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Hensen's node from vitamin A-deficient quail embryo induces chick limb bud duplication and retains its normal asymmetric expression of Sonic hedgehog (Shh).

Both Hensen's node, the organizer center in chick embryo, and exogenous retinoic acid are known to induce limb duplication when grafted or applied to the host chick limb bud. Retinoic acid is known to be present in the node and has been proposed as the putative morphogen for chick limb development. Here, we report that Hensen's node from vitamin A-deficient quail embryo induces limb duplication in the host chick embryo similar to that induced by the node from vitamin A-sufficient control embryos. We also demonstrate that the expression of Sonic hedgehog (Shh), recently shown to be the mediator of polarizing activity in the chick limb bud, is not affected by the endogenous vitamin A status of the embryo. Furthermore, whole-mount in situ hybridization revealed asymmetry of Shh expression in the Hensen's node of both vitamin A-sufficient and -deficient quail embryos. Retinoids were not detectable in the eggs from which vitamin A-deficient embryos were obtained. Extracts from normal embryos induced a level of expression of reporter gene equivalent to the presence of 3.4 pg of active retinoids per embryo, while those from vitamin A-deficient embryos induced a baseline level of reporter gene expression similar to that of the controls. Our studies suggest that endogenous retinoic acid is not involved in Shh expression nor in regulating its asymmetry during normal early avian embryogenesis and support the current view that endogenous retinoic acid may not be a direct morphogen for limb bud duplication.