Summary 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 [1]. Here, we utilize Raldh2-/- and Raldh3-/- mouse embryos lacking RA synthesis [2] to demonstrate that RA signaling is not required for limb expression of Shh and Meis2. We demonstrate that RA action is required outside the limb field in the body axis during forelimb induction, but that RA is unnecessary at later stages when hindlimb budding and patterning occurs. 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 [3, 4] and heart development [5]. 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.

Two isomers of retinoic acid (RA) may be necessary as ligands for retinoid signaling: all-trans-RA for RA receptors (RARs) and 9-cis-RA for retinoid X receptors (RXRs). This was explored by using retinaldehyde dehydrogenase (Raldh)2 ؊/؊ mouse embryos lacking mesodermal RA synthesis that display early growth arrest unless rescued by all-trans-RA administration. Because isomerization of all-trans-RA to 9-cis-RA can occur, it is unclear whether both ligands are needed for rescue. We show here that an RAR-specific ligand can rescue Raldh2 ؊/؊ embryos as efficiently as all-trans-RA, whereas an RXR-specific ligand has no effect. Further, whereas all-trans-RA was detected in embryos, 9-cis-RA was undetectable unless a supraphysiological dose of all-trans-RA was administered, revealing that 9-cis-RA is of pharmacological but not physiological significance. Because 9-cis-RA is undetectable and unnecessary for Raldh2 ؊/؊ rescue, and others have shown that 4-oxo-RA is unnecessary for mouse development, all-trans-RA emerges as the only ligand clearly necessary for retinoid receptor signaling.

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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.