1. Cancer Biology
  2. Stem Cells and Regenerative Medicine
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Interplay of opposing fate choices stalls oncogenic growth in murine skin epithelium

  1. Madeline Sandoval
  2. Zhe Ying
  3. Slobodan Beronja  Is a corresponding author
  1. Division of Human Biology, Fred Hutchinson Cancer Research Center, United States
  2. Molecular and Cellular Biology Graduate Program, University of Washington, United States
Research Article
Cite this article as: eLife 2021;10:e54618 doi: 10.7554/eLife.54618
5 figures, 1 table and 3 additional files

Figures

Figure 1 with 1 supplement
HrasG12V induces progenitor cell renewal in single cells.

(A) Schematic of Hrasll-G12V and R26mT/mG mice. Hrasll-G12V mouse changes expression of wild-type (WT)-Hras allele to mutant-Hras allele upon addition of Cre. R26mT/mG mouse changes expression of mTomato to mGFP after Cre activation. CreER expression is induced by tamoxifen. (B) Schematic of single-cell induction and imaging experiment. (C) Epidermal progenitor cells can undergo three distinct division types. (D) Representative images of basal progenitor cell divisions in Cre-activated R26mT/mG epidermis captured using intravital imaging. Scale bar is 10 µm. (E) Quantification of division choices of single cells in adult epidermis. At least 75 cells were scored per animal. n ≥ 3 animals per genotype. (F) Rate of renewal significantly increases in single HrasG12V progenitor cells. At least 75 cells were scored per animal. n = 3 animals per genotype. (H) Quantification of basal cell numbers in WT and HrasG12V clones at 24 weeks. Each point represents one clone. Twenty total clones were scored from n ≥ 3 animals per genotype. (G) Representative images of basal cells of WT and HrasG12V clones at 24 weeks. Scale bar is 10 µm. For (E,F,H), the center line represents the mean; errors bars represent the s.d. Two-tailed Student’s t-test was used. n.s. denotes p value > 0.05; **** denotes p value < 0.0001.

Figure 1—figure supplement 1
Hras G12V expression has no impact on apoptosis or senescence in skin epithelium.

(A) Quantification of caspase 3+ cells in adult epidermis. >1500 cells were counted per animal. n = 3 animals per genotype. (B) No senescence is detected in HrasG12V clones. Scale bar is 10 µm. For (A), the center line represents the mean and errors bars represent the s.d. Two-tailed Student’s t-test was used. n.s. denotes p value > 0.05.

Cell fate identification (CFI) assay reveals dynamic changes in progenitor renewal in expanding HrasG12V clones.

(A) Schematic of CFI assay. EdU-only cells are assessed for location in epidermis (basal or suprabasal layer) as indicator of cell fate choice. (B) Representative images of tissues stained for EdU and BrdU expression, 30 min, 2.5 hr, and 24 hr post-EdU injection. Cells at the 24 hr time point are fully committed to progenitor or differentiated fate. Scale bar is 25 µm. Inset scale bar is 10 µm. At least 2000 total cells were counted per animal. n ≥ 2 animals per time point. (C) Representative images of cell divisions as seen in CFI assay. Blue arrow marks symmetric differentiation division. (D) Cell fate choices scored by CFI assay are not significantly different from cell fate choices scored via intravital imaging. At least 50 cells were scored per animal. n ≥ 3 animals per condition. (E) Schematic of clone activation. Tissues were processed in 2-week intervals following tamoxifen injection (blue arrows). (F–H) Division choices of progenitor cells in activated clones. At least 30 divisions were counted per animal. n ≥ 3 animals per condition. (I) Rate of renewal in HrasG12V and WT clones initially differs significantly, but as HrasG12V clones reach 24 weeks, renewal rates drop to near-homeostatic levels. More than 30 total cells were counted per animal. n ≥ 3 animals per condition. (J) EdU-incorporation over 2 hr in WT and HrasG12V epidermis. At least 300 cells were counted per animal. n = 3 animals per condition. (K) HrasG12V clones expand significantly over time. At least 25 clones were counted per animal. n ≥ 3 animals per condition. For (F–K), the center point represents the mean; errors bars represent the s.d. Two-tailed Student’s t-test was used. n.s. denotes p value > 0.05; * denotes p value < 0.05; ** denotes p value < 0.01; *** denotes p value < 0.001; **** denotes p value < 0.0001.

Figure 3 with 1 supplement
HrasG12V clones develop intraclone heterogeneity over time.

(A) Representative images of basal cell population of HrasG12V clones at weeks 2 and 10. Scale bar is 10 µm. (B) Density of individual HrasG12V clones. Dashed gray line represents average density in wild-type (WT) clones. At least 25 clones were counted per animal. n ≥ 2 animals per timepoint. (C) Outlines of individual HrasG12V clones show a circular morphology. Scale bars are 25 µm. (D) Quantification of circularity of individual HrasG12V clones. At least 25 clones were counted per animal. n ≥ 3 animals per timepoint. (E) Quantification of edge cells and inner cells in HrasG12V clones. At least 52 clones were counted per animal. n ≥ 3 animals per time point. (F) Proportion of edge cells in HrasG12V clones decreases as the clone expands. At least 52 clones were counted per animal. n ≥ 3 animals per timepoint. (G) Edge cells undergo significantly more renewing divisions and significantly fewer differentiating divisions compared to inner cells in week 10 HrasG12V clones. A total of 147 divisions were counted. n = 4 animals. (H) Rate of renewal in edge cells and inner cells of week 10 HrasG12V clones. A total of 147 divisions were counted from four animals. (I) WT cells adjacent to an HrasG12V clone undergo significantly fewer renewing divisions compared to a non-adjacent WT cell. A total of 160 divisions were counted. n = 5 animals. (J) The renewal rate of WT cells adjacent to an HrasG12V clone is significantly lower compared to WT cells not located adjacent to an HrasG12V clone. A total of 160 divisions were counted. n = 5 animals. (K) Early phase HrasG12V clones expand. In contrast, the inner region of late phase HrasG12V clones undergo high rates of differentiation, but the clone does not collapse because the edge cells are more renewing and compensate for the inner cells. In response to the high rate of renewal in oncogenic edge cells, the neighboring WT cells undergo a high rate of differentiation. For (B,D) each point represents a single clone. For (B, D–J); the center point represents the mean; errors bars represent the s.d. Two-tailed Student’s t-test was used. n.s. denotes p value > 0.05; * denotes p value < 0.05; ** denotes p value < 0.01; *** denotes p value < 0.001.

Figure 3—figure supplement 1
Circularity and cell fate dynamics of wild-type (WT) epidermal clones.

(A) Outlines of individual wild-type (WT) clones show a circular morphology. Scale bars are 25 µm. (B) Quantification of circularity of individual WT clones. At least 22 clones were counted per animal. n = 2 animals per timepoint. (C) Quantification of edge and inner cell compartments of WT clones. At least 30 clones were counted per animal. n = 2 animals per timepoint. (D) Division choices of edge and inner cells of large areas of activated WT tissue. At least 24 clones were counted per animal. n = 3 animals. (E) Quantification of renewal rate of inner and edge compartments of large areas of activated WT tissue. At least 24 clones were counted per animal. n = 3 animals. For (B–E), the center line represents the mean and errors bars the s.d. Two-tailed Student’s t-test was used. n.s. denotes p value > 0.05.

HrasG12V-induced differentiation is replicated in E18.5 epidermis and serves as the basis for in vivo genetic screen.

(A) Representative images of clonal and field activation of HrasG12V in developing epidermis. Scale bar is 25 µm. Inset scale bar is 25 µm. (B) HrasG12V expression can be induced with LV-Cre (high or low titer) or K14-Cre. Changes in HrasG12V expression yield differences in tissue hyperplasia. (C) Rate of renewal decreases in tissues which broadly express HrasG12V (LV-Cre field and K14Cre field). At least 75 cells were counted from each animal per genotype. n ≥ 3 animals. (D) Diagram of shRNA screen methodology. A lentiviral pool targeting Ras effectors was injected into E9.5 embryos. The epidermis was harvested at E18.5, digested into single cells and separated by FACS based on low or high expression of α6 Itg. Each population was profiled for shRNA enrichment. (E,F) Needle plots of shRNA’s enriched in basal layer compared to suprabasal layer in wild-type (WT) and HrasG12V epidermis. shRNAs that are enriched in basal layer (α6 Itghigh population) relative to suprabasal layer (α6 Itglow population) inhibit differentiation. The indicated shRNAs are candidate (Rassf5) or validated (Pik3ca) regulators of HrasG12V-induced differentiation in addition to potential positive regulators of renewal in HrasG12V epidermis (Rassf4, Rgl2, Rgs14). n ≥ 3 animals per genotype. For (C), the center point represents the mean; errors bars represent the s.d. Two-tailed Student’s t-test was used. n.s. denotes p value > 0.5, *** denotes p value < 0.001.

Figure 5 with 1 supplement
Rassf5 is a necessary and sufficient driver of HrasG12V-induced differentiation.

(A) Rassf5 mRNA is increased in isolated HrasG12V E18.5 basal cells. shRNAs targeting Rassf5 efficiently knock down transcript expression. n = 3 animals per genotype. (B) Immunoblot of Rassf5 expression in HrasG12V E18.5 epidermis. (C) LV-Cre-shRassf5 construct for simultaneous knock down of Rassf5 and induction of HrasG12V expression. (D) Quantification of differentiation using EdU-BrdU pulse/chase differentiation assay. Depletion of Rassf5 promotes renewal in HrasG12V epidermis. At least 75 cells were counted per animal. n ≥ 3 animals per genotype. (E) Diagram of construct containing Rassf5 open-reading frame (ORF) fused with tdTomato (Rassf5-tdT) and co-expressing Cre. (F) Representative images of HrasG12V clone and HrasG12V/Rassf5-tdT clone in E18.5 epidermis. Overexpression of Rassf5 yields clones with reduced basal compartment and extensive suprabasal compartment. Scale bar is 25 µm. (G) Whole mount images of HrasG12V clone and HrasG12V/Rassf5-tdT clone in E18.5 epidermis. Scale bar is 25 µm. (H) Quantification of basal cell numbers in E18.5 HrasG12V and HrasG12V/Rassf5-tdT clones. Twenty-two clones were counted in total. n = 3 animals per genotype. (I) Quantification of the ratio of suprabasal surface area to basal surface area of HrasG12V and HrasG12V/Rassf5-tdT clones in E18.5 epidermis. Twenty-two clones were counted in total. n = 3 animals per genotype. (J) Difference between the renewal rate of edge and inner cells is lost in HrasG12V/shRassf5 clones. Animals are >10 weeks old. Red dash lines represent renewal rate of edge and inner cells from week 10 clones. At least 120 cells were counted per genotype. n = 3 animals. (K) Knock down of Rassf5 in adult HrasG12V epidermis significantly increases renewal rate. At least 75 cells were quantified per animal. n ≥ 3 animals per genotype. (L) LV-rtTA-T2A-CreER and LV-TRE-Rassf5-tdT constructs for inducible expression of HrasG12V and Rassf5. (M) Schematic of Rassf5 overexpression in adult HrasG12V clones. Developing embryos were broadly transduced with LV-rtTA-T2A-CreER and sporadically transduced with LV-TRE-Rassf5-tdT. At P21, tamoxifen injection induced expression of HrasG12V, and at P24, doxycycline injection induced expression of Rassf5. Tissues were processed at P27. (N) Clones expressing HrasG12V/Rassf5 have a reduced basal cell population compared to clones expressing HrasG12V. n = 3 animals per condition. A total of 200 clones per condition were counted. (O) There are proportionally fewer HrasG12V clones composed of only differentiated cells compared to HrasG12V/Rassf5. At least 59 clones were counted per animal. n = 3 animals per condition. For (H,I,N), each dot represents an individual clone. For (A,D,H–K,N,O), the center line represents the mean and errors bars the s.d. Two-tailed Student’s t-tests were used. n.s. denotes p value > 0.05. * denotes p value < 0.05; ** denotes p value < 0.01*** denotes p value < 0.001; **** denotes p value < 0.0001.

Figure 5—figure supplement 1
Rassf5 depletion does not induce apoptosis or senescence in Hras G12V epidermal clones.

(A) Real-time PCR quantification of Rassf5 transcripts from cultured keratinocytes. Asterisks mark shRNAs that demonstrate efficient Rassf5 knock down and were used in additional experiments. Statistics were based on three experiments. (B) Quantification of caspase 3+ cells in adult epidermis. >1500 cells were counted per animal. n = 3 animals per genotype. (C) Senescence-associated β-galactosidase staining does detect senescence in oncogenic tissues with Rassf knockdown. For (A,B), the center line represents the mean and errors bars the s.d. Two-tailed Student’s t-test was used. n.s. denotes p value > 0.05; * denotes p value < 0.05.

Tables

Key resources table
Reagent type (species)
or resource
DesignationSource or referenceIdentifiersAdditional information
Genetic reagent (Mus musculus)C57BL/6JJackson LaboratoriesStock #: 000664
Cell line (Homo sapiens)293-FTInvitrogenCat #: R70007
AntibodyPerCP/Cyanine5.5 rat monoclonal anti-human/mouse CD49fBioLegendCat #: 3136171:50
AntibodyChicken polyclonal anti-GFPAbcamCat #: ab13970 RRID:AB_300798IF 1:1000
AntibodyMouse monoclonal anti-BrdUInvitrogenCat #: MoBU-1IF: 1:100
AntibodyMouse monoclonal anti-GAPDHProteintechCat #: 60004–1-Ig RRID:AB_2737588WB: 1:2000
AntibodyPeroxidase AffiniPure goat polyclonal Anti-Mouse IgG (H+L)Jackson ImmunoresearchCat #: 115-035-003WB: 1:5000
AntibodyRabbit polyclonal anti-K10BioLegendCat #: Poly19054IF: 1:1000
AntibodyRabbit polyclonal anti-Nore1ADonated by G. ClarkCat #: PAS17071WB: 1:1000
AntibodyRabbit polyclonal anti-RFPRocklandCat #: 6000-401-379 RRID:AB_11182807IF: 1:1000
AntibodyRat monoclonal anti-NidogenSanta Cruz BiotechnologyCat #: sc-33706 RRID:AB_627519IF: 1:1000
Commercial assay or kitBCA Protein Assay KitThermo FisherCat #: 23225
Commercial assay or kitClick-iT EdU cell proliferation kit for imaging, Alexa Fluor 555Thermo Fisher ScientificCat. #: C10337
Commercial assay or kitDNeasy Blood and Tissue KitQiagenCat #: 69504
Commercial assay or kitiScript Reverse Transcription SupermixBio-RadCat #: 1708840
Commercial assay or kitPower SYBR Green PCR Master MixThermo Fisher ScientificCat. #: 4367660
Commercial assay or kitSuperSignal West Femto Maximum Sensitivity SubstrateThermo FisherCat #: 34094
Commercial assay or kitZero Blunt TOPO kitInvitrogenCat #: 450245
Recombinant DNA reagentFLAG-Nore1AddgeneCat #: 1975 RRID:Addgene_1975
Recombinant DNA reagenttdTomato-C1AddgeneCat #: 54653
RRID:Addgene_54653
Software, algorithmPrismGraphPadRRID:SCR_002798
Software, algorithmZen BlackZeissRRID:SCR_018163
OtherM.O.M. bufferVector LabsCat #: BMK-220

Additional files

Source data 1

Quantification and statistical analyses of shRNA enrichment and depletion in progenitor and differentiated cells of wild type and HrasG12V epidermis.

Relative enrichment of each shRNA targeting putative regulators of progenitor renewal in epidermal basal progenitors relative to differentiated suprabasal cells, as quantified by DESeq2 analyses.

https://cdn.elifesciences.org/articles/54618/elife-54618-data1-v2.xlsx
Supplementary file 1

Regulators of progenitor renewal in wild type and HrasG12V epidermis.

Candidates regulators of progenitor renewal as identified by DESeq2 analyses of shRNA enrichment in epidermal basal progenitors relative to differentiated suprabasal cells. Positive fold-change indicates shRNA enrichment in basal progenitors.

https://cdn.elifesciences.org/articles/54618/elife-54618-supp1-v2.xlsx
Transparent reporting form
https://cdn.elifesciences.org/articles/54618/elife-54618-transrepform-v2.pdf

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