Gene expression, polarising activity and skeletal patterning in reaggregated hind limb mesenchyme

The developing chick limb has two major signalling centres; the apical ectodermal ridge maintains expression of several important genes and outgrowth of the limb, and the polarising region specifies the pattern of skeletal elements along the anteroposterior axis. We have used reaggregated leg grafts (mesenchyme dissociated into single cells, placed in an ectodermal jacket and grafted to a host) to study patterning in a system where the developmental axes are severely disrupted. Reaggregates from different regions of leg mesenchyme developed correspondingly different digits, giving a system in which skeletal phenotype could be compared with the expression of genes thought to be important in patterning. We found that posterior third and whole leg reaggregates gave rise to different digits, yet expressed the same combination of HoxD, Bmp-2 and shh genes throughout their development. Anterior thirds initially only express the 3' end of the HoxD cluster but activate the more 5' members of the cluster sequentially over a period of 48 hours, a period during which Bmp-2 is activated but no shh or Fgf-4 expression could be detected. Our results suggest that there are two independent mechanisms for activating the HoxD complex, one polarising region-dependent and one independent, and that shh expression may not be necessary to maintain outgrowth and patterning once a ridge has been established.