Erythroid differentiation regulator-1 induced by microbiota in early life drives intestinal stem cell proliferation and regeneration

Abstract

Gut microbiota and their metabolites are instrumental in regulating intestinal homeostasis. However, early-life microbiota associated influences on intestinal development remain incompletely understood. Here we demonstrate that co-housing of germ-free (GF) mice with specific-pathogen free (SPF) mice at weaning (exGF) results in altered intestinal gene expression. Our results reveal that one highly differentially expressed gene, erythroid differentiation regulator-1 (Erdr1), is induced during development in SPF but not GF or exGF mice and localizes to Lgr5⁺ stem cells and transit amplifying (TA) cells. Erdr1 functions to induce Wnt signaling in epithelial cells, increase Lgr5⁺ stem cell expansion, and promote intestinal organoid growth. Additionally, Erdr1 accelerates scratch-wound closure in vitro, increases Lgr5⁺ intestinal stem cell regeneration following radiation-induced injury in vivo, and enhances recovery from dextran sodium sulfate (DSS)-induced colonic damage. Collectively, our findings indicate that early-life microbiota controls Erdr1-mediated intestinal epithelial proliferation and regeneration in response to mucosal damage.

Fig. 1. Early-life microbiota regulates Erdr1 expression.

a Experimental schematic of the exGF model: Germ-free (GF) mice were colonized with microbiota beginning at day 21 by co-housing with SPF mice for 3 weeks. b Volcano plots showing log2 fold-change of total colon gene expression in SPF mice compared with exGF or GF mice. Genes that were significantly decreased in exGF or GF compared with SPF colons are highlighted on red, while those increased in exGF or GF are highlighted in blue. c qPCR analysis of Erdr1 using whole small and large intestinal tissue from SPF, exGF, and GF mice. n = 2 (duodenum/jejunum/ileum from exGF), n = 4 (duodenum/jejunum/ileum from SPF and GF, colon). d ChIP analysis for histone H3 acetylation (H3Ac) in the Erdr1 promoter region using total colon tissue. n = 4; two independent experiments. All data are presented as mean ± SD; *P < 0.05, **P < 0.01, ***P < 0.001 by one-way ANOVA with Tukey’s multiple comparison test.

Fig. 2. Erdr1 is expressed in Lgr5⁺ intestinal stem cells (ISCs) and transit amplifying (TA) cells.

a RNAscope staining for Erdr1 within small and large intestine from SPF, exGF, and GF mice. Green indicates Erdr1 RNA. b 3-color RNAscope staining of longitudinal sections of small intestine: green, Erdr1; pink, Lgr5; white, Lyz1; blue, DAPI. c 3-color RNAscope staining of transverse sections of small intestine, as in b.

Fig. 3. Erdr1 increases growth of intestinal organoids.

a Expression of Erdr1 in intestinal small and large organoids derived from SPF, exGF and GF mice. n = 4. b ChIP analysis for histone H3 acetylation (H3Ac) in the Erdr1 promoter region using large intestine (LI) organoids derived from SPF, exGF, and GF mice. n = 4; two independent experiments. c Representative images of LI organoids derived from SPF, exGF, and GF mice and cultured ±Erdr1 (1 μg) for 6 days. d Organoid efficiency of LI organoids cultured ±Erdr1. n = 4; representative of three experiments. e Frequency of budding organoids cultured ±Erdr1 for 6 days. n = 4; representative of three experiments. f Surface area of LI organoids cultured ±Erdr1. n = 21; representative of three experiments. All data are presented as mean ± SD; *P < 0.05, **P < 0.01, ***P < 0.001 by unpaired, two-tailed t-tests.

Fig. 4. Erdr1 induces ISC signature gene expression and Lgr5⁺ ISCs.

a Expression of stem cell signature genes (Lgr5, Olmf4, Smoc2, Ascl2, Tnfrsf19) in LI organoids from SPF mice cultured ±Erdr1 for 6 days. n = 4 (Olfm4), n = 5 (Smoc2, Ascl2, Tnfrsf19), n = 6 (Lgr5); representative of three experiments. b–d Frequency and cell numbers of Lgr5-EGFP⁺ cells (live cells, EpCAM1⁺, EGFP⁺) in SI organoids cultured ±Erdr1 for 6 days. n = 4; representative of three experiments. All data are presented as mean ± SD; *P < 0.05, **P < 0.01, ***P < 0.001 by unpaired, two-tailed t-tests.

Fig. 5. Erdr1 enhances Wnt signaling in intestinal epithelial cells and organoids.

a The relative luciferase activity measured by the TOP/FOP-flash assay in SKCO-15 human intestinal epithelial cells stimulated ±Erdr1. n = 6; representative of two experiments. b Expression of Wnt signaling pathway target genes (Myc, Axin2, Ephb2, Sox9) in LI organoid cultures ±Erdr1 (1 μg/mL). n = 4; representative of three experiments. All data are presented as mean ± SD; *P < 0.05, **P < 0.01, ***P < 0.001 by unpaired, two-tailed t-tests.

Fig. 6. Erdr1 accelerates wound closure in mouse and human intestinal epithelial cells.

Scratch-wound assay using Mode-K mouse intestinal epithelial cells cultured ±Erdr1 (1 μg/mL) for 36 h (a picture; b % wound closure). n = 5; representative of three experiments. c–d Cell cycle analysis of Mode-K cells cultured ±Erdr1. n = 3; representative of three experiments (c histogram; d % cell cycle). e–g Ki-67 staining of Mode-K cells stimulated ±Erdr1. n = 3; representative of three experiments. h–i Wound closure of Mode-K cells treated with Erdr1 siRNA or control siRNA (k picture; l % wound closure). n = 7 (control siRNA), n = 8 (Erdr1 siRNA); representative of two experiments. All data are presented as mean ± SD; *P < 0.05, **P < 0.01, ***P < 0.001 by unpaired, two-tailed t-tests.

Fig. 7. Erdr1 promotes the regeneration of Lgr5⁺ ISCs following radiation-induced injury.

a Representative images of EGFP staining in ileum sections from Lgr5-EGFP reporter mice housed in SPF conditions. Mice were injected ±Erdr1 daily following 10 Gy irradiation. b Quantification of Lgr5-EGFP⁺ cells per crypt. n = 60; representative of three experiments. c Representative images of Ki-67 staining in ileum and colon sections (pink, Ki-67; blue, DAPI). d Quantification of Ki-67 positive cells per crypt from the ileum (top) and colon (bottom). n = 60; representative of three experiments. All data are presented as mean ± SD; *P < 0.05, **P < 0.01, ***P < 0.001 by one-way ANOVA with Tukey’s multiple comparison test.

Fig. 8. Erdr1 promotes recovery from dextran sulfate sodium (DSS)-induced colitis.

a DAI score of mice treated for 5 days with DSS, followed by normal drinking water for 4 days. Erdr1 was administered at day 4, 6, and 8; anti-Erdr1 antibody was administrated at day 4 and day 7. b Colon images from mice treated in a. c Colon length from mice treated in a. d Representative H&E stained sections and e histology scores from mice as treated in a. n = 11 (control), n = 4 (Erdr1), n = 5 (anti-Erdr1 Ab); representative of two experiments. All data are presented as mean ± SD; *P < 0.05, **P < 0.01, by one-way ANOVA with Tukey’s multiple comparison test.