Extended Data Fig. 1: Asymmetric cell divisions from embryo to adult life. | Nature Aging

Extended Data Fig. 1: Asymmetric cell divisions from embryo to adult life.

From: Distinct types of stem cell divisions determine organ regeneration and aging in hair follicles

Extended Data Fig. 1

a, Schematic representation of cell divisions. Symmetric cell divisions (SCDs) of stem cells generate two identical cells by orienting the mitotic spindle parallel to the underlying basement membrane. In contrast, the asymmetric cells divisions (ACDs) of stem cells generate a self-renewing stem cell and a differentiating cell by orienting the mitotic spindle perpendicular to the underlying basement membrane. By analyzing the cell division axes using phospho-histone H3 (PH3) with γ-tubulin (γ—TUB) (red asterisk, metaphase) or Survivin with DAPI (orange asterisk, anaphase), SCDs or ACDs are defined as cell division angle 0–30o or 60–90o against basement membrane, respectively. b, Schematic representation of spindle orientation in E16.5 embryonic basal cells (n = 21 cells). E16.5 embryonic basal cells undergo perpendicular cell division or parallel cell division as previously described (Lechter & Fuchs, 2005, Nature 437, 275–280; Williams et al., 2014, Nature Cell Biology, 16 758–769). c, Representative IF images of PH3, γ—TUB and PARD3 in E16.5 embryonic basal cells. Perpendicularly dividing cells against the basement membrane show polarized PARD3 expression at the apical side (arrowheads) (assumed as asymmetric cell division), while parallelly dividing cells against the basement-membrane show non-polarized PARD3 expression (assumed as symmetric cell division). d, Schematic representation of spindle orientation in bulge (n = 68 cells) and hair germ basal cells (n = 43 cells) and HFs with several stem cell markers at postnatal day 23 (p23). HFs at early anagen stage consist of four different types with different stem cell markers. The bulge and hair germ basal cells at p23 undergo perpendicular cell division or parallel cell division. e, f, Representative IF images of PH3, γ—TUB and PARD3 (e) or NUMB (f) in bulge basal cells (Bg) and in hair germ cells (Hg) at p23. In bulge basal cells, perpendicularly dividing cells showed polarized PARD3 expression at the apical side (arrowheads) (assumed as asymmetric cell division), while parallelly dividing cells showed non-polarized PARD3 expression (assumed as symmetric cell division). However, in hair germ cells, perpendicularly dividing cells do not show polarized PARD3 expression at the apical side. g, Quantification of percent cells with polarized localization of NUMB and PARD3 in Bg and Hg areas (Bg, n = 52; Hg, n = 32 cells). h, Quantification of percent cells with polarized expression of NUMB and PARD3 in Bg, sBg and bulb areas (Bg, n = 18; sBg, n = 55; Bulb, n = 6 cells). In Bg and sBg basal cells, cells dividing at a perpendicular angle (60–90°) significantly showed polarized expression (g, h). i, IF images of ITGA6, COL17A1 and LHX2 in HFs at p25. At this stage, old bulge and hair germ cells proliferate and generate new bulge cells. j, IF images of ITGA6, KRT6 and KRT75 in HFs from p25 to p28. k, Representative IF images of GFP, COL17A1 and Survivin at p23 from GFP-labeled HFSCs at p21. Perpendicularly dividing Survivin+ GFP cells generate COL17A1+ cells. l, Schematic representation of spindle orientation in bulge (n = 38) and sub-bulge (n = 67 cells) HFs with stem cell markers at 3 days after HC induction by depilation at 7–9 wo (n = 4 mice). m, Quantification of cell division angles in bulge basal cells at p23 or p25 or 3 days after HC induction at 7–9 wo. Bulge basal cells at 3 days after HC induction showed a similar cell division pattern to p25 bulge basal cells. n, 3D reconstructed IF images of Survivin in GFP labeled-HFSCs after HC induction. Perpendicular cell division patterns are subdivided as inner cell division and surface cell division. o, The frequency of type of cell division (n = 17 HFs) in (n). p, 3D reconstructed IF images of KRT75 and Survivin in HFSCs after HC induction. HFSCs occasionally perpendicularly divided and differentiated into KRT75+ cells at 3 days after HC induction (n, o, p). q, Schematic representation of hair regeneration after depilation at 8–11 wo. r, Representative IF images of PH3 in the bulge and hair germ areas at 3 days after HC induction. s, Quantification of the number of PH3+ mitotic cells in the bulge areas at 3 days after HC induction (8 wo, n = 30 HFs; 26 mo, n = 60 HFs). PH3+ mitotic cells in the bulge were significantly decreased by aging. t, Quantification of the postulated number of cell division angles in the bulge and sub-bulge areas at 3 days after HC induction. Data were calculated by the number of actual mitotic cells (Extended Data Fig. 1s) and the ratio of cell division angles (Fig. 1h). u, IF images of NUMB in HFSCs from young (8w) and from aged (26 mo) mice at 3 days after depilation. Arrowheads; polarized numb expression. v, Quantification of percent cells with polarized expression in Bg (young (7–12 wo), n = 9 mice, n = 35 cells; aged (24–27 mo), n = 3 mice, n = 17 cells) and in sBg (young (7–12 wo), n = 9 mice, n = 14 cells; aged (24–27 mo), n = 3 mice, n = 15 cells) areas. Aged HFSCs significantly lost their polarized expression. w, 3D reconstructed IF images of GFP, KRT10 and KRT75 in GFP labeled-HFSCs from young (11 wo) and from aged (25 mo) mice at 3 days after depilation. x, y, Representative IF images (x) and 3D reconstructed IF images (y) of GFP and KRT10 without hair depilation in GFP labeled-HFSCs from young and from aged mice. Frequency of KRT10+ HFs in the GFP+ HFSCs are shown at the bottom (n = 8–10 images in each group). Arrows, normal HFSCs; arrowheads; aged epidermally differentiated HFSCs. White line and fan-shaped line and upper number, spindle angle against basal cell layer (o) ; white dashed line, basal cell layer. Error bars, means ± SEM; Chi-square test (g, h, t, v) or two-tailed Mann-Whitney U-test (s).

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