867a GATA3 is Essential for Proper Morphogenesis of the Pylorus

Abstract

BACKGROUND: Kruppel-like factor 5 (KLF5) is a pro-proliferative transcription factor that is highly expressed in dividing epithelial cells at the crypt base of the intestinal epithelium. KLF5 promotes proliferation In Vitro and In Vivo and is induced bymitogens and various stress stimuli. To study the role of KLF5 in maintaining intestinal homeostasis, we characterized the morphology of mice with conditional deletion of Klf5 in the gut using the villin-Cre (VilCre) recombinase system. METHODS: Mice carrying floxed alleles of Klf5 (Klf5fl/fl) were crossed with Vil-Cre;Klf5fl/+ mice to generate Vil-Cre;Klf5fl/fl mice. Klf5fl/fl littermates served as controls. Mice were sacrificed at 8 weeks of age and the small intestines and colon removed for histological examination. Immunohistochemical staining was used to examine the expression of Klf5, the proliferation marker, Ki67 and various markers of differentiation. RESULTS: Immunohistochemical staining of Klf5 in the small and large bowels of the VilCre;Klf5fl/fl mice showed that deletion of the floxed Klf5 alleles was variable, with the most complete deletion occurring in the distal small intestine and proximal colon. Initial characterization of the mice indicated that the Vil-Cre;Klf5fl/fl mice were leaner than the Klf5fl/fl littermates, weighing 18% less than genderand age-matched controls (n=5, P = 0.01). Histological staining of intestinal and colonic tissues from Vil-Cre;Klf5fl/fl mice revealed a disruption of normal crypt architecture and a regenerative phenotype, particularly in the colon. Staining with the proliferation marker, Ki67, showed proliferating cells scattered throughout the epithelia of the small and large intestines, with a phenotype similar to that of EphB2/EphB3-null mice. Paneth cells likewise migrated upward from the crypt base. Immunohistochemical staining for EphB2 and EphB3 in Klf5-deleted tissues revealed reduced expression of both receptors and loss of a normal expression gradient within the crypts. Reduction of EphB2 and EphB3 correlated with reduced expression of the differentiation markers intestinal alkaline phosphatase and carbonic anhydrase I. CONCLUSION: Deletion of Klf5 in the gut epithelium results in dramatic changes in morphology that correlate with reduced expression of Wnt targets, EphB2 and EphB3. The boundary between proliferating and differentiation zones are lost, resulting in scattering of proliferating cells throughout the epithelium and reduced differentiation. Thus, KLF5 is critical for maintenance of normal crypt architecture in the intestinal mucosa due to its effects on epithelial cell positioning and differentiation.