Regulated spindle orientation buffers tissue growth in the epidermis

4 figures and 1 additional file

Figures

Figure 1 with 3 supplements
Mitotic spindles reorient in response to changes in proliferation.

(A) Proliferation of control and K14-rtTA;TRE-Cdkn1b embryonic epidermis measured by pHH3 incorporation after treatment with doxycycline from e14.5–16.5. (B–C) Radial histograms of mitotic spindle orientation for e16.5 control (n = 61) and Cdkn1b (n = 59) embryonic epidermis. (D) Proliferation of adult backskin epidermis, control or TPA-treated (10 μl of 0.4 mM TPA, applied daily for 5 days), as measured by BrdU incorporation. n > 150 for each of three mice. (E–F) Radial histograms of mitotic spindle orientation for control (n = 20) and TPA-treated (n = 41) epidermis. Three or more mice were examined for each condition. *p<0.05, ***p<0.001.

https://doi.org/10.7554/eLife.48482.002
Figure 1—figure supplement 1
Image of a dividing basal epidermal cell demonstrating the method of spindle angle measurement.

We measure the angle formed by a line through the spindle poles and one along the basement membrane.

https://doi.org/10.7554/eLife.48482.003
Figure 1—figure supplement 2
Proliferation and spindle orientation differences between back and footpad skin.

(A) Proliferation in WT backskin and footpad as measured by BrdU incorporation. n > 300 cells for each of 3 mice. p<0.001. (B,C) H and E images of WT backskin and footpad epidermis, showing their difference in thickness. (D–F) Radial histograms of mitotic spindles in WT backskin (n = 22), footpad (n = 78), and footpad from a K14-rtTA;TRE-Cdkn1b (n = 24) mouse epidermis. Note that the data in (D) is the same as the data presented in Figure 2A.

https://doi.org/10.7554/eLife.48482.004
Figure 1—figure supplement 3
Expansion of keratin 10 (K10) positive cell layers in epidermis treated with TPA.

K10 (red), nuclei (blue), and the basement membrane is marked by β4-integrin staining in green.

https://doi.org/10.7554/eLife.48482.005
Figure 2 with 1 supplement
Oncogenic KRAS alters footpad epidermis spindle orientation in a NuMA-dependent manner.

(A,B) Radial histogram of mitotic spindles in adult backskin 21 days after tamoxifen-induced recombination in control (A) and K5CreER; KRASG12D mice (B). (C,D) Radial histogram of mitotic spindles in adult footpad epidermis, 21 days after tamoxifen-induced recombination in control (C) and K5CreER; KRASG12D mice, (n = 78 cells), and (D) (n = 64 cells). (E) Radial histogram of mitotic spindles in adult footpad 21 days after tamoxifen-induced recombination in K5CreER; KRASG12D;NuMAΔMTBD mice (n = 57 cells). Note that the data in 2C is the same as that presented in Figure 1—figure supplement 2E.

https://doi.org/10.7554/eLife.48482.006
Figure 2—figure supplement 1
Radial histogram of cell division orientation in the footpad epidermis from NuMA mutant mice (K14-Cre;NuMAMTBD/MTBD).

n = 41 cells.

https://doi.org/10.7554/eLife.48482.007
Figure 3 with 1 supplement
Loss of regulated spindle orientation synergizes with oncogenic KRAS to cause tissue overgrowth.

(A) Images of K5CreER; KRASG12D and K5CreER; NuMAΔMTBD; KRASG12D mice 21 days after recombination with tamoxifen, with inset of footpad and anal-genital region. (B,C)) Kaplan-Meier Survival plot of K5CreER; KRASG12D and K5CreER; NuMAΔMTBD; KRASG12D mice (B) and K5CreER; p53-/- and K5CreER; p53-/-; NuMAΔMTBD mice (C). n = 12 mice for K5CreER; NuMAΔMTBD; KRASG12D and 10 for other genotypes. (D,E) H and E images of K5CreER; KRASG12D (D) and K5CreER; NuMAΔMTBD; KRASG12D (E) footpad epidermis. (F,G) Immunofluorescence images of K5CreER; KRASG12D (F) and K5CreER; NuMAΔMTBD; KRASG12D (G) footpad epidermis showing localization of K5/K14+ basal and K10+ suprabasal epidermal layers. Scale bar = 50 μm. (H) Quantitation of basement membrane length divided by tissue length in indicated genotypes (n = 3 mice/condition). (I) Quantitation of proliferation, as assayed by BrdU incorporation, in control and KRASG12D mice. (n > 300 cells, three mice/condition). (J) Image showing co-localization of keratin 10 (red) and histone H2B (green) in a basal cell from a K10-rtTA; TRE-H2B-GFP mouse. (K) Quantitation of Keratin 10 positive basal cells in paw and backskin with indicated genotypes. n > 300 cells/mouse, three mice/condition. (L) Fluorescence intensity (normalized) of β4-integrin in control and KRASG12D expressing footpad epidermis (n = 3 mice/condition).

https://doi.org/10.7554/eLife.48482.008
Figure 3—figure supplement 1
Effect of TPA treatment on control and NuMAΔMTBD ear skin.

Images show K10 (green) stained ear skin epidermis with the basement membrane noted with a dotted line. These mice were topically treated with TPA for ten days (treatment every second day). The graph indicates the basement membrane length/tissue length of the skin.

https://doi.org/10.7554/eLife.48482.009
Author response image 1
Effect of combined TPA treatment and Cdkn1b expression on the epidermis.

Top – Basement membrane (red) and nuclei (blue) staining of control, TPA-treated, and TPA-treated/Cdkn1b-expressing epidermis. Note the increased thickness of the epidermis with TPA treatment and the dramatic changes in tissue architecture when in TPA + Cdkn1b. Bottom – Image showing the epidermal integrity defects in the TPA + Cdkn1b treated skin (basement membrane is red, krt14 is green, and nuclei are blue).

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  1. Angel Morrow
  2. Julie Underwood
  3. Lindsey Seldin
  4. Taylor Hinnant
  5. Terry Lechler
(2019)
Regulated spindle orientation buffers tissue growth in the epidermis
eLife 8:e48482.
https://doi.org/10.7554/eLife.48482