IGF-I and insulin in the acquisition of limb-forming ability by the embryonic lateral plate.

Acquisition of limb-forming ability by discrete regions of the lateral plate of the chick embryo is dependent on a medial-lateral inductive signaling cascade moving sequentially from the area of Hensen's node to the somitic mesoderm, the intermediate mesoderm, and then to the prospective limb-forming regions of the lateral plate. IGF-I and insulin are expressed by medial tissues as they are influencing the prospective limb-forming regions of the lateral plate. Here we report that IGF-I and insulin, but not FGF-2 or FGF-4, induce the formation of limb bud-like structures in vitro from prospective limb regions before they have acquired the ability to form limbs independent of medial tissues, and also induce the formation of limb bud-like structures from the prospective flank. The limb bud-like structures induced by IGF-I and insulin possess a thickened cap of ectoderm along their distal tips that resembles the apical ectodermal ridge (AER) and this thickened distal apical ectoderm expresses the AER-characteristic homeobox-containing gene Msx-2. Like in normal limb buds, a population of highly proliferating cells which express the homeobox-containing gene Msx-1 are localized in the mesoderm directly subjacent to the thickened AER-like structures induced by IGF-I and insulin. However, the limb bud-like structures induced by IGF-I and insulin do not express sonic hedgehog, which encodes a secreted signaling molecule that has been implicated in regulating the anteroposterior patterning of the developing limb bud. IGF-I- and insulin-treated prospective limb explants give rise to rudimentary limbs containing identifiable skeletal elements when grafted into the coelom or to somites of host embryos. Overall, these results suggest that IGF-I and insulin may be endogenous signals produced by medial tissues that are involved in conferring limb-forming ability to the lateral plate and may promote the initial outgrowth of limb buds and possibly induce the AER. However, other signals are necessary to promote the expression of genes such as sonic hedgehog that regulate the patterning of the developing limb.