Different regions of the skin vary in their characteristics such as thickness, pigmentation, innervation, and presence, size and density of hair follicles and sweat glands, that are reflected in differential responses to injury and disease. As examples, androgenetic alopecia is limited to the scalp; acne predominates in facial skin; psoriasis is often most prominent in extensor regions; palmoplantar keratoderma is limited to palms and soles; and vitiligo can appear in symmetrical patterns. While regional characteristics of the skin are established during fetal development, positional information must be retained in the skin throughout life to allow for maintenance of regional characteristics and their re-establishment in wound healing. Positional information is known to reside in the skin dermis, but its molecular basis is poorly understood. To address this question, we propose the following Specific Aims. AIM 1: To identify candidate factors and areas of chromatin involved in establishing skin heterogeneity in embryogenesis we will analyze transcriptional profiles through single cell RNA-seq, and chromatin structure via single cell ATAC-seq, in distinct regions of developing skin to identify those that show region-specific expression or openness, respectively. AIM 2: (i) To determine which of these candidate factors and chromatin areas may also be responsible for maintaining regional skin heterogeneity in adult life, we will perform the same analyses on the corresponding areas of adult skin. (ii) We hypothesize that epigenetic mechanisms contribute to maintenance of regional skin characteristics. To test this, we will first identify patterns of DNA methylation and histone modifications that characterize developing dermis in specific skin regions by carrying out Bisulfite-seq to reveal sites of DNA methylation, and CUT&RUN for histone modifications that mark enhancers and active, repressed, or poised genes. We will then ask which of these patterns are maintained in adult dermal cells from the same regions. AIM 3: To test the functions of candidate regulators in directing and maintaining region-specific differentiation programs, we will use inducible genetic tools to delete the corresponding genes in developing or adult mouse dermis in vivo. Together, these experiments provide a comprehensive and unbiased approach to identify novel mechanisms that establish and maintain skin heterogeneity. Improved understanding of these mechanisms has potential to reveal new therapeutic targets in wound healing and in common and rare diseases that affect specific skin regions and have a major negative impact on quality of life; data obtained in this project will also inform strategies for generating specific skin types, including hair follicle- and sweat gland-bearing skin, for reparative skin grafting.