(A, B) Dnmt1loxP/loxP(control, n=5) and Dnmt1loxP/loxP;Villin-CreERT2 (Dnmt1 mutant, n=9) mice were tamoxifen treated at four weeks of age, and weighed every day over a 17-day period. Dnmt1 mutants lost a significant amount of weight by day 11, but recovered to near-starting weight by day 16. *p<0.05, Student’s t-test. (B) All Dnmt1 mutants survive the 17-day time-course described in (A), similar to controls. (C–D) Hematoxylin and Eosin staining of control and Dnmt1 mutant intestines. One week post-ablation, Dnmt1 mutants exhibit loss of crypt integrity, vacuolization of the epithelium, and an increase in crypt fission (D) compared to controls (C). (E–F) Immunohistochemistry confirms loss of Dnmt1 protein in mutants one week following tamoxifen treatment (F) relative to control intestine (E). (G-H) Immunofluorescent staining for Ki67 (red), which marks proliferating cells, and γH2AX (green), which marks DNA double-strand breaks as a marker of chromosomal instability. One week following Dnmt1 ablation, mutant crypts display increased levels of γH2AX foci (H, yellow arrows) relative to controls (G). Dnmt1 mutants also display slightly enlarged crypts (Ki67 in H versus G), as described previously (Sheaffer et al., 2014). (I–J) Immunofluorescent TUNEL staining (red), which marks apoptotic nuclei, and E-cadherin (green), to outline the intestinal epithelium. One week after tamoxifen treatment, Dnmt1 mutants display increased crypt cell apoptosis (J, yellow arrows) compared to controls (I). (K–L) Crypts were isolated from paraffin-embedded tissue by laser capture microdissection, and the methylation levels of LINE1 loci and the imprinting control region of H19 were determined by targeted bisulfite sequencing. One week after tamoxifen treatment, methylation of LINE1 (K) and H19 (L) are significantly decreased in Dnmt1 mutants compared to controls (n=4 per genotype). ***p<0.001, *p<0.05, Student’s t-test. For data and p values, refer to Figure 1—source data 1. Error bars represent S.E.M. Scale bars are 50 μm. For all staining, n=3 biological replicates.