Defining the heterogeneity of cell lineages in the inter-follicular epidermis

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Abstract Despite the crucial importance of the inter-follicular epidermis (IFE) for the essential body barrier function, molecular characterization of the stem cells (SCs) within the basal layer has not been achieved. This impairs our ability to study the mechanisms that specifically control IFE SCs for proper homeostasis and wound healing and to understand how these mechanisms may be affected in skin disease and aging. The IFE SCs have been traditionally identified in vivo as DNA label retaining cells (LRCs) while transit amplifying (TA) or progenitor cells were considered non-LRCs. However, LRC and non-LRC markers to unambiguously test this model in vivo had been lacking. Using our H2B-GFP pulse-chase transgenic mouse system, we label IFE LRCs and non-LRCs, define mRNA expression profiles, and find that these cells are molecularly distinct. In our first set of preliminary data, we demonstrate that, contrary to the expectation that SC are frequently dividing cells, both of our IFE cellular subsets of LRCs (marked by Dlx1CreER) and of non-LRCs (marked by Slc1a3CreER) act as two independent SCs in long-term lineage tracing. Collectively, our data support a model in which the IFE is a heterogeneous tissue, being composed of molecularly distinct domains or territories, which are spatially patterned relative to each other and to skin landmarks. These territories are enriched in LRCs and non-LRCs, regenerate at different rates, and contain distinct SCs and differentiated cells that can be defined as molecularly discrete IFE cellular subsets. We provide LRC and non-LRC-enriched markers and genetic labeling tools that define novel IFE cellular subsets, which will enable us to rigorously examine the newly uncovered IFE heterogeneity. The specific objectives are to: (i) examine the organization of IFE territories throughout life, and determine relative SC potential of newly uncovered IFE cellular subsets; (ii) validate and refine markers of IFE heterogeneity from our newly uncovered undifferentiated and differentiated IFE cellular subsets; and (iii) unravel mechanism of IFE heterogeneity by focusing on two transcription factors we identified in our LRC versus non-LRC IFE subsets. Collectively, our data will constitute a key for understanding previously un-recognized cellular and molecular heterogeneity within the IFE and provide a new entry point into SC regulation in this essential, yet poorly understood skin compartment.