The heterogeneity and compartmentalization of stem cells is a common principle in many epithelia, and is known to function in epithelial maintenance, but its other physiological roles remain elusive. Here we show transcriptional and anatomical contributions of compartmentalized epidermal stem cells in tactile sensory unit formation in the mouse hair follicle. Epidermal stem cells in the follicle upper-bulge, where mechanosensory lanceolate complexes innervate, express a unique set of extracellular matrix (ECM) and neurogenesis-related genes. These epidermal stem cells deposit an ECM protein called EGFL6 into the collar matrix, a novel ECM that tightly ensheathes lanceolate complexes. EGFL6 is required for the proper patterning, touch responses, and αv integrin-enrichment of lanceolate complexes. By maintaining a quiescent original epidermal stem cell niche, the old bulge, epidermal stem cells provide anatomically stable follicle–lanceolate complex interfaces, irrespective of the stage of follicle regeneration cycle. Thus, compartmentalized epidermal stem cells provide a niche linking the hair follicle and the nervous system throughout the hair cycle.

31The heterogeneity and compartmentalization of stem cells is a common principle in many 32 epithelia, and is known to function in epithelial maintenance, but its other physiological 33 roles remain elusive. Here we show transcriptional and anatomical contributions of 34 compartmentalized epidermal stem cells (EpSCs) in tactile sensory unit formation in the 35 hair follicle (HF). EpSCs in the follicle upper-bulge, where mechanosensory lanceolate 36 complexes (LCs) innervate, express a unique set of extracellular matrix (ECM) and 37 neurogenesis-related genes. These EpSCs deposit an ECM protein EGFL6 into the collar 38 matrix, a novel ECM that tightly ensheathes LCs. EGFL6 is required for the proper 39 patterning, touch responses, and av integrin-enrichment of LCs. By maintaining a quiescent 40 original EpSC niche, the old bulge, EpSCs provide anatomically stable HF-LC interfaces, 41 irrespective of the stage of follicle regeneration cycle. Thus, compartmentalized EpSCs 42 provide a niche linking the HF and the nervous system throughout the hair cycle. 43 44 Results 95 Upper-bulge EpSCs are molecularly specialized for HF-nerve interactions 96We first examined the global transcriptional features of distinct EpSC populations in the 97 HF. To this end, we established FACS-based cell purification methods using several eGFP 98 reporter mouse lines that label different SC compartments to isolate cellular subpopulations 99 resident in the lower-isthmus (Lgr6 + ), upper-bulge (Gli1 + ), mid-bulge (CD34 + ), and hair 100 germ (Cdh3 + ) as well as unfractionated basal EpSCs (a6 integrin + ) ( Figure 1A and B, Figure 101

Inter-tissue interaction is fundamental to multicellularity. Although the basement membrane (BM) is located at tissue interfaces, its mode of action in inter-tissue interactions remains poorly understood, mainly because the molecular and structural details of the BM at distinct inter-tissue interfaces remain unclear. By combining quantitative transcriptomics and immunohistochemistry, we systematically identify the cellular origin, molecular identity and tissue distribution of extracellular matrix molecules in mouse hair follicles, and reveal that BM composition and architecture are exquisitely specialized for distinct inter-tissue interactions, including epithelial–fibroblast, epithelial–muscle and epithelial–nerve interactions. The epithelial–fibroblast interface, namely, hair germ–dermal papilla interface, makes asymmetrically organized side-specific heterogeneity in the BM, defined by the newly characterized interface, hook and mesh BMs. One component of these BMs, laminin α5, is required for hair cycle regulation and hair germ–dermal papilla anchoring. Our study highlights the significance of BM heterogeneity in distinct inter-tissue interactions.

21Inter-tissue interaction is fundamental to multicellularity. Although the basement 22 membrane (BM) is located at tissue interfaces, its mode of action in inter-tissue 23 interactions remains poorly understood, mainly because the molecular and structural 24 details of the BM at distinct inter-tissue interfaces remain unclear. By combining 25 quantitative transcriptomics and immunohistochemistry, we systematically identify the 26 cellular origin, molecular identity and tissue distribution of extracellular matrix molecules 27 in mouse hair follicles, and reveal that BM composition and architecture are exquisitely 28 specialized for distinct inter-tissue interactions, including epidermal-fibroblast, 29 epidermal-muscle and epidermal-nerve interactions. The epidermal-fibroblast interface, 30 namely, hair germ-dermal papilla interface, makes asymmetrically organized side-31 specific heterogeneity in BM, defined by the newly characterized interface, hook and 32 mesh BMs. One component of these BMs, laminin a5, is required for the topological and 33 functional integrity of hair germ-dermal papilla interactions. Our study highlights the 34 significance of BM heterogeneity in distinct inter-tissue interactions. tissues, where it serves to compartmentalize and also tightly integrate tissues 1, 2 . The 40 BM has several crucial roles: i) it provides structural support to cells that is essential for 41 the development of organ structures; ii) it signals to cells through adhesion receptors 42 such as integrins; iii) it controls the tissue distributions and activities of soluble growth 43 factors; and iv) its mechanical characteristics influence cell behavior 3, 4, 5 . Thus, the 44 composition and structure of the BM play critical roles in cell proliferation, 45 differentiation, migration, survival, polarity and positioning, underpinning many 46 fundamental biological phenomena, including the developmental patterning, inter-tissue 47 interactions and stem cell niche formation. 48The BM is composed of a large variety of molecules that exhibit 49 spatiotemporal expression patterns during development and homeostasis, indicating that 50 individual cell types are exposed to tailor-made BM niches 6, 7, 8 . In mammals, the entire 51 set of ECM molecules, called the matrixome or matrisome, is encoded by 52 approximately 300 ECM genes and there are also approximately 800 ECM-associated 53 genes, such as those encoding ECM-modifying enzymes and growth factors 7, 9 54 (http://matrisomeproject.mit.edu/). Although information about the unique distribution, 55 biochemical activities and in vivo functions of individual BM molecules has been 56 accumulated, the entire molecular landscape of the BM composition, including its 57 cellular origins, tissue localizations and pattern-forming processes, in all organs remains 58 largely unknown. One major reason for the difficulty in obtaining a comprehensive 59 Tsutsui et al. 4 understanding of the ECM's composition lies in the biochemical properties of ECM 60 proteins, including their la...

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