Fig. 4. Overproliferation and failure to induce apoptosis generate excessive epithelial cells in Foxf mutants. (A-C) Low (top) and high (bottom) magnifications. Hematoxylin and Eosin staining of E18.5 small intestine sections from wild-type (A), Foxf1^-/+; Foxf2^-/+ (B) and Foxf2^-/- (C). Normal villi (A) are covered by a smooth monolayer of highly polarized epithelial cells with basal nuclei. Villi in the compound heterozygote (B) are large, club-shaped with multilayered epithelia, whereas in Foxf2 null mutants (C), the epithelial cells detach from the mesenchyme and adhesion between epithelial cells from adjacent villi create inter-villus cross linking and luminal obstruction. (D-F) Altered distribution of E-cadherin immunostaining reveals loss of epithelial polarity. In normal, polarized epithelial cells (D), E-cadherin is confined to the lateral membrane, where adherence junctions connect adjacent cells, and basal and apical membranes lack this cell-adhesion molecule. In the compound Foxf heterozygote (E), internal layers of epithelial cells have ubiquitous membrane-associated staining, but the outer layer has apical membranes free of E-cadherin. In Foxf2^-/- small intestine (F) the epithelial cells show many signs of lost polarity, including E-cadherin staining along the entire circumference. (G-I) Dissolution of the proliferation boundary in Foxf mutants. Immunostaining for PCNA – a proliferation marker – identifies actively cycling cells, which in wild-type E18.5 small intestine epithelium (G) are confined to the intervillus pockets (predecessors of crypts of Lieberkühn). In Foxf mutants (H,I), the boundary between the basal proliferative and the distal post-mitotic compartments is missing, and PCNA-positive epithelial cells are found throughout the villi. (J-M) Partial resistance to apoptosis in Foxf2^-/- intestinal epithelium. TUNEL assay (red nuclei, apoptotic cells) of E18.5 wild-type (J,K) and Foxf2^-/- (L,M) small intestine. (J) Most parts of the normal intestine show no or few apoptotic cells at this stage. (K) Where local slippage between mesenchyme and epithelium causes poor contact between epithelial cells and basement membrane, apoptosis is induced in the affected cells. (L) In the light of the severe ECM deficiency and the beginning separation between epithelium and mesenchyme (arrowheads), the Foxf2^-/- intestinal epithelium contains surprisingly few apoptotic cells. (M) Not until the final stages of tissue disintegration in the worst affected areas, where the epithelial cells detach completely from the villus core, does the frequency of apoptotic cells increase significantly.