RDH10 Oxidation of Vitamin A Is a Critical Control Step in Synthesis of Retinoic Acid during Mouse Embryogenesis

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 null 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.

Diffusible signals and epigenetic timing cooperate in late proximo-distal limb patterning

“…Indeed, previous rescue experiments in which RA was administered at different stages and doses confirmed the special sensitivity of proximal limb segments and Meis gene expression to RA availability (Niederreither et al, 2002). Regarding the Rdh10 analysis, while Rdh10-null mutants die around E10.5-E12.5, the model analyzed by Cunningham et al (Cunningham et al, 2013) is a hypomorph mutant called T-Rex, the lethal phase of which is E13.5-E14.5 (Rhinn et al, 2011;Sandell et al, 2012), and therefore T-Rex mutants contain significant amounts of functional RA, which were not detected by the reporter. Relevant to this discrepancy, the RA sensitivity assays aimed to calibrate the reporter (Cunningham et al, 2013) were carried out in vitro by whole embryo exposure to RA, a situation very different to in vivo RA delivery.…”

“…However, the apparently normal Meis gene expression and PD hindlimb patterning in mice deficient for the enzyme RDH10 (part of the RA synthesis pathway) have challenged the view of RA as an important signal for limb patterning (Cunningham et al, 2013). Further studies, however, indicate that the mutant mouse used in that study (T-rex) bears a hypomorphic allele that allows embryo development up to embryonic day (E) 13-E14 (Sandell et al, 2012;Sandell et al, 2007), whereas Raldh2-null embryos (which are expected to have a greater deficiency of RA) only reach E9 (Niederreither et al, 1999). Therefore the T-rex mutants in fact contain functional RA levels, and their validity for studying the role of RA in limb development is thus questionable (see also Discussion).…”

Cyp26 enzymes are required to balance the cardiac and vascular lineages within the anterior lateral plate mesoderm

“…Therefore, we favor the hypothesis that a modest increase in RA signaling, due to Cyp26 deficiency, is the maximal effect achievable from endogenously produced RA. However, as numerous feedback mechanisms of RA exist to maintain appropriate embryonic RA levels (Niederreither et al, 1997;Dobbs-McAuliffe et al, 2004;Emoto et al, 2005;Sandell et al, 2007Sandell et al, , 2012White et al, 2007;D'Aniello et al, 2013), it is also feasible that diminished raldh2 expression, which occurs in Cyp26-deficient embryos (Emoto et al, 2005), contributes to the attenuation of RA levels. Thus, our data support a model where loss of Cyp26 enzymes results in endogenous increases in RA that promote atrial cell specification independently of effects on ventricular cell specification.…”

Retinoic acid receptor regulation of epimorphic and homeostatic regeneration in the axolotl

“…RAR/RXR complexes work as transcriptional repressors with no ligand and as activators in the presence of RA ligand (Rochette-Egly and Germain, 2009). Limiting RA concentration, inhibiting RAR signaling, or inhibiting RA metabolism has detrimental effects on limb development in chicks (Roselló-Díez et al, 2011;Stratford et al, 1996), zebrafish (Grandel et al, 2002) and mammals (Dranse et al, 2011;Lohnes et al, 1994;Niederreither et al, 2002;Sandell et al, 2012Sandell et al, , 2007Williams et al, 2009;Yashiro et al, 2004). The role of RA during limb regeneration is less clear (Blum and Begemann, 2013), although several lines of evidence support an active role.…”