Hardy, Adrian Paul; (1996) Development of reaggregated chick hindlimb buds. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

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. In this study, reaggregated leg grafts (mesenchyme dissociated into single cells, placed in an ectodermal jacket and grafted to a host) were used to study patterning in a system where the developmental axes were 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. 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, or polarising activity, could be detected. These results suggest that there are at least 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. The study also examined over-expression of a number of gene products in reaggregates, using avian retroviruses. Overexpression of BMP-2 resulted in the grafts forming large amounts of cartilage, almost to the exclusion of most other tissues. Conversely, over-expression of FGF-4 resulted in the grafts almost completely failing to differentiate into tissues typical of the chick limb, instead forming bags of undifferentiated mesenchyme.

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