Figure 4. Overexpressing Cdc42 V2 in mouse intestinal epithelium affects differentiation.

(A) A schematic diagram showing the strategy of developing a Cdc42 V2^Tg mouse allele, which conditionally expressed a Flag-tagged Cdc42-V2 in a Cre-dependent manner. A loxP-stop-loxP-3xFlag-V2-bGHpoly(A) cassette was inserted downstream of a chick actin (CAG) promoter. (B) Western blots for Flag detected Flag-V2 expression in duodenum, jejunum, ileum, and colon of V2^Tg driven by Vil-Cre. HEK293 cells expressing Flag-V2 were used as positive controls. (C) Immunohistochemistry for Flag detected Flag-V2 expression in V2^Tg mouse IECs. Images are representative of 3 independent mice per genotype. (D) Western blots for Cdc42 showed that the transgenic expression of V2 (upper band) in a V2^Tg line was approximately 40% of endogenous Cdc42 (empty arrowhead). (E) H&E staining of mouse intestines. V2^Tg small intestines showed longer villi and more crypts; KO showed blunted villi. (F) Alcian blue staining of mouse intestinal sections of indicated genotypes. (G) Quantification of cyclooxygenases 1–positive (Cox1⁺) cell number per crypt-villus unit. (H) Quantification of Alcian blue–positive cells per crypt-villus unit. (I) Immunofluorescence staining for lysozyme (red), E-cad (green), and DAPI (blue). (J) Alkaline phosphatase (AP) staining showed representative AP⁺ inclusion bodies in KO IECs. Scale bar: 100 μm. (K) Quantification of mislocalized Paneth cells. (L) Quantification of AP⁺ inclusion bodies. Data in G, H, K, and L were quantified from multiple intestinal sections of a total of 3 animals per genotype. Please also see Supplemental Figures 3 and 4.