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Par3–mInsc and Gαi3 cooperate to promote oriented epidermal cell divisions through LGN

Abstract

Asymmetric cell divisions allow stem cells to balance proliferation and differentiation. During embryogenesis, murine epidermis expands rapidly from a single layer of unspecified basal layer progenitors to a stratified, differentiated epithelium. Morphogenesis involves perpendicular (asymmetric) divisions and the spindle orientation protein LGN, but little is known about how the apical localization of LGN is regulated. Here, we combine conventional genetics and lentiviral-mediated in vivo RNAi to explore the functions of the LGN-interacting proteins Par3, mInsc and Gαi3. Whereas loss of each gene alone leads to randomized division angles, combined loss of Gnai3 and mInsc causes a phenotype of mostly planar divisions, akin to loss of LGN. These findings lend experimental support for the hitherto untested model that Par3–mInsc and Gαi3 act cooperatively to polarize LGN and promote perpendicular divisions. Finally, we uncover a developmental switch between delamination-driven early stratification and spindle-orientation-dependent differentiation that occurs around E15, revealing a two-step mechanism underlying epidermal maturation.

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Figure 1: LGN promotes perpendicular divisions in a developmentally restricted manner.
Figure 2: Early stratification is driven by delamination.
Figure 3: Loss- or gain-of-function in spindle orientation genes does not alter early stratification behaviour.
Figure 4: mInsc cKOs show impaired LGN localization and randomized spindle orientation.
Figure 5: Pard3 knockout or knockdown phenocopies loss of mInsc.
Figure 6: Gαi3 promotes apical LGN localization and perpendicular divisions.
Figure 7: Gαi3 and mInsc act cooperatively to localize LGN to the apical cortex.
Figure 8: Model of epidermal stratification mechanisms.

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Acknowledgements

We thank N. Stokes, D. Oristian and A. Aldeguer (Fuchs laboratory) and T. Anthony Curtis (Williams laboratory), for their expert technical assistance. We thank K. Byrd, K. Lough, and members of the Williams and Fuchs laboratories for critical reading of the manuscript and K. Lough for valuable input into the model presented in Fig. 8. We are grateful to S. Ohno and T. Hirose (both at Yokohama City University of Medicine, Japan) for sharing the Pard3 floxed mouse line. S.E.W. was supported by an American Cancer Society postdoctoral fellowship and E.F. is an investigator in the Howard Hughes Medical Institute. Work in the Fuchs laboratory was supported by a grant from the National Institutes of Health (E.F. R37-27883).

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S.E.W. designed and conducted experiments and analysed the data under the supervision of E.F. L.A.R. performed the imaging and analysis for the lineage tracing experiments. M.P.P. and J.A.K. provided mInsc mice before publication. S.E.W. and E.F. wrote the manuscript. All authors critically read and contributed to the manuscript.

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Correspondence to Elaine Fuchs.

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Integrated supplementary information

Supplementary Figure 4 Maturation of differentiation markers in developing epidermis.

(a) Beginning around E13.5, at the stage when suprabasal cells (except the periderm) are absent, sporadic basal cells coexpress the spinous keratin, K10, along with basal keratins K5 and K14 (not shown). Note also that at this age, the basement membrane marker and hemidesmosome constituent β4-integrin is broadly and diffusely expressed throughout basal cells at E13.5, while it progressively becomes more basally restricted at later ages. (b) Sections of wild-type E14.5 back skin from anterior (less differentiated) to posterior (more differentiated). In anterior regions of single-layered epidermis, K10 and K5 are broadly coexpressed, while β4-integrin remains diffuse. In areas where β4-integrin begins to show some apical enrichment and suprabasal cells are present, K10 becomes more restricted to suprabasal cells, though the basal keratin K5 is diffusely coexpressed there. In posterior regions, the segregation of K10 and K5 becomes more apparent. (c) At E15.5, β4-integrin becomes more restricted to the epidermal-dermal boundary, while K10 and K5 are expressed in opposing domains. An exception is the appearance of sporadic cells positioned in the basal layer which coexpress K10 and K4 (arrows). We suggest that these are cells undergoing differentiation by delamination rather than asymmetric cell division.

Supplementary Figure 5 Both Notch and LGN are dispensable for early stage stratification.

(a) (Top left) Schematic of lentiviral Notch reporter construct. Nuclear H2B-RFP is expressed under a constitutive reporter to mark cells transduced with the reporter construct, while cytosolic GFP reveals cells in which Notch signalling has been activated. At E17.5 (right), robust Notch/GFP+ cells are observed in suprabasal (K10+, blue) layers. In contrast, at E15.5 (bottom left), few cells show detectable Notch activity, and those that do are weakly positive, and appear in both basal (arrow) and suprabasal (arrowhead) layers. (b) Quantification of the percentage of cells expressing the Notch reporter construct (RFP+) which show detectable Notch activity (GFP+). At E17.5, there is a strong bias towards suprabasal Notch activity, while at E15.5 Notch activity is lower overall, and present equally in basal and suprasal cells. (c,d) Spinous differentiation as indicated by K10 (green) at E15.5 (c) and E16.5 (d) in shScramble control (top), LGN knockdown (middle) and Rbpj knockout (bottom) sections of back skin. Note that the formation of the initial spinous layer is not impacted by impairing spindle orientation or Notch. Unlike controls, however, differentiation fails to progress in these mutants. RFP (red) indicates H2B-mRFP1 in top and middle panels, and Cre-mRFP1 in bottom panels. Rbpj loss at this age was confirmed by absence of the target gene Hes1 in suprabasal layers (not shown17). Scale bars: 50 μm.

Supplementary Figure 6 Characterization of shRNA knockdown efficiency in vitro and in vivo.

(a) Quantification of mInsc knockdown efficiency in keratinocytes stably-transduced with retroviral mInsc, necessary due to low endogenous levels of mInsc in low calcium conditions. (b) Quantification of Pard3 knockdown in keratinocytes. Red letters indicate those with the highest levels of knockdown (94–97%), subsequently used for in vivo studies. (c) Quantification of mRNA knockdown efficiency in keratinocytes for Gnai2 (left) and Gnai3 (right) shRNA clones. Those with the strongest knockdown (>97%) are shown in red letters, and were subsequently used in vivo. Bars are the mean ± s.d. (d) Back skin sections from E17.5 epidermis showing specific loss of Par3 expression in mosaic Pard3 knockout (top left) or knockdown tissue. (e) Confirmation of knockdown efficiency by immunofluorescence in E17.5 back skin. In addition to its polarized localization in mitotic basal cells, Gαi3 is also localized to cell membranes suprabasally, like Par3 (top panel). This localization is specifically lost in RFP+ regions of mosaic knockdown tissue (bottom panel). (f) Gαii3 is normally enriched apically in mitotic basal cells (left, see also Fig. 5b), but following Gnai3 knockdown, LGN cortical expression is frequently reduced (middle row, weaker hairpin) or delocalized (bottom row, stronger hairpin).

Supplementary Table 1 Chi-square analyses of division angle distributions.
Supplementary Table 2 Clone distribution of lentiviral lineage tracing.
Supplementary Table 3 Effect of Par3 loss on mInsc apical localization.

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Williams, S., Ratliff, L., Postiglione, M. et al. Par3–mInsc and Gαi3 cooperate to promote oriented epidermal cell divisions through LGN. Nat Cell Biol 16, 758–769 (2014). https://doi.org/10.1038/ncb3001

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