Developmental regulation of villin gene expression in the epithelial cell lineages of mouse digestive and urogenital tracts.

The expression of villin, an actin-binding protein and major structural component of the brush border of specialized absorptive cells, was studied during mouse embryogenesis. We show that the ontogeny of villin expression is limited to the epithelial cell lineages of the digestive and uro-genital tracts and accounts for the tissue-specific expression observed in adult mice. This spatiotemporal pattern of villin expression is distinctive in sequence, intensity, regional distribution and polarization. During the development of the primitive gut, villin is faintly and discontinuously expressed in the invaginating foregut but it is expressed in every cell bordering the hindgut pocket. Later, villin expression increases along the developing intestine and concentrates in the brush border of the epithelium bordering the villi. In gut derivatives, villin is present in liver and pancreas primordia but only biliary and pancreatic cells maintain a faint villin expression as observed in adults. In the urogenital tract, mesonephric tubules are the first mesodermal derived structures to express villin. This expression is maintained in the ductuli efferents, paradidymis and epo?phoron. Villin then appears in the proximal metanephric tubules and later increases and concentrates in the brush border of the renal proximal tubular epithelial cells. Thus villin expression can be considered as an early marker of the endodermal cell lineage during the development of the digestive system. Conversely, during the development of the excretory and genital system, villin is only expressed after the mesenchyme/epithelium conversion following the appearance of tubular structures. These observations emphasize the multiple levels of regulation of villin gene activity that occur during mouse embryogenesis and account for the strict pattern of tissue-specific expression observed in adults. In the future, regulatory elements of the villin gene may be used to target the early expression of oncogenes to the digestive and urogenital tracts of transgenic mice.     

From the structure to the function of villin, an actin-binding protein of the brush border

Villin, a calcium-regulated actin-binding protein, modulates the structure and assembly of actin filaments in vitro. It is organized into three domains, the first two of which are homologous. Villin is mainly produced in epithelial cells that develop a brush border and which are responsible for nutrient uptake. Expression of the villin structural gene is precisely regulated during mouse embryogenesis and is restricted in adults, to certain epithelia of the gastrointestinal and urogenital tracts. The function of villin has been assessed by transfecting CV1 cells with a human cDNA encoding wild-type villin or mutant villin. Synthesis of large amounts of villin in cells which do not normally produce this protein induces the growth of microvilli on the cell surface and the redistribution of F-actin, concomitant with the disappearance of stress fibers. The complete villin sequence is required for the morphogenic effect. These results suggest that villin plays a key role in the morphogenesis of microvilli.     

F9 teratocarcinoma aggregates express villin upon differentiation into visceral endoderm-like cells

The visceral endoderm of the mouse embryo is a polarized epithelium which has recently been shown to express villin, a major actin binding component of absorptive epitheliums. We report here that villin is induced during differentiation of aggregates of the mouse embryonal carcinoma F9, an in vitro system widely used to study extraembryonic endoderm differentiation. Identical results were obtained with a variant of F9 which carries an immortalizing vector. Villin is coexpressed with F-actin and with alpha-foetoprotein, in most of the visceral endoderm-like cells lining the aggregates. This system is potentially useful to study (i) the induction of villin expression and (ii) the establishment of polarity in the visceral endoderm epithelium.     

Expression of villin in the mouse oviduct and the seminiferous ducts

Summary Villin, a 95-kD cytoskeletal protein selectively expressed in the microvilli of some absorptive cells was localized immunohistochemically in the oviduct and the seminiferous excretory ducts of the mouse. Villin was found in the proximal part of the oviduct, comprising the preampulla, ampulla, and part of the isthmus. Distal to the isthmus the oviductal cells lining the junctura and the intrauterine colliculus tubaris were devoid of villin. No villin could be detected in the uterine cells.Ductuli efferentes, connecting the rete testis with the epididymis were the only portion of the male seminiferous ductal system expressing villin. The cells lining the epididymis and the vas deferens were devoid of villin. These data show that villin is selectively expressed in male and female reproductive systems and that it is limited to anatomically defined proximal portions of the reproductive ducts.     

Dimerization and actin-bundling properties of villin and its role in the assembly of epithelial cell brush borders

Villin is a major actin-bundling protein in the brush border of epithelial cells. In this study we demonstrate for the first time that villin can bundle actin filaments using a single F-actin binding site, because it has the ability to self-associate. Using fluorescence resonance energy transfer, we demonstrate villin self-association in living cells in microvilli and in growth factor-stimulated cells in membrane ruffles and lamellipodia. Using sucrose density gradient, size-exclusion chromatography, and matrix-assisted laser desorption ionization time-of-flight, the majority of villin was identified as a monomer or dimer. Villin dimers were also identified in Caco-2 cells, which endogenously express villin and Madin-Darby canine kidney cells that ectopically express villin. Using truncation mutants of villin, site-directed mutagenesis, and fluorescence resonance energy transfer, an amino-terminal dimerization site was identified that regulated villin self-association in parallel conformation as well as actin bundling by villin. This detailed analysis describes for the first time microvillus assembly by villin, redefines the actin-bundling function of villin, and provides a molecular mechanism for actin bundling by villin, which could have wider implications for other actin cross-linking proteins that share a villin-like headpiece domain. Our study also provides a molecular basis to separate the morphologically distinct actin-severing and actin-bundling properties of villin.     

Differential localization of villin and fimbrin during development of the mouse visceral endoderm and intestinal epithelium

The apical surface of transporting epithelia is specially modified to absorb nutrients efficiently by amplifying its surface area as microvilli. Each microvillus is supported by an underlying core of bundled actin filaments. Villin and fimbrin are two actin-binding proteins that bundle actin filaments in the intestine and kidney brush border epithelium. To better understand their function in the assembly of the cytoskeleton during epithelial differentiation, we examined the pattern of villin and fimbrin expression in the developing mouse using immunofluorescence and immunoelectron microscopy. Villin is first detected at day 5 in the primitive endoderm of the postimplantation embryo and is later restricted to the visceral endoderm. By day 8.5, villin becomes redistributed to the apical surface in the visceral endoderm, appearing in the gut at day 10 and concentrating in the apical cytoplasm of the differentiating intestinal epithelium 2-3 days later. In contrast, fimbrin is found in the oocyte and in all tissues of the early embryo. In both the visceral endoderm and gut epithelium, fimbrin concentrates at the apical surface 2-3 days after villin; this redistribution occurs when the visceral endoderm microvilli first contain organized microfilament bundles and when microvilli first begin to appear in the gut. These results suggest a common mechanism of assembly of the absorptive surface of two different tissues in the embryo and identify villin as a useful marker for the visceral endoderm.     

Shigella flexneri infection is dependent on villin in the mouse intestine and in primary cultures of intestinal epithelial cells

Villin is an actin-binding protein present in intestinal and kidney brush borders. Villin has been shown to present in vitro Ca(2+)-dependent bundling and severing F-actin properties. The study of villin knock-out mice allowed us to show that while bundling of F-actin microfilaments is unaffected, this protein is important for the reorganization of the actin cytoskeleton elicited by various signals during both physiological and pathological conditions. Here, we studied the role of villin during infection by Shigella flexneri, the causative agent of bacillary dysentery. This bacterium induces the reorganization of the host actin cytoskeleton to penetrate into epithelial cells and spread from cell to cell. In vivo, we show that unlike newborn vil+/+ mice, which are sensitive to Shigella invasion, resulting in a destructive inflammatory response of the intestinal mucosa following intragastric inoculation, newborn vil-/- mice appear fully resistant to infection. Using primary cultures of intestinal epithelial cells derived from vil+/+ or vil -/- mice, we demonstrate that villin plays an essential role in S. flexneri entry and cell-to-cell dissemination. Villin expression is thus critical for Shigella infection through its ability to remodel the actin cytoskeleton.     

Assembly of the intestinal brush border: appearance and redistribution of microvillar core proteins in developing chick enterocytes

The assembly of the intestinal microvillus cytoskeleton during embryogenesis in the chick was examined by immunochemical and light microscopic immunolocalization techniques. For these studies, affinity-purified antibodies reactive with three major cytoskeletal proteins of the adult intestinal microvillus, fimbrin, villin, and the 110-kD subunit of the 110K-calmodulin protein complex were prepared. Immunocytochemical staining of frozen sections of embryonic duodena revealed that all three proteins were present at detectable levels at the earliest stages examined, day 7-8 of incubation (Hamilton/Hamburger stages 25-30). Although initially all three proteins were diffusely distributed throughout the cytoplasm, there was a marked asynchrony in the accumulation of these core proteins within the apical domain of the enterocyte. Villin displayed concentrated apical staining by embryonic day 8 (stage 28), while the apical concentration of fimbrin was first observed at embryonic day 10 (stage 37). Diffuse staining of the enterocyte cytoplasm with the anti-110K was observed throughout development until a few days before hatch. By embryonic day 19-21 110K staining was concentrated at the cell periphery (apical and basolateral). The restricted apical localization characteristic of 110K in the adult brush border was not observed until the day of hatching. Immunoblot analysis of whole, solubilized embryonic duodena confirmed the presence of 110K, villin, and fimbrin throughout development and indicated substantial increases in all three proteins, particularly late in development. Immunoblot staining with anti-110K also revealed the presence of a high molecular mass (200 kD) immunoreactive species in embryonic intestine. This 200-kD form was absent from isolated embryonic enterocytes and may be a component of intestinal smooth muscle.     

The phenotype of triparental hepatoma cell hybrids depends on the fusion sequence used to generate them

Villin is a major protein of the microfilament bundle which makes up the core of each microvillus of the brush border of the intestinal epithelial cell. Using antibodies to villin in indirect immunofluorescence microscopy on isolated cells and on frozen tissue sections, the protein is readily detectable in the microvilli of the brush border of both intestinal and renal epithelial cells. However, villin could not be detected in tissue culture cells either by immunofluorescence microscopy or by immune replica procedures. When native villin was microinjected into such cells and its distribution visualized by immunofluorescence microscopy, the protein was found to be associated with microfilamentous structures. Moreover, preferential association of the villin into the microfilaments at the leading edges of the living cell was observed. Since villin behaves in vitro as a calcium-regulated F-actin bundling protein, we discuss the possibility that villin is immunologically distinct but functionally related to putative calcium-regulatory factors assumed to be present in cultured cells.     

Sequence of human villin: a large duplicated domain homologous with other actin-severing proteins and a unique small carboxy-terminal domain related to villin specificity

Villin is a calcium-regulated actin-binding protein that caps, severs, and bundles actin filaments in vitro. This 92,500-D protein is a major constituent of the actin bundles within the microvilli of the brush border surface of intestinal and kidney proximal tubule cells. Villin is a very early marker of cells involved in absorption and its expression is highly increased during intestinal cell differentiation. The amino acid sequence deduced from the cDNA sequence revealed that human villin is composed of three domains. The first two domains appear as the result of a duplication: their structural organization is similar. We can then define a basic unit in which a slightly hydrophilic motif is followed by three hydrophobic motifs, similar between themselves and regularly spaced. The duplicated domain is highly homologous to three other actin-severing proteins and this basic structure represents the whole molecule in severin and fragmin, while two basic units compose gelsolin. The third domain which is carboxy terminal is villin specific: it is unique among actin modulating proteins so far known. It could account for its actin-binding properties (dual regulation by calcium of severing and bundling activities). We propose that it may also be related to the subcellular localization of villin in different epithelial cell types.     

Villin as a structural marker to study the assembly of the brush border

Summary Brush borders which are localized at the apical face of enterocytes, are composed of thousands of stiff microvilli containing bundles of microfilaments made of actin. Their assembly occurs during terminal differentiation of the enterocytes when these cells migrate along the villus of the intestinal mucosa. The cell line HT 29 derived from a human colonic adenocarcinoma whose differentiation can be induced, can also be used as a model to study in culture the assembly of the intestinal brush border.Villin is one of the actin binding proteins found in microvilli which compose brush borders. Villin is expressed in the adult and in the embryo before the appearance of the brush border. Villin can be used as a tissue-specific marker for normal diffentiated and undifferentiated cells derived from gastrointestinal tractus in the adult as well as in the embryo. Since villin is a good marker for intestinal cells and plays a structural role in the assembly of the brush border we have analysed its expression and its localization in HT 29 cells. In HT 29 cells, as in the tissue, villin is synthesized at low levels before the appearance of the brush border. The high rate of synthesis and the recruitement of villin at the apical pole of the cells can be correlated with the existence of a well developed brush border.     

Villin function in the organization of the actin cytoskeleton. Correlation of in vivo effects to its biochemical activities in vitro.

Villin is an actin-binding protein of the intestinal brush border that bundles, nucleates, caps, and severs actin in a Ca(2+)-dependent manner in vitro. Villin induces the growth of microvilli in transfected cells, an activity that requires a carboxyl-terminally located KKEK motif. By combining cell transfection and biochemical assays, we show that the capacity of villin to induce growth of microvilli in cells correlates with its ability to bundle F-actin in vitro but not with its nucleating activity. In agreement with its importance for microfilament bundling in cells, the KKEK motif of the carboxyl-terminal F-actin-binding site is crucial for bundling in vitro. In addition, substitutions of basic residues in a second site, located in the amino-terminal portion of villin, impaired its activity in cells and reduced its binding to F-actin in the absence of Ca(2+) as well as its bundling and severing activities in vitro. Altogether, these findings suggest that villin participates in the organization and stabilization of the brush border core bundle but does not initiate its assembly by nucleation of actin filaments.