Sonic hedgehog signaling during digit pattern duplication after application of recombinant protein and expressing cells

HoxD expression and cartilage pattern formation were compared after application of a recombinant amino-terminal peptide of Sonic hedgehog protein (Shh-N) and implantation of cells expressing the Sonic hedgehog (Shh) gene. During digit duplication after implantation of a Shh-N-soaked bead, BMP-2 and Patched expression was transiently induced in the anterior limb mesenchyme 20 h after grafting, but was reduced to the basal level 48 h after grafting. On the contrary, when Shh-expressing cells were grafted to the anterior limb bud, expression domains of the BMP-2 and Patched genes were initially induced in the restricted region in close proximity to the grafted cells. Induced expression of BMP-2 and Patched was maintained in the anterior-peripheral region of the limb bud for 42 h after grafting. In either case, HoxD12 and HoxD13 were consistently induced in the anterior-distal limb mesenchyme, accompanying mirror-image duplication of the digit pattern. Induction and maintenance of HoxD expression were consistent with the resultant digit pattern. A steep gradient of Shh activity provided by Shh-expressing cells is most adequate to induce complete digit pattern, as compared to the shallow gradient provided by Shh-N protein released from a bead. These results suggest that positional identity is respecified by Shh-N activity within the first 24 h during digit duplication, and that Shh-N on its own is not acting as a long-range signaling molecule to determine positional identity at a distance in the limb bud.