Figure 1. BMP restricts Lgr5⁺ stem cell expansion independently of Wnt/β-catenin during intestinal homeostasis.

(a) Vil-CreER;Lgr5-EGFP and Vil-CreER;Lgr5-EGFP;Bmpr1a^fl/fl mice were analysed 1 week after 5-day tamoxifen administration. Proximal jejunum sections were stained for p-Smad1/5/8 (for BMP signalling activity) and EGFP (for stem cells). Nuclei were counter-stained with 4′,6-diamidino-2-phenylindole (DAPI). The lower panels show enlargements of the upper panels. Images are representative of n=6 mice per genotype. Quantifications of crypt length and Lgr5⁺ cell number is shown aside, and the data represent mean±s.e.m. of n=6 mice per genotype. ***P<0.001 by Student's t-tests. (b,c) In situ hybridization of Olfm4 and immunohistochemical staining of Sox9 in Bmpr1a^fl/fl and Vil-CreER;Bmpr1a^fl/fl mice at day 12. Images are representative of n=6 mice per genotype. (d) Immunofluorescence images showing Lgr5⁺ stem cells 12 days after the first injection in Lgr5-CreERT2;Bmpr1a^fl/fl mice (n=5 mice per genotype). Dotted lines delineate the normal Lgr5⁺ stem cell pool. Images are representative of three independent experiments. (e) BrdU incorporation in ISCs and transient amplifying cells after a 2 h pulse in Vil-CreER;Bmpr1a^fl/fl and Bmpr1a^fl/fl mice at day 12. Images are representative of n=6 mice per genotype. The asterisks indicate the replicating CBC cells at the base of crypt. (f) In vitro colony formation assay of GFP^high cells sorted from Vil-CreER;Bmpr1a^fl/fl and Bmpr1a^fl/fl mice at day 12. Data represent mean±s.e.m. of n=3 experiments. (g) Lineage tracing in the jejunum of Lgr5-CreERT2;td-Tomato mice and Lgr5-CreERT2;td-Tomato;Bmpr1a^fl/fl mice. Representative images of td-Tomato immunofluorescence at day 3.5 after induction were shown (n=4 mice per genotype). (h) β-catenin immunofluorescence staining of proximal jejunum sections from Bmpr1a^fl/fl and Vil-CreER;Bmpr1a^fl/fl mice at day 12. The right panels show enlargements of boxed areas. Nuclei were counter-stained with DAPI. Images are representative of n=6 mice per genotype. (i) Quantitative RT–PCR analysis of Bmpr1a, Id1, Axin2, EphB2 and Cd44 expression in intestinal crypts from Vil-CreER;Bmpr1a^fl/fl and control mice at day 12 after induction. Data represent mean±s.e.m. of n=5 mice per genotype. *P<0.05, ***P<0.001 by Student's t-tests. Scale bars, 50 μm. CBC, crypt columnar cell; EGFP, enhanced green fluorescent protein.