Creation and analysis of villin-deficient mice.

Abstract

The small intestine is functionally dependent on the presence of the brush border, a tightly packed array of microvilli forming the apical surface of absorptive cells. The core of each microvillus is composed of a bundle of actin filaments cross-linked by two proteins, fimbrin and villin. In addition to its bundling activity, villin can nucleate, cap and sever actin filaments in a calcium-dependent manner, suggesting that it might play an important role in brush border morphogenesis. The results of two in vitro studies further support this conclusion. Transfection of a villin cDNA into fibroblasts, which normally do not possess microvilli or express villin, resulted in a reorganization of the cytoskeleton with the subsequent production of microvilli (Friedrich, et al., Cell 59:461, 1989). Second, transfection of a villin antisense cDNA into the intestinal cell line CaCo2 blocked the formation of microvilli (Costa de Beauregard, et al. EMBO J 14:409, 1995). These experiments suggest that villin is both necessary and sufficient for microvillar formation in cultured cells. To determine whether this is also true in vivo, we created a null allele of the villin gene by targeted recombination in mouse embryonic stem cells. The targeting construct was designed such that a successful recombination also places a $\beta$-galactosidase gene under the control of the villin promoter. These experiments allowed us to determine if villin is required in vivo, and provided a marker to follow villin expression throughout development and after birth. Analysis of embryos from 6.5 to 10.5 dpc confirmed correct temporal and spatial expression of the villin/$\beta$-galactosidase transgene. Within the developing intestine, loss of the villin protein appeared to have no significant effect on the process of microvillar initiation and development. Adult villin-deficient mice were viable, and exhibited morphologically normal microvilli in colon, kidney and liver. In the small intestine, however, subtle defects in the ultrastructure of microvillar actin core bundles were detected. These data demonstrate that villin is not essential for the formation and maintenance of functional microvilli in vivo.