PROJECT SUMMARY The digit tips of many mammals, including humans, innately regenerate following amputation. Multi-tissue digit tip regeneration requires coordinated proliferation and interaction of many cell types in a structure termed the ‘blastema’. Significant progress has been made in defining the origin and cellular composition of the mouse digit tip blastema, but there is a gap in our understanding of how the heterogeneous blastema becomes a correctly organized and shaped composite tissue. This proposal focuses on defining the molecular mechanism(s) of patterning the regenerating mouse digit tip. In general, broad similarities between the regenerative blastema and the embryonic limb bud underlie the hypothesis that gene networks required for embryonic limb patterning are re-deployed in the regenerative blastema. While studies support this hypothesis in some non-mammalian models of limb regeneration, our data demonstrate that this is not true for dorsal- ventral patterning during mouse digit tip regeneration. This suggests that mammalian and non-mammalian limb-derived blastemas may employ different mechanisms for re-establishing limb tissue morphology. We propose that the mammalian digit tip blastema utilizes non-developmental patterning mechanisms and receives molecular patterning cues from the nail epithelium. In this proposal, we focus on the dorsal-ventral and proximal-distal anatomical axes; we aim to determine the patterning mechanism(s) in the mouse digit tip blastema and to determine how manipulation of these pathways during regeneration impacts morphology. Toward this, we will model and molecularly define proximal-distal patterning genes using computational and genetic approaches. We will also use grafting studies to determine the tissue patterning autonomy of the digit tip blastema. Finally, we will visualize putative dorsal-ventral electrochemical gradients in the blastema and perform gain and loss of function studies to manipulate these signals. Collectively, the data generated from this project will reveal patterning mechanisms for mammalian digit tip regeneration and highlight how this may be fundamentally different from non-mammalian limb regeneration models. This research will ultimately inform blastema induction efforts for mammalian full limb amputations because exogenous patterning information may need to be provided.