The epidermis is a proliferative tissue that turns over repeatedly throughout life. Remarkably, in aged sun-exposed skin, homeostasis and the architecture of the epidermis is maintained despite prevalent clones of cells containing oncogenic mutations (Martincorena et al., 2015). Recent work has highlighted oncogene-induced differentiation and active elimination of mutant clones as two mechanisms of epidermal robustness, suggesting there are multiple modes of response to perturbation (Brown et al., 2017; Ying et al., 2018). Homeostasis requires that gain of cells (proliferation) must be balanced with loss of cells (differentiation or cell death). One possible mechanism to control homeostasis is regulated spindle orientation and asymmetric cell divisions. These divisions do not increase progenitor number, but rather commit one daughter to differentiation. Embryonic epidermal progenitors can divide parallel to the basement membrane, resulting in two basal daughter cells, which we term symmetric divisions. These cells can also divide perpendicular to the basement membrane (Lechler and Fuchs, 2005; Poulson and Lechler, 2010; Smart, 1970; Williams et al., 2011). Perpendicular divisions are termed asymmetric divisions because they contribute one daughter cell to the differentiated suprabasal cell layer and do not alter the number of basal progenitor cells. Because of these properties, regulated spindle orientation is a candidate for providing resilience to homeostatic perturbation. In the embryonic epidermis, asymmetric divisions are used to drive stratification of the skin (Lechler and Fuchs, 2005; Smart, 1970; Williams et al., 2011). However, in adult backskin most divisions are planar (symmetric) and the utility of regulated spindle orientation has not been explored (Ipponjima et al., 2016; Rompolas et al., 2016). Here, we examine the possibility that regulated spindle orientation can buffer the effects of perturbed homeostasis.

We began by examining whether spindle orientation was altered when developmental or adult homeostatic proliferation was experimentally altered. In the embryonic mouse epidermis, where proliferation is high, the ratio of perpendicular to planar divisions is approximately 70:30 (Lechler and Fuchs, 2005; Smart, 1970). To inhibit basal cell cycle progression, we used a basal epidermal keratin 14 promoter to drive a doxycycline-inducible allele of Cdkn1b, a CDK1 inhibitor (K14-rtTA;tetO-CDKN1b) (Pruitt et al., 2013). Quantitation of the percentage of cells expressing the mitotic marker phospho-histone H3 (pHH3) revealed a significant decrease in basal cell proliferation (Figure 1A). This also resulted in a significant reduction in perpendicular divisions, from 70% to 35%, with a concomitant increase in planar divisions (from 22% to 42%) (Figure 1B,C). We measured spindle angles as shown in Figure 1—figure supplement 1. This data is consistent with the need for increased rates of planar divisions to maintain the surface area of the epidermis in a rapidly growing embryo.

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In contrast to the embryo, the proliferation rate in the adult backskin is low. Live imaging has shown that most divisions occur symmetrically and that basal cells delaminate from the basement membrane to populate suprabasal cell layers (Ipponjima et al., 2016; Rompolas et al., 2016). Consistent with this observation, we see that the majority of mitotic spindles are planar in adult backskin (Figure 1E). Notably, there is regional variability in epidermis from different anatomical areas in both proliferation and spindle orientation. The epidermis of the footpad, which is thicker, has both a higher proliferation index and increased numbers of perpendicular divisions (Figure 1—figure supplement 2). Similar to the embryonic epidermis, expression of Cdkn1b caused a shift to more planar cell divisions in the paw epidermis (Figure 1—figure supplement 2E).

To induce hyperproliferation of adult backskin epidermis we topically applied the mitogen TPA. This resulted in thickening of the epidermis and a 6-fold increase in proliferation rate as measured by BrdU incorporation (Figure 1D). Notably, this treatment increased the percentage of perpendicular divisions from 20% to 40% (Figure 1E,F). Therefore, one of the responses to TPA-induced hyperproliferation in the epidermis is an increase in the rate of divisions that give rise to differentiated progeny, as seen by staining for the differentiation marker, keratin 10 (Figure 1—figure supplement 3). Together these data demonstrate correlations between proliferation and spindle orientation, and support the hypothesis that cells tune their division orientation in response to the needs of the surrounding tissue as a mechanism to promote robust homeostasis.

To directly determine whether regulated spindle orientation was important for epidermal homeostasis we examined the effects of mutating the nuclear mitotic apparatus protein, NuMA, which is essential for spindle orientation in embryonic skin (Williams et al., 2011; Seldin et al., 2016). We did not see significant phenotypes when we disrupted NuMA’s spindle orientation function in adult epidermis for short times (data not shown), consistent with findings that adult backskin homeostasis is normally maintained by delamination and differentiation rather than by regulated spindle orientation (Rompolas et al., 2016).

Given that tissues responded to TPA-induced hyperproliferation by increasing perpendicular divisions, we next asked whether this also occurred in response to an oncogenic perturbation. Mutations that activate Ras family members can drive squamous cell carcinoma and a significant percentage of squamous tumors carry Ras mutations (Daya-Grosjean et al., 1993; Pierceall et al., 1991; South et al., 2014). Unexpectedly, we found that expression of KRAS^G12D (at endogenous levels and only in the epidermis; K5CreER;KRAS^G12D/+) had distinct effects in backskin and footpad epidermis. While there was no significant change in division orientation ratios in the backskin, active K-Ras resulted in an increase in perpendicular divisions in the footpads (Figure 2A–D). These data rule out the possibility that active K-Ras inhibits regulated spindle orientation/asymmetric cell divisions as an oncogenic mechanism. It raised the alternative possibility that the increased perpendicular divisions may protect the tissue against overgrowth. We therefore addressed the molecular requirements for spindle orientation in skin expressing active K-Ras. We combined a NuMA mutation that disrupts embryonic spindle orientation with the KRAS^G12D oncogene (Seldin et al., 2016; Silk et al., 2009). This resulted in a significant shift of divisions from perpendicular to planar, consistent with NuMA being required for spindle orientation in this context (Figure 2E). On their own, mutations in NuMA resulted in a skewing of spindle orientations in the pawskin (Figure 2—figure supplement 1).

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The consequence of perturbing regulated spindle orientation in epidermis expressing active KRAS was dramatic. Within two weeks of recombination, large overgrowths appeared on the footpads and anogenital regions of the double mutant mice (K5Cre^ER;KRAS^G12D/+;NuMA^MTBDfl/fl) (Figure 3A). These features were not found in single KRAS^G12D or NuMA mutants, although KRAS^G12D alone was sufficient for formation of oral tumors similar to those that appeared in the double mutants. Most mice died within 3–4 weeks after recombination (Figure 3B).

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Histologic analysis of the double mutant paws demonstrated massive tissue overgrowth and papilloma formation (Figure 3D–G). Despite this dramatic change in tissue architecture, the basement membrane remained intact and differentiation was normal at early time points. Thus, this combination of mutations is not sufficient for invasion, at least in the short time period we were able to assay. The massive tissue overgrowth led to lethality, likely due, at least in part, to blockage of the oral cavity.

Similar to the loss of NuMA function in the adult epidermis, loss of spindle orientation alone is not detrimental in developing fly wings (Nakajima et al., 2013). However, tumors form when combined with additional mutations that inhibited apoptosis. Notably, we saw no genetic interaction when mutant p53 was combined with the NuMA^ΔMTBD deletion (Figure 3C). These data are consistent with the fact that apoptosis is not a major determinant of tissue homeostasis in the epidermis under normal conditions, and demonstrates a specificity for disrupted spindle organization working synergistically with only a subset of oncogenic drivers.

Histologic analysis revealed expansion of the stem cell pool in the KRAS^G12D; NuMA^ΔMTBD epidermis. To accommodate the increase in cell numbers, the basement membrane became highly involuted (Figure 3D–G). We quantitated this as basement membrane length per tissue length and found a 2–4 fold increase in this measure, corresponding to a 4–16 fold increase in area (Figure 3H). As cell density was not notably altered, this demonstrates an expansion of the basal stem cell pool. These data are consistent with regulated spindle orientation buffering the effects of oncogenic K-Ras. Similarly, we found that low-dose TPA treatment caused a subtle yet significant increase in basement membrane length in NuMA^ΔMTBD mice as compared to control mice (Figure 3—figure supplement 1).

Active K-Ras has both proliferative and anti-differentiative effects in different tissues. Because increased proliferation can drive increased asymmetric divisions (Figure 1), we asked whether the changes observed in K-Ras mutant skin could be due to oncogene-induced hyperproliferation. Notably, we found that constitutively active KRas expression is not sufficient to drive hyperproliferation in the paw skin, nor did it do so in combination with a mutation in NuMA (Figure 3I).

Previous work has suggested that active Ras can inhibit differentiation when overexpressed in the epidermis (Dajee et al., 2002). However, this has not been examined either in the adult pawskin or under endogenous levels of expression. In adults, cells commit to differentiation, detach from the basement membrane, and move upward. A small proportion of basal cells express the differentiation marker keratin 10 (K10) and can be fluorescently labeled in mice using established mouse lines (K10-rtTA;TRE-H2B-GFP) (Muroyama and Lechler, 2017; Figure 3J). In the pawskin, we found that ~10% of basal cells were labeled with H2B-GFP, and this fell to 0.4% two days after removal of doxycycline, consistent with these cells entering differentiation (n>200 cells). Additionally, H2B-GFP labeled-basal cells incorporated EdU at a lower rate than surrounding basal cells (28 ± 2.4% for K10^-ve basal cells verses 9 ± 3.0% for K10^+ve basal cells, n=3 mice, >400 cells/mouse). We quantitated differentiation by analyzing the percentage of basal cells that express keratin 10. Using the K10-rtTA;TRE-H2B-GFP genetic labeling system we found that differentiation was suppressed in KRAS^G12D expressing cells (Figure 3K). This was most dramatic in the paw epidermis, but was also observed in backskin to a lesser extent. While the mechanism underlying this suppression is not clear, we also found that the relative intensity of β4-integrin was increased in the mutant pawskin, in a similar manner to what was observed when K-Ras was overexpressed in the embryo (Dajee et al., 2002; Figure 3J). Robust adhesion and signaling through integrins is known to promote progenitor cell fate, and thus may contribute to the changes in differentiation that we observed.

Together, our data demonstrate that the epidermis can respond to changes in homeostasis by altering mitotic spindle orientation. Increasing perpendicular (asymmetric) divisions in response to either increased proliferation or decreased differentiation allows homeostasis to be reset without dramatically affecting tissue architecture. Our genetic analysis clearly shows that this is essential to prevent tissue overgrowth in an oncogenic background. In addition, we found that the epidermis responded differently to the same oncogenic insult at different anatomic sites. This likely reflects the differences in proliferation, differentiation, and rates of asymmetric cell division rates in different regions of the epidermis and suggests that distinct ‘second hits’ may be needed to collaborate with oncogenes in tissues with different mechanisms of achieving homeostasis.

Our data are consistent with the idea that both regulated spindle orientation and delamination/differentiation can be used to buffer basal cell number. Disruption of either one was insufficient to induce homeostatic defects due to compensation by the other. However, blockade of both homeostatic buffers resulted in tissue overgrowth and progenitor cell expansion. Notably, squamous cell carcinomas have been reported to have increased rates of perpendicular divisions as compared to normal epidermis (Beck et al., 2011; Siegle et al., 2014). It will be important to see whether regulated spindle orientation is protective against tumor development in these contexts.

There are no large datasets associated with this manuscript.

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Thank you for submitting your article "Regulated spindle orientation buffers tissue growth in the epidermis" for consideration by eLife. Your article has been reviewed by three peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by Anna Akhmanova as the Senior Editor. The reviewers have opted to remain anonymous.

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

Summary:

The short report by Morrow et al. sheds light on a highly interesting question of how the epidermis maintains its homeostasis and remains morphologically normal despite a large amount of oncogenic mutations. They report that this is achieved by regulation of spindle orientation (parallel or perpendicular) in dividing cells. While in adult murine back the spindle is primarily oriented parallel to the basal membrane, there is a shift towards perpendicular orientation and thus asymmetric division in embryonic skin and the footpad, both of which display a higher proliferation rate compared to adult back skin.

Morrow et al. further demonstrate that Cdkn1b expression (in embryonic epidermis) reduces, whereas TPA-treatment or oncogenic KRAS induces perpendicular spindle orientation/asymmetric cell division, and that NuMA is a key regulator.

Overall, the manuscript is conceptually interesting and provides insights into how tissue is maintained in homeostasis or upon pharmacological or genetic insults. Although it would have been valuable to know more on the mechanism of the orientation, it is a short report and future studies will further clarify the mechanism. There are few concerns that should be addressed prior publication.

Essential revisions:

1) The authors use Cdkn1b overexpressing embryonic epidermis to demonstrate that hyperproliferation induces perpendicular-cell division (Figure 1A-C). Does epidermal Cdkn1b expression also affect spindle orientation and proliferation in adult skin (back and footpad)? As it is well known that embryonic and adult skin are differently regulated in homeostasis, they should show the data in adult skin (TPA-treat back skin and/or footpad skin) using the mouse line (K14-rtTA;TRE-Cdkn1b) to show that hyperproliferation induces perpendicular cell division in adult epidermis.

2) The authors mention that NuMA mutation does not affect adult epidermis morphology. Is this true for both back skin and footpad epidermis? How does NuMA mutation alone affect the division rate and orientation of cells in the footpad?

3) Figure 3I: Surprisingly, oncogenic KRas seems to reduce proliferation of basal cells. What is the explanation for that? Is there increased proliferation in suprabasal layers? How does oncogenic KRas induce perpendicular cell division without hyperproliferation? Does it suppress delamination? The authors mention that differentiation is suppressed. However, as stated before, altered spindle orientation correlated with increased proliferation. It would be better to provide the data showing the delamination rate in WT, KRAS^G12D, and KRAS^G12D/NuMA^ΔMTBD mice. Furthermore, Authors should also show quantitation of proliferation in KRAS^G12D/NuMA^ΔMTBD skin.

4) How do the authors reconcile the work from Lim et al. (Science, 2014) showing that the mechanism of probabilistic fate and neutral clonal competition rather than asymmetric cell division dominates the footpad skin?

Essential revisions:

1) The authors use Cdkn1b overexpressing embryonic epidermis to demonstrate that hyperproliferation induces perpendicular-cell division (Figure 1A-C). Does epidermal Cdkn1b expression also affect spindle orientation and proliferation in adult skin (back and footpad)? As it is well known that embryonic and adult skin are differently regulated in homeostasis, they should show the data in adult skin (TPA-treat back skin and/or footpad skin) using the mouse line (K14-rtTA;TRE-Cdkn1b) to show that hyperproliferation induces perpendicular cell division in adult epidermis.

We quantitated spindle orientation in paw skin after Cdkn1b expression (Krt14- rtTA;TRE-Cdkn1b) and found that, like in the embryonic epidermis, there was a switch to more planar cell divisions (Figure 1—figure supplement 2). We did not perform this experiment in backskin because of the very low levels of proliferation upon Cdkn1b expression and due to the fact that spindle orientation is largely planar under control conditions.

We also attempted the suggested experiment – to determine whether Cdkn1b expression in the basal epidermis of the adult is able to reverse the effects of TPA on spindle orientation. However, the combination of TPA treatment and Cdkn1b expression resulted in dramatic effects on the skin. There was thickening of the skin and massive cellularization of the dermis, likely an inflammatory response. There were also epidermal integrity defects (cell-cell separations). Due to these findings, we did not quantitate division orientation.

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2) The authors mention that NuMA mutation does not affect adult epidermis morphology. Is this true for both back skin and footpad epidermis? How does NuMA mutation alone affect the division rate and orientation of cells in the footpad?

We have included the data on division rate (Figure 3I) and spindle orientation (Figure 2—figure supplement 1). The division rate is not statistically different from controls. There is a skew to more random divisions in the NuMA mutant, as expected. Because these parameters are unaltered and the architecture looks normal, we have not included histological images in the manuscript.

3) Figure 3I: Surprisingly, oncogenic KRas seems to reduce proliferation of basal cells. What is the explanation for that? Is there increased proliferation in suprabasal layers? How does oncogenic KRas induce perpendicular cell division without hyperproliferation? Does it suppress delamination? The authors mention that differentiation is suppressed. However, as stated before, altered spindle orientation correlated with increased proliferation. It would be better to provide the data showing the delamination rate in WT, KRAS^G12D, and KRAS^G12D/NuMA^ΔMTBD mice. Furthermore, Authors should also show quantitation of proliferation in KRAS^G12D/NuMA^ΔMTBD skin.

The change in proliferation rate in the KRas did not reach statistical significance in our first experiments. We repeated these results in combination with the NuMA mutant proliferation data (requested and discussed above) and again, we found no statistically significant difference (Figure 3I). We did not note any change in suprabasal proliferation.

There are several possibilities for how KRas alters division orientation. First, we cannot rule out a direct effect of KRas on division orientation. However, based on the decrease in differentiation/delamination that we observed, one possibility is that the epidermis responds to this homeostatic perturbation by increasing perpendicular divisions.

We have now included the proliferation data for the KRas/NuMA double mutant as suggested (Figure 3I). There was no significant change from control, Kras, or NuMA alone. We did not perform the suggested experiment using the K10-rtTA;TRE-H2b-GFP system to analyze differentiation as this would have required multiple rounds of matings that was not possible in the revision timeline.

4) How do the authors reconcile the work from Lim et al. (Science, 2014) showing that the mechanism of probabilistic fate and neutral clonal competition rather than asymmetric cell division dominates the footpad skin?

We do not view these works as being contradictory. A cell that undergoes asymmetric divisions and differentiation can also undergo neutral clone competition. It is an interesting question whether the kinetics of clone competition would change when you alter delamination/ACD rates in a tissue, but it is beyond the scope of this manuscript to address this.

Author details

1. Angel Morrow

   Department of Dermatology, Duke University, Durham, United States

   Present address

   Novozymes, Durham, United Kingdom

   Contribution

   Data curation, Formal analysis, Investigation, Methodology, Writing—review and editing

   Contributed equally with

   Julie Underwood

   Competing interests

   No competing interests declared

2. Julie Underwood

   Department of Dermatology, Duke University, Durham, United States

   Contribution

   Data curation, Investigation, Methodology, Writing—review and editing

   Contributed equally with

   Angel Morrow

   Competing interests

   No competing interests declared

3. Lindsey Seldin

   1. Department of Dermatology, Duke University, Durham, United States
   2. Department of Cell Biology, Duke University, Durham, United States

   Present address

   Department of Cell and Developmental Biology, Vanderbilt University, Nashville, United States

   Contribution

   Data curation, Formal analysis, Investigation, Methodology, Writing—review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-4995-1152

4. Taylor Hinnant

   1. Department of Dermatology, Duke University, Durham, United States
   2. Department of Cell Biology, Duke University, Durham, United States

   Contribution

   Data curation, Investigation, Methodology

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-8912-6851

5. Terry Lechler

   1. Department of Dermatology, Duke University, Durham, United States
   2. Department of Cell Biology, Duke University, Durham, United States

   Contribution

   Conceptualization, Data curation, Formal analysis, Supervision, Funding acquisition, Writing—original draft, Project administration, Writing—review and editing

   For correspondence

   terry.lechler@duke.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-3901-7013

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