p53 promotes revival stem cells in the regenerating intestine after severe radiation injury

Abstract

Ionizing radiation induces cell death in the gastrointestinal (GI) epithelium by activating p53. However, p53 also prevents animal lethality caused by radiation-induced acute GI syndrome. Through single-cell RNA-sequencing of the irradiated mouse small intestine, we find that p53 target genes are specifically enriched in regenerating epithelial cells that undergo fetal-like reversion, including revival stem cells (revSCs) that promote animal survival after severe damage of the GI tract. Accordingly, in mice with p53 deleted specifically in the GI epithelium, ionizing radiation fails to induce fetal-like revSCs. Using intestinal organoids, we show that transient p53 expression is required for the induction of revival stem cells and is controlled by an Mdm2-mediated negative feedback loop. Together, our findings reveal that p53 suppresses severe radiation-induced GI injury by promoting fetal-like reprogramming of irradiated intestinal epithelial cells.

Fig. 1. Revival stem cells and fetal-like CBC cells are induced after irradiation.

A Workflow schematic for profiling the single-cell transcriptome of regenerating intestinal epithelium and generating a reference and query map. The reference map was generated using intestinal epithelial cells from p53 WT mice (p53^FL/+ and p53 WT) at indicated time points after 12 Gy SBI (step 1). The reference map was used to query the transcriptional profile of intestinal epithelial cells from p53^FL/− mice (step 2). (IR: irradiated). (Created with BioRender.com). B UMAP representation of the reference map, including all time points with intestinal epithelial cell clusters labeled based on their transcriptional profile. C UMAP plots showing changes in cellular clusters between non-irradiated (NR) and two or three days after 12 Gy SBI (IR D2/D3), highlighting the emergence and expansion of revival stem cells (revSC) and fetal-like CBC cells (FCC) clusters after radiation exposure. D UMAP plots showing the mRNA expression pattern of Ly6a, Clu, and Mki67 in revSC and FCC clusters at different time points after SBI. E Representative smFISH images of Ly6a and Clu expression in mouse intestinal tissue sections at day 3 post-SBI, with white and pink arrowheads indicating Ly6a+Clu- and Ly6a+Clu+ cells, respectively (scale bar = 50 μm). Data from one experiment (n = 4 mice, n = 15–20 intestinal tissue areas/mouse). F Percentage of double Ly6a and Clu positive cells within the Clu (orange bar) and Ly6a (red bar) cell populations. Data represents quantification of 15 tissue sections across 4 mice (n = 4). G Percentage of Ly6a and Clu positive cells in intestinal tissue sections from NR mice (0 h) (n = 3) or 48 h (n = 3), 60 h (n = 3) and 72 h (n = 4) after 12 Gy of SBI. The boxplot represents the interquartile range (IQR) of the data, with the median indicated. The whiskers represent the highest and lowest values within 1.5 times the IQR. Each dot represents the average of 15 tissue areas quantified in each mouse. Statistical significance was calculated using a one-way ANOVA test followed by a Post hoc Tukey’s HSD test.

Fig. 2. p53 transcriptional targets are enriched in revSCs and FCC cells during regeneration.

A UMAP displaying the p53 transcriptional gene signature expression across epithelial intestinal cell clusters in p53 WT mice, including all time points. B Heatmap showing the average expression of selected gene markers across crypt base columnar cells (CBC), transit amplifying (TA), fetal-like CBC cells (FCCs), and revival stem cells (revSCs) cell populations. C UMAPS showing the expression of individual p53 transcriptional gene targets across all epithelial intestinal cell clusters in non-irradiated (NR) and 2 days (IR D2) and 3 days (IR D3) after 12 Gy SBI. RevSC and FCC clusters are highlighted at IR D3.

Fig. 3. p53 is required to induce revSC and FCC cells during intestinal regeneration.

A Schematic illustrating the generation of a query map (p53 KO: Villin^Cre; p53^FL/−) compared with the reference map (p53 WT: Villin^Cre; p53^FL/+ and WT). (Created with BioRender.com). B UMAP plots depicting changes in cellular composition in mice with p53 WT or p53 KO intestinal epithelium in non-irradiated (d0) or two days (IR D2) after 12 Gy SBI. C Representative images from smFISH of Clu and Ly6a expression in mice with p53 WT or p53 KO intestinal epithelium in non-irradiated (NR) or 48 h and 60 h after irradiation. Scale bar = 50 μm. D Percentage of double Clu and Ly6a-positive cells quantified from C. The boxplot represents the IQR of the data, with the median indicated. The whiskers represent the highest and lowest values within 1.5 times the IQR. Each dot represents the average of 15 tissue areas quantified for each mouse (0 h: n = 3, 48 h: n = 3, and 60 h: n = 3). Statistical significance was assessed using a two-sided linear regression model for overall significance, and pairwise comparisons were made using the Bonferroni correction method (p val = 0.0003159). E Schematic indicating that small intestinal organoids were treated with 4-hydroxytamoxifen (4-OHT) prior to irradiation (3 or 5 Gy) in control (DMSO) conditions or during p53-inhibition by Pifithrin-α (Pf-α) treatment. F Representative immunofluorescence images of organoids showing Clu and Clu progeny (Yellow) and Lgr5 (green) on the 5^th day of regeneration, grown in media containing either DMSO or Pf-α. White arrows indicate the absence of Clu+ cells and progeny in the crypts of irradiated organoids treated with Pf-α. (n = 3 experiments, 2 technical replicates). Scale bar = 100 and 50 μm. G Quantification of the percent of organoids displaying lineage-traced. Each dot represents an individual experiment (n = 3 experiments), with the number of organoids counted per experiment and per condition ranging from 17–106 (n = 17–106). The bars represent the mean percentage of organoids with more than 50% tdTOM-positive, and the error bars indicate the standard error of the mean. Statistical significance was calculated using a one-way ANOVA test followed by a Post-hoc Tukey’s HSD test.

Fig. 4. Constitutive expression of p53 impairs intestinal tissue regeneration after irradiation.

A Immunofluorescence of Clu-GFP (green), P53 (red), and Epcam (yellow) on intestinal tissue sections 1, 2, and 3 days after 12 Gy SBI. Scale bar = 50 μm. B Quantification of the percentage of cells with nuclear P53 from A. Data from one experiment (3 mice/timepoint, n = 3) is shown, with each dot representing a single cross-section. The boxplot represents the interquartile range (IQR) of the data, with the median indicated. The whiskers extend from the box to the highest and lowest values within 1.5 times the IQR. Statistical significance was calculated using one-way ANOVA test followed by a Post-hoc Tukey’s HSD test. C UMAP plots showing the Mdm2 mRNA expression in revSC and FCC populations at day 0, 2, and 3 days after 12 Gy SBI. D Representative smFISH of Mdm2 transcript in mouse intestinal tissue sections from non-irradiated (NR) or 3 days (IR D3) after 12 Gy SBI. Scale bar = 50 μm. E Percentage of Mdm2 positive cells quantified in intestinal tissue sections represented in D. The bars represent the mean value of the data for each condition, with error bars indicating the standard deviation from the mean. Each dot represents the average value of five tissue areas that were quantified for each mouse (non-irradiated: (n = 2) or irradiated: (n = 3)). Statistical significance was calculated using a two-sided t test comparison. F Schematic indicating that small intestinal organoids were treated with 4-OHT to induce a lineage trace form Clu+ cells before irradiation (3 or 5 Gy), all prior to passaging. Organoid regeneration was tracked over a 4-day period, with growth under control (DMSO) or Nutlin-3 treatment. G Representative immunofluorescence images of Clu and Clu progeny (yellow), Lgr5 (green), and p53 (red), indicating that under continuous p53 activation (Nutlin-3), organoids show an inability to recover from IR conditions by day 4 of regeneration. (n = 2 experiments, 2 technical replicates/experiment). Scale bar = 50 μm. H Bright field images 1 and 2 passages after irradiation, indicating that organoids show an inability to recover from continuous p53 activation, with culture collapse evident after the initial treatments. (n = 3 experiments, 2 technical replicates/experiment). Scale bar = 500 μm.

Fig. 5. p53 TAD domains are required to prevent radiation-induced GI syndrome.

A Gene set enrichment analysis (GSEA) showing top upregulated or downregulated gene sets two days after irradiation (IR) in p53 WT or p53 KO intestinal epithelial cells. P values were calculated using the empirical Bayes moderated t test from the Limma package. B UMAPs showing the expression of the DNA Repair and G2M Checkpoint gene signatures across epithelial cell clusters in intestinal tissues of p53 WT mice 3 days after 12 SBI. C Schematic of p53 protein domains showing the locations of the transactivation domains (TAD) mutation residues (upper) and schematic of p53 alleles of the Villin^Cre mice used in this study (lower). TAD1 and TAD2: transactivation domains 1 and 2. OD: oligomerization domain. D Representative images of immunohistochemistry staining of p21 protein on intestinal tissue sections from p53 WT and p53 mutants non-irradiated (No IR) or 4 hours after 13.3 Gy SBI. Scale bars = 50 μm. E Quantification of p21 staining from D. p21-positive cells within the +4 to +10 region of the crypt were counted from intestinal tissue sections from p53^FL/+ (NR: n = 3, IR: n = 9), p53^FL/FL(NR: n = 3, IR: n = 12), p53^LSL,25,26/FL (NR: n = 10, IR: n = 7) and p53^{LSL,25,26,53,54/FL} (NR: n = 2, IR: n = 5) mice. For each tissue section, 10–15 crypts were quantified. The bars represent the mean value of the data for each treatment within each genotype, with error bars indicating the standard deviation from the mean. Statistical significance was calculated using a two-sided t test. F Kaplan–Meier curves of p53 WT (p53^FL/+) or p53 mutants (p53^FL/FL, p53^LSL-25,26/FL, and p53^{LSL-25,26,53,54/FL}) mice following 13.4 Gy of SBI (n = 69). P val < 0.0001 is calculated using a log-rank test. G The schematic illustrates the evaluation of intestinal permeability in p53 WT and KO after IR. (Created with BioRender.com). Dextran-FITC concentration in the blood of p53 WT (NR: n = 6, IR: n = 8) and p53 KO (NR: n = 1, IR: n = 7) mice at day 5 after 12 Gy of SBI. The bars represent the mean value of the Dextran-FITC concentration for each genotype, with error bars indicating the standard deviation (SD) from the mean. Statistical significance was calculated using a two-sided t test.