embryos have been identified surviving as late as E14.5 [23,28]. The residual production of RA and the less severe phenotype of Rdh10trex embryos compared to Aldh1a2 mutant embryos indicate that some RDH activity remains intact in the Rdh10trex embryos, mediated possibly by hypomorphic activity of the Rdh10trex point mutant enzyme, by other RDH enzymes, or by ADH enzymes. In order to further our understanding of the regulation of RA synthesis during embryonic development, it is important to know which enzymes catalyze the RDH reaction and whether the relevant enzymes are RDH10 Control of RA Synthesis redundant and ubiquitous within the embryo or if they are instead expressed in a regulated fashion that may have bearing on the control of production of this important signaling molecule. To clarify the enzymatic regulation of Vitamin A metabolism and RA synthesis during embryogenesis, we have generated new mouse models that disrupt this process and comprise a graded series of RA deficiency. Here we describe a novel targeted knockout allele of Rdh10. We show that eliminating RDH10 activity results in severe embryonic abnormalities, more extreme than the phenotype of the original Rdh10trex point mutant. In this report we also introduce a new point mutant allele of Aldh1a2, generated in a chemical mutagenesis screen, which disrupts the RALDH reaction and exhibits a phenotype similar to previously described targeted knockout alleles of Aldh1a2. Both the Rdh10 knockout embryos and the Aldh1a2 point mutant embryos exhibit similar shortened anteroposterior axes, defects in embryo turning, dilated and un-looped hearts, small somites, and forelimb bud agenesis. Each results in embryo death by ,E10.5. Using the RARElacZ reporter transgene, we show that RA synthesis in Rdh10 embryos is severely reduced at E9.5 and is almost completely eliminated at the earlier critical E8.0–E8.5 stage of development. The severity of the Rdh10 phenotype indicates that the RDH mediated oxidation of retinol to retinal during Vitamin A metabolism is carried out primarily by RDH10 and that neither ADH family members nor other enzymes contribute significantly to the RDH step of RA synthesis during early embryogenesis. In this report we also find that Rdh10 gene expression is elevated when RA levels are reduced, providing evidence that Rdh10 functions as a control point for feedback regulation of the RA synthesis pathway in mammals. These data demonstrate that the initial RDH reaction of Vitamin A metabolism, mediated primarily by RDH10, plays as critical a role in embryogenesis and is as important in the control and regulation of RA production as is the subsequent RALDH reaction. Results Graded retinoid deficiency phenotypes: Rdh10trex vs. Aldh1a2gri The previously reported mouse Rdh10trex point mutation disrupts RDH10 function and causes numerous embryonic defects culminating in embryo death between E.10.5–14.5 [23]. In Rdh10trex/trex mutant embryos RA synthesis is reduced but not completely eliminated, as residual RA signaling is detected in the trunk neural tube. This raised the possibility that Rdh10trex/trex may be a severe hypomorph rather than a complete null. For comparison with other mutants in the present study, we examined Rdh10trex/trex embryos at E9.5 and E10.5 (Fig. 1A,B,D,E). Although the Rdh10trex/trex phenotype is somewhat variable at E9.5, all embryos have numerous discernible abnormalities. First and second pharyngeal arches form in most Rdh10trex/trex mutant embryos, but third and more posterior pharyngeal arches are absent or fused to the second arch (Fig. 1B,E). Otic vesicles are usually small, duplicated and displaced posteriorly (Fig. 1B). Mutant embryos exhibit cardiac abnormalities, including heart edema and looping defects (Fig. 1B). At E10.5 a reduction in size of the forelimb bud and absence of posterior pharyngeal arches are clearly evident (Fig. 1E). This constellation of defects observed in the Rdh10trex/trex embryos is consistent with retinoid deficiency but is less severe than that observed in embryos in which RALDH2 function has been eliminated. As part of the same chemical mutagenesis screen that yielded the Rdh10trex mutation, we also generated a mutant originally PLoS ONE | www.plosone.org 2 February 2012 | Volume 7 | Issue 2 | e30698