The histone variant macroH2A confers functional robustness to the intestinal stem cell compartment
Fig 5
Regeneration and DNA damage response in macroH2A DKO intestine.
(A) Left: representative images of Ki-67 immmunohistochemistry within macroH2A WT and DKO proximal jejunum 3 days after exposure of mice to 12 Gy whole body γ-irradiation. 10x objective. Right: quantitation of Ki67+ nascent crypt foci per mm. N = 3 mice per condition, mean ± SD. (B) Top: post-IR lineage tracing scheme: macroH2A WT or DKO Hopx-CreERT2::Rosa26-LSL-tdTomato mice were injected with 2mg tamoxifen 48h and 24h prior to treatment with 12 Gy whole-body gamma irradiation, and 72h later sacrificed for analysis. Bottom: representative immunofluorescence of tdTomato lineage tracing (red) counterstained with DAPI (blue) within macroH2A WT and DKO crypts 72 hours after γ-irradiation. 30x objective (C) Left: quantitation of tdTomato tracing events per 500μm, N = 3 mice per condition, mean ± SD. Right: quantitation of tdTomato tracing events per 500μm normalized to percentage of Hopx-tdTomato+ ISCs during homeostasis (values in Fig 4A), N = 3 mice per condition, mean ± SD. (D) Experimental scheme highlighting the timing of DNA damage and apoptosis analysis (24h post IR) and regeneration and lineage tracing analysis (72h post IR) (E) Left: flow cytometry plots of SSC-A vs. cleaved caspase-3 content within total crypt epithelium or Hopx-tdTomato+ subpopulations of macroH2A WT and DKO proximal jejunal crypt cells 24 hours after γ-irradiation. Right: quantitation of total crypt epithelium CC3 positivity and Hopx-tdTomato+/CC3 double positivity as defined by boxed subpopulation on left. N = 3 mice per condition, mean ± SD. *p<0.05, ***p<0.0005, ns = not significant, Student’s t-test. Scale bars = 100μm.