Insulin-like growth factor I and II and insulin-like growth factor binding protein-2 RNAs are expressed in adjacent tissues within rat embryonic and fetal limbs.

Since the rapid proliferation of cells in a directed manner is a necessary component of limb formation, the distribution of locally produced mitogenic molecules within the developing limb is of considerable interest. We have used in situ hybridization to localize transcripts for both insulin-like growth factor binding protein-2 (IGFBP-2) and its ligands, the insulin-like growth factors I and II (IGF-I and IGF-II), within limb buds of rat embryos 10-16 days after conception (equivalent to stages 1-12 of mouse limb morphogenesis, Wanek et al, 1989. J. Exp. Zool. 249, 41-49). The mRNA for IGFBP-2 is very abundant in an anterior-posterior strip of ectoderm along the distal edge of the limb bud (the progenitor of the apical ectodermal ridge or AER) from as early as limb stage 1 (Embryonic Day 10) and is much less abundant in the rest of the limb ectoderm. A high level of IGFBP-2 expression continues to characterize the AER following its definitive appearance (stage 3) and throughout its existence (until stage 7). This is a period of rapid outgrowth during which the rate of mesodermal cell division is highest in cells nearest to the AER. The AER is known to have mitogenic activity in vitro and to direct limb outgrowth in vivo, but, until recently, few putative molecular correlates of these activities have been detected. The transcripts for IGF-I and IGF-II are also present at high abundance in developing limbs, especially in mesodermally derived cells. IGF-I mRNA is abundant in presumptive limb mesoderm from the beginning of limb outgrowth (just before stage 1), but is very low or undetectable in much of the rest of the embryo, while IGF-II mRNA becomes very abundant in limb mesoderm at stage 2. The distribution in limbs of both IGF-I and IGF-II mRNA changes dramatically during outgrowth and differentiation, so that their expression characterizes complementary populations of cells by stage 11. Taken together, these data suggest that IGFs and the IGF binding proteins, which may modulate IGF action, contribute to limb outgrowth and patterning.