Fig. 3. Tissue disintegration due to ECM deficiency in E18.5 Foxf2^-/- intestine. (A,B) Hematoxylin and Eosin stained sections of colon from wild type (A) and Foxf2^-/- (B). The mutant has a distended distal colon and the mesodermal layers separate from each other and from the epithelium (top in close-up) because of poor cell adhesion. (C-K) Immunostaining with antisera against a sheet forming collagen (type IV), a fibrillar collagen (type I) and SMA. Both collagens are reduced throughout the length of the intestine (from duodenum to rectum) in Foxf2^-/-. The mutant intestinal wall (I) is flimsy in appearance compared with the wild type (H), owing to lack of collagen fibers. Both the small intestine (I) and colon (E; here from a proximal, non-distended part) has a smaller diameter in the mutant than in wild-type littermates (C,H). Foxf1^-/+; Foxf2^-/+ (D; proximal colon) also has reduced amounts of ECM components, but less extreme than in Foxf2^-/-. (L,M) Immunostaining with anti-SMA in wild type (L) and Foxf1^-/+; Foxf2^-/+ (M) E18.5 small intestine shows ectopic expression of SMA in intravillus mesenchyme of the mutant. (N-Q) Cell-autonomous requirement for Foxf proteins for collagen expression in intestinal fibroblasts. Primary fibroblasts were prepared from E18.5 wild-type intestine and transfected with a plasmid expressing GFP (N,O) or a dominant-negative Foxf protein fused to GFP (P,Q). After 24 hours, cells were fixed and stained with an antiserum against collagen I (red). Collagen staining is seen in cytoplasmic vesicles and fibers between the cell and the glass substrate. In cells expressing the dominant-negative Foxf protein, identified by their green nuclear fluorescence, collagen staining is reduced dramatically. Blue, DAPI nuclear staining.