Andrei Molotkov

Retinoic acid generated by Raldh2 in mesoderm is required for mouse dorsal endodermal pancreas development

Studies on nonmammalian vertebrate embryos have indicated that retinoic acid (RA) is required for pancreas development. We have analyzed mouse embryos carrying a null mutation of the gene encoding retinaldehyde dehydrogenase 2 (Raldh2), which controls RA synthesis. Raldh2−/− embryos specifically lack expression of Pdx1 (a homeobox gene required for pancreas development) and Prox1 in dorsal endodermal but not ventral endodermal pancreatic precursor tissues. Ventral endodermal expression of Hex is not affected in Raldh2−/− embryos, indicating that liver specification is not dependent upon RA. Also, expression of Foxa2 across the dorsoventral axis of the endoderm is not affected in Raldh2−/− embryos, indicating that a lack of RA does not cause a general defect in foregut endoderm development. Comparison of wild‐type and Raldh2−/− embryos carrying an RA‐reporter transgene demonstrates that RA activity is normally present throughout the endoderm except in the ventral‐most region but is totally missing in endoderm of Raldh2−/− embryos. Thus, Raldh2 expressed in adjacent splanchnic lateral plate mesoderm provides an RA signal to dorsal endoderm. Dorsal Pdx1 expression is rescued in Raldh2−/− embryos by low‐dose maternal administration of RA, which preferentially restores RA‐reporter expression in the dorsal endoderm. Our findings demonstrate a specific role for RA in mouse embryos as a mesodermally synthesized signal needed for dorsal endodermal expression of Pdx1 during development of the dorsal pancreatic lineage. Developmental Dynamics 232:950–957, 2005.

Cytosolic retinoid dehydrogenases govern ubiquitous metabolism of retinol to retinaldehyde followed by tissue-specific metabolism to retinoic acid

The ability of vitamin A (retinol) to control growth and development depends upon tissue-specific metabolism of retinol to retinoic acid (RA). RA then functions as a ligand for retinoid receptor signaling. Mouse genetic studies support a role for cytosolic alcohol dehydrogenases (ADH) in the first step (oxidation of retinol to retinaldehyde) and a role for cytosolic retinaldehyde dehydrogenases (RALDH) in the second step (oxidation of retinaldehyde to RA). Mice lacking ADH3 have reduced survival and a growth defect that can be rescued by dietary retinol supplementation, whereas the effect of a loss of ADH1 or ADH4 is noticed only in mice subjected to vitamin A excess or deficiency, respectively. Also, genetic deficiency of both ADH1 and ADH4 does not have additive effects, verifying separate roles for these enzymes in retinoid metabolism. As for the second step of RA synthesis, a null mutation of RALDH2 is embryonic lethal, eliminating most mesodermal RA synthesis, whereas loss of RALDH1 eliminates RA synthesis only in the embryonic dorsal retina with no obvious effect on development. Analysis of RA-rescued RALDH2 mutants has also revealed that RALDH3 and at least one additional enzyme produce RA tissue-specifically in embryos. Collectively, these genetic findings indicate that metabolism of retinol to retinaldehyde is not tissue-restricted as it is catalyzed by ubiquitously-expressed ADH3 (a low activity form) as well as by tissue-specifically expressed ADH1 and ADH4 (high activity forms). In contrast, further metabolism of retinaldehyde to RA is tissue-restricted as all enzymes identified are tissue-specific. An important concept to emerge is that selective expression of enzymes catalyzing the second step is what limits the tissues that can completely metabolize retinol to RA to initiate retinoid signaling.

Targeted Disruption of Aldh1a1 (Raldh1) Provides Evidence for a Complex Mechanism of Retinoic Acid Synthesis in the Developing Retina

ABSTRACT Genetic studies have shown that retinoic acid (RA) signaling is required for mouse retina development, controlled in part by an RA-generating aldehyde dehydrogenase encoded by Aldh1a2 (Raldh2) expressed transiently in the optic vesicles. We examined the function of a related gene, Aldh1a1 (Raldh1), expressed throughout development in the dorsal retina. Raldh1−/− mice are viable and exhibit apparently normal retinal morphology despite a complete absence of Raldh1 protein in the dorsal neural retina. RA signaling in the optic cup, detected by using a RARE-lacZ transgene, is not significantly altered in Raldh1−/− embryos at embryonic day 10.5, possibly due to normal expression of Aldh1a3 (Raldh3) in dorsal retinal pigment epithelium and ventral neural retina. However, at E16.5 when Raldh3 is expressed ventrally but not dorsally, Raldh1−/− embryos lack RARE-lacZ expression in the dorsal retina and its retinocollicular axonal projections, whereas normal RARE-lacZ expression is detected in the ventral retina and its axonal projections. Retrograde labeling of adult Raldh1−/− retinal ganglion cells indicated that dorsal retinal axons project to the superior colliculus, and electroretinography revealed no defect of adult visual function, suggesting that dorsal RA signaling is unnecessary for retinal ganglion cell axonal outgrowth. We observed that RA synthesis in liver of Raldh1−/− mice was greatly reduced, thus showing that Raldh1 indeed participates in RA synthesis in vivo. Our findings suggest that RA signaling may be necessary only during early stages of retina development and that if RA synthesis is needed in dorsal retina, it is catalyzed by multiple enzymes, including Raldh1.

RALDH3, a retinaldehyde dehydrogenase that generates retinoic acid, is expressed in the ventral retina, otic vesicle and olfactory pit during mouse development.

The enzymes that generate retinoic acid during development have been identified as members of the aldehyde dehydrogenase (ALDH) family. The developmental expression patterns of two ALDHs that function as retinaldehyde dehydrogenases, RALDH1 and RALDH2, have been described. Here we report the cloning and expression of a third retinaldehyde dehydrogenase from the mouse called RALDH3 that shares 94% amino acid sequence identity to a human retinaldehyde dehydrogenase previously named ALDH6. In mouse embryos, RALDH3 expression is first noticed in the ventral optic eminence at E8.75, then in the optic vesicle/cup, otic vesicle, and olfactory placode/pit from E9.5 to E11.5. Expression in the developing eye is primarily localized in the ventral retina, thus indicating that RALDH3 represents the V1 dehydrogenase activity described there earlier. From E8.5 to E10.5 RALDH3 expression is distinct from that of RALDH1 or RALDH2, thus indicating a unique role in sensory organ development.

Retinoic acid guides eye morphogenetic movements via paracrine signaling but is unnecessary for retinal dorsoventral patterning

Retinoic acid (RA) is required for patterning of the posterior nervous system, but its role in the retina remains unclear. RA is synthesized in discrete regions of the embryonic eye by three retinaldehyde dehydrogenases (RALDHs) displaying distinct expression patterns. Overlapping functions of these enzymes have hampered genetic efforts to elucidate RA function in the eye. Here, we report Raldh1, Raldh2 and Raldh3 single, double and triple null mice exhibiting progressively less or no RA synthesis in the eye. Our genetic studies indicate that RA signaling is not required for the establishment or maintenance of dorsoventral patterning in the retina, as we observe normal expression of Tbx5 and ephrin B2 (Efnb2) dorsally, plus Vax2 and Ephb2 ventrally. Instead, RA is required for the morphogenetic movements needed to shape the developing retina and surrounding mesenchyme. At early stages, Raldh2 expressed in mesenchyme and Raldh3 expressed in the retinal pigmented epithelium generate RA that delivers an essential signal to the neural retina required for morphogenetic movements that lead to ventral invagination of the optic cup. At later stages, Raldh1 expressed in dorsal neural retina and Raldh3 expressed in ventral neural retina (plus weaker expression of each in lens/corneal ectoderm) generates RA that travels to surrounding mesenchyme, where it is needed to limit the anterior invasion of perioptic mesenchyme during the formation of corneal mesenchyme and eyelids. At all stages, RA target tissues are distinct from locations of RA synthesis, indicating that RALDHs function cell-nonautonomously to generate paracrine RA signals that guide morphogenetic movements in neighboring cells.

Stimulation of retinoic acid production and growth by ubiquitously expressed alcohol dehydrogenase Adh3

Influence of vitamin A (retinol) on growth depends on its sequential oxidation to retinal and then to retinoic acid (RA), producing a ligand for RA receptors essential in development of specific tissues. Genetic studies have revealed that aldehyde dehydrogenases function as tissue-specific catalysts for oxidation of retinal to RA. However, enzymes catalyzing the first step of RA synthesis, oxidation of retinol to retinal, remain unclear because none of the present candidate enzymes have expression patterns that fully overlap with those of aldehyde dehydrogenases during development. Here, we provide genetic evidence that alcohol dehydrogenase (ADH) performs this function by demonstrating a role for Adh3, a ubiquitously expressed form. Adh3 null mutant mice exhibit reduced RA generation in vivo, growth deficiency that can be rescued by retinol supplementation, and completely penetrant postnatal lethality during vitamin A deficiency. ADH3 was also shown to have in vitro retinol oxidation activity. Unlike the second step, the first step of RA synthesis is not tissue-restricted because it is catalyzed by ADH3, a ubiquitous enzyme having an ancient origin.

Retinoic acid promotes limb induction through effects on body axis extension but is unnecessary for limb patterning.

Retinoic acid (RA) is thought to be a key signaling molecule involved in limb bud patterning along the proximodistal or anteroposterior axes functioning through induction of Meis2 and Shh, respectively. Here, we utilize Raldh2-/- and Raldh3-/- mouse embryos lacking RA synthesis to demonstrate that RA signaling is not required for limb expression of Shh and Meis2. We demonstrate that RA action is required outside of the limb field in the body axis during forelimb induction but that RA is unnecessary at later stages when hindlimb budding and patterning occur. We provide evidence for a model of trunk mesodermal RA action in which forelimb induction requires RA repression of Fgf8 in the developing trunk similar to how RA controls somitogenesis and heart development. We demonstrate that pectoral fin development in RA-deficient zebrafish embryos can be rescued by an FGF receptor antagonist SU5402. In addition, embryo ChIP assays demonstrate that RA receptors bind the Fgf8 promoter in vivo. Our findings suggest that RA signaling is not required for limb proximodistal or anteroposterior patterning but that RA inhibition of FGF8 signaling during the early stages of body axis extension provides an environment permissive for induction of forelimb buds.

Non-cell-autonomous retinoid signaling is crucial for renal development.

In humans and mice, mutations in the Ret gene result in Hirschsprungs disease and renal defects. In the embryonic kidney, binding of Ret to its ligand, Gdnf, induces a program of epithelial cell remodeling that controls primary branch formation and branching morphogenesis within the kidney. Our previous studies showed that transcription factors belonging to the retinoic acid (RA) receptor family are crucial for controlling Ret expression in the ureteric bud; however, the mechanism by which retinoid-signaling acts has remained unclear. In the current study, we show that expression of a dominant-negative RA receptor in mouse ureteric bud cells abolishes Ret expression and Ret-dependent functions including ureteric bud formation and branching morphogenesis, indicating that RA-receptor signaling in ureteric bud cells is crucial for renal development. Conversely, we find that RA-receptor signaling in ureteric bud cells depends mainly on RA generated in nearby stromal cells by retinaldehyde dehydrogenase 2, an enzyme required for most fetal RA synthesis. Together, these studies suggest that renal development depends on paracrine RA signaling between stromal mesenchyme and ureteric bud cells that regulates Ret expression both during ureteric bud formation and within the developing collecting duct system.

Requirement of mesodermal retinoic acid generated by Raldh2 for posterior neural transformation

Studies in amphibian embryos have suggested that retinoic acid (RA) may function as a signal that stimulates posterior differentiation of the nervous system as postulated by the activation-transformation model for anteroposterior patterning of the nervous system. We have tested this hypothesis in retinaldehyde dehydrogenase-2 (Raldh2) null mutant mice lacking RA synthesis in the somitic mesoderm. Raldh2(-/-) embryos exhibited neural induction (activation) as evidenced by expression of Sox1 and Sox2 along the neural plate, but differentiation of spinal cord neuroectodermal progenitor cells (posterior transformation) did not occur as demonstrated by a loss of Pax6 and Olig2 expression along the posterior neural plate. Spinal cord differentiation in Raldh2(-/-) embryos was rescued by maternal RA administration, and during the rescue RA was found to act directly in the neuroectoderm but not the somitic mesoderm. RA generated by Raldh2 in the somitic mesoderm was found to normally travel as a signal throughout the mesoderm and neuroectoderm of the trunk and into tailbud neuroectoderm, but not into tailbud mesoderm. Raldh2(-/-) embryos also exhibited increased Fgf8 expression in the tailbud, and decreased cell proliferation in tailbud neuroectoderm. Our findings demonstrate that RA synthesized in the somitic mesoderm is necessary for posterior neural transformation in the mouse and that Raldh2 provides the only source of RA for posterior development. An important concept to emerge from our studies is that the somitic mesodermal RA signal acts in the neuroectoderm but not mesoderm to generate a spinal cord fate.

Raldh2 expression in optic vesicle generates a retinoic acid signal needed for invagination of retina during optic cup formation

Three retinaldehyde dehydrogenase genes (Raldh1, Raldh2, and Raldh3) expressed in unique spatiotemporal patterns may control synthesis of retinoic acid (RA) needed for retina development. However, previous studies indicate that retina formation still proceeds normally in Raldh1‐/‐ mouse embryos lacking RA synthesis in the dorsal neural retina at the optic cup stage. Here, we demonstrate that Raldh2‐/‐ embryos lacking RA synthesis in the optic vesicle exhibit a failure in retina invagination needed to develop an optic cup. This was also observed in Raldh1‐/‐:Raldh2‐/‐ double mutants, which develop similarly. Both mutants retain RA activity in the lens placode associated with Raldh3 expression, but this RA activity is insufficient to induce optic cup formation. Maternal RA administration at the optic vesicle stage rescues optic cup formation in Raldh2‐/‐ and Raldh1‐/‐:Raldh2‐/‐ embryos, demonstrating that Raldh1 is not required during rescue of optic cup development. The optic cup of rescued Raldh1‐/‐:Raldh2‐/‐ embryos exhibits normal RA activity and this is associated with Raldh3 expression in the retina and lens. Thus, RA signaling initiates in the optic vesicle in response to Raldh2 but can be maintained during optic cup formation by a gene other than Raldh1, most likely Raldh3. Loss of optic vesicle RA signaling does not effect expression of early determinants of retina at the optic vesicle stage (Pax6, Six3, Rx, Mitf). Our findings suggest that RA functions as one of the signals needed for invagination of the retina to generate an optic cup. Developmental Dynamics 231:270–277, 2004.