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We present evidence for the existence of two phases of retinoic acid (RA) signaling required for vertebrate limb development. Limb RA synthesis is under the control of retinaldehyde dehydrogenase-2 (Raldh2) expressed in the lateral plate mesoderm, which generates a proximodistal RA signal during limb outgrowth. We report that Raldh2(-/-) embryos lack trunk mesodermal RA activity and fail to initiate forelimb development. This is associated with deficient expression of important limb determinants Tbx5, Meis2, and dHand needed to establish forelimb bud initiation, proximal identity, and the zone of polarizing activity (ZPA), respectively. Limb expression of these genes can be rescued by maternal RA treatment limited to embryonic day 8 (E8) during limb field establishment, but the mutant forelimbs obtained at E10 display a significant growth defect associated with a smaller apical ectodermal ridge (AER), referred to here as an apical ectodermal mound (AEM). In these RA-deficient forelimbs, a ZPA expressing Shh forms, but it is located distally adjacent to the Fgf8 expression domain in the AEM rather than posteriorly as is normal. AER formation in Raldh2(-/-) forelimbs is rescued by continuous RA treatment through E10, which restores RA to distal ectoderm fated to become the AER. Our findings indicate the existence of an early phase of RA signaling acting upstream of Tbx5, Meis2, and dHand, followed by a late phase of RA signaling needed to expand AER structure fully along the distal ectoderm. During ZPA formation, RA acts early to activate expression of dHand, but it is not required later for Shh activation.

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.