activity [33]. Loss of RALDH activity in Aldh1a2gri/gri mutant embryos causes a severe reduction in RA signaling (Fig. 5 A–B), as has been observed for previously reported knockout alleles of Aldh1a2 [8,27]. Whereas wild type littermate embryos have strong RA signaling throughout the trunk and within the forebrain, homozygous Aldh1a2gri/gri mutant embryos are almost completely devoid of RA signaling. A small residual amount of RA signaling can be detected in mutant embryos in the forebrain, consistent with the RA signaling in previously reported alleles [8,27]. In the mutants we examine here, there is also a very small amount of residual RA signaling detected within the trunk, specifically within the neural tube region at the level of the first few somites. These data demonstrate that the Aldh1a2gri mutation is either a allele or extreme hypomorph. The residual RA signaling observed within the forebrain of mutant embryos corresponds to the signal Figure 5. Distribution of RA signaling in Rdh10 and Aldh1a2 mutant embryos. Embryos carrying the RARE-lacZ reporter transgene were collected at E8.5, E9.5 or E10.5 and stained for b-galactosidase activity to visualize RA signaling. Blue staining indicates presence of RA. (A–B) Aldh1a2gri/gri and littermates at E10.5. Arrow indicates small amount of RA signaling detected in neural tube of mutant embryos, n = 13. (C–F) Rdh10delta/delta and littermates at E8.5, n = 5. (G–J) Rdh10delta/delta and littermate embryo at E9.5, n = 6. Embryos shown in lateral (G–H) or dorsal (I–J) view. doi:10.1371/journal.pone.0030698.g005 RDH10 Control of RA Synthesis in previously reported knockout alleles of Raldh2 and likely results from activity mediated by RALDH1 and RALDH3. The small amount of RA signaling detected in the trunk of Aldh1a2gri/gri mutant embryos, which has also been observed in one of the established Aldh1a2 knockout alleles [34], may result from a tiny fraction of wild type activity remaining in the Aldh1a2gri allele or from variation in the phenotype on different strain backgrounds. Analogous to the RA attenuation resulting from disruption of Aldh1a2, loss of RDH10 function in the Rdh10delta/delta mutants also elicits a dramatic reduction in RA signaling (Fig. 5 C–J). At E8.5 RA signaling in mutant embryos is essentially absent (Fig. 5 C–F). At E9.5 residual RA signaling can be detected in Rdh10delta/delta embryos within the trunk neural tube region, the extent and pattern being slightly broader than is seen for the Aldh1a2gri/gri mutants (Fig. 5 G–J). Residual RA signaling within the forebrain, which is observed in Aldh1a2gri/gri mutants, is completely abolished in Rdh10delta/delta embryos. For the Rdh10delta/delta mutant embryos, as with the Rdh10trex/trex mutant embryos, the residual RA signaling in the trunk region is localized to the neural tube. Rdh10delta/delta embryos also retain a small amount of residual signaling in the heart and tissue dorsal to the heart. Rdh10delta/delta embryos have variable residual RA signaling that is somewhat reflective of their variability in phenotype. These data indicate that, at the critical E8.0–E8.5 stage of development, RDH10 is essential for nearly all RDH activity within the early developing embryo. At E9.5 and later stages, the residual RA detected indicates that other enzymes can contribute a small amount of RDH activity to metabolism of Vitamin A into RA and the small amount of RA produced results in signaling concentrated within the trunk neural tube. Rdh10 transcription is upregulated by retinoid deficiency The severe phenotype of the Rdh10bgeo/bgeo and Rdh10delta/delta mutants and dramatic reduction in RA synthesis, indicate that RDH10 is responsible for most RDH activity within the early embryo and raises the possibility that the RDH reaction could function as a control point in converting Vitamin A to RA. We therefore sought to determine if embryo RA levels feed back to regulate transcription of the Rdh10 gene. To that end we evaluated Rdh10 expression in Aldh1a2gri/gri mutant embryos in which RA production is severely limited. Rdh10 expression levels were assessed by quantitative RT-PCR of E9.5 Aldh1a2gri/gri mutant embryos and their wild type and heterozygous littermates (Fig. 6 A). The RA deficient Aldh1a2gri/gri embryos have significantly elevated Rdh10 expression levels relative to wild type embryos, with expression levels in homozygous mutant embryos ranging between 1.3-fold and 1.8-fold higher than wild type and heterozygous littermates. The elevated Rdh10 transcript in Aldh1a2gri/gri mutant embryos was expressed in the normal spatial distribution within the embryo at E8.5 (Fig. 6B). These data showing elevation of Rdh10 expression in RA-deficient Aldh1a2gri/gri mutant embryos clearly demonstrate that Rdh10 transcription is regulated by feedback from the metabolic product RA in mice. Interestingly, Rdh10 contains a potential RAR-RXR (RARE) sequence within exon 5, however to date it is not known whether this is a functional motif nor if it acts in a negative or positive fashion in response to retinoid signaling as has been observed for other RAREs. Nonetheless, our results provide compelling evidence that the RDH reaction is an important control point in regulating metabolism of Vitamin A into RA in mammals and sets the stage for future examination of the complexities of feedback and feedforward enzymatic regulation during this process. PLoS ONE | www.plosone.org 7 February 2012 | Volume 7 | Issue 2 | e30698