Asymmetric cell divisions promote Notch-dependent epidermal differentiation

Abstract

Stem and progenitor cells use asymmetric cell divisions to balance proliferation and differentiation. Evidence from invertebrates shows that this process is regulated by proteins asymmetrically distributed at the cell cortex during mitosis: Par3-Par6-aPKC, which confer polarity, and Gα(i)-LGN/AGS3-NuMA-dynein/dynactin, which govern spindle positioning. Here we focus on developing mouse skin, where progenitor cells execute a switch from symmetric to predominantly asymmetric divisions concomitant with stratification. Using in vivo skin-specific lentiviral RNA interference, we investigate spindle orientation regulation and provide direct evidence that LGN (also called Gpsm2), NuMA and dynactin (Dctn1) are involved. In compromising asymmetric cell divisions, we uncover profound defects in stratification, differentiation and barrier formation, and implicate Notch signalling as an important effector. Our study demonstrates the efficacy of applying RNA interference in vivo to mammalian systems, and the ease of uncovering complex genetic interactions, here to gain insights into how changes in spindle orientation are coupled to establishing proper tissue architecture during skin development.

Figure 1. Spindle orientation defects following LGN, Numa1, and Dctn1 depletion

a, Immunodetection of anaphase-telophase pronuclei. with spindle midbody marker Survivin. b, Apical colocalisation of ACD components during mitosis. c,d shRNA knockdown efficiencies in keratinocytes and epidermis (n=3 separate experiments). e, Representative axes of division (lines) in E16.5 transduced anaphase/telophase cells. f, Radial histogram quantification of data from (e), n’s are indicated. g, Cell-autonomous elimination of ACDs upon LGN, Numa1, or Dctn1 knockdown. h–i, Interdependence of Gαi3/LGN/NuMA cortical localisation. j,k Misalignment of angles between LGN crescent centre and centrosomal axis (spindle) upon Numa1 knockdown (each dot indicates a single data point). Scale bars: 10µm. Error bars: S.D. (c, d); S.E.M (k). Dotted lines denote basement membrane (thick); cell boundaries (thin).

Figure 2. Impaired stratification in vitro and in vivo in when ACDs are impaired

a, Quantification of differentiation (K10) in shLGN-1617-transduced and rescued keratinocytes (n=8 fields/condition). b, Skin barrier defects in ACD knockdown embryos. c, Epidermal ultrastructure. Layers: BL, basal; SL, spinous; GL, granular; SC, stratum corneum (bar, 10µm).. Late-stage differentiation defects in LGN knockdowns are shown at higher magnification (bar, 2µm; Gr, keratohyalin-granules; Nu, nuclei). d, Quantifications revealing ~17% increase in basal nuclei density (~36% more basal cells/mm) in E17.5 shLGN-1617 epidermis. Whiskers indicate minimum and maximum values; boxes span 25–75 percentiles, centre bar denotes median value; +marks designate mean, n>20 sections/condition. e, Measurements of epidermal thinning in knockdowns (n>3 embryos/condition). Error bars represent S.D.

Figure 3. Differentiation defects following LGN, Numa1, and Dctn1 depletion

a,b, Reduced terminal differentiation in E17.5 ACD knockdowns. Basally-transduced regions are identified by H2B-mRFP1, always most intense in suprabasal progeny. Note correlation of repressed differentiation with transduction (RFP⁺; line demarcates low/high infection boundary). c,d, Partial restoration of shLGN-1617 epidermal defects upon transducing full-length(FL)LGN or LGNΔC (n>15 fields; n>6 embryos/condition). e–h, EYFP-mInsc enhancement of LGN-dependent ACDs. e, EYFP-mInsc and LGN immunolocalisation in mitotic cells of E17.5 shScramble or shLGN-1617 epidermis after EYFP-mInsc. co-transduction. f, Quantifications of division axes (n’s indicated). (g,h) LGN-dependent enhancement of spinous-layer thickness upon mInsc overexpression (n>10 fields; n>3 embryos/condition). Scale bars: 50 µm (a–c, g), 10 µm (e). Error bars are S.E.M.

Figure 4. Loss of LGN or Numa1 impairs suprabasal Notch activation

a, qPCR vs. microarray comparisons of Notch pathway gene expression in E14–E15 wild-type epidermis. b,c, Diminished Hes1 and full-length Notch3 in shLGN-1617-transduced epidermis. Line (b) demarcates low/high-infection boundary. d, Decreased Notch3 (p=0.0133) and Hes1 (p=0.0169) mRNAs in E18 shLGN-1617 suprabasal cells. Note also dampened suprabasal:basal Notch1 (p=0.20), Notch2 (p=0.19). e, Lentiviral Notch reporter for coordinate shRNA-knockdown. f,g Abrogation of Notch reporter expression (EGFP⁺), concomitant with differentiation defects, in E17.5 RBPJ cKO and shLGN-1617 epidermis. h, Effects of LGN/Numa1 knockdown on Notch reporter activity (n>24 fields; >3 embryos/condition). i, Reduced activity in P0 Notch reporter transgenics transduced with shLGN-1617;H2B-mRFP1. Error bars in a,d represent S.D; S.E.M. in h. Scale bars: 50µm. For qPCR (a,d), n’s are triplicates from 2 separate experiments.

Figure 5. Genetic interaction between ACD and Notch pathways

a–c, Normal LGN localization and ACDs in RBPJ mutants (each dot represents one data point in b, n’s indicated in c). d,e, Analyses of differentiation defects in E17.5 headskins from control or RBPJ^fl/fl embryos transduced at E9.5 with shLGN-1617;H2B-mRFP1 (LGN1617), shScramble;NLS-Cre-mRFP1 (RBPJ+scramble), or shLGN-1617;NLS-Cre-mRFP1 (RBPJ+LGN1617). Comparable defects in double and single mutants/knockdowns, suggest a common pathway for RBPJ and LGN. f–h, Restoring Notch signalling rescues shLGN-1617 differentiation defects. Headskin (f); backskin (g,h). Combinations of single and double mutant clones (separated by vertical lines) expressing shLGN-1617 (red) and active NICD (GFP, pseudocolored in blue) were generated by co-infecting E9.5 Rosa-Lox-stop-Lox-NICD-IRES-GFP-knockin embryos with shScramble/shLGN-1617;H2B-mRFP1 and NLS-Cre. Scale bars: 10µm (a); 50µm (d, f, g). Error bars represent S.D. (b), S.E.M. (e,h). p values from two-tailed student’s t-tests are indicated; ns: not statistically significant. For e,h, n>10 fields; n>3 embryos.