Shh acts as a mitogen and cell survival factor in many adult processes during normal tissure renewal and in many types of cancer, but acts as a morphogen in several developmental contexts. Whether and how the mitogenic role of Shh is integrated with the morphogenetic role in developmental contexts is still poorly understood. In the limb, Shh regulates both digit number and identity of different digits (A-to-P, thumb to pinky). Shh is thought to act as a morphogen forming a gradient along the limb AP axis, with higher concentrations specifying more posterior digit types. We have determined the time-requirements for Shh function in limb (using a tamoxifen-regulated Cre to remove Shh at different times in mice). To perform this analysis, we generated and characterized a conditional Cre recombinase line selectively expressed in early limb mesoderm, neural crest, gut and tailbud. This line provides an excellent tool available to the scientific community to illuminate different temporal roles of key developmental regulators in several important developmental models using mouse mutants, as well as for genetic lieage tracing studies in mice. We found that Shh has separable, dual functions in limb: an early transient role controlling digit pattern, and an ongoing role regulating cell mass (survival and proliferation) that determines the total digit number. We find an invariant order of digit loss that is not consistent with predictions from current morphogen gradient models. The order of digit loss correlates inversely with the order in which digit primordia first form, and the phenotype severity at different times correlates with degree of apoptosis and decrease in mitotic index. Our results suggest that Shh regulates digit pattern only very early and transiently but is required over an extended time mainly to ensure cell survival and/or proliferation. If the limb bud has fewer cells, fewer digit primordia can form but those that do form are normal. The Gli3 transcription factor is the major downstream effector of Shh signaling in the nucleus. In collaborative studies with Dr. Alex Joyner (Sloan Kettering Inst.), the time-requirements for Gli3 function have also been analyzed and the results of this study support our model, also suggesting that the requirement for Shh in growth to expand digit numbers requires a much more prolonged time than the requirement for patterning. To test this model for Shh function, we have assessed whether restoring survival and/or proliferation in mutant embryos after later-stage Shh deletion can rescue digit formation. To rescue cell survival, the compound mutant for the pro-apoptotic Bcl2 family members Bax/Bak (which play roles in normal interdigital apoptosis) has been introduced to inactivate the intrinsic death pathway. Myc has been identified as a direct target of Hedgehog signaling that regulates proliferation in several systems. To attempt rescue of proliferation, we are employing a RosaMycER transgene (from Dr. Gerard Evan) to provide a proliferative impulse whose timing, duration and level of action can be closely regulated by tamoxifen. Our preliminary results indicate that both normal digit number and pattern (morphogenesis) can be rescued by simply restoring cell survival and proliferation in Shh mutant embryos. These results challenge the view that Shh behaves as a classic morphogen and suggest that the roles of Shh during organ morphogenesis and during tumorigenesis may be very similar. Having found that Shh loss in limb causes G1-arrest,in parallel we are also analyzing how Shh regulates the cell cycle. Understanding the proliferative and anti-apoptotic actions of Shh may provide a reference for deciphering Shh roles in cancer. To learn how proliferation and recruitment of mesenchymal cells relate to the alternating order in which condensations form and integrate our results with other work on Shh function, we are developing tools to image very early condensations, and to trace cell lineage genetically (collaboratively with Dr. Stephen Lockett, NCI). For example, do condensations form via spatially restricted proliferation of precursors in an alternating sequence, or by alternating foci of cell adhesion? We are also using genetic tools including mutants that act to alter digit patterns at very early times to learn when and how the alternating sequence of digit formation is determined. These tools will help unravel how the observed alternating A-P order of condensation is regulated, and whether this sequence is evolutionarily conserved among different vertebrates. We are also analyzing whether another hedgehog signal (Indian Hedgehog, Ihh), produced later when cartilage begins to form, plays any role in determining the final morphology or identity of each digit together with Shh.We will assess the time requirements for all hedgehog ligands during early limb development by removing an essential pathway component (smoothened, smo) downstream of signal-receptor interaction at different times using a conditional mutant allele. In a complementary collaborative study,we are also analyzing the converse, gain-of-function phenotype of constitutively active smo (with Dr.Sohyun Ahn, NICHD).
