Healing following full-thickness skin damage repairs the defect but does not restore normal skin structures and function. Even with the best clinical management currently available, the skin that is formed is often scarred, dry, fragile, and at risk for repeated skin breakdown. Our long-term goal is to study proteins that regulate signaling pathways in dermal fibroblasts that promote skin regeneration and improve healing. TSC2 is a protein that inhibits mTORC1 and other growth signaling pathways, and it appears to regulate hair follicle morphogenesis and skin regeneration. Fibroblast-like cells with loss of TSC2, derived from angiofibromas and forehead plaques in patients with tuberous sclerosis complex (TSC), induced hair follicle formation, angiogenesis, and lymphangiogenesis in grafted skin equivalents. The capacity of these cells to induce de novo hair follicle formation overcomes a barrier to studies of skin regeneration, and increased vessels are expected to promote wound healing. Our objective is to identify the molecular basis for the regenerative characteristics of fibroblasts with loss of TSC2. Our specific aims are: 1) to identify the cellular contexts in which loss of TSC2 promotes skin regeneration, 2) to determine the signaling pathway alterations in fibroblasts deficient for TSC2 that promote skin regeneration, and 3) to determine the mechanisms by which TSC2-null cells interact with their cellular microenvironment to promote skin regeneration. In aim 1, in vivo hair follicle formation, angiogenesis, and lymphangiogenesis will be measured using cells with loss or presence of TSC2, including TSC2-null cells from TSC patients, human fibroblasts with stable knockdown of TSC2 using shRNA, and Tsc2-null mouse fibroblasts obtained by lineage-specific deletion of Tsc2. In aim 2, the pathways controlling the effects of TSC2-null cells on hair follicle and vessel formation will be identified by knocking dow raptor, a component of mTORC1. Preliminary data indicates cross-talk between TSC2 and TGF signaling, and the basis for this interaction will be elucidated. In aim 3, the in vivo effect of certain paracrine factors will be blocked to define their contributions to angiogenesis and lymphangiogenesis promoted by TSC2-null cells. Completion of the proposed studies will provide mechanistic information that will be critical to design new therapeutic interventions to promote skin regeneration over repair. In particular, they are expected to provide new strategies to promote hair follicle formation and vascularization in bioengineered skin.