Asymmetric cell divisions promote stratification and differentiation of mammalian skin

Abstract

The epidermis is a stratified squamous epithelium forming the barrier that excludes harmful microbes and retains body fluids. To perform these functions, proliferative basal cells in the innermost layer periodically detach from an underlying basement membrane of extracellular matrix, move outward and eventually die. Once suprabasal, cells stop dividing and enter a differentiation programme to form the barrier. The mechanism of stratification is poorly understood. Although studies in vitro have led to the view that stratification occurs through the delamination and subsequent movement of epidermal cells, most culture conditions favour keratinocytes that lack the polarity and cuboidal morphology of basal keratinocytes in tissue. These features could be important in considering an alternative mechanism, that stratification occurs through asymmetric cell divisions in which the mitotic spindle orients perpendicularly to the basement membrane. Here we show that basal epidermal cells use their polarity to divide asymmetrically, generating a committed suprabasal cell and a proliferative basal cell. We further demonstrate that integrins and cadherins are essential for the apical localization of atypical protein kinase C, the Par3-LGN-Inscuteable complex and NuMA-dynactin to align the spindle.

Figure 1. Asymmetric cell divisions govern stratification and differentiation during epidermal development

a–d, Images of embryonic skin showing the orientation of mitoses relative to the basement membrane (white dotted line), separating epidermis (epi in a) from dermis (der). Arrows indicate the mitotic cells in E12.5 epidermis; a (anaphase) and b (metaphase). DAPI marks the DNA in a. Indirect immunofluorescence with tubulin antibodies marks the spindle in b. Transgenic Centrin-GFP (green) marks spindle poles in c (metaphase, parallel spindle orientation), and d (anaphase, perpendicular spindle orientation). e, Quantification of division planes in epidermis during development. SL, single-layered E12.5 epidermis, ML, multi-layered E12.5 epidermis, T, total E12.5 epidermis. f, Parallel spindle orientation of an anaphase cell in p63 KO epidermis at E18.5; DAPI staining. g, h, Antibodies against β4 integrin (red) label the base of the basal cells. Antibodies against Ki67 (green) show that not all suprabasal cells at E15.5 (g) have withdrawn from a proliferative state; however, Ki67 is confined to basal cells at E18.5 (h). i, Antibodies against phosphohistone H3 (green) reveal the presence of mitotic suprabasal keratinocytes at E15.5. Red staining shows β4 integrin. j, Keratin 1 immunofluorescence (green) shows that suprabasal cells have turned on this differentiation marker at E15.5. Red staining shows β4 integrin. k, Magnified view of a keratin-1-positive (green) suprabasal mitotic cell in anaphase. Scale bars are 10 μm.

Figure 2. Mitotic apical localization of a mInsc–LGN–Par3 complex

a, mInsc interacts with LGN in cultured cells. Keratinocytes were transfected with K14 promoter driven full-length LGN (LGN-Flag) and mInsc (GFP-mInsc). Reciprocal co-immunoprecipitations (IPs) were performed. The extract (Ext) lane contains 10% of the input protein for the co-immunoprecipitation. b, Endogenous mInsc, LGN and Par3 form a complex in epidermis from E14.5 embryos. Protein extracts were prepared from embryos treated with nocodazole for 5 h to enrich for mitotic cells. Extracts were immunoprecipitated with anti-mInsc or anti-Par3 antibody as indicated, and immunoblots of the IPs were then performed with LGN, Par3 and mInsc antibodies. c, Epidermal protein lysates were prepared from E14.5 embryos treated with dimethylsulphoxide (DMSO; vehicle) or nocodazole for 5 h and probed with the indicated antibodies. d–l, Epifluorescence (GFP) or indirect immunofluorescence with antibodies colour-coded as indicated. DAPI (blue) was used to label chromatin. d–f, Apical crescents of LGN (d, e) and mInsc-GFP (f) are present in most mitotic basal cells of E15.5 embryonic epidermis (d) and adult (e, f). epi, epidermis; der, dermis. Note that e and f show co-localization of LGN and mInsc-GFP to the apical cortex of an anaphase cell. g, One E14.5 spindle pole was always located directly underneath the LGN crescent. h, Par3 localizes at E15.5 to the apical cortex of mitotic basal keratinocytes. The white dashed line demarcates basement membrane in e and f and borders of the anaphase cell in h. i, Mouse skin keratinocytes were stained with anti-LGN and β-tubulin antibodies. j, Keratinocytes were transfected with K14-GFP-mInsc and labelled simultaneously with DAPI to localize the DNA and identify mitotic cells. The image is a compressed Z-stack of confocal sections to view the entire cell. k, l, Keratinocytes were stained for DAPI and Par3 in mitotic (k) and interphase (l) cells. Scale bars are 10 μm, except in g, h, in which they are 5 μm.

Figure 3. Polarized mitotic localization of NuMA and dynactin

a, Mitotic keratinocyte stained simultaneously for NuMA (green) and microtubules (β-tubulin; red). b, E15.5 skin section stained with anti-NuMA antibodies reveals localization of NuMA (green) to spindle poles (arrowhead) and a cortical apical crescent (arrow). White dotted line denotes basement membrane. c, LGN (green) and NuMA (red) localize to the same crescent. K14-GFP-LGN-transfected keratinocytes were stained with antibodies against NuMA. d, Keratinocytes were treated with nocodazole to disrupt microtubules and then stained as in c. e, An anaphase keratinocyte stained for the p150glued subunit (green) of the dynactin complex. f, Co-localization of NuMA (green) and p150glued (red) in a mitotic keratinocyte. DNA is stained with DAPI (blue).

Figure 4. Cadherin and integrin requirements for LGN–NuMA localization and spindle orientation

a–d, LGN localization (green staining) was determined in embryonic skin from knockout (KO) mice lacking β4 integrin (a), β1 integrin (b), desmoplakin (c) and α-catenin (d). e, f, NuMA localization (green) in β1-integrin KO (e) and α-catenin KO (f) epidermis. g, LGN (green) and h, NuMA (green) localization in sections from embryos treated with monastrol for 5 h. i, j, Schematic representation of LGN localization in WT (i) and β1 KO (j) epidermis. Each dot represents the centre of an LGN crescent in a single mitotic cell. k, Schematic representation of NuMA localization in α-catenin KO epidermis. l, Quantification of cells with apical cortical localization of LGN. Note that α-catenin (α-cat) and p63 KOs do not show cortical localization of LGN. Error bars represent standard deviation. m–p, Schematic representation of spindle orientation in embryo skin from WT mice (m) and in β1 KO (n), α-catenin KO (o) and p63 KO (p) mice. Each line represents the spindle axis of a single late metaphase or anaphase cell. q–s, Immunolocalization of PKC-ζ (green) in WT (q), β1-integrin KO (r) and α-catenin KO epidermis (s). Red staining shows β4 integrin. Scale bars are 10 μm, except in e, f, in which they are 5 μm.