Development 109,449-459(1990)Printed in Great Britain © The Company of Biologists Limited J990

449

Cytoskeletal protein and mRNA accumulation during brush border

formation in adult chicken enterocytes

KARL R. FATHt, STEVEN D. OBENAUFf, and DAVID R. BURGESS*

Department of Cell Biology and Anatomy, University of Miami School of Medicine, 1600 NW 10th Avenue, Miami, Florida 33101, USA

•Author for all communications.t These authors contributed equally to the contents of this paper and should be considered joint first authors

Summary

We have explored the development of the brush borderin adult chicken enterocytes by analyzing the cytoskel-etal protein and mRNA levels as enterocytes arise fromcrypt stem cells and differentiate as they move towardthe villus. At the base of the crypt, a small population ofcells contain a rudimentary terminal web and a few shortmicrovilli with long rootlets. These microvilli appear toarise from bundles of actin filaments which nucleate onthe plasma membrane. The microvilli apparentlyelongate via the addition of membrane supplied byvesicles that fuse with the microvillus and extend themembrane around the actin core. Actin, villin, myosin,tropomyosin and spectrin, but not myosin I (previouslycalled 110 kD; see Mooseker and Coleman, J. Cell Biol.108, 2395-2400, 1989) are already concentrated in theluminal cytoplasm of crypt cells, as seen by immunoflu-orescence. Using quantitative densitometry of cDNA-hybridized RNA blots from cells isolated from crypts,villus middle (mid), or villus tip (tip), we found a 2- to3-fold increase in villin, calmodulin and tropomyosin

steady-state mRNA levels; an increase parallel to mor-phological brush border development. Actin, spectrinand myosin mRNA levels did not change significantly.ELISA of total crypt, mid and tip cell lysates show thatthere are no significant changes in actin, myosin, spec-trin, tropomyosin, myosin I, villin or a-actinin proteinlevels as the brush border develops. The G-/F-actinratio also did not change with brush border assembly.We conclude that, although the brush border is not fullyassembled in immature enterocytes, the major cytoskel-etal proteins are present in their full concentration andalready localized within the apical cytoplasm. Thereforebrush border formation may involve reorganization of apool of existing cytoskeletal proteins mediated by theexpression or regulation of an unidentified key pro-tein(s).

Key words: crypt, differentiation, microvilli, rootlets,terminal web, villus.

Introduction

Two model systems that have been useful for under-standing the organization and assembly of actin cyto-skeletons are skeletal muscle, with its striking sarco-meric pattern, and the red blood cell, with its relativelysimple cortical cytoskeleton. We have learned muchabout cytoskeletal protein gene expression in thesecells, and how the proteins are arranged to generatecontractile forces and how they stabilize the membranecytoskeleton. The analysis of the developmental as-sembly of the red blood cell and muscle cytoskeletonhas led to an understanding of the regulation ofcytoskeletal formation at the gene transcriptional andpost-translational levels. Studies with myoblasts duringskeletal muscle differentiation show that the levels ofmRNAs encoding sarcomeric-specific proteins (eg. tro-ponin, myosin heavy chains) increase many-fold andthe levels of mRNAs encoding the non-muscle cytoskel-

etal proteins decrease as myoblasts fuse (Shani et al.1982; Wade and Kedes, 1989). In the erythrocyte,cortical cytoskeleton differentiation is controlled by asimilar co-ordinate activation of specific genes; how-ever, the assembly of the spectrin heterodimer isregulated by post-translational modifications and differ-ential levels of a'- and /3-spectrin synthesis (Blikstad etal. 1983; Moon and McMahon, 1987).

The simplicity and stereotypic arrangement of thebrush border cytoskeleton have made the enterocyteanother excellent model for analyzing the organizationand development of an actin-based cytoskeleton (seereviews by Mooseker, 1985; Burgess, 1987). The ma-ture brush border is structurally divided into two parts;the microvilli and the terminal web. A microvilluscontains a core bundle of actin filaments, which extendsinto the terminal web as the rootlet. The actin-bindingproteins villin, myosin I and fimbrin are associated withthe core filaments, and, in addition to tropomyosin, are

450 K. R. Fath, S. D. Obenauf and D. R. Burgess

present in the rootlets. The terminal web contains ameshwork of actin filaments cross-linked with myosin,nonerythroid spectrins (fodrin and TW 260/240), a-actinin and tropomyosin. Although much is knownabout the structure of the adult brush border, little isknown about its assembly. Research in brush bordercytoskeleton formation has focused on embryonicchickens, rats and mice (Chambers and Grey, 1979;Rochette-Egly and Haffen, 1987; Shibayama etal. 1987;Maunoury et al. 1988; Stidwill and Burgess, 1986;Takemura et al. 1988; Ezzell et al. 1989), and mostrecently on cell lines (Dudouet et al. 1987). In theembryo, mitotic stem cells are found over the entirelength of the villus (Overton and Shoup, 1964) whereasin the adult, mitotic cells are restricted to lower regionsof the crypt. Those embryonic cells destined to becomeabsorptive cells initially form a few, short, wide micro-villi with no terminal web. Next the rootlets increase inlength, and the number of microvilli and the number ofcore filaments increase. Finally, the microvilli grow totheir mature length and the terminal web completesformation 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 theadult, for in comparison to the embryo, it is morefeasible to isolate sufficient cells at differing degrees ofdevelopment for biochemical and structural analysis.The adult intestinal epithelium is a continually differen-tiating tissue, constantly renewed by division of the oneto four undifferentiated stem cells that reside in crypts(Potten and Morris, 1988; Gordon, 1989). Cells ceasedivision and differentiate as they migrate out of thecrypts onto the villus. After 2-3 days they are shed fromthe villus tip into the intestinal lumen (Cheng andLeBlond, 1974). Although little is known about themorphological changes during the differentiation of theadult enterocyte, several previous works, which werenot focused on the cytoskeleton, have suggested thatthey are similar to those during embryonic development(Brown, 1962; Trier and Rubin, 1965).

In this paper, we report ultrastructural and immuno-cytochemical studies of adult chicken brush borderdevelopment. To correlate structural changes with theexpression of cytoskeletal proteins, we measured thesteady-state cytoskeletal protein and mRNA levels inisolated crypt and villus cells. We found that a primitivebrush border is already formed in the basal crypt, earlyin the cell's life history, but while cytoskeletal proteinmRNAs increase somewhat, the corresponding levelsof the proteins do not change significantly duringdevelopment of the mature brush border.

Materials and methods

Electron microscopyThe proximal loops of adult White Leghorn chicken duodenawere rinsed in saline (0.15 M NaCl) and fixed in 3% glutaral-dehyde, 0.2 % tannic acid, 0.1 M NaPO4 (pH 7.0) for 2 h in thedark at room temperature (RT). After a rinse in 10% sucrosein 0.1M NaPO4, the tissue was postfixed for 2h with 0.5%

OsO4, 0.8% KjFe(CN)6 in 0.1M NaPO4 on ice in the dark.The tissue was rinsed for 15 min in water, en bloc stained withaqueous uranyl acetate, dehydrated through a graded seriesof ethanols into propylene oxide and embedded in Epon/Araldite. Thin sections were stained with uranyl acetate andlead citrate, and observed with a JEOL 100CX electronmicroscope operated at 60 kV.

Proteins and antibodiesVillin was purified from calcium-extracted brush bordersaccording to the methods of Bretscher and Weber (1978).Rabbit antisera directed against villin was prepared by intra-cutaneous injection of 200 /.ig of protein in complete Freund'sadjuvant (Cappel Laboratories) followed 14 days later by200 /.ig of protein in incomplete Freund's adjuvant. Rabbitswere bled 14 days after the final antigen injection. The villinantisera were shown to be monospecific on Western blots oftotal cell and brush border proteins (data not shown).Antiserum to chicken actin was obtained from ICN Immuno-Biologicals and antisera to erythrocyte spectrin and chickengizzard o^actinin were obtained from Sigma. Antisera raisedagainst bovine brain fodrin used for immunofluorescence wasa generous gift from Dr K. Burridge. Antibodies to brushborder myosin and tropomyosin have been previously charac-terized (Broschat et al. 1983; Broschat and Burgess, 1986).Pure chicken brush border myosin I (originally referred to asthe 110 kD protein) and antisera were a generous gift from DrJ. Collins.

ImmunofluorescenceThe proximal duodenal loop from an adult chicken was cutopen, rinsed in saline, and fixed on ice for lh with 3.7%formaldehyde in Solution I (75 mM KC1, 1 ITIM EGTA, 0.1 ITIMMgCl2, 10 mM Imidazole, pH6.9). The fixed tissue wascryoprotected with 1M sucrose, lmM EGTA, 0.1M Tris-Cl,pH7.4 and frozen in OCT (Tissue Tek, Miles Lab.) byimmersion in liquid nitrogen. Frozen sections 5-7 jim(Reichert-Jung) thick were collected on uncoated glass slidesand stored at -80°C. Slides were thawed at RT in Tris-buffered saline (TBS: 50mM 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 andblocked for 30 min with 3 % BSA (Sigma, Fraction V) in TBS.The tissue was stained with the primary antibody for 1 h at37°C, rinsed in TBS and stained with a 1/50 dilution ofFrrC-goat anti-rabbit IgG (Cappel) for 30 min at 37°C. Allantibodies were diluted in TBS containing 0.3% BSA. Toobserve the distribution of polymerized actin, sections werestained with 6.6 /JM rhodamine-phalloidin (MolecularProbes). The slides were observed with a Leitz Laborlux Smicroscope equipped with epifluorescence optics. Micro-graphs were photographed at El 1600 on T-Max 400 film anddeveloped with T-Max developer (Kodak). Nonimmune con-trols showed only very dim, diffuse staining and so were notincluded as figures.

Isolation of intestinal epithelial cells.Enterocytes from the villus tip (tip), villus middle (mid) andcrypt 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 inhyperosmotic saline and collecting fractions at selected timepoints. In these conditions, cells slough off at the basementmembrane and are separated from the lamina propria andmuscularis mucosa. The identity and purity of the cellfractions was determined with electron and phase-contrast

Brush border development 451

microscopy, and by determination of alkaline phosphataseactivity (Burgess et al. 1989).

Quantification of cytoskeletal protein levels in isolatedcell extractsEnzyme-linked immunosorbent assays (ELISA) were used toexamine changes in protein levels in enterocytes isolated fromthe crypt, mid and tip. Briefly, isolated cells were homogen-ized in Solution I containing 1 M guanidine hydrochloride,cleared of particulatesby centrifugation at lOOOOgfor lOmin,and 0.5f/g adsorbed to 96-well plates (Costar). The plateswere blocked with 3 % gelatin in TBS-0.05 % Tween-20 andincubated with antisera. Titration curves with varying anti-body or antigen concentrations were used to determineoptimal cell extract and antibody dilutions. All determi-nations were performed in the linear range of each antibody/antigen curve. After washing, the bound antibodies weredetected using Protein A conjugated with horseradish peroxi-dase (BioRad, Richmond, CA.) and the color developed with2,2 azino-di-[3-ethylbenzthiazoline-6-sulfonic acid]. Plateswere quantified using a Bio-Tek autoplate reader at 405 nmand the levels standardized with purified proteins. The valuesreported are the means from cell fractions isolated from threechickens.

DNA probes and nick translationThe following chicken cDNA clones were used as probes forRNA hybridization experiments: /S-smooth muscle tropomyo-sin in pUC8 (Helfman et al. 1984); /3-actin in pUC18 (Patersonet al. 1984); smooth muscle myosin in pBR325 (Molina et al.1987); calmodulin in pBR322 (Putkey et al. 1983); cr-spectrinin pUCB (Birkenmeier et al. 1985); villin in pBSM13+(Bazari et al. 1988). All probes were labelled with P by nicktranslation (Maniatis et al. 1982).

RNA preparation and blottingTotal RNA was prepared from crypt, mid and tip cellfractions using the guanidinium isothiocyanate method ofChirgwin et al. (1979). The duodena from three chickens werepooled for each of three cell isolation preparations. For dotblots, 3 ng of total RNA were denatured at 65°C for 15min in7.5xSSC (lxSSC is 0.15M NaCl, 0.015M sodium citrate)containing 4.16 M formaldehyde and dotted onto nitrocellu-lose using a microfiltration apparatus (Bio-Rad). For North-ern blots, 5-10 ^g of total RNA per lane were electrophor-esed on 1 % formaldehyde gels and transferred tonitrocellulose or Nytran (Schleicher and Schuell) by capillaryblotting using 20xSSC as transfer buffer (Maniatis et al.1982). The filters were hybridized at 42°C for at least 20 h in50 % formamide and 0.825 M Na+ by the methods of Maniatiset al. (1982). The final filter wash was at 65°C in lxSSC, 0.1 %SDS. The filters were exposed to preflashed Kodak XAR-5film (Laskey and Mills, 1977) with an intensifying screen at-70°C.

Relative mRNA levels were determined by scanning auto-radiograms with an E-C scanning densitometer coupled to aWaters digital automatic integrator. The linearity of responseof the photographic emulsion was verified (r=0.994) using adot blot containing a 50-fold concentration range of tip RNAhybridized with the /3-actin probe. Only films with signalswithin the linear range of the film and densitometer were usedin our determinations. To assess the validity of densitometricanalysis, several hybridized blots were also quantified byreading punched-out dots in a liquid scintillation counter. Theresults of densitometry and scintillation counting were ident-ical (data not shown).

MiscellaneousProtein concentrations were determined using the hot Lowrymethod (Schacterle and Pollack, 1973). Concentrations of G-and F-actin in guanidine hydrochloride extracts of whole cellswere determined by the DNase-inhibition assay (Blikstad etal. 1978) as previously described (Stidwill and Burgess, 1986)using a Zeiss PM6 spectrophotometer. The assay was cali-brated with actin purified from chicken breast muscle(Spudich and Watt, 1971).

Results

Ultrastructure of the developing brush border inintestinal crypt cellsTo understand the development of the brush border, westudied the ultrastructure of the enterocytes in the cryptbase, where the cells differentiate, and in the uppercrypt and villus base where the brush border was morecompletely formed. Although the cells develop pro-gressively as they migrate up the crypt, we observed noabsolute synchrony in brush border formation; how-ever, adjacent cells were in similar stages of maturation.The cells in the lower, mitotic zone of the crypt wereshort, cuboidal, with centrally located nuclei. Theapical cytoplasm was dome-shaped and extended intothe lumen (Fig. 1A-D). The most-primitive-appearingluminal cytoskeleton contained loose bundles of micro-filaments emanating from the apical plasma membranewhich was slightly bulging on the surface (Fig. 1A).These large groups of filaments, which were not wellbundled, extended up to 1/im into the cytoplasm.These cells also contained adherens junctions with acircumferential ring of microfilaments (marked with anasterisk in Fig. 1A). These apical loose filaments thenappeared to collect into 43-48 nm-wide, well-packedbundles (Fig. IB). Next a small number of short primi-tive microvilli (less than 1 pm long) formed which werecommonly clustered in the middle luminal surface andran at angles not normal to the cell surface (Fig. 1C).These microvilli always contained a central bundle ofmicrofilaments that extended approximately 1 /m\ intothe apical cytoplasm as rootlets. Polyribosomes,coated- and membranous vesicles were distributedthroughout the apical cytoplasm and approached theluminal membrane. Numerous microvilli contained twoor three core filament bundles which remained separatethroughout the length of the microvillus (Fig. 1D,E).This separation is most apparent in cross-sections ofmicrovilli which show that individual bundles are tightlypacked and remain segregated from the other bundleswithin the same microvillus (inset Fig. ID). Commonlythe core microfilaments near the forming microvillus tipappeared less tightly bundled than in the lower micro-villus and rootlet. These microvilli contained lateralarms that extended from the core filaments to theplasma membrane (arrowheads in Fig. ID). Althoughit was often difficult to clearly discern these lateral armsin longitudinal sections, (as is often the situation inunextracted, mature microvilli), their periodicity alongthe core bundle was similar to that in mature microvilli(33-35nm). The next step in microvillus formation

452 K. R. Fath, S. D. Obenauf and D. R. Burgess

seemed to be the insertion of new membrane betweenindividual core filament bundles resulting in microvillicontaining a single core filament bundle (Fig. 1F,G).Arrows in Fig. ID indicate two membrane-fusion pro-files that appear as examples of membrane insertionbetween microfilament cores. Higher up the crypt themicrovilli were thinner, more numerous and ran moreperpendicular to the cell surface (Fig. IF). Thesemicrovilli contained core actin filaments that were moreordered than those in the lower crypt and the terminalweb was more highly cross-linked. Subsequently, the

microvilli elongated and increased in number so thatthere was little free apical membrane between themicrovilli (Fig. 1G). These longer microvilli extendedrootlets that were still longer than the microvilli andsignificantly longer than the 0.5^an-long rootlets ofmature microvilli. To aid in comparison of these root-lets with those of the mature brush border, the scale barin Fig. 1 was included as 0.5jum. It was at this matu-ration stage that the terminal web stratified andexcluded polyribosomes and other organelles from theapical cell cytoplasm (Fig. 1G).

Brush border development 453

Fig. 1. Transmission electron micrographs of thedeveloping brush border in the intestinal crypt. The apicalcytoplasm of enterocytes at different stages of brush borderassembly in the lower (A-F) and upper regions of the (G)crypt. (A) Actin microfilaments extend from the apicalplasma membrane into the cytoplasm from dome-shapedregions of the luminal surface. These cells have arudimentary terminal web with a zonula adherenscircumferential ring (asterisk). (B) This cell contains apicalmicrofilaments, which are bundled amongst a network offilaments comprising the early terminal web. (C) Themicrofilament bundles in this cell are longer and extend intoshort microvilli which run at acute angles relative to the cellsurface. (D) A cell containing short, fat microvilli some ofwhich contain two or more core microfilament bundles.These bundles appear to remain separated throughout thelength of the microvillus. This segregation is evident incross-sections of several microvilli (inset). The arrowheadsindicate examples of the lateral (myosin I) cross-bridgesbetween the core filaments and the microvillus membrane.The arrows mark two membrane-fusion profiles referred toin the results. (E) Another cell at approximately the samedevelopmental stage as the cell in D, showing that near themicrovillus tip the core filaments are more loosely bundled(arrow). (F) The microvilli of this more mature cell containonly one core microfilament bundle and the filaments in thisbundle are more ordered than those in lower regions of thecrypt. (G) These microvilli, from a cell in the upper regionof the crypt, are more numerous, taller and straighter thanthose in lower regions. The terminal web is now highlycross-linked and most cellular organelles are excluded fromthe apical cytoplasm. Bar, 0.5 f.tm.

Localization of cytoskeletal proteins byimmunofluorescenceTo determine which brush border cytoskeletal proteinswere present in crypt cells, we explored the distributionof major brush border cytoskeletal proteins with immu-nofluorescence microscopy on frozen sections. Becausethe crypts are generally cut in cross-section, they appearas rings with the apical cytoplasm towards the center.We found that the terminal web proteins tropomyosin,spectrin and myosin were concentrated in the apicalcytoplasm of crypt cells (Fig. 2A,C,E). Tropomyosinwas also seen in lesser concentration at the basolateralmembrane (Fig. 2A). Spectrin appeared to be excludedfrom the forming brush border at the apical cell-celljunctions (Fig. 2C). The protein vinculin was onlyobserved at the adherens junctions where the circum-ferential bundle of actin filaments approach the plasmamembrane (data not shown). In the enterocytes on thevillus, the distribution of tropomyosin, spectrin andmyosin was similar to their distributions in the cryptcells (Fig. 2B,D,F).

The microvillar proteins actin and villin were concen-trated in the crypt cell apex (Fig. 3A,C). Villin was alsodiffusely distributed throughout the apical cytoplasm.Whereas actin was also found in cells in the laminapropria, villin was restricted to the epithelium. Actinand villin were also concentrated near the apical mem-brane in enterocytes on the villus (Fig. 3B,D). Incontrast to the other major cytoskeletal proteins, thelateral arm protein myosin I was diffusely distributed

apical to the nucleus and not concentrated at the apicalplasma membrane (Fig. 3E). Although we observedmicrovillar core filament lateral arms in the electronmicroscope (Fig. ID), the majority of myosin I was notconcentrated in the crypt brush border. By the time thecells have migrated to the villus, myosin I was concen-trated in microvilli (Fig. 3F). Myosin I antisera alsorecognized unidentified structures filling the middleportion of occasional crypt and villar cells, whichappeared to be in the Golgi zone of goblet cells.Attempts to block this binding with higher concen-trations of non-specific proteins were not successful.

Cytoskeletal mRNA levels during brush bordermaturationWe wished to determine if the formation and assemblyof the brush border cytoskeleton correlated withchanges in the levels of mRNAs that encode theconstituent cytoskeletal proteins. Using enterocytesisolated from crypts, villus middle (mid) or villus tip(tip), we isolated total RNA and performed Northernand dot blot analysis with radiolabeled cDNA clones.Autoradiographs were quantified, using a scanningdensitometer and the percentage of each message in thethree cell fractions determined. We used total RNA forthese measurements since it has been shown that theRNA/DNA ratio and percent of RNA as poly(A)+ donot change significantly from crypt cells to fully differ-entiated tip cells (Morrison and Porteous, 1980; ourunpublished results). To test cDNA specificity, total tipcell RNA was hybridized on Northern blots (Fig. 4A,4B lane T). All probes hybridized to mRNAs ofappropriate molecular mass; the sizes of the mRNAsare listed in the legend of Fig. 4. Fig. 4B is an exampleof a Northern blot used to quantify actin mRNA levelsin crypt, mid and tip enterocytes. On blots hybridizedwith the other cDNAs, we observed no mRNA degra-dation 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 respectivemessages (Fig. 4), dot blots were also used to quantifymRNA levels. Using quantitative densitometry of dotand Northern blots (Fig. 5), we found a 3-fold increasein villin mRNA (P=0.02 by Students Mest) from cryptto tip; a 2-fold increase in calmodulin mRNA (P=0.03);and a 2-fold increase in /8-tropomyosin mRNA(P=0.003). However, cv-spectrin and /3-actin mRNAlevels did not change significantly, while myosin mRNAlevels decreased 3-fold, but only with a low degree ofsignificance (P=0.06).

Cytoskeletal protein levels during brush borderdevelopmentTo determine if the observed changes in mRNA levelscorrelated with changes in the steady-state levels of therespective proteins, we measured the levels of thecytoskeletal proteins in the cell fractions. ELISAs wereused to measure total cell protein levels from guani-dine-extracted crypt, mid and tip enterocytes. Serialdilutions of cell extracts and antigen versus antiserawere used to find the optimal concentrations of each

454 K. R. Fath, S. D. Obenauf and D. R. Burgess

Fig. 2. Fluorescent light micrograph of the distribution of the terminal web proteins tropomyosin, spectrin, and myosin.Immunofluorescent localization of tropomyosin (A,B). spectrin (C,D) and myosin (E,F) in frozen sections of crypts (A,C,E)and villi (B,D,F). Note that all three proteins are concentrated at the apical surface of the crypt and villus cells. Spectrin(C,D) is excluded from the cell-cell junctions and so appears as a discontinuous apical ring or bar. Bar, in A,C,E,F; 10 ^m.Bar, in B,D; 25/tm.

that gave a linear signal increase with increasing con-centrations of cell proteins. We found (Fig. 6) nostatistically significant changes in the steady-state levelsof actin, myosin, spectrin, tropomyosin, o--actinin, ormyosin I when comparing crypt, mid or tip cells. Thelevel of villin was a little over 2-fold higher in mid cellsthan crypt cells (P=0.06); however, the total increasealong the crypt-tip axis was not statistically significant.

G- and F-aclin levels during brush border developmentIn addition to transcriptional and translational regu-lation of cytoskeletal protein expression, brush borderformation may be regulated by the state of assembly ofactin, a principal structural element of the brush bor-der. To discover if increases in polymerized actincorrelated with the assembly of the brush border, we

measured G- and F-actin levels using the DNase-inhibition assay. Although the microvilli are muchlonger in the villus tip than in the crypt, we detected nosignificant increase in relative levels of total cellularpolymerized actin in crypt, mid and tip cells. The meanpercentage 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 havepreviously measured in differentiated adult enterocytes(Stidwill and Burgess, 1986).

Discussion

In this paper, we report our studies on intestinal brushborder development in the adult chicken. We find thatonly a small subpopulation of cells in the basal crypt is

Brush border development 455

Fig. 3. Fluorescent light micrograph of the distribution of the microvillar proteins actin, villin, and myosin I. Distribution ofactin (A,B) using rhodamine-phalloidin and distribution of villin (C,D) and myosin I (E,F) using antibodies in frozensections of crypts (A,C,E) and villi (B,D,F). Note that actin and villin are concentrated at the apical plasma membrane incrypts (A,C) while myosin I is diffusely distributed in the apical cytoplasm (E). Actin, villin and myosin I are concentratedin the brush border of villus cells (B,D,F). Bar, in A,C,E; 10urn."Bar, in B,D,F; 25 fan.

structurally undifferentiated. These cells likely corre-spond to the crypt stem cells, which give rise toabsorptive columnar, goblet and enteroendocrine cells(Cheng and LeBlond, 1974; Potten and Morris, 1988;Gordon, 1989). As in embryonic development, theadult undifferentiated cells initially contain a few, shortmicrovilli, which gradually increase in number andlength. The terminal web, except for the zonula adher-ens circumferential ring, which is already present inimmature cells, forms and stratifies near the end ofmaturation. Until the terminal web forms, polyribo-somes and membranous vesicles are found in thecytoplasm adjacent to the apical plasma membrane.Along the crypt-tip axis, we detected 2- to 3-foldincreases in the steady-state level of villin, /3-tropomyo-sin and calmodulin mRNAs; no change in the levels of

spectrin and actin mRNAs; and a less statisticallysignificant 3-fold decrease in myosin message. Thisconstant level of actin mRNA expression is consistentwith in situ hybridization studies in the mouse intestine,which report a uniform density of actin mRNA alongthe crypt-tip axis (Cheng and Bjerknes, 1989). Bycontrast to the changes in mRNA levels, the corre-sponding levels of these cytoskeletal proteins did notchange. Although the cytoskeletal protein levelsremained constant, the levels of luminal membraneenzymes such as oligosaccharidases, peptidases andalkaline phosphatase (Quaroni, 1984; Weiser et al.1986) and the level of phosphotyrosine-containing pro-teins (Burgess et al. 1989) change dramatically asenterocytes migrate to the upper crypt and onto thevillus. These changes, among others, have been noted

456 K. R. Fath, S. D. Obenauf and D. R. Burgess

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A BFig. 4. cDNA probe specificity. (A) A composite of fiveNorthern blots of 10 ng total villus tip RNA hybridized to32P-labelled, nick-translated calmodulin (CaM), smoothmuscle myosin, /3-smooth muscle tropomyosin (TM), a-spectrin, and villin cDNAs. The approximate mass of themRNAs were: CaM, 1.4 kb; myosin, 6.3 kb; TM, 2 kb;spectrin, 7 kb; and villin, 3.3 kb. The final wash was done inlxSSC, 0.1 % SDS at 65°C. Molecular size markers inkilobases are shown on the left. (B) A Northern blot of10 we of tip (T), mid (M) or crypt (C) total RNA hybridizedto P-labelled /3-actin cDNA. The probe hybridized to asingle band of 2.3 kb in all fractions. This blot is an exampleof those used in densitometric scanning and illustrates thatthe mRNA was not degraded during cell isolation.Although the level of actin message is higher in the tip cellson this blot, the mean crypt-tip levels from threeexperiments are not significantly different (see Fig. 5).

in isolated villus cell fractions and confirm the validityof the cell isolation paradigm for quantifying changes incytoskeletal protein expression during enterocyte dif-ferentiation.

The high levels of major cytoskeletal proteins instructurally immature crypt cells may constitute a suf-ficient soluble pool which is then reorganized to formthe brush border. This supposition is supported bystudies on changes in the distribution of myosin I, villinand fimbrin in embryonic chicken intestinal epithelia(Shibayama et al. 1987). They found a large quantity ofthese proteins diffusely distributed in the apical cyto-plasm prior to brush border assembly. Other studieshave shown large amounts of caldesmon (a Ca2+-regulated actin-binding protein in the terminal web),myosin I and spectrin in enterocytes prior to brushborder assembly during embryogenesis in rat intestine(Rochette-Egly and Haffen, 1987); for spectrin in adulthuman colon crypts (Younes et al. 1989); and for villinin cultured human colonic adenocarcinoma HT-29 celllines (Dudouet etal. 1987). In the differentiated entero-cyte, continual synthesis of cytoskeletal proteins andhigh mRNA levels are likely required to maintain thestrictly regulated microvillar length and terminal weborganization where there is a rapid turnover of brush

border cytoskeletal proteins (Cowell and Danielsen,1984; Stidwill et al. 1984); whereas correspondingly highsynthetic levels may be necessary in undifferentiatedcells for initial brush border assembly.

Our results may also comment on the mechanisms ofmicrovillus assembly and suggest both similarities anddifferences between initial formation during embryo-genesis and their formation in crypt cells in the adult.These structural analyses show that crypt stem cells,like embryonic cells, have domed apical surfaces withsparse microvilli situated at angles not normal to thesurface and lack a well-formed terminal web (Overtonand Shoup, 1964; Chambers and Grey, 1979; Takemuraet al. 1988). Another similarity is that villin, actin,tropomyosin and myosin concentrate at the apicalsurface prior to or coincident with microvillus assembly,but myosin I remains diffuse until brush borders arewell formed (Rochette-Egly and Haffen, 1987; Shi-bayama et al. 1987; Maunoury et al. 1988; Ezzell et al.1989). The most obvious difference between embryonicand adult microvillus formation is that crypt cellsinitially form short microvilli with very long rootlets.Our findings are consistent with the model for microvil-lus formation first elaborated by Tilney and Cardell(1970), which proposes that core actin filaments nu-cleate on the membrane. We found cells not yetexpressing microvilli that contained many actin fila-ments streaming from the apical membrane into theapical cytoplasm (Fig. 1A). Often these filaments werecollected into bundles with the same diameter asforming microvillar cores. These bundles perhaps nu-cleate new actin filaments, which become microvillicores. Although the early microvilli contain discreteactin filament bundles, it is not until later when theterminal web begins to assemble that the core actinfilaments are packed into ordered arrays (cf. Fig. IEand F). Consistent with work on embryos (Shibayamaet al. 1987; Ezzell et al. 1989), this increased order mayresult from the new expression of the bundling proteinfimbrin in the microvillar core. Studies have suggestedthat actin bundles containing both villin and fimbrinmore closely resemble the microvillar core than fila-ments bundled by villin alone (Matsudaira et al. 1983;Glenney et al. 1981). Because the rootlets are at leasttwice as long as they will be when mature, and the earlymicrovilli contain myosin I lateral crossbridges(Fig. ID), we propose that as myosin I binds to theactin bundle and plasma membrane, it zips the mem-brane around the microvillus core. Since myosin I canbind to membranes (Adams and Pollard, 1989), it maybe transported to the luminal surface by vesicles coatedwith myosin I, which fuse with the membrane as theylink the microfilaments to the membrane. In effect,such vesicle fusion would elongate the microvillus.Because this 'zipping-up' appears to occur concomitantwith formation of the terminal web filaments, togetherthey may serve to flatten the cell surface, anchor themicrovillus rootlets and orient the microvilli perpen-dicular to the surface.

Previous studies concerning cytoskeletal protein ex-pression during enterocyte differentiation have been

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MyosinSpectrinTropomyosin

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Fig. 5. Cytoskeletal protein mRNA levels along the crypt-tip axis. The steady-state levels of each mRNA in the crypt, midand tip cell fractions was determined by quantitative densitometry of Northern and dot blots. Shown are the mean valuesfrom 3 enterocyte preparations. (A) The levels of mRNAs encoding microvillar proteins. Along the crypt-tip axis the levelof villin mRNAs increased 3-fold (P=0.02); calmodulin mRNA levels increased 2-fold (P=0.03); and the level of actinmessages did not change significantly. (B) The levels of mRNAs encoding terminal web proteins. From crypt to tip the levelof /S-tropomyosin mRNA increased 2-fold (p=0.003) while changes in ospectrin and myosin mRNA levels were notstatistically significant.

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Fig. 6. Cytoskeletal protein levels along the crypt-tip axis. The levels of cytoskeletal proteins were determined using ELISAof guanidine hydrochloride extracts of crypt, mid and tip enterocytes isolated from 3 chickens. (A) Along the crypt-tip axis,we found no significant changes in the levels of the microvillar proteins actin and myosin I. The level of villin increased alittle over 2-fold from crypt to tip (P=0.06), however, the net increase from crypt to tip was not significant. (B) The levels ofthe terminal web proteins myosin, spectrin, tropomyosin and a--actinin did not change significantly along the crypt-tip axis.

focused largely on villin, in part because it is a usefulmarker for microvilli in intestinal cells. Robine et al.(1985) have explored the distribution of villin in the ratintestine. In agreement with our results, in the lightmicroscope they found villin concentrated in the apicalregions of crypt and tip cells. However, by Western blot

analysis they found 10-fold more villin protein in tipthan in crypt cells; corresponding mRNA levels werenot reported. By comparison, we found a 3-fold mRNAincrease and at most a 2-fold protein increase along thecrypt-tip axis using sensitive ELISA methods. The2-fold villin increase we measured in the chicken

458 K. R. Fath, S. D. Obenauf and D. R. Burgess

intestine, however, is consistent with a similar increasenoted in the villus of the mouse intestine (Boiler et al.1990).

The human colon adenocarcinoma cell line HT-29and its subclone HT29-18, both of which contain cellscapable of becoming enterocytes and goblet cells, havebeen useful in exploring enterocyte differentiation invitro (Dudouet et al. 1987; Huet et al. 1987). As thedifferentiated HT29-18 cells are selected, the level ofvillin mRNA and protein increase 10-fold (Dudouet etal. 1987). A subclone, HT29-18-Q, which maintainssome of its differentiated phenotype including a rudi-mentary brush border and higher basal villin levels, canbe stimulated to fully differentiate and form a maturebrush border (Huet etal. 1987). This full differentiationin HT29-18-C! cells is accompanied by only a 20%increase in villin levels. The differentiation of theHT29-18-C! clone may therefore be similar to thedifferentiation of developing crypt cells.

Since our results indicate that cytoskeletal proteinsand F-actin levels do not change during differentiation,we suggest that the expression of a minor componentmay regulate microvillus formation. This componentmay be integral to the bundling and stabilization ofactin filaments in the microvillus core or it may be amembrane-associated protein that nucleates microvillarassembly. Low levels of this component would generatefew microvilli, whereupon increased synthesis wouldpermit the utilization of the abundant pool of cytoskel-etal proteins to allow immediate, synchronous, andabundant microvillar formation. A similar model hasbeen proposed in sea urchin embryos in which thequantal and limited synthesis of tektins regulates theassembly of cilia (Stephens, 1989).

Our study suggests that the changes in cytoskeletalgene expression in crypt stem cells developing intoimmature 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 maynot be sensitive enough to detect dramatic changes in asubpopulation of structurally undifferentiated cryptcells. The exploration of these rapid changes willrequire in situ hybridization to localize these rare cellsand to quantify relative changes in mRNA levels asbrush border formation is initiated. This approach hasbeen used to study actin and villin mRNA distributionalong the crypt-tip axis in the mouse small intestine;unfortunately no note was made of high levels of thesemRNAs in specific crypt cells (Boiler et al. 1990; Chengand Bjerknes, 1989). While we have tried to infercytoskeletal gene activity by measuring the steady-statelevels of specific messages, our analysis reflects onlymessage accumulation. Nuclear runoff analyses must bedone to accurately measure transcription rates of thecytoskeletal genes in undifferentiated versus differen-tiated 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 partby a University of Miami Institutional Fellowship. We aregrateful to Drs C. Birkenmeier, D. Helfman, P. Matsudaira,

A. Means, D. Paterson, and J. Robbins for kindly providingtheir cDNA clones. We thank Dr K. Broschat for providingthe brush border villin and tropomyosin and their antisera; DrJ. Collins for myosin I and myosin I antisera; and Dr K.Burridge for the fodrin antisera. We would especially like tothank Ms Jessie Singer for her capable work with the RNAblots and helpful comments.

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{Accepted 1 March 1990)