Fig 2: Reduced Notch signaling, but increased Wnt signaling in Ccn1-deficient mice.Ccn1?Lgr5 mice were analyzed at day 5-post corn oil or TM. a Immunofluorescence staining of Notch1 intracellular domain (N1-ICD, red) in the crypt of the jejunum. DAPI is counterstaining. Representative images are shown from three independent replicates. Bars: 20 µm. b Immunoblot analysis of indicated protein expression (N1-ICD, N2-ICD, Jag1, DLL1, and DLL2) in the jejunum. ß-actin as a loading control. Representative images are shown from at least three replicates. c qPCR analysis of Notch target genes (Hes1, Hey1, and HeyL) and Atoh1. Data represent the mean ± SEM of four biological replicates. One-sided t-test. ***p < 0.001. d Immunofluorescence staining of active ß-catenin in the crypt of Ccn1?Lgr5 mice. Images are representatives of three independent replicates. Bars: 20 µm. e Immunoblot analysis of active and total ß-catenin. Images are representative of three replicates. f, g qPCR analysis of Wnt target genes (Axin2, Cd44, Ephb2, and Myc; f), Wnt ligands (Wnt3a, Wnt5a, and Wnt3b; g), and a secreted Wnt antagonist Dkk1 (g). Data represent the mean ± SEM of four biological replicates. One-sided t-test. *p < 0.05; **p < 0.01; ***p < 0.001. Source data and the exact p-values are provided in a Source Data file.

Fig 3: A diagram of CCN1 regulating Notch and Wnt signaling through distinct integrins avß3/avß5-mediated signaling pathways.CCN1 expressed at the luminal side of the crypt base binds to integrins avß3/avß5 on, presumably, Paneth cells and Lgr5 + ISCs, which elicits two distinct signaling pathways bifurcating downstream of FAK. For the regulation of Wnt signaling, FAK recruits and activates Src, leading to YAP stabilization and activation through phosphorylation at tyrosine 357. Active p-YAP (Y357) can enter the nucleus, where it promotes TEADs-dependent transcription of target genes, such as a secreted Wnt antagonist Dkk1, which interferes with Wnt binding to its cognate receptor Frizzled by sequestering a Wnt co-receptor LRP5/6. Concurrently, YAP-TEADs can further increase the expression of another target gene Ccn1 in the crypt, suggesting a regulatory loop between CCN1 and YAP. On the other hand, integrin-FAK signaling results in NF-?B activation and subsequent induction of Jag1, a Notch ligand, which promotes Notch signaling important for ISC fate decision in the crypt base.