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# Epithelial geometry regulates spindle orientation and progenitor fate during formation of the mammalian epidermis.

### Author information

- 1
- Department of Molecular Biology, Princeton University, Princeton, United States.

### Abstract

The control of cell fate through oriented cell division is imperative for proper organ development. Basal epidermal progenitor cells divide parallel or perpendicular to the basement membrane to self-renew or produce differentiated stratified layers, but the mechanisms regulating the choice between division orientations are unknown. Using time-lapse imaging to follow divisions and fates of basal progenitors, we find that mouse embryos defective for the planar cell polarity (PCP) gene, *Vangl2*, exhibit increased perpendicular divisions and hyperthickened epidermis. Surprisingly, this is not due to defective Vangl2 function in the epidermis, but to changes in cell geometry and packing that arise from the open neural tube characteristic of PCP mutants. Through regional variations in epidermal deformation and physical manipulations, we show that local tissue architecture, rather than cortical PCP cues, regulates the decision between symmetric and stratifying divisions, allowing flexibility for basal cells to adapt to the needs of the developing tissue.

© 2019, Box et al.

#### KEYWORDS:

cell geometry; developmental biology; epidermis; mouse; oriented cell division; planar cell polarity; regenerative medicine; skin; stem cells; stratification

**A**) Schematic of E14.5 skin depicting several planar cell polarity components and example division orientations. Dotted line represents focal plane for live imaging. (

**B,C**) Stills from time-lapse movies of

*Vangl2*E14.5 skin explants expressing K14-H2B-GFP, showing examples of planar (

^{WT}**B**) and perpendicular (

**C**) division orientations. Top panels are the planar view of the basal layer of the epidermis, bottom panels are XZ dimension. Scale bar = 10 μm. See also and . (

**D**) Example and quantification of division angles in live epidermal explants at E14.5.

*Vangl2*, n = 310 divisions pooled from three embryos.

^{WT}*Vangl2*, n = 271 divisions from three embryos. Modified Kuiper’s Test, p=1.7523e-15. (

^{Lp/Lp}**E**) Distribution of division orientations in live epidermal explants at E14.5. Planar: Θ <= 20°, unpaired two-tailed t-test p=0.027; oblique: 20°>Θ>=70°, p=0.030; perpendicular: 70°>Θ>=90°, p=0.427. n = 3 explants from each genotype. (

**F**) Angular frequency of division angles quantified from fixed whole mount skins dissected from embryos e13.5 – e16.5. E13.5:

*Vangl2*, n = 208 divisions from three embryos;

^{WT}*Vangl2*, n = 198 divisions from two embryos; p=2.8648e-08. E14.5:

^{Lp/Lp}*Vangl2*, n = 164 divisions from three embryos;

^{WT}*Vangl2*, n = 166 divisions from three embryos; p=1.5118e-18. E15.5:

^{Lp/Lp}*Vangl2*, n = 152 divisions from three embryos;

^{WT}*Vangl2*, n = 116 divisions from three embryos; p=0.3482. E16.5:

^{Lp/Lp}*Vangl2*, n = 132 divisions from three embryos;

^{WT}*Vangl2*, n = 115 divisions from three embryos; p=0.1785.

^{Lp/Lp}**A, B**) The relationship between division angle and final cell positions. Daughter cells were followed for 1.5 hr after cell division, and assigned a positional fate: symmetric or asymmetric. Each dot represents a single division event. (

**C**) Representation of the same data in (

**A,B**), showing the distribution of fates for each division orientation. For planar divisions: symmetric fates = 94% [

*Vangl2*], 78% [

^{WT}*Vangl2*]. Oblique divisions: symmetric fates = 41% [

^{Lp/Lp}*Vangl2*], 21% [

^{WT}*Vangl2*]. Perpendicular divisions: asymmetric fates = 89% [

^{Lp/Lp}*Vangl2*], 94% [

^{WT}*Vangl2*]. (

^{Lp/Lp}**D**) Combining all division orientations,

*Vangl2*embryos display an overall bias toward asymmetric final cell positions. Unpaired two-tailed t-test, p=0.017. For (

^{Lp/Lp}**A–D**), n = 284 divisions pooled across three

*Vangl2*embryos; n = 238 divisions pooled from three

^{WT}*Vangl2*embryos. (

^{Lp/Lp}**E**) Sagittal sections of E18.5 skin from

*Vangl2*and

^{WT}; K14-H2B-GFP*Vangl2*embryos. Involucrin (red) labels the outer stratified layers and nuclei are labeled with Hoechst. Scale bar = 50 μm. (

^{Lp/Lp}; K14-H2B-GFP**F**) Quantification of skin thickness at E18.5. n = 10 images for each of three embryos per genotype. Bars represent means of the three embryos, error bars are SEM. Unpaired two-tailed t-test, p=0.017. (

**G**) XZ panels from whole mount images of

*Vangl2*and Vangl2 mutant skins at E14.5 – E16.5, expressing membrane-tdTomato or immunostained for E-Cadherin. Yellow dotted lines outline the outermost epidermal layer. Scale bars = 10 μm. (

^{WT}**H**) Quantification of skin thickness at E14.5 – E16.5. n = 30 measurements per genotype per stage. Bars represent means, error bars are SD. E14.5: unpaired two-tailed t-test, p=0.9340; E15.5: unpaired two-tailed t-test, p=0.0003; E16.5: unpaired two-tailed t-test, p<0.0001.

**A**) Examples from

*Vangl2*embryos of cells dividing in a planar orientation with unilateral (left) and absent (right) LGN localization. In all images, Hoechst labels the nuclei, cell membranes are marked with E-Cadherin (red) and LGN is white. Yellow dashed lines outline the dividing cell, and yellow arrowheads mark LGN localization. (

^{WT}**B**) Frequency of LGN localization patterns in planar cell divisions in

*Vangl2*embryos at E14.5. n = 36 cells (metaphase-telophase). ‘Not correlated’ LGN refers to a detectable signal that does not correlate with division plane (i.e., basal in a perpendicularly dividing cell). (

^{WT}**C**) Examples from

*Vangl2*embryos of cells dividing in a perpendicular orientation with apical (left) and absent (right) LGN. (

^{ΔTm/Lp}**D**) Frequency of LGN localization patterns in all dividing cells in

*Vangl2*and

^{WT}*Vangl2*embryos at E14.5. n = 99 and 107 cells, respectively (metaphase-telophase). All scale bars = 10 μm.

^{ΔTm/Lp}**A**) Example image of apical LGN localization in a

*Vangl2*E16.5 embryo. Shown is a mitotic cell in metaphase, poised to divide perpendicularly. In all images, Hoechst labels the nuclei, cell membranes are marked with E-Cadherin (red) and LGN is white. Yellow dashed lines outline the dividing cell, and yellow arrowheads mark LGN localization.

^{WT}**A**) Representative images of basal layer labeled with E-Cadherin from E14.5

*Vangl2*and

^{WT}*Vangl2*embryos. (

^{Lp/Lp}**B**) Quantification of cross sectional areas of basal cells. Each dot represents average area of all cells in a single field of view. n = 10 total fields of view from three embryos per genotype. Bars are mean with SEM. Unpaired two-tailed t-test, p=0.0057 (

**C**) Quantification of cell height along apical-basal axis. 300 cells were measured from across three embryos per genotype. Whiskers indicate minimum and maximum values, + indicates the mean. Unpaired two-tailed t-test, p<0.0001. (

**D**) Quantification of distribution of polygon classes, as defined by the number of each cell’s neighbors. n = 10 total fields of view from three embryos per genotype. Error bars denote SD. (

**E**) Quantification of cell density, as number of cells per field of view (1200 μm

^{2}). Each dot represents a single field of view. n = 10 fields of view across three embryos per genotype. Bars are mean with SD. Unpaired two-tailed t-test, p=0.0001. (

**F**) Proliferation rates, quantified as the number of mitotic cells in a field of view as a percentage of total number of cells. n = 10 fields of view across three embryos. Whiskers denote min-max values, mean shown as ‘+.’ Unpaired two-tailed t-test, p=0.9987. (

**G**) Still images from a time-lapse movie of E14.5

*Vangl2*explant showing an example of a basal cell dividing within the plane of the epidermis, along its longest interphase axis. Red line denotes orientation of the longest XY axis of the cell. See also . (

^{WT}; K14-Cre; mTmG**H**) Still images from a time-lapse movie of an E14.5 skin explant from

*Vangl2*embryo showing a cell dividing in a perpendicular orientation. Daughter cells are marked with asterisks in the XZ view. See also . (

^{ΔTm/Lp}; mTmG**I,J**) Relationship between height:width aspect ratio and division orientation. H:W ratios were measured from interphase cells at the time just before the onset of mitotic rounding. Width is defined as the longest planar axis of the cell. Each dot corresponds to a single division event, and divisions were binned according to the angle of the division plane in cytokinesis. Bars are mean with SD. (

**I**) n = 72 divisions pooled from three E14.5 embryos. One-way ANOVA, p<0.0001. Tukey’s Multiple Comparison Test: planar vs oblique, p<0.05; planar vs perpendicular, p<0.05; oblique vs perpendicular, p>0.05. (

**J**) n = 66 divisions from two E14.5

*Vangl2*embryos. One-way ANOVA, p<0.0001. Tukey’s Multiple Comparison Test: planar vs oblique, p<0.05; planar vs perpendicular, p<0.05; oblique vs perpendicular, p<0.05. All scale bars = 10 μm.

^{ΔTm/Lp}**A**) Representative images of basal layer labeled with membrane-tdTomato from E13.5

*Vangl2*and

^{WT}*Vangl2*embryos. (

^{Lp/ΔTm}**B**) Quantification of cross sectional areas of basal cells. Each dot represents average area of all cells in a single field of view. n = 12 total fields of view from three

*Vangl2*embryos and two

^{WT}*Vangl2*embryos. Bars are mean with SEM. Unpaired two-tailed t-test, p<0.0001. (

^{Lp/ΔTm}**C**) Quantification of cell height along apical-basal axis. n = 300 cells from across three

*Vangl2*embryos and two

^{WT}*Vangl2*embryos. Whiskers indicate minimum and maximum values, + indicates the mean. Unpaired two-tailed t-test, p<0.0001. (

^{Lp/ΔTm}**D**) Quantification of distribution of polygon classes, as defined by the number of each cell’s neighbors. n = 12 fields of view per genotype. Error bars denote SD. (

**E**) Quantification of cell density, as number of cells per field of view (700 μm

^{2}). Each dot represents a single field of view. n = 12 fields of view per genotype. Bars are mean with SD. Unpaired two-tailed t-test, p<0.0001. (

**F**) Proliferation rates, quantified as the number of mitotic cells in a field of view as a percentage of total number of cells. n = 12 fields of view per genotype. Whiskers denote min-max values, mean shown as ‘+.’ Unpaired two-tailed t-test, p=0.4416. (

**G**) Representative images of basal layer labeled with membrane-tdTomato from E15.5

*Vangl2*and

^{WT}*Vangl2*embryos. (

^{ΔTm/ΔTm}**H**) Quantification of cross sectional areas of basal cells. Each dot represents average area of all cells in a single field of view. n = 15 total fields of view from three embryos per genotype. Bars are mean with SEM. Unpaired two-tailed t-test, p=0.2314. (

**I**) Quantification of cell height along apical-basal axis. n = 300 cells from across three embryos per genotype. Whiskers indicate minimum and maximum values, + indicates the mean. Unpaired two-tailed t-test, p<0.0001. (

**J**) Quantification of distribution of polygon classes, as defined by the number of each cell’s neighbors. n = 15 fields of view per genotype. Error bars denote SD. (

**K**) Quantification of cell density, as number of cells per field of view (700 μm

^{2}). Each dot represents a single field of view. n = 15 fields of view per genotype. Bars are mean with SD. Unpaired two-tailed t-test, p=0.6472. (

**L**) Proliferation rates, quantified as the number of mitotic cells in a field of view as a percentage of total number of cells. n = 15 fields of view per genotype. Whiskers denote min-max values, mean shown as ‘+.’ Unpaired two-tailed t-test, p=0.7641. All scale bars = 10 μm.

**A**) Relationship between division angle and height:width ratio in E14.5

*Vangl2*skin explants. Slope = 0.006090 ± 0.0009291; R

^{WT}^{2}= 0.3803. Pearson’s correlation coefficient R = 0.62. (

**B**) Relationship between division angle and height:width ratio in E14.5

*Vangl2*skin explants. Slope = 0.005308 ± 0.0009498; R

^{Lp/ΔTm}^{2}= 0.3280. Pearson’s correlation coefficient R = 0.57.

**A**) Representative images of the basal layer of

*Vangl1*control and

^{fl/fl}; Vangl2^{fl/fl}; mTmG*Vangl1*embryos. Brightness of Vangl2 panel in

^{fl/fl}, Vangl2^{fl/fl}; K14-Cre; mTmG*Vangl1*was increased to show lack of Vangl2 staining. Scale bars = 10 μm. (

^{fl/fl}; Vangl2^{fl/fl}; K14-Cre; mTmG**B**) Quantification of basal cell cross-sectional areas. Each dot represents average area of all cells in a single field of view. n = 15 fields of view across three embryos per genotype. Bars are mean with SEM. Unpaired two-tailed t-test, p=0.9209. (

**C**) Quantification of cell height along apical-basal axis. n = 300 cells per genotype. Whiskers indicate minimum and maximum values, + indicates the mean. Unpaired two-tailed t-test, p=0.4209. (

**D**) Quantification of cell density, as number of cells per field of view (700 μm

^{2}). Each dot represents a single field of view. n = 15 fields of view across three embryos per genotype. Bars are mean with SD. Unpaired two-tailed t-test, p=0.7840. (

**E**) Division orientations in

*Vangl1*control embryos. n = 211 divisions, from 45 images across three embryos. (

^{fl/fl}; Vangl2^{fl/fl}; mTmG**F**) Division orientations in

*Vangl1*embryos. n = 181 divisions, from 45 images across three embryos. Modified Kuiper’s Test, p=0.3743.

^{fl/fl}; Vangl2^{fl/fl}; K14-Cre; mTmG**A**) Representative images of the basal layer of

*Fz6*and

^{WT}*Fz6*embryos expressing membrane-tdTomato. Scale bars = 10 μm. (

^{-/-}**B**) Quantification of basal cell cross-sectional areas. Each dot represents average area of all cells in a single field of view. n = 15 fields of view across three embryos per genotype. Bars are mean with SEM. Unpaired two-tailed t-test, p=0.1357. (

**C**) Quantification of cell height along apical-basal axis. n = 300 cells per genotype. Whiskers indicate minimum and maximum values, + indicates the mean. Unpaired two-tailed t-test, p=0.4283. (

**D**) Quantification of cell density, as number of cells per field of view (700 μm

^{2}). Each dot represents a single field of view. n = 15 fields of view across three embryos per genotype. Bars are mean with SD. Unpaired two-tailed t-test, p=0.1739. (

**E**) Division orientations in

*Fz6*embryos. n = 167 divisions, from three embryos. (

^{WT}**F**) Division orientations in

*Fz6*embryos. n = 167 divisions, from three embryos. Modified Kuiper Test, p=0.0644.

^{-/-}**A**) Representative images of the basal layer of

*Vangl2*and

^{WT}*Vangl2*embryos with or without

^{ΔTm/Lp}*K14-Vangl2-GFP*. Green = 488 channel, Greyscale = Celsr1, Red = membrane tdTomato. Rose plots quantify Celsr1 polarity in the basal layer. Red line indicates the average magnitude and direction of polarity. Rose plots were generated from three images per genotype. All scale bars = 10 μm. (

**B**) Quantification of cell surface areas. Each dot represents average surface area of all cells in a single field of view. n = 15 fields of view across three embryos per genotype. Bars are mean with SEM. Unpaired two-tailed t-test, p<0.0001. (

**C**) Quantification of cell height along apical-basal axis. n = 300 cells per genotype. Whiskers indicate minimum and maximum values, + indicates the mean. Unpaired two-tailed t-test, p<0.0001. (

**D**) Quantification of cell density, as number of cells per field of view (700 μm

^{2}). Each dot represents a single field of view. n = 15 fields of view across three embryos per genotype. Bars are mean with SD. Unpaired two-tailed t-test, p<0.0001. (

**E**) Division orientations in

*Vangl2*embryos. n = 134 divisions, from 45 images across three embryos. (

^{WT}; K14-Vangl2-GFP**F**) Division orientations in

*Vangl2*embryos. n = 159 divisions, from 45 images across three embryos. Modified Kuiper Test, p=1.6201e-19.

^{ΔTm/Lp}; K14-Vangl2-GFP**A**) Schematic of an E13.5

*Vangl2*embryo, depicting the lateral, intermediate, and midline regions. (

^{WT}**B**) Quantification of cell height along apical-basal axis. n = 300 cells per genotype. Whiskers indicate minimum and maximum values, + indicates the mean. Lateral vs Intermediate, unpaired two-tailed t-test, p<0.0001; Lateral vs Midline, unpaired two-tailed t-test, p<0.0001; Intermediate vs Midline, unpaired two-tailed t-test, p<0.0001. (

**C**) Quantification of basal cross-sectional areas. Each dot represents average surface area of all cells in a single field of view. Two images per region were analyzed, from each of three embryos, for a total of 6 measurements per region. Bars are mean with SEM. Lateral vs Intermediate, unpaired two-tailed t-test, p=0.4872; Lateral vs Midline, unpaired two-tailed t-test, p<0.0001; Intermediate vs Midline, unpaired two-tailed t-test, p<0.0001. (

**D**) Quantification of cell elongations. Each dot represents the average elongation value of all cells in a field of view. Two images per region were analyzed, from each of three embryos, for a total of 6 measurements per region. Bars represent mean with SEM. Lateral vs Intermediate, unpaired two-tailed t-test, p=0.0125; Lateral vs Midline, unpaired two-tailed t-test, p=0.2256; Intermediate vs Midline, unpaired two-tailed t-test, p=0.0432. (

**E**) Quantification of cell density, as number of cells per image (875 μm

^{2}). Each dot represents a single field of view. Two images per region were analyzed, from each of three embryos, for a total of 6 measurements per region. Bars represent mean with SD. Lateral vs Intermediate, unpaired two-tailed t-test, p=0.8567; Lateral vs Midline, unpaired two-tailed t-test, p<0.0001; Intermediate vs Midline, unpaired two-tailed t-test, p<0.0001. (

**F–H**) Representative images of cell divisions in the lateral, intermediate, and midline regions in E13.5

*Vangl2*embryos. Hoechst and membrane-tdTomato mark the nuclei and cell membranes, respectively. (

^{WT}; K14-H2B-GFP**I–K**) Division orientations in the lateral, intermediate, and midline regions. Lateral: n = 99 divisions pooled from three embryos; Intermediate: n = 109 divisions pooled from three embryos; Midline: n = 50 divisions pooled from three embryos. Modified Kuiper’s Test: lateral vs intermediate, p=7.2638e-04; lateral vs midline, p=9.8559e-06; intermediate vs midline, p=1..5851e-04. (

**L**) Distribution of planar vs oblique + perpendicular division orientations in the lateral, intermediate, and midline regions.

**A**) Quantification of cell height along apical-basal axis. n = 300 cells per region. Whiskers indicate minimum and maximum values, + indicates the mean. Unpaired two-tailed t-test, p=0.9412. (

**B**) Quantification of basal cross-sectional areas. Each dot represents average surface area of all cells in a single field of view. Three images per region were analyzed, from each of two embryos, for a total of 6 measurements per region. Bars are mean with SEM. Unpaired two-tailed t-test, p=0.3670. (

**C**) Quantification of cell density, as number of cells per image (875 μm

^{2}). Each dot represents a single field of view. Two images per region were analyzed, from each of three embryos, for a total of 6 measurements per region. Bars represent mean with SD. Unpaired two-tailed t-test, p=0.3385. (

**D**) Quantification of cell elongations. Each dot represents the average elongation value of all cells in a field of view. Three images per region were analyzed, from each of two embryos, for a total of 6 measurements per region. Bars represent mean with SEM. Unpaired two-tailed t-test, p=0.2755. (

**E,F**) Representative images of cell divisions in the lateral and intermediate regions in

*E13.5 Vangl2*embryos. Hoechst and membrane-tdTomato mark the nuclei and cell membranes, respectively. (

^{Lp/ΔTm}; K14-H2B-GFP**G,H**) Division orientations in the lateral and intermediate regions. Lateral: n = 87 divisions pooled from two embryos; Intermediate: n = 111 divisions pooled from two embryos. Modified Kuiper’s test, p=0.2250. (

**I**) Distribution of planar vs oblique + perpendicular division orientations in the lateral and intermediate regions.

**A**) Representative images of the basal layer of E14.5

*Vangl2*unstretched and stretched skin explants. White, membrane-tdTomato. Scale bars = 10 μm. (

^{ΔTm/Lp}**B**) Quantification of cell elongation in unstretched vs stretched skins. Each dot represents the average elongation value of all cells in a field of view. n = three images from each of three separate experiments. Bars are mean with SEM. Unpaired two-tailed t-test, p<0.0001. (

**C**) Quantification of cell height along apical-basal axis. n = 450 cells per genotype. Whiskers indicate minimum and maximum values, + indicates the mean. Unpaired two-tailed t-test, p<0.0001. (

**D**) Division orientations in unstretched E14.5

*Vangl2*explants. n = 48 divisions pooled from three explants. (

^{ΔTm/Lp}**E**) Division orientations in stretched E14.5

*Vangl2*embryos. n = 57 divisions pooled from three explants. Modified Kuiper’s Test, p=3.9430e-06.

^{ΔTm/Lp}**A**) Representative images of the basal layer of E15.5

*Vangl2*unstretched and stretched skin explants with vectors illustrating direction and magnitude of cell elongation. White, membrane-tdTomato. Scale bars = 10 μm. (

^{WT}**B**) Quantification of cell elongation in unstretched vs stretched skins. Each dot represents the average elongation value of all cells in a field of view. n = three images from each of two separate embryos/experiments. Bars are mean with SEM. Unpaired two-tailed t-test, p=0.0001. (

**C**) Quantification of cell height along apical-basal axis. n = 450 cells per genotype. Whiskers indicate minimum and maximum values, + indicates the mean. Unpaired two-tailed t-test, p<0.0001. (

**D**) Division orientations in control E15.5

*Vangl2*explants. n = 61 divisions pooled from two explants. (

^{WT}**E**) Division orientations in stretched E15.5

*Vangl2*skin explants. n = 50 divisions pooled from two explants. Modified Kuiper’s Test, p=1.0967e-08.

^{WT}*Vangl2*mutant, but this difference was not significant (Chi-squared test for bin 1.0-1.5 p=0.52; bin 0.5-1.0 p=0.35), but this could perhaps be due to the low sample size within each bin. Thus, I don’t think we can conclude with our data set whether Vangl2 affects the spindle by variables other than shape.

^{Lp/Lp}*Vangl2*embryos to be even greater, suggesting our results aren’t artifacts of how we binned division planes. We decided to keep the original binning of these data and preserve the original language used in the text. We also provide a comparison of the data binned in different ways in .

^{Lp/Lp}### Conflict of interest statement

KB, BJ, DD No competing interests declared