Resolving the Hierarchy and Regulation of Sebaceous Gland Stem Cells

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PROJECT SUMMARY Sebaceous glands (SGs) are appendages of mammalian skin that produce and secrete a mixture of lipids, known as sebum. Sebum production is crucial for skin maintenance and deviations in normal sebaceous lipid production are implicated in several skin pathologies including acne vulgaris and alopecia. Sebum production is carried out by sebocytes, the constituent cell population of SGs. Sebocytes undergo a unique maturation process which culminates with the rupture of the cell membrane. This process, termed holocrine secretion, facilitates the secretion of sebum from SGs, but consequently results in sebocyte death. As lipid turnover is a function of sebocyte turnover, sebocyte replenishment is critical to the functional maintenance of SGs. The lifelong turnover of sebocytes requires a continuous supply of cells to replenish and maintain the gland, which suggests the involvement of stem cells in the maintenance of SG tissues. However, the intrinsic and extrinsic mechanisms that regulate activity of the presumptive SG stem cells are not well defined. This is in part due to the limitations of static histological analyses. Such experimental approaches can only capture single snapshots in time, which are insufficient for delineating a hierarchical organization in a tissue with a rapid turnover rate. Furthermore, the overlapping expression of stem cell-associated genes, like Sox9, between the hair follicle and SGs has limited our ability to interpret the functional role of these markers in SG physiology uncoupled from the hair. To overcome these limitations, I have combined genetic lineage-tracing tools with live imaging approaches based on two- photon microscopy. The overall goal of this study is to implement an integrative approach by combining a live imaging system with state-of-the-art genomics tools to 1) characterize stem cell dynamics in SG homeostasis and regeneration by live imaging, and 2) analyze how sebocyte fate is influenced by the key molecular signal, Sox9. In my preliminary experiments, I found that cells from the hair follicle isthmus, an adjacent compartment, made cell contributions to SGs, and that skin-specific knockouts of Sox9 resulted in SG atrophy. I hypothesize that sebocytes are replenished by a heterogeneous stem cell population from the isthmus and that SOX9 is required for their regulation and SG physiology. Aim 1 of this proposal is designed to resolve the sebocyte lineage hierarchy and test the necessity of the isthmus in SG regeneration. Aim 2 of this proposal will address the functional role of SOX9 in regulating sebocyte fate. This research offers an innovative approach to study SG stem cells within the natural tissue environment by combining cutting-edge imaging technologies and genetic tools. The goal of this proposal is to uncover the fundamental mechanisms of SG maintenance by resolving the sebocyte lineage and molecular pathways that regulate SG stem cell activity in the skin.