Extended Data Fig. 5: Placode cell fate is determined by cell position but not cell division orientation.

a, b, Bar plots were converted from the stacked bar plot in Fig. 2a, b, respectively. Statistical analysis was performed by one-way ANOVA followed by Tukey’s test. c, Upper and lower daughter cells after perpendicular division in late placode basal layer are marked by LHX2, while the SOX9⁺ suprabasal nuclei are not labelled with LHX2. This suggests that upper daughter cells hold transcriptional similarity to the basal layer and remain in the pseudo-stratified basal layer. E14.5 dorsal HFs in embryonic skin tissue (not explants) were immunostained for SOX9 and LHX2. Cell division was identified by chromosome condensation. Dashed box shows magnified region (right panels). BM, basement membrane; DP, dermal papilla; U, upper daughter cell; L, lower daughter cell. Scale bars, 50 μm. d, e, Localization of SOX9⁺ cells in HF placodes in vivo. E14.5 dorsal HFs (d) and E12.0 whisker HFs (e) in embryonic skin tissues (not explants) were immunostained for SOX9 and CDH3. Hair placodes were detected with CDH3 expression or Fucci-G1 probe fluorescence signals. SOX9 expressions were detected not only in flat suprabasal cells (cyan arrowheads) but also in basal cells (yellow arrowheads) located at the periphery of the hair placode. Scale bars, 50 μm. f, Photographs of ex vivo cultured dorsal skin of E12.5 K14-rtTA;TetO-H2B-eGFP;Fucci-G1 mice on days 0, 2, 3 and 10. Yellow open arrowheads and filled arrowheads indicate first-wave and second-wave hair placodes, respectively. Dashed box shows magnified region (right). Scale bars, 100 μm. g, Immunostaining of dorsal HFs in day-9 ex vivo culture of E12.5 dorsal skin. The following tissue compartments were detected: bulge stem cells (NFATc1⁺SOX9⁺NPNT⁺KRT15⁺), sebaceous glands (LipidTOX⁺), hair matrix (CDH3⁺), dermal sheaths (SMA⁺), dermal papilla cells (SOX2⁺) and melanocyte (TRP2⁺). Scale bars, 50 μm. h, Immunostaining of day-9 ex vivo cultured dorsal HFs derived from E12.5 embryos for hair cell layer markers shown in Extended Data Fig. 2d. Ex vivo developing dorsal HFs had distinct cellular layers characteristic of HFs except for the medulla, which is formed in mature HFs. Scale bars, 50 μm. i, Lineage tracking data of long-term continuous imaging of dorsal HF development from the placode stage to the hair germ stage, which correspond to the bottom panels in Fig. 2c. Origin of prospective bulge SCs (red) was located at the periphery of the hair placode as observed in the whisker HF placode. Scale bars, 50 μm. j, Bee swarm plot of the distances of dorsal hair placode cells from the placode centre. HFs used for measurement were from cultured dorsal HFs of K14-rtTA;TetO-H2B-eGFP;Fucci-G1 and Sox9^IRES-eGFP/+;R26-H2B-mCherry  mice. Values were scaled based on the diameter of each placode. Different epithelial lineages were aligned in a concentric manner in the placode. Two-sided nested t-test was used. k, Fate of basal and suprabasal cells in the dorsal HF placode analysed in i. Cell fates at the hair germ stage are shown. Summarized data are shown in the left panel, and the data for corresponding replicate HFs are shown in the right panels. Two-sided Fisher’s exact test was used. l, Stacked bar plots showing the lineage distribution of placode basal cells in the dorsal HF at the hair germ stage. Cells grouped in the black bar in k were examined. Summarized data are shown in the left panel, and the data for corresponding replicate HFs are shown in the right panel. m–o, Replicate HFs related to Fig. 2d–f are shown in m–o. m, Fate of basal and suprabasal cells in pre-placodes of Sox9-IRES-eGFP reporter derived dorsal skin explants. Fate of GFP⁺ cells at hair placode stage are shown. GFP⁺ cell lineages were determined at hair germ stage. n, Fate of basal and suprabasal cells in placodes of Sox9-IRES-eGFP reporter-derived dorsal skin explants. Fate of GFP⁺ cells at the hair germ stage are shown. o, Lineage distribution of GFP⁺ basal cells at hair germ stage. Cells grouped in the black bar in n were examined. p, Lineage-tracing strategy of Sox9⁺ cells in ex vivo cultured dorsal skin derived from E12.5 Sox9^creERt2/+;R26R-H2B-mCherry^fl/+;K14-H2B-eGFP (top panel) and snapshot images of the culture (bottom panels). Yellow spots, one of tracked basal cell lineages; Cyan spots, one of tracked suprabasal cell lineages. 4-OHT, 4-hydroxytamoxifen. Scale bars, 50 μm. q, Fate of basal and suprabasal cells in the dorsal HF placode analysed by lineage-tracing with Sox9-creER. Fates of H2B–mCherry⁺ cells at the hair germ stage are shown. Summarized data are shown in the left panel, and the data for corresponding replicate HFs are shown in the right panels. Two-sided Fisher’s exact test was used. r, Lineage distribution of H2B–mCherry⁺ basal cells at hair germ stage. Cells grouped in the black bar in q were examined. Summarized data are shown in the left panel, and the data for corresponding replicate HFs are shown in the right panels. Each value in the graph is the mean ± s.d. from three independent experiments, one or two HF each. Numbers of analysed cell lineages are summarized in Supplementary Table 2. See also ‘Statistical analysis and reproducibility’ in Methods and Source Data.

Source data