Extended Data Fig. 1: Origin and early lineage of bulge stem cells cannot be traced by expression of known adult stem cell markers.

a–g, Immunohistochemistry of developing whisker hair follicles (HFs) for known stem cell (SC) markers at E12.0 (a, early hair placode), E12.5 (b, late hair placode), E13.0 (c, hair germ), E14.0 (d), E15.0 (e), E17.0 (f) and post-natal day (P)5 (g). LHX2 was expressed in the basal cells of the placode, while K15 and SOX9 were detected in suprabasal and peripheral basal cells of the placode (a). At the late placode stage (E12.5), SOX9 was strongly expressed in the flat suprabasal cells of the placode (arrows in b) and also in some basal cells located near the placode periphery (arrowheads in b). NFATc1 was undetectable throughout these stages (a, b). Thus, at the onset of HF morphogenesis, the expression patterns of adult SC markers vary and there is no clear region in which all SC markers overlap. From the hair germ stage (E13.0) onward, NFATc1 appeared in whisker HFs, and its expression was always restricted to the upper region of the HF (c–g). NFATc1-positive epithelial cells formed a compartment with pseudo-stratified (see h) and bulge-like morphology (d–g) with gradual loss of the expression of mitotic marker Ki67 during follicle development (a–g), which are typical characteristics of adult bulge HFSCs. SOX9 and LHX2 expression also became restricted to the middle to upper part of the HF after E14.0 (d–g). The expression patterns of SC markers in E17.0 follicles were almost equivalent to those seen in the upper half of P5 mature follicles, in which a large bulge-like epithelial structure was evident (brackets in f, g). Taken together, these results indicate that basal epithelial cells acquire compartmentalized SC marker expression patterns of the mature bulge by E17.0 in whisker HFs. The origin and early lineage of bulge SCs cannot be traced by following the expression of known adult SC markers because of their wide variation in expression patterns of the hair placode, hair germ and bulbous peg stages. Thus, at present, there is no marker that can exclusively label the origin and early lineage of prospective SCs from the placode stage. Scale bars, 100 μm. h, Three-dimensional-reconstructed z-stack images from whisker HFs derived from KRT5-cre;R26R-Lyn-Venus;R26-H2B-mCherry at E14.0. Cell membranes were sparsely labelled by Venus. Arrowheads indicate the pseudo-stratified epithelium. Scale bar, 50 μm. i, Summary of known SC marker expressions in the developing whisker HF. j–n, Immunohistochemistry of developing dorsal HFs for known SC markers at E14.5 (j, hair placode), E15.5 (k, hair germ), E16.5 (l), E17.5 (m) and E19.5 (n). SOX9-positive cells were localized in the suprabasal layer (arrows in j) and in the basal layer near the placode periphery (arrowheads in j) in the dorsal HF placode as observed in the whisker HF placode. Morphogenetic events and marker expression patterns closely resemble those of whisker HFs. Scale bars, 50 μm.