Cytoskeletal protein and mRNA accumulation during brush border formation in adult chicken enterocytes

Two model systems that have been useful for understanding the organization and assembly of actin cytoskeletons are skeletal muscle, with its striking sarcomeric pattern, and the red blood cell, with its relatively simple cortical cytoskeleton. We have learned much about cytoskeletal protein gene expression in these cells, and how the proteins are arranged to generate contractile forces and how they stabilize the membrane cytoskeleton. The analysis of the developmental assembly of the red blood cell and muscle cytoskeleton has led to an understanding of the regulation of cytoskeletal formation at the gene transcriptional and post-translational levels. Studies with myoblasts during skeletal muscle differentiation show that the levels of mRNAs encoding sarcomeric-specific proteins (eg. troponin, myosin heavy chains) increase many-fold and the levels of mRNAs encoding the non-muscle cytoskeletal proteins decrease as myoblasts fuse (Shani et al. 1982; Wade and Kedes, 1989). In the erythrocyte, cortical cytoskeleton differentiation is controlled by a similar co-ordinate activation of specific genes; however, the assembly of the spectrin heterodimer is regulated by post-translational modifications and differential levels of α- and β-spectrin synthesis (Blikstad et al. 1983; Moon and McMahon, 1987).

The simplicity and stereotypic arrangement of the brush border cytoskeleton have made the enterocyte another excellent model for analyzing the organization and development of an actin-based cytoskeleton (see reviews by Mooseker, 1985; Burgess, 1987). The mature brush border is structurally divided into two parts; the microvilli and the terminal web. A microvillus contains a core bundle of actin filaments, which extends into the terminal web as the rootlet. The actin-binding proteins villin, myosin I and fimbrin are associated with the core filaments, and, in addition to tropomyosin, are present in the rootlets. The terminal web contains a meshwork of actin filaments cross-linked with myosin, nonerythroid spectrins (fodrin and TW 260/240), a-actinin and tropomyosin. Although much is known about the structure of the adult brush border, little is known about its assembly. Research in brush border cytoskeleton formation has focused on embryonic chickens, rats and mice (Chambers and Grey, 1979; Rochette-Egly and Haffen, 1987; Shibayama et al. 1987; Maunoury et al. 1988; Stidwill and Burgess, 1986; Takemura et al. 1988; Ezzell et al. 1989), and most recently on cell lines (Dudouet et al. 1987). In the embryo, mitotic stem cells are found over the entire length of the villus (Overton and Shoup, 1964) whereas in the adult, mitotic cells are restricted to lower regions of the crypt. Those embryonic cells destined to become absorptive cells initially form a few, short, wide microvilli with no terminal web. Next the rootlets increase in length, and the number of microvilli and the number of core filaments increase. Finally, the microvilli grow to their mature length and the terminal web completes formation and stratifies (Chambers and Grey, 1979; Stidwill and Burgess, 1986; Shibayama et al. 1987; Takemura et al. 1988).

We chose to study brush border development in the adult, for in comparison to the embryo, it is more feasible to isolate sufficient cells at differing degrees of development for biochemical and structural analysis. The adult intestinal epithelium is a continually differentiating tissue, constantly renewed by division of the one to four undifferentiated stem cells that reside in crypts (Potten and Morris, 1988; Gordon, 1989). Cells cease division and differentiate as they migrate out of the crypts onto the villus. After 2–3 days they are shed from the villus tip into the intestinal lumen (Cheng and LeBlond, 1974). Although little is known about the morphological changes during the differentiation of the adult enterocyte, several previous works, which were not focused on the cytoskeleton, have suggested that they are similar to those during embryonic development (Brown, 1962; Trier and Rubin, 1965).

In this paper, we report ultrastructural and immunocytochemical studies of adult chicken brush border development. To correlate structural changes with the expression of cytoskeletal proteins, we measured the steady-state cytoskeletal protein and mRNA levels in isolated crypt and villus cells. We found that a primitive brush border is already formed in the basal crypt, early in the cell’s life history, but while cytoskeletal protein mRNAs increase somewhat, the corresponding levels of the proteins do not change significantly during development of the mature brush border.

The proximal loops of adult White Leghorn chicken duodena were rinsed in saline (0.15 M NaCl) and fixed in 3% glutaraldehyde, 0.2% tannic acid, 0.1 M NaPO₄ (pH 7.0) for2h in the dark at room temperature (RT). After a rinse in 10% sucrose in 0.1M NaPO₄, the tissue was postfixed for 2h with 0.5% OsO₄, 0.8% K3Fe(CN)₆ in 0.1M NaPO₄ on ice in the dark. The tissue was rinsed for 15 min in water, en bloc stained with aqueous uranyl acetate, dehydrated through a graded series of ethanols into propylene oxide and embedded in Epon/ Araldite. Thin sections were stained with uranyl acetate and lead citrate, and observed with a JEOL 100CX electron microscope operated at 60 kV.

Villin was purified from calcium-extracted brush borders according to the methods of Bretscher and Weber (1978). Rabbit antisera directed against villin was prepared by intra-cutaneous injection of 200 μg of protein in complete Freund’s adjuvant (Cappel Laboratories) followed 14 days later by 200 μg of protein in incomplete Freund’s adjuvant. Rabbits were bled 14 days after the final antigen injection. The villin antisera were shown to be monospecific on Western blots of total cell and brush border proteins (data not shown). Antiserum to chicken actin was obtained from ICN Immuno-Biologicals and antisera to erythrocyte spectrin and chicken gizzard n-actinin were obtained from Sigma. Antisera raised against bovine brain fodrin used for immunofluorescence was a generous gift from Dr K. Burridge. Antibodies to brush border myosin and tropomyosin have been previously characterized (Broschat et al. 1983; Broschat and Burgess, 1986). Pure chicken brush border myosin I (originally referred to as the 110 kD protein) and antisera were a generous gift from Dr J. Collins.

The proximal duodenal loop from an adult chicken was cut open, rinsed in saline, and fixed on ice for Ih with 3.7% formaldehyde in Solution I (75 mM KC1, 1 mM EGTA, 0.1 mM MgC1₂, 10 mM Imidazole, pH 6.9). The fixed tissue was cryoprotected with 1M sucrose, 1mM EGTA, 0.1M Tris-Cl, pH 7.4 and frozen in OCT (Tissue Tek, Miles Lab.) by immersion in liquid nitrogen. Frozen sections 5–7 μm (Reichert-Jung) thick were collected on uncoated glass slides and stored at −80°C. Slides were thawed at RT in Trisbuffered saline (TBS: 50IDM Tris-HCl, 150ITIM NaCl, 0.1% NaN₃, pH7.6), then permeabilized for 3min with 0.2% Triton X-100 in TBS. The sections were rinsed in TBS and blocked for 30 min with 3% BSA (Sigma, Fraction V) in TBS. The tissue was stained with the primary antibody for 1 h at 37°C, rinsed in TBS and stained with a 1/50 dilution of FITC-goat anti-rabbit IgG (Cappel) for 30 min at 37°C. All antibodies were diluted in TBS containing 0.3% BSA. To observe the distribution of polymerized actin, sections were stained with 6.6μM rhodamine-phalloidin (Molecular Probes). The slides were observed with a Leitz Laborlux S microscope equipped with epifluorescence optics. Micrographs were photographed at El 1600 on T-Max 400 film and developed with T-Max developer (Kodak). Nonimmune controls showed only very dim, diffuse staining and so were not included as figures.

Enterocytes from the villus tip (tip), villus middle (mid) and crypt were isolated by a modification of previous methods (Weiser, 1973; Breimer et al. 1981; Burgess et al. 1989). Briefly, this method entailed stirring intestinal pieces in hyperosmotic saline and collecting fractions at selected time points. In these conditions, cells slough off at the basement membrane and are separated from the lamina propria and muscularis mucosa. The identity and purity of the cell fractions was determined with electron and phase-contrast microscopy, and by determination of alkaline phosphatase activity (Burgess et al. 1989).

Enzyme-linked immunosorbent assays (ELISA) were used to examine changes in protein levels in enterocytes isolated from the crypt, mid and tip. Briefly, isolated cells were homogenized in Solution I containing 1 M guanidine hydrochloride, cleared of particulates by centrifugation at 10000g for 10 min, and 0.5μg adsorbed to 96-well plates (Costar). The plates were blocked with 3% gelatin in TBS-0.05% Tween-20 and incubated with antisera. Titration curves with varying antibody or antigen concentrations were used to determine optimal cell extract and antibody dilutions. All determinations were performed in the linear range of each antibody/ antigen curve. After washing, the bound antibodies were detected using Protein A conjugated with horseradish peroxidase (BioRad, Richmond, CA.) and the color developed with 2,2 azino-di-[3-ethylbenzthiazoline-6-sulfonic acid]. Plates were quantified using a Bio-Tek autopíate reader at 405 nm and the levels standardized with purified proteins. The values reported are the means from cell fractions isolated from three chickens.

The following chicken cDNA clones were used as probes for RNA hybridization experiments: β-smooth muscle tropomyosin in pLJC8 (Helfman et al. 1984); /Tactin in pUC18 (Paterson et al. 1984); smooth muscle myosin in pBR325 (Molina et al. 1987); calmodulin in pBR322 (Putkey et al. 1983); a-spectrin in pUCB (Birkenmeier et al. 1985); villin in pBSM13+ (Bazari et al. 1988). All probes were labelled with *P by nick translation (Maniatis et al. 1982).

Total RNA was prepared from crypt, mid and tip cell fractions using the guanidinium isothiocyanate method of Chirgwin et al. (1979). The duodena from three chickens were pooled for each of three cell isolation preparations. For dot blots, 3μg of total RNA were denatured at 65°C for 15 min in 7.5×SSC (I×SSC is 0.15M NaCl, 0.015M sodium citrate) containing 4.16 M formaldehyde and dotted onto nitrocellulose using a microfiltration apparatus (Bio-Rad). For Northern blots, 5–10 μg of total RNA per lane were electrophoresed on 1% formaldehyde gels and transferred to nitrocellulose or Nytran (Schleicher and Schuell) by capillary blotting using 20xSSC as transfer buffer (Maniatis et al. 1982). The filters were hybridized at 42°C for at least 20 h in 50% formamide and 0.825 M Na⁺ by the methods of Maniatis et al. (1982). The final filter wash was at 65°C in IxSSC, 0.1% SDS. The filters were exposed to preflashed Kodak XAR-5 film (Laskey and Mills, 1977) with an intensifying screen at −70°C.

Relative mRNA levels were determined by scanning autoradiograms with an E-C scanning densitometer coupled to a Waters digital automatic integrator. The linearity of response of the photographic emulsion was verified (r=0.994) using a dot blot containing a 50-fold concentration range of tip RNA hybridized with the β-actin probe. Only films with signals within the linear range of the film and densitometer were used in our determinations. To assess the validity of densitométrie analysis, several hybridized blots were also quantified by reading punched-out dots in a liquid scintillation counter. The results of densitometry and scintillation counting were identical (data not shown).

Protein concentrations were determined using the hot Lowry method (Schacterle and Pollack, 1973). Concentrations of Gand F-actin in guanidine hydrochloride extracts of whole cells were determined by the DNase-inhibition assay (Blikstad et al. 1978) as previously described (Stidwill and Burgess, 1986) using a Zeiss PM6 spectrophotometer. The assay was calibrated with actin purified from chicken breast muscle (Spudich and Watt, 1971).

To understand the development of the brush border, we studied the ultrastructure of the enterocytes in the crypt base, where the cells differentiate, and in the upper crypt and villus base where the brush border was more completely formed. Although the cells develop progressively as they migrate up the crypt, we observed no absolute synchrony in brush border formation; however, adjacent cells were in similar stages of maturation. The cells in the lower, mitotic zone of the crypt were short, cuboidal, with centrally located nuclei. The apical cytoplasm was dome-shaped and extended into the lumen (Fig. 1A-D). The most-primitive-appearing luminal cytoskeleton contained loose bundles of microfilaments emanating from the apical plasma membrane which was slightly bulging on the surface (Fig. 1A). These large groups of filaments, which were not well bundled, extended up to 1μm into the cytoplasm. These cells also contained adherens junctions with a circumferential ring of microfilaments (marked with an asterisk in Fig. 1A). These apical loose filaments then appeared to collect into 43–48 nm-wide, well-packed bundles (Fig. 1B). Next a small number of short primitive microvilli (less than 1 μm long) formed which were commonly clustered in the middle luminal surface and ran at angles not normal to the cell surface (Fig. 1C). These microvilli always contained a central bundle of microfilaments that extended approximately 1 μm into the apical cytoplasm as rootlets. Polyribosomes, coated- and membranous vesicles were distributed throughout the apical cytoplasm and approached the luminal membrane. Numerous microvilli contained two or three core filament bundles which remained separate throughout the length of the microvillus (Fig. 1D,E). This separation is most apparent in cross-sections of microvilli which show that individual bundles are tightly packed and remain segregated from the other bundles within the same microvillus (inset Fig. 1D). Commonly the core microfilaments near the forming microvillus tip appeared less tightly bundled than in the lower microvillus and rootlet. These microvilli contained lateral arms that extended from the core filaments to the plasma membrane (arrowheads in Fig. 1D). Although it was often difficult to clearly discern these lateral arms in longitudinal sections, (as is often the situation in unextracted, mature microvilli), their periodicity along the core bundle was similar to that in mature microvilli (33–35nm). The next step in microvillus formation seemed to be the insertion of new membrane between individual core filament bundles resulting in microvilli containing a single core filament bundle (Fig. 1F,G). Arrows in Fig. 1D indicate two membrane-fusion profiles that appear as examples of membrane insertion between microfilament cores. Higher up the crypt the microvilli were thinner, more numerous and ran more perpendicular to the cell surface (Fig. 1F). These microvilli contained core actin filaments that were more ordered than those in the lower crypt and the terminal web was more highly cross-linked. Subsequently, the microvilli elongated and increased in number so that there was little free apical membrane between the microvilli (Fig. 1G). These longer microvilli extended rootlets that were still longer than the microvilli and significantly longer than the 0.5 μm-long rootlets of mature microvilli. To aid in comparison of these rootlets with those of the mature brush border, the scale bar in Fig. 1 was included as 0.5μm. It was at this maturation stage that the terminal web stratified and excluded polyribosomes and other organelles from the apical cell cytoplasm (Fig. 1G).

To determine which brush border cytoskeletal proteins were present in crypt cells, we explored the distribution of major brush border cytoskeletal proteins with immunofluorescence microscopy on frozen sections. Because the crypts are generally cut in cross-section, they appear as rings with the apical cytoplasm towards the center. We found that the terminal web proteins tropomyosin, spectrin and myosin were concentrated in the apical cytoplasm of crypt cells (Fig. 2A,C,E). Tropomyosin was also seen in lesser concentration at the basolateral membrane (Fig. 2A). Spectrin appeared to be excluded from the forming brush border at the apical cell-cell junctions (Fig. 2C). The protein vinculin was only observed at the adherens junctions where the circumferential bundle of actin filaments approach the plasma membrane (data not shown). In the enterocytes on the villus, the distribution of tropomyosin, spectrin and myosin was similar to their distributions in the crypt cells (Fig. 2B,D,F).

The microvillar proteins actin and villin were concentrated in the crypt cell apex (Fig. 3A,C). Villin was also diffusely distributed throughout the apical cytoplasm. Whereas actin was also found in cells in the lamina propria, villin was restricted to the epithelium. Actin and villin were also concentrated near the apical membrane in enterocytes on the villus (Fig. 3B,D). In contrast to the other major cytoskeletal proteins, the lateral arm protein myosin I was diffusely distributed apical to the nucleus and not concentrated at the apical plasma membrane (Fig. 3E). Although we observed microvillar core filament lateral arms in the electron microscope (Fig. 1D), the majority of myosin I was not concentrated in the crypt brush border. By the time the cells have migrated to the villus, myosin I was concentrated in microvilli (Fig. 3F). Myosin I antisera also recognized unidentified structures filling the middle portion of occasional crypt and villar cells, which appeared to be in the Golgi zone of goblet cells. Attempts to block this binding with higher concentrations of non-specific proteins were not successful.

We wished to determine if the formation and assembly of the brush border cytoskeleton correlated with changes in the levels of mRNAs that encode the constituent cytoskeletal proteins. Using enterocytes isolated from crypts, villus middle (mid) or villus tip (tip), we isolated total RNA and performed Northern and dot blot analysis with radiolabeled cDNA clones. Autoradiographs were quantified, using a scanning densitometer and the percentage of each message in the three cell fractions determined. We used total RNA for these measurements since it has been shown that the RNA/DNA ratio and percent of RNA as poly(A)⁺ do not change significantly from crypt cells to fully differentiated tip cells (Morrison and Porteous, 1980; our unpublished results). To test cDNA specificity, total tip cell RNA was hybridized on Northern blots (Fig. 4A, 4B lane T). All probes hybridized to mRNAs of appropriate molecular mass; the sizes of the mRNAs are listed in the legend of Fig. 4. Fig. 4B is an example of a Northern blot used to quantify actin mRNA levels in crypt, mid and tip enterocytes. On blots hybridized with the other cDNAs, we observed no mRNA degradation during cell isolation, and although not shown, the 18S and 28S rRNA were also intact in all samples. Because the cDNAs hybridized only to their respective messages (Fig. 4), dot blots were also used to quantify mRNA levels. Using quantitative densitometry of dot and Northern blots (Fig. 5), we found a 3-fold increase in villin mRNA (P=0.02 by Students t-test) from crypt to tip; a 2-fold increase in calmodulin mRNA (P=0.03); and a 2-fold increase in β-tropomyosin mRNA (P=0.003). However, α-spectrin and β-actin mRNA levels did not change significantly, while myosin mRNA levels decreased 3-fold, but only with a low degree of significance (P=0.06).

To determine if the observed changes in mRNA levels correlated with changes in the steady-state levels of the respective proteins, we measured the levels of the cytoskeletal proteins in the cell fractions. ELISAs were used to measure total cell protein levels from guanidine-extracted crypt, mid and tip enterocytes. Serial dilutions of cell extracts and antigen versus antisera were used to find the optimal concentrations of each that gave a linear signal increase with increasing concentrations of cell proteins. We found (Fig. 6) no statistically significant changes in the steady-state levels of actin, myosin, spectrin, tropomyosin, α-actinin, or myosin I when comparing crypt, mid or tip cells. The level of villin was a little over 2-fold higher in mid cells than crypt cells (P=0.06); however, the total increase along the crypt-tip axis was not statistically significant.

In addition to transcriptional and translational regulation of cytoskeletal protein expression, brush border formation may be regulated by the state of assembly of actin, a principal structural element of the brush border. To discover if increases in polymerized actin correlated with the assembly of the brush border, we measured G- and F-actin levels using the DNase-inhibition assay. Although the microvilli are much longer in the villus tip than in the crypt, we detected no significant increase in relative levels of total cellular polymerized actin in crypt, mid and tip cells. The mean percentage of actin that was polymerized in the crypt, mid and tip cells was 69, 68, and 73, respectively (n=7). The level in the tip is comparable to those we have previously measured in differentiated adult enterocytes (Stidwill and Burgess, 1986).

In this paper, we report our studies on intestinal brush border development in the adult chicken. We find that only a small subpopulation of cells in the basal crypt is structurally undifferentiated. These cells likely correspond to the crypt stem cells, which give rise to absorptive columnar, goblet and enteroendocrine cells (Cheng and LeBlond, 1974; Potten and Morris, 1988; Gordon, 1989). As in embryonic development, the adult undifferentiated cells initially contain a few, short microvilli, which gradually increase in number and length. The terminal web, except for the zonula adherens circumferential ring, which is already present in immature cells, forms and stratifies near the end of maturation. Until the terminal web forms, polyribosomes and membranous vesicles are found in the cytoplasm adjacent to the apical plasma membrane. Along the crypt-tip axis, we detected 2- to 3-fold increases in the steady-state level of villin, β-tropomyosin and calmodulin mRNAs; no change in the levels of spectrin and actin mRNAs; and a less statistically significant 3-fold decrease in myosin message. This constant level of actin mRNA expression is consistent with in situ hybridization studies in the mouse intestine, which report a uniform density of actin mRNA along the crypt-tip axis (Cheng and Bjerknes, 1989). By contrast to the changes in mRNA levels, the corresponding levels of these cytoskeletal proteins did not change. Although the cytoskeletal protein levels remained constant, the levels of luminal membrane enzymes such as oligosaccharidases, peptidases and alkaline phosphatase (Quaroni, 1984; Weiser et al. 1986) and the level of phosphotyrosine-containing proteins (Burgess et al. 1989) change dramatically as enterocytes migrate to the upper crypt and onto the villus. These changes, among others, have been noted in isolated villus cell fractions and confirm the validity of the cell isolation paradigm for quantifying changes in cytoskeletal protein expression during enterocyte differentiation.

The high levels of major cytoskeletal proteins in structurally immature crypt cells may constitute a sufficient soluble pool which is then reorganized to form the brush border. This supposition is supported by studies on changes in the distribution of myosin I, villin and fimbrin in embryonic chicken intestinal epithelia (Shibayama et al. 1987). They found a large quantity of these proteins diffusely distributed in the apical cytoplasm prior to brush border assembly. Other studies have shown large amounts of caldesmon (a Ca²⁺-regulated actin-binding protein in the terminal web), myosin I and spectrin in enterocytes prior to brush border assembly during embryogenesis in rat intestine (Rochette-Egly and Haffen, 1987); for spectrin in adult human colon crypts (Younes et al. 1989); and for villin in cultured human colonic adenocarcinoma HT-29 cell lines (Dudouet et al. 1987). In the differentiated enterocyte, continual synthesis of cytoskeletal proteins and high mRNA levels are likely required to maintain the strictly regulated microvillar length and terminal web organization where there is a rapid turnover of brush border cytoskeletal proteins (Cowell and Danielsen, 1984; Stidwill et al. 1984); whereas correspondingly high synthetic levels may be necessary in undifferentiated cells for initial brush border assembly.

Our results may also comment on the mechanisms of microvillus assembly and suggest both similarities and differences between initial formation during embryogenesis and their formation in crypt cells in the adult. These structural analyses show that crypt stem cells, like embryonic cells, have domed apical surfaces with sparse microvilli situated at angles not normal to the surface and lack a well-formed terminal web (Overton and Shoup, 1964; Chambers and Grey, 1979; Takemura et al. 1988). Another similarity is that villin, actin, tropomyosin and myosin concentrate at the apical surface prior to or coincident with microvillus assembly, but myosin I remains diffuse until brush borders are well formed (Rochette-Egly and Haffen, 1987; Shibayama et al. 1987; Maunoury et al. 1988; Ezzell et al. 1989). The most obvious difference between embryonic and adult microvillus formation is that crypt cells initially form short microvilli with very long rootlets. Our findings are consistent with the model for microvillus formation first elaborated by Tilney and Cardell (1970), which proposes that core actin filaments nucleate on the membrane. We found cells not yet expressing microvilli that contained many actin filaments streaming from the apical membrane into the apical cytoplasm (Fig. 1A). Often these filaments were collected into bundles with the same diameter as forming microvillar cores. These bundles perhaps nucleate new actin filaments, which become microvilli cores. Although the early microvilli contain discrete actin filament bundles, it is not until later when the terminal web begins to assemble that the core actin filaments are packed into ordered arrays (cf. Fig. 1E and F). Consistent with work on embryos (Shibayama et al. 1987; Ezzell et al. 1989), this increased order may result from the new expression of the bundling protein fimbrin in the microvillar core. Studies have suggested that actin bundles containing both villin and fimbrin more closely resemble the microvillar core than filaments bundled by villin alone (Matsudaira et al. 1983; Glenney et al. 1981). Because the rootlets are at least twice as long as they will be when mature, and the early microvilli contain myosin 1 lateral crossbridges (Fig. 1D), we propose that as myosin I binds to the actin bundle and plasma membrane, it zips the membrane around the microvillus core. Since myosin I can bind to membranes (Adams and Pollard, 1989), it may be transported to the luminal surface by vesicles coated with myosin I, which fuse with the membrane as they link the microfilaments to the membrane. In effect, such vesicle fusion would elongate the microvillus. Because this ‘zipping-up’ appears to occur concomitant with formation of the terminal web filaments, together they may serve to flatten the cell surface, anchor the microvillus rootlets and orient the microvilli perpendicular to the surface.

Previous studies concerning cytoskeletal protein expression during enterocyte differentiation have been focused largely on villin, in part because it is a useful marker for microvilli in intestinal cells. Robine et al. (1985) have explored the distribution of villin in the rat intestine. In agreement with our results, in the light microscope they found villin concentrated in the apical regions of crypt and tip cells. However, by Western blot analysis they found 10-fold more villin protein in tip than in crypt cells; corresponding mRNA levels were not reported. By comparison, we found a 3-fold mRNA increase and at most a 2-fold protein increase along the crypt-tip axis using sensitive ELISA methods. The 2-fold villin increase we measured in the chicken intestine, however, is consistent with a similar increase noted in the villus of the mouse intestine (Boller et al. 1990).

The human colon adenocarcinoma cell line HT-29 and its subclone HT29-18, both of which contain cells capable of becoming enterocytes and goblet cells, have been useful in exploring enterocyte differentiation in vitro (Dudouet et al. 1987; Huet et al. 1987). As the differentiated HT29-18 cells are selected, the level of villin mRNA and protein increase 10-fold (Dudouet et al. 1987). A subclone, HT29-18-Q, which maintains some of its differentiated phenotype including a rudimentary brush border and higher basal villin levels, can be stimulated to fully differentiate and form a mature brush border (Huet et al. 1987). This full differentiation in HT29-18-Ci cells is accompanied by only a 20% increase in villin levels. The differentiation of the HT29-18-C] clone may therefore be similar to the differentiation of developing crypt cells.

Since our results indicate that cytoskeletal proteins and F-actin levels do not change during differentiation, we suggest that the expression of a minor component may regulate microvillus formation. This component may be integral to the bundling and stabilization of actin filaments in the microvillus core or it may be a membrane-associated protein that nucleates microvillar assembly. Low levels of this component would generate few microvilli, whereupon increased synthesis would permit the utilization of the abundant pool of cytoskeletal proteins to allow immediate, synchronous, and abundant microvillar formation. A similar model has been proposed in sea urchin embryos in which the quantal and limited synthesis of tektins regulates the assembly of cilia (Stephens, 1989).

Our study suggests that the changes in cytoskeletal gene expression in crypt stem cells developing into immature absorptive cells may occur rapidly and, consequently, in only a few cells at any point in time. Therefore, our assaying of all crypt cells together may not be sensitive enough to detect dramatic changes in a subpopulation of structurally undifferentiated crypt cells. The exploration of these rapid changes will require in situ hybridization to localize these rare cells and to quantify relative changes in mRNA levels as brush border formation is initiated. This approach has been used to study actin and villin mRNA distribution along the crypt-tip axis in the mouse small intestine; unfortunately no note was made of high levels of these mRNAs in specific crypt cells (Boller el al. 1990; Cheng and Bjerknes, 1989). While we have tried to infer cytoskeletal gene activity by measuring the steady-state levels of specific messages, our analysis reflects only message accumulation. Nuclear runoff analyses must be done to accurately measure transcription rates of the cytoskeletal genes in undifferentiated versus differentiated cells.

This study was supported by grants from NIH (DK 31643) and the Florida ACS to D.R.B. K.R.F. was supported in part by a University of Miami Institutional Fellowship. We are grateful to Drs C. Birkenmeier, D. Helfman, P. Matsudaira, A. Means, D. Paterson, and J. Robbins for kindly providing their cDNA clones. We thank Dr K. Broschat for providing the brush border villin and tropomyosin and their antisera; Dr J. Collins for myosin I and myosin I antisera; and Dr K. Burridge for the fodrin antisera. We would especially like to thank Ms Jessie Singer for her capable work with the RNA blots and helpful comments.