The role of Sonic Hedgehog in regulatingproliferation, cell survival, and cell-cycle exit in thezebrafish fin buds and neural-plate derived tissues

Abstract

The development of multicellular organisms depends on the integration between pattern formation and the regulation of cell number. The secreted signaling protein Sonic Hedgehog (Shh) has been implicated in directing both of these processes, suggesting it may participate in achieving this integration. The role of Shh in directing pattern formation in vertebrate model systems, including the zebrafish, has been well characterized during the last decades. Among the organs in which the patterning function of Shh has been best studied are the limb buds, the retina, and the neural tube. I therefore chose to study the role of Shh in regulating cell proliferation, cell death, and cell survival in these organs of the zebrafish. In addition, I also examined the interaction between Shh and several other factors directing cell proliferation and cell death, including the secreted signaling protein Fgf, and the transcription factor p53. In the context of the zebrafish paired fin buds, I focused on the interplay between Shh and the Fgf signaling pathways. Shh directs pattern formation along the anterior/posterior axis of the vertebrate limb, whereas several Fgfs in combination direct pattern formation along the proximal/distal axis of the limb. In addition, Shh and Fgf signaling pathways in the limb bud are mutually interdependent. Therefore, I aimed to determine the relative importance of each pathway for proliferation in this organ. In zebrafish shh mutants, both proliferation and Fgf signaling in the pectoral fin buds are initially normal, but later are strongly reduced. Furthermore, pharmacological inhibition of Hh signaling for short periods has little effect on either Fgf signaling, or on cell-cycle gene expression, whereas long periods of inhibition lead to the downregulation of both. By contrast, even short periods of pharmacological inhibition of Fgf signaling lead to strong disruption of proliferation in the fin buds, without affecting Shh signaling. Activation of Fgf signaling by implantation of FGF4-soaked beads into shh mutant pectoral fin buds leads to the rescue of cell-cycle gene expression and proliferation in these organs. These results show that the role of Shh in this process is indirect, and is mediated by its effect on Fgf signaling. By contrast, the activity of the Fgf pathway affects proliferation directly and independently of its effect on Shh. In neural-plate derived tissues such as the retina and the neural tube, Shh is essential for survival of cells during development. Here I identify p53 as the mediator of cell death in shh mutant since in the absence of Shh activity, p53 target genes are induced, and p53 loss leads to suppression of apoptosis in shh mutants. p53 induces apoptosis in the absence of Shh signaling by activating expression of the pro-apoptotic target genes puma and bax1, which induce the intrinsic pathway of apoptosis and whose level of expression correlates with the severity of apoptotic phenotypes. In support of the hypothesis that p53 activation results from loss of Shh signaling, p53 target gene expression and apoptosis are both suppressed by over-expression of dominant-negative PKA in shh mutants. To monitor p53 activation in living zebrafish embryos, I constructed a transgenic line expressing fluorescent protein under the control of the p53-driven promoter. Indeed, p53 reporter expression correlates very well with apoptosis levels in vivo, except in the early retina. Furthermore, p53 reporter can be induced by genotoxic drugs and colocalises with active-Caspase3. p53 reporter-positive cells were also found defective in their cell cycle progression at 48 hpf. Consistent with this result, proliferation assays on the double shh p53 mutant revealed that loss of p53 rescues normal cell-cycle exit and increases the rate of mitosis in the shh mutant retina. Moreover, differentiation of amacrine cells and photoreceptors was rescued in the double shh p53 mutant retina. These results show that in the absence of shh, p53 is required for the induction of apoptosis, and also regulates proliferation, cell-cycle exit and differentiation in the retina. Taken together, my results show that Shh plays an important role in regulating both proliferation and cell survival during vertebrate development, and that it affects these processes distinctly in different tissues. In the context of the paired fin buds Shh directs pattern formation and in addition promotes cell proliferation, via activation of the Fgf signaling pathway. In neural-plate derived tissues Shh not only plays a patterning role, but also promotes cell survival and proliferation by antagonizing activation of the transcription factor p53.