RDH10 Oxidation of Vitamin A Is a Critical Control Step in Synthesis of Retinoic Acid during Mouse Embryogenesis Lisa L. Sandell1,2*, Megan L. Lynn 1 , Kimberly E. Inman1, William McDowell1, Paul A. Trainor1,3* 1 Stowers Institute for Medical Research, Kansas City, Missouri, United States of America, 2 Department of Molecular, Cellular and Craniofacial Biology, University of Louisville, Louisville, Kentucky, United States of America, 3 Department of Anatomy and Cell Biology, University of Kansas Medical Center, Kansas City, Kansas, United States of America Abstract Retinoic Acid (RA) is a small lipophilic signaling molecule essential for embryonic development and adult tissue maintenance. Both an excess of RA and a deficiency of RA can cause pathogenic anomalies, hence it is critical to understand the mechanisms controlling the spatial and temporal distribution of RA. However, our current understanding of these processes remains incomplete. Vitamin A is metabolized to RA via two sequential enzymatic reactions. The first requires retinol dehydrogenase (RDH) activity to oxidize Vitamin A (retinol) to retinal, and the second requires retinaldehyde activity (RALDH) to oxidize retinal into RA. The first reaction has previously been attributed to the alcohol dehydrogenase (ADH) family, whose genes are ubiquitously or redundantly expressed. Consequently, the specificity of RA synthesis was thought to reside exclusively at the level of the second reaction. To better understand the metabolism of Vitamin A into RA during embryogenesis, we generated new mouse models that disrupt this process. Here we describe a new targeted knockout of Rdh10 in which RA synthesis is severely impaired, particularly at critical early embryonic stages. We also introduce a new mutant allele of Aldh1a2. Both mutations produce similar developmental defects resulting in lethality around embryonic day 10.5 (E10.5). The severity of the Rdh10 phenotype demonstrates that embryonic oxidation of retinol is carried out primarily by RDH10 and that neither ADHs nor other enzymes contribute significantly to this reaction. We also show that reduced RA production results in upregulation of Rdh10. These data demonstrate that RDH10 plays a critical role in mediating the rate limiting RDH step of Vitamin A metabolism and functions as a nodal point in feedback regulation of RA synthesis. Moreover, RDH10-mediated oxidation of retinol plays as important a role in the control and regulation of RA production during embryogenesis as does the subsequent RALDH-mediated reaction. Citation: Sandell LL, Lynn ML, Inman KE, McDowell W, Trainor PA (2012) RDH10 Oxidation of Vitamin A Is a Critical Control Step in Synthesis of Retinoic Acid during Mouse Embryogenesis. PLoS ONE 7(2): e30698. doi:10.1371/journal.pone.0030698 Editor: Domingos Henrique, Instituto de Medicina Molecular, Portugal Received October 8, 2011; Accepted December 23, 2011; Published February 2, 2012 Copyright: 2012 Sandell et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Research in the Trainor laboratory is supported by the Stowers Institute for Medical Research and the National Institute of Dental and Craniofacial Research (R01 DE 016082). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: pat@stowers.org (PAT); lisa.sandell@louisville.edu (LLS) Introduction Retinoic acid (RA) is a derivative of Vitamin A (retinol) that plays an essential role in many vertebrate biological processes including energy metabolism, brain function, immune response, reproduction and embryonic development (reviewed in [1,2,3, 4,5,6]). The processes of embryonic growth and patterning are particularly dependent on this small molecule, as can be appreciated by the striking abnormalities and midgestation lethality that occur when RA metabolism is limited or disrupted during development in mice [7,8,9,10] or other mammals [11,12,13,14]. The defects arising from RA perturbation occur because RA controls the transcription of a variety of essential developmental genes by virtue of its function as a ligand that binds to retinoic acid receptors (RAR). The RAR, which form heterodimers with retinoid receptors (RXR), activate or repress gene activity when RA is bound (reviewed in [15]). Owing to the importance of RA in regulating essential developmental processes, it is critical to understand the mechanisms that regulate the metabolism of this key compound. Unlike many signaling molecules whose function is modulated by changes in RNA transcription or protein activity, RA is a small molecule whose level and tissue distribution are regulated by biochemical synthesis and by degradation. It has been known for some time that synthesis of RA from the dietary precursor retinol occurs via two sequential enzymatic reactions - first retinol is oxidized to form retinal and, second, retinal is oxidized to form RA [16]. The first reaction is facilitated by enzymes with retinol dehydrogenase (RDH) activity and the second reaction is carried out by enzymes with retinaldehyde dehydrogenase activity (RALDH). The RALDH second step of RA synthesis is carried out by RALDH1, RALDH2 and RALDH3, which are encoded by Aldh1a1, Aldh1a2 and Aldh1a3, respectively [7,8,9,10]. RALDH2 is the primary enzyme responsible for conversion of retinal to RA during early embryogenesis, mediating the major part of RA synthesis within the early developing embryo [8]. Loss of RALDH2 function eliminates RA signaling within the trunk region and results in severe developmental abnormalities, including shortened anteroposterior axis, defects in embryo turning, dilated and un-looped hearts, small somites, absence of forelimb buds and lethality by embryonic day 10.5 (E10.5). Loss of RALDH3 disrupts RA signaling in the embryonic head, resulting in defects in formation of the nose and eye and lethality at birth. PLoS ONE | www.plosone.org 1 February 2012 | Volume 7 | Issue 2 | e30698