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.

Retinoic acid (RA) generated by Raldh2 in paraxial mesoderm is required for specification of the posterior hindbrain, including restriction of Hoxb1 expression to presumptive rhombomere 4 (r4). Hoxb1expression requires 3′ and 5′ RA response elements for widespread induction up to r4 and for r3/r5 repression, but RA has previously been detected only from r5-r8, and vHnf1 is required for repression of Hoxb1 posterior to r4 in zebrafish. We demonstrate in mouse embryos that an RA signal initially travels from the paraxial mesoderm to r3, forming a boundary next to the r2 expression domain of Cyp26a1 (which encodes an RA-degrading enzyme). After Hoxb1 induction, the RA boundary quickly shifts to r4/r5, coincident with induction of Cyp26c1 in r4. A functional role for Cyp26c1 in RA degradation was established through examination of RA-treated embryos. Analysis of Raldh2–/– and vHnf1–/– embryos supports a direct role for RA in Hoxb1 induction up to r4 and repression in r3/r5, as well as an indirect role for RA in Hoxb1 repression posterior to r4 via RA induction of vHnf1 up to the r4/r5 boundary. Our findings suggest that Raldh2 and Cyp26 generate shifting boundaries of RA activity, such that r3-r4 receives a short pulse of RA and r5-r8 receives a long pulse of RA. These two pulses of RA activity function to establish expression of Hoxb1 and vHnf1 on opposite sides of the r4/r5 boundary.

Somitogenesis requires bilateral rhythmic segmentation of paraxial mesoderm along the anteroposterior axis 1 . The location of somite segmentation depends on opposing signalling gradients of retinoic acid (generated by retinaldehyde dehydrogenase-2; Raldh2) anteriorly and fibroblast growth factor (FGF; generated by Fgf8) posteriorly 2,3 . Retinoic-acid-deficient embryos exhibit somite leftright asymmetry [4][5][6] , but it remains unclear how retinoic acid mediates left-right patterning. Here, we demonstrate that retinoic-acid signalling is uniform across the left-right axis and occurs in node ectoderm but not node mesoderm. In Raldh2 −/− mouse embryos, ectodermal Fgf8 expression encroaches anteriorly into node ectoderm and neural plate, but its expression in presomitic mesoderm is initially unchanged. The late stages of somitogenesis were rescued in Raldh2 −/− mouse embryos when the maternal diet was supplemented with retinoic acid until only the 6-somite stage, demonstrating that retinoic acid is only needed during node stages. A retinoic-acid-reporter transgene marking the action of maternal retinoic acid in rescued Raldh2 −/− embryos revealed that the targets of retinoic-acid signalling during somitogenesis are the node ectoderm and the posterior neural plate, not the presomitic mesoderm. Our findings suggest that antagonism of Fgf8 expression by retinoic acid occurs in the ectoderm and that failure of this mechanism generates excessive FGF8 signalling to adjacent mesoderm, resulting initially in smaller somites and then left-right asymmetry.In the 'clock and wave front' model of somitogenesis, a moving wavefront of Fgf8 gene expression in the tailbud regresses posteriorly as the body axis extends, and mesodermal segmentation occurs just anterior to the Fgf8-expression domain 7,8 . The anterior extent of Fgf8 expression is limited by retinoic acid that is generated in the presomitic mesoderm and antagonizes Fgf8 expression in the tailbud 2,9 . Retinoic acid synthesized in the presomitic mesoderm by RALDH2 is required to maintain bilateral symmetry between the left and right somite columns [4][5][6] . Presomitic mesoderm in mouse Raldh2 −/− embryos displays left-right asymmetric expression of Hes7 and Lfng, genes involved in oscillator function 4 , suggesting that a loss of retinoic acid allows left-right asymmetry to occur in presomitic mesoderm where it normally does not occur. However, lateral-plate mesoderm in Raldh2 −/− embryos still maintains asymmetric expression of Nodal and Pitx2, genes required for left-right asymmetry 10 . Retinoic acid acts as a buffer to prevent left-right asymmetry from occurring in presomitic mesoderm, but its mechanism of action is unclear. Genetic studies in mice have demonstrated that oxidation of retinol to retinaldehyde is ubiquitously catalysed by several alcohol dehydrogenases 11 , whereas oxidation of retinaldehyde to retinoic acid occurs in a tissue specific manner, under the control of Raldh2 (Aldh1a2) 12,13 . All-trans-retinoic acid synthesized by RALDH2 is t...

Distinct progenitor cell populations exist in cardiac mesoderm important for patterning of the heart. During heart tube formation in mouse, Tbx5 is expressed in progenitors located more laterally, whereas Isl1 and Fgf8 are expressed in progenitors located more medially. Signals that drive mesodermal progenitors into various cardiac lineages include Fgf8, which functions to induce Isl1. Studies in chick and zebrafish have shown that retinoic acid restricts the number of cardiac progenitors, but its role in mammalian cardiac development is unclear. Here, we demonstrate that Raldh2 ؊/؊ mouse embryos lacking retinoic acid signaling exhibit a posterior expansion of the cardiac Fgf8 expression domain as well as an expansion of Isl1 expression into mesoderm lying posterior to the cardiac field. We provide evidence that retinoic acid acts specifically in the posterior-medial region of the cardiac field to establish the heart posterior boundary potentially by reducing Fgf8 expression which restricts the Isl1 domain.