Autoregulation of Shh expression and Shh induction of cell death suggest amechanism for modulating polarising activity during chick limb development

The polarising region expresses the signalling molecule sonic hedgehog (Shh ), and is an embryonic signalling centre essential for outgrowth and patter ning of the vertebrate limb. Previous work has suggested that there is a bu ffering mechanism that regulates polarising activity, Little is known about how the number of Shh-expressing cells is controlled but, paradoxically, t he polarising region appears to overlap with the posterior necrotic zone, a region of programmed cell death. We have investigated how Shh expression a nd cell death respond when levels of polarising activity are altered, and s how an autoregulatory effect of Shh on Shh expression and that Shh affects cell death in the posterior necrotic zone. When we increased Shh signalling , by grafting polarising region cells or applying Shh protein beads, this l ed to a reduction in the endogenous Shh domain and an increase in posterior cell death. In contrast, cells in other necrotic regions of the limb bud, including the interdigital areas, were rescued from death by Shh protein. A pplication of Shh protein to late limb buds also caused alterations in digi t morphogenesis, When we reduced the number of Shh-expressing cells in the polarising region by surgery or drug-induced killing, this led to an expans ion of the Shh domain and a decrease in the number of dead cells, Furthermo re, direct prevention of cell death using a retroviral vector expressing Bc l2 led to an increase in Shh expression. Finally, we provide evidence that the fate of some of the Shh-expressing cells in the polarising region is to undergo apoptosis and contribute to the posterior necrotic zone during nor mal limb development. Taken together, these results show that there is a bu ffering system that regulates the number of Shh-expressing cells and thus p olarising activity during limb development. They also suggest that cell dea th induced by Shh could be the cellular mechanism involved. Such an autoreg ulatory process based on cell death could represent a general way for regul ating patterning signals in embryos.