Differential Localization of Villin and Fimbrin During Development of the Mouse Visceral Endoderm and Intestinal Epithelium

Development 106, 407-419 (1989) 407 Printed in Great Britain © The Company of Biologists Limited 1989

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

ROBERT M. EZZELL1*, MARK M. CHAFEL1 and PAUL T. MATSUDAIRA1'2 lWhitehead Institute for Biomedical Research and 2Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02142, USA

*To whom correspondence should be addressed at: Whitehead Institute for Biomedical Research, 9 Cambridge Center, Cambridge, Mass. 02142, USA

Summary

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

Introduction duct-lining cells of the pancreas, liver and epididymis (Robineef al. 1985). In absorptive epithelium, microvilli promote efficient Fimbrin is thought to be the primary actin filament uptake of nutrients by increasing the apical surface cross-linker of the microvillus core because, in vitro, area. The actin cytoskeleton plays a structural role in fimbrin cross-links actin filaments into uniformly polar- maintaining surface area by stabilizing these fingerlike ized bundles structurally similar to those observed projections. In the intestine and kidney proximal tubule within microvilli (Glenney et al. 1981; Matsudaira et al. brush border, actin filaments in the microvillus core are 1983). Unlike villin, fimbrin is present in a variety of organized by two proteins, villin and fimbrin (Moo- nonbrush-border-containing cell types (Bretscher & seker, 1985). Villin is a member of a family of actin- Weber, 1980). It is found in highly ordered actin- severing proteins that regulate the length and assembly containing structures, such as microvilli, microspikes of actin filaments in a Ca -dependent manner (Stossel and stereocilia, as well as in less organized actin et al. 1985; Pollard & Cooper, 1986; Matsudaira & network in membrane ruffles. Janmey, 1988): at submicromolar levels of Ca2+, villin Since villin and fimbrin play an important role in the cross-links actin filaments into bundles; at Ca2+ concen- structural organization of actin filaments in brush bor- trations above 1 fun, villin binds to and caps the barbed, der microvilli, determining the spatial and temporal fast-assembly end of the filament; above 10 ^m Ca2+, distribution of these two actin-binding proteins during villin severs actin filaments. Not all microvilli contain development is important in understanding microvilli villin. Villin is mainly found in the microvilli of intesti- assembly and the role of actin-associated proteins in nal and kidney brush borders (Bretscher et al. 1981; epithelial morphogenesis. Shibayama et al. (1987) re- Drenckhahn et al. 1983; Rodman et al. 1986) and in the cently examined the appearance of villin and fimbrin in 408 R. M. Ezzell, M. M. Chafel and P. T. Matsudaira the developing chicken and found that both proteins are membranes (Millipore Corp.) to which were absorbed elec- present in the gut at the earliest stage examined (day 7 trophoretically purified villin or fimbrin. Bound antibodies of incubation). However, villin and fimbrin accumu- were eluted with O-lM-glycine-HCl (pH2-7), quickly lated asynchronously in the apical cytoplasm of the adjusted to pH7-5 with lM-sodium phosphate (pH8-5), and differentiating epithelium: villin displayed concentrated dialysed against PBS containing 0-02 % NaN3. The specificity of the antibodies for their respective anti- apical staining by day 8, whereas fimbrin became gens was assessed using immunoblots. Adult chicken and apically concentrated at day 10. Shibayama et al. (1987) mouse intestinal epithelial cells, isolated using the procedure proposed that the early appearance and differential of Matsudaira & Burgess (1979), and visceral yolk sacs apical accumulation of villin and fimbrin in the gut play manually dissected from 10-5-day mouse embryos were pre- a role in microvillus assembly and growth during pared for electrophoresis by sonicating for 10s in 50 vol. of a development. sample buffer consisting of 2% SDS, 20% glycerol, and Using immunocytochemistry at the light and electron 0-2M-dithiothreitol in 50 mM-Tris-glycine buffer (pH6-8), microscopic levels, we have extended these obser- boiled for 5min, and centrifuged for lOmin at 14 000 g to vations in the mouse embryo to determine how early in remove insoluble material. Samples were electrophoresed on 5 %-15 % SDS-polyacrylamide minigels (Matsudaira & Bur- mammalian development villin and fimbrin appear and gess, 1978), and then electrophoretically transferred onto to correlate the distribution of these proteins with the 0-22 /jm pore size nitrocellulose (NC) paper (Schleicher & assembly of the microvillus cytoskeleton. We find that Schuell, Inc., Keene, NH) for 2h at 0-5 A in a transfer buffer villin is sequentially expressed in two tissues that have consisting of 10mM-3-(cyclohexylamino)-l-propanesulfonic similar absorptive functions: first, in the visceral endo- acid (pHll) and 10% methanol. After washing in Tris- derm (an extraembryonic tissue derived from the primi- buffered saline, pH7-4 (TBS) with 0-1% Tween-20 (three tive endoderm) and then in the intestine (derived from times, 15min each), nonspecific protein binding was blocked the fetal or definitive endoderm). Our results confirm by incubating the NC paper in a blocking solution consisting the recent report by Maunoury et al. (1988) describing of 5 % bovine serum albumin (BSA) and 2 % nonfat dry milk villin distribution and expression during endoderm in TBS for 2h at 37°C. The NC paper was incubated in affinity-purified antibodies (2 jig ml"1 in blocking solution development in the mouse. The pattern of villin ex- containing 0-2% NP-40) overnight at 4°C and washed three pression in the visceral endoderm and the gut correlates times (15min each) in TBS with 0-1 % NP-40. The NC paper with lineage tracer analysis showing that the visceral was incubated again in blocking solution for 1 h at 37 °C prior endoderm does not contribute to the definitive gut of to incubating in I-Protein A (Amersham Corp.) in blocking the embryo (reviewed by Rossant, 1986). In contrast, buffer containing 0-2 % NP-40. After washing three times in fimbrin is present throughout development, beginning TBS with 0-1 % NP-40, the NC paper was dried and exposed with the primary oocyte. Fimbrin becomes concen- to X-ray film. trated after villin in the apical cytoplasm of the visceral endoderm, when the microvilli are straight and micro- Fluorescence microscopy filaments are organized into bundles, and in the devel- Ovaries, embryos and intestines were fixed for 6h on ice in oping gut during the appearance of microvilli. 4% paraformaldehyde in PBS containing 2mM-EGTA (PBS-EGTA). For fixation, 8-5 day and earlier embryos were left in the decidua and later-stage embryos were dissected out of the deciduum. After fixation, tissues were rinsed in Materials and methods PBS-EGTA, tumbled end over end in 0-6M-sucrose in PBS-EGTA for 2-6 h at 4°C, and then embedded and frozen Mice and embryos (in an isopentane-liquid N2 bath) in OCT compound (Miles, Inbred 8- to 12-week-old FVB strain mice were used for this Naperville, IL) for cryostat sectioning. Frozen sections study. The age of the embryos was determined by designating (4—6 /an thick) were mounted on polylysine-coated micro- midday on the day the vaginal plug was found as day 0-5 of scope slides and extracted with acetone for 2min at — 20°C gestation. Embryos younger than day 5 were obtained by prior to staining with antibodies. The sections were not superovulating mice by intraperitoneal injection of pregnant allowed to dry during the extraction and staining procedure. mare serum gonadotropin (5i.u.) followed 48 h later by For antibody staining, the sections were first covered with a human chorionic gonadotropin (5i.u.). Later stage embryos blocking solution (3% BSA in PBS-EGTA), incubated for were obtained from mated nonhormone-primed females. lh at 37°C, and rinsed in PBS. The sections were then incubated for lh at 37°C in affinity-purified antibodies Production and characterization of antibodies (20/igmP1 in blocking solution) to either villin or fimbrin or Villin and fimbrin were purified from chicken intestinal with normal rabbit IgG, washed three times (15 min each) in epithelial cells using the methods of Matsudaira et al. (1985) PBS-EGTA in staining dishes using magnetic stirring, and and Glenney et al. (1981). Antisera reactive with villin incubated for lh at 37°C in fluorescein-conjugated donkey (R200.2) and fimbrin (R163.3) were prepared by subcu- anti-rabbit IgG (Jackson Immunoresearch) diluted 1:100 in taneous injection of villin headpiece (Matsudaira et al. 1985) blocking solution. Slides were washed in PBS-EGTA and a or whole fimbrin, respectively, into rabbits using standard drop of lmgml"1 p-phenylenediamine (SIGMA) in a 9:1 procedures. The mouse monoclonal anti-actin antibody C4, mixture of glycerol and PBS-EGTA was added to each prepared against chicken gizzard actin (Lessard, 1988), was a section before application of a coverslip and sealing with clear gift from Drs James Lessard and Nancy Sawtell (Children's nail polish. Hospital Research Foundation, Cincinatti, OH). For localization of filamentous (F-) actin, acetone-extracted Affinity-purified antibodies were prepared by first precipi- sections were stained for 30 min at ambient temperature with tating immunoglobulins from antisera with 45 % ammonium rhodamine-phalloidin (Molecular Probes, Junction City, sulfate, dialyzing against PBS and incubating with Immobilon OR) diluted 1:20 in PBS-EGTA, and washed in Actin-binding proteins in epithelial differentiation 409

PBS-EGTA. All slides were examined with a Zeiss Axiophot cells and visceral yolk sac from day 10-5 mouse embryos microscope and photographed using Kodak TMAX 400 film (Fig. 1). Antibodies to chicken villin and fimbrin were developed at 1000 ASA with Kodak TMAX developer. cross-reactive with a single band of the appropriate Electron microscopy and ultrastructural molecular mass on immunoblots of electrophoretically immunocytochemistry transferred proteins. Villin has a molecular mass of 95 kd in chickens and 93 kd in mouse, whereas we find For ultrastructural studies, early embryos and intestines dissected from later-stage embryos were fixed for 2-16 h in that fimbrin is 68 kd in both the chicken and mouse 3 % glutaraldehyde, 1-5 % paraformaldehyde and 1-5 % acro- intestine and in mouse 10-5 day visceral yolk sac. In the lein in 0-lM-sodium cacodylate (pH7-2). Specimens were rat, villin has a molecular mass of 91 kd (Alicea & rinsed three times inO-lM-sodium cacodylate (pH7-2) for lh, Mooseker, 1988). and postfixed in 2% OSO4 in 0-lM-sodium cacodylate (pH7-2) on ice. After rinsing four times (15 min each) in cold Localization of villin and fimbrin in the early embryo distilled water, specimens were stained for 1 h at 4°C in 1 % The distribution of villin during early development was aqueous uranyl acetate, rinsed again in cold distilled water determined by immunocytochemical staining of frozen and dehydrated through a graded ethanol series. Dehydration sections of embryos. Villin was first detected soon after was continued in a 1:1 mixture of propylene oxide and ethanol for 15 min, followed by two changes of 100 % propyl- implantation at day 5 as a diffuse cytoplasmic staining of ene oxide for 15 min each. Specimens were embedded in a the primitive endoderm, a layer of cells lining the Polybed 812-Araldite mixture (Polysciences, Inc.) by first blastocoelic surface of the inner cell mass (Fig. 2A and infiltrating in a 3:1 mixture of propylene oxide and resin for 2B). Villin was present in the primitive endoderin cells 1 h, a 1:1 mixture for 2 h, and finally a 100 % resin mixture for bordering the 'ventral' surface of the inner cell mass and 12-24 h. Resin was polymerized at 50°C for 3-4 days. not in the primitive endoderm cells along the lateral To determine areas to be examined in the electron micro- sides of the inner cell mass. By day 6, the ventral scope, \)jm thick sections were cut with glass knives from primitive endoderm cells had differentiated into the manually trimmed blocks, stained with 1% toluidine blue in visceral endoderm, which continued to express villin 1 % sodium borate for 4 min at 70°C, rinsed in distilled water, (Fig. 2C). In contrast, primitive endoderm cells along and examined by light microscopy. The blocks were then the lateral sides of the inner cell mass become motile retrimmed, and ultrathin (silver) sections were cut with a and migrate along the inner surface of the trophecto- diamond knife. Sections collected on 200-mesh grids were stained with 1 % aqueous uranyl acetate, rinsed in distilled derm; there they differentiate into the parietal endo- water, and stained with 0-31 % lead citrate using the pro- derm, which contains no detectable villin. By 6-5 days, cedure of Venable & Coggeshall (1965). the inner cell mass had developed into an elongated, For ultrastructural immunocytochemistry, embryos were cylinder-shaped embryo (the embryonic ectoderm), fixed for 16 h at 4°C in a periodate-lysine-paraformaldehyde fixative (pH6-9) as described by McLean & Nakane (1974), anti-vlllln anti-flmbrin rinsed for 30 min in PBS-EGTA, dehydrated in a graded series of dimethyrformamide, and embedded in Lowicryl K4M using the rapid embedding procedure of Altman et al. (1984). Blocks were surveyed for areas to be stained with antibody by examining thick sections stained with toluidine blue (see above). For antibody staining, gold thickness 95 K- sections were mounted on 200-mesh Formvar-coated nickel grids, rehydrated for 10 min with PBS-EGTA, incubated for 1 h at ambient temperature in the blocking solution used for immunofluorescence staining (see above) and stained overnight at 4°C with 40 jig ml"1 affinity-purified rabbit anti-villin or anti-fimbrin antibody, or with 100/igmP1 anti-actin mouse monoclonal IgG C4 (all in blocking solution). Grids were then rinsed three times (10 min each) in PBS-EGTA and stained with 5-8 nm gold-conjugated goat anti-rabbit IgG (for villin and fimbrin) or goat anti-mouse IgG (for actin) for 2h at ambient temperature. The gold-conjugated antibodies were purchased from Janssen Pharmaceuticals. Afterward the grids were rinsed three times (10 min each) in PBS-EGTA and three times (5 min each) in distilled water. Specimens were counterstained in 1 % uranyl acetate for 10 min, rinsed in 12 3 12 3 distilled water, and dried. All specimens were examined in a Fig. 1. Specificity of antibodies for chicken and mouse villin Philips 410 LS electron microscope. and fimbrin on immunoblots. Samples were electrophoresed on 5%-15% SDS-polyacrylamide gels, transferred to NC paper and incubated with affinity-purified antibodies to Results chicken villin (left panel) and chicken fimbrin (right panel). Lanes 1, adult chicken intestinal epithelial cells; lanes 2, Specificity of antibodies to villin and fimbrin adult mouse intestinal epithelial cells; lanes 3, visceral yolk The specificity of the affinity-purified antibodies used in sac from 10-5 day mouse embryo. Villin is 95 kd in chicken these studies was ascertained on immunoblots of SDS- and 93 kd in mouse, whereas fimbrin is 68 kd in both solubilized adult chicken and mouse intestinal epithelial chicken and mouse. 410 R. M. Ezzell, M. M. Chafel and P. T. Matsudaira surrounded by visceral endoderm. Villin was found in blots of day 10-5 visceral yolk sac (see Fig. 1), villin the visceral endoderm, but not in the underlying continued to be expressed in the visceral endoderm as it embryonic and extraembryonic ectoderm (Fig. 2D and differentiated into the epithelial lining of the visceral 2E). yolk sac. During this period, the embryonic endoderm, As seen in both immunofluorescence of embryos which is contiguous to the visceral yolk sac, begins to from 7 to 9 days of development (Fig. 3) and immuno- fold into the gut. Examination of 8-5 day embryos

Fig. 2. Early-stage embryos stained with anti-villin antibodies examined with immunofluorescence (A, C and D) and phase- contrast optics (B and E). (A and B) Day 5 embryo showing villin in the primitive endoderm (PrE) bordering the 'ventral' or lower surface of the inner cell mass (ICM). Villin is not expressed in PrE cells (above white arrows in (A)) that are loosely associated with the lateral sides of the ICM (indicated with arrow in (B)). At 6 days (C) villin is expressed in the visceral endoderm (VE) surrounding the primitive ectoderm; the white arrow indicates the proamniotic cavity. In the 6-5 day embryo (D and E), villin is present in the VE in contract with both the embryonic ectoderm (ECT) and extraembryonic ectoderm (Ext ECT) but is not expressed in the parietal endoderm (PE). The fluorescence in the surrounding decidual cells are nonspecific since staining is observed in adjacent sections stained with normal rabbit IgG followed by fluorescein- conjugated secondary antibody (not shown). Bars, 50 fxm. Actin-binding proteins in epithelial differentiation 411 shows villin to be present in the visceral yolk sac but cytoplasm of the visceral endoderm and, by day 8-5, the absent in the invaginating gut (Fig. 3B). The abrupt apical localization became more prominent (compare decrease in villin staining marks a boundary between Figs 2D and 3B, right panel). This same distribution the visceral endoderm and the differentiating embry- pattern is seen for villin in the epithelial lining of the onic endoderm. adult gut (see Fig. 7). Staining of the visceral yolk sac At day 6-5 villin was concentrated in the apical with rhodamine-phalloidin, a compound that selec-

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Fig. 3. Sections of an 8-5 day embryo at the level of the foregut (left) and hindgut (right) stained with hematoxylin and eosin (A), anti-villin antibodies (B), and rhodamine-phalloidin (C). At this stage of development the visceral endoderm has differentiated into the epithelial lining of the visceral yolk sac (VYS), which is contiguous with the invaginating foregut (arrow in A, left) and future hindgut (bracket in A, right). Villin is expressed in the VYS but is absent in both the foregut and hindgut. At right, box in A correspond to area shown in B, and C is a portion of the VYS near the hindgut. The localization of villin in the apical cytoplasm of the visceral endoderm layer of the VYS (arrows in B, right) correlates with the concentration of F-actin in the apical surface of the visceral endoderm (arrows in C). Bars, 200/mi for A (left and right) and B (left), and 50/an for B and C (right). 412 R. M. Ezzell, M. M. Chafel and P. T. Matsudaira

tively binds F-actin (Barak et al. 1981), reveals a fimbrin and actin correlates with the appearance of rigid corresponding accumulation of F-actin in the apical microvilli containing filament bundles (see Fig. 5C). A surface in the visceral endoderm (Fig. 3C). similar organization of villin and fimbrin was found in Fimbrin was expressed throughout development, the kidney proximal tubule brush border (Rodman et beginning with the preovulatory oocyte (Figs 4A-5D). al. 1986). Control experiments showed that when pre- Examination of sections of ovaries stained with anti- immune primary antibody was used, labeling of sections fimbrin antibodies showed bright fluorescence in with gold particles was diffuse and minimal (not oocytes at all stages of growth. In primary oocytes, shown). fimbrin shows a punctate distribution in the cytoplasm and a thin, submembranous concentration. In later- Localization of villin and fimbrin in the developing stage oocytes, fimbrin exhibits a diffuse cytoplasmic intestine distribution localized to the cell cortex. Staining of The pattern of villin and fimbrin distribution in the oocytes with rhodamine-phalloidin revealed a similar developing intestine was similar to that seen in the cortical concentration of F-actin (not shown); sugges- visceral endoderm. Villin was first detected in the ting that fimbrin is associated with submembranous intestinal epithelium at day 10, after the embryonic actin filaments and may be involved in bundling actin endoderm had formed a tube and closed off from the filaments in the microvilli covering the surface of the visceral endoderm (Fig. 7). No villin was seen in the mouse oocyte (Wassarman & Josefowicz, 1978). Fol- underlying mesenchyme or connective tissues. The licle cells surrounding the oocyte and adjacent ovarian villin-staining pattern in the intestinal cells at this stage tissues also stain with anti-fimbrin antibodies, but the of development was mottled and was localized to the fluorescence intensity is weak and diffuse. Neither cell boundaries. Fimbrin was more evenly distributed oocytes nor ovarian tissues stained with anti-villin throughout the cell but was slightly more concentrated antibodies. along the basal surface of the epithelium (Fig. 7A). By After implantation at day 5, fimbrin was found in all day 12-5, the intestine had become eliptical in cross embryonic and extraembryonic tissues of the embryo section. During this time, villin became concentrated at (Fig. 4E). At 8-5 days diffuse cytoplasmic fimbrin the apical or luminal surface of the intestine. Fimbrin staining was found in the epithelial lining of the 8-5 day remained distributed in the cytoplasm and did not foregut (Fig. 4F), which does not express villin until ~2 display apical localization until day 15-5, 2-3 days after days later in development (see Fig. 7). At the same the redistribution of villin. The distribution of villin and time, fimbrin becomes redistributed to the apical cyto- fimbrin at this stage of development resembles its plasm of the visceral endoderm. This localization to the localization in the adult gut (Fig. 7D and 7d). apical surface became more prominent by day 10-5 (compare Fig. 4G and 4H). Ultrastructural morphology of the developing mouse intestine Ultrastructural localization of villin and fimbrin during Before day 16 of development, the apical surface of the differentiation of the visceral endoderm intestinal epithelium was sparsely populated by micro- At the ultrastructural level, the apical surface of the villi, and those present were short and protruded from visceral endoderm changed in morphology during de- the cell at various angles (Fig. 8A and 8B). By day 16-5, velopment (Fig. 5). At day 6-5, the visceral endoderm there were numerous microvilli, each containing a was covered by long, bulbous projections that re- bundle of microfilaments (Fig. 8C); however, com- sembled microvilli, morphology similar to that ob- pared with the microvilli of the adult brush border served by Hogan & Newman (1984) and Ishikawa et al. (Fig. 8D), the embryonic microvilli were short and their (1986) using the scanning electron microscope to exam- actin bundles did not extend into the apical cytoplasm. ine the surface of the mouse visceral endoderm. By day There also was no network of filaments or well-defined 8-5, these structures were more compact and contained junctional complexes that delineated the terminal web. loose arrays of filaments. At day 10-5 and day 12-5, the The apical accumulation of villin and fimbrin in the microvilli were straight and microfilament bundles were intestinal epithelium before the appearance of micro- visible. villi (see Fig. 7) may account for the compact mor- To investigate further the distribution of villin, fim- phology of the microvilli when they first appear in the brin and actin in the visceral endoderm, Lowicryl- gut. embedded sections of the visceral endoderm were stained with antibodies to villin, fimbrin or actin, followed by gold-labeled secondary antibodies. Fig. 6 Discussion shows the ultrastructural distribution of villin, fimbrin and actin in the microvilli and apical cytoplasm of 8-5 Our results demonstrate that the microvillar core pro- day and 10-5 day visceral endoderm. Although diffusely teins villin and fimbrin are differentially localized dur- distributed in the apical cytoplasm, all three proteins ing mouse development but display identical patterns of were also associated with the cytoplasmic surface of the accumulation in both the extraembryonic visceral endo- microvillar membranes in 8-5 day visceral endoderm. derm and the embryonic intestinal epithelium. Fimbrin By day 10-5, these proteins were concentrated in the is present in the preovulatory oocyte and is detected microvillus core. The change in distribution of villin, throughout development in various embryonic tissues. Actin-binding proteins in epithelial differentiation 413

Fig. 4. Distribution of fimbrin in preovulatory oocytes (A-D), 5 day embryo (E), 8-5 day foregut (F), and 8-5 day (G) and 10-5 day (H) visceral yolk sac. A primary oocyte examined with immunofluorescence (A) and phase-contrast optics (B) shows a punctate distribution of fimbrin in the cytoplasm and a ring of submembranous staining. Dark oval in center of oocyte in A is the germinal vesicle, which does not stain with fimbrin antibodies. In a later-stage oocytes (C,D) fimbrin is concentrated in the cortical cytoplasm. Fimbrin is expressed throughout the postimplantation embryo (E), including in the inner cells mass (1CM) and surrounding maternal decidual cells. Fimbrin is also present in the epithelial lining of the foregut (arrows in F), which does not express villin until 2 days later, at ~day 10 (see Fig. 7). The concentration of fimbrin in the apical surface of the visceral endoderm does not become prominent until day 10-5 (arrows in G and H), when the visceral endoderm microvilli are straight and filament bundles are visible (see Fig. 5C). Bars, 20nm for A-D, 50^m for E-H. 414 R. M. Ezzell, M. M. Chafel and P. T. Matsudaira

Fig. 5. Electron microscopic examination of the visceral endoderm microvilli at days 6-5 (A), 8-5 (B), 10-5 (C), and 12-5 (D). The microvilli at day 6-5 are loosely structured, but at day 8-5 are more defined and contain loosely organized filaments. At days 10-5 and 12-5, the microvilli are straight and contain organized microfilament bundles. The presence of numerous coated pits and vesicles at the apical surface reflects the absorptive and transport functions of the visceral endoderm. Bar, 1 /an.

In contrast, villin is first detected in the primitive Villin is a marker of the visceral endoderm endoderm before gastrulation and later in the intestinal The inner cell mass gives rise to the embryonic tissues, epithelium after the gut has sealed into a tube. As these including the gut endoderm, whereas the surrounding tissues differentiate, villin accumulates at the apical primitive endoderm gives rise to two extraembryonic surface of the cells within 2-3 days after it first appears, cell types: the visceral endoderm, which remains associ- and fimbrin redistributes to the apical surface 2-3 days ated with the differentiating inner cell mass; and the later. A similar asynchrony in the redistribution of villin parietal endoderm, which colonizes the inner surface of and fimbrin was observed by Shibayama et al. (1987) the trophectoderm (Gardner, 1983, 1984; Rossant, studying the chicken gut. The expression of villin during 1986). When Cockroft & Gardner (1987) examined the mouse development has also been examined by developmental potential of the visceral endoderm by Maunoury et al. (1988). They also observe that villin injecting genetically marked visceral endoderm cells first appears in the visceral endoderm followed later by into blastocysts, they found the injected cells had the gut; and immunoblot and Northern blot analysis of differentiated into visceral and parietal endoderm but extraembryonic and embryonic tissues revealed, re- did not become part of the embryo. Lawson etal. (1986) spectively, a single polypeptide of 93 kd and an mRNA and Lawson & Pederson (1987), injecting various cells of 3-4 kb in length. Fig. 9 summarizes our findings and of the postimplantation embryo with horseradish per- relates the appearance and distribution of villin and oxidase, showed that the definitive endoderm arises fimbrin to the differentiation of the visceral endoderm from the embryonic ectoderm and that the visceral and the gut. Our results provide information about endoderm contributes few, if any, cells to the gut. The functional relationships between villin-containing cells presence of villin in the visceral endoderm and its derived from embryonic and extraembryonic lineages, absence in the undifferentiated embryonic gut (see and suggest a mechanism for the assembly of the Fig. 3) is consistent with data showing that the visceral microvillus cytoskeleton. endoderm does not contribute cells to the fetal gut. Actin-binding proteins in epithelial differentiation 415 8.5 DAY

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Fig. 6. Ultrastructural localization of villin (A), fimbrin (B), and actin (C) in the visceral endoderm microvilli from an 8-5day (upper panel) and 10-5 day (lower panel) embryo. Villin, fimbrin and actin in 8-5 day visceral endoderm are associated with the cytoplasmic surface of the microvillus membranes (arrows in upper panel); whereas, at day 10-5, these proteins are concentrated in the microvillus core (arrows in lower panel). No labeling is observed with either normal rabbit or normal mouse IgG, followed by the appropriate gold-conjugated secondary antibody (not shown). Bar, 0-5 /im. 416 R. M. Ezzell, M. M. Chafel and P. T. Matsudaira

Fig. 7. Distribution of villin (A-D) and fimbrin (a-d) in the developing duodenum at days 10-5 (A, a), 12-5 (B, b), and 15-5 (C, c) and in the adult duodenum (D, d). Villin first appears in the newly formed gut at ~day 10. At day 12-5, villin is concentrated in the apical cytoplasm; staining becomes more pronounced at day 15-5 as the lumen folds. Fimbrin does not concentrate in the apical cytoplasm until day 15-5, 2-3 days after villin redistributes to the apical surface. Arrow in lower right corner of (c) points to smooth muscle surrounding gut, which stains with anti-fimbrin antibodies. Bars, 50 fim. Actin-binding proteins in epithelial differentiation All

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Fig. 8. Electron microscopic examination of the apical surface of the differentiating intestinal epithelium at 14-5 (A), 15-5 (B), and 16-5 days (C) and in the adult duodenum (D). Microvilli in the gut first appear as compact, short stubs at day 14-5 and increase in length and density during development. Arrows in C and D point to occluding junctions. Bar, 1/OTI.

Our studies, and those of Maunoury et al. (1988), Role of villin and fimbrin in microvillus assembly show that villin is a valuable marker for the visceral The identical sequence in the accumulation of villin and endoderm and can be used in conjunction with other fimbrin at the apical surface in the visceral endoderm markers to investigate the differentiation of extra- and in the intestinal epithelium (see Fig. 9) suggests embryonic tissues in the mouse embryo. For example, that the two tissues employ a common mechanism for alpha-fetoprotein is detected in visceral endoderm cells assembling the microvillar cytoskeleton during devel- that are in contact with the embryonic, but not the opment. As discussed by Shibayama et al. (1987) for the extraembryonic, ectoderm (Dziadek, 1978; Dziadek & chicken, this mechanism may first involve the growth of Adamson, 1978). In comparison, ENDO C, a member actin filaments from membrane nucleation sites and the of the cytokeratin family of intermediate filament cross-linking of the newly assembled filaments into proteins, is detected by the monoclonal antibody bundles. This scheme is consistent with the order of TROMA-3 only in parietal endoderm cells, whereas a appearance and actin-binding activities of villin and second monoclonal antibody, TROMA-1, recognizes a fimbrin. Villin, as a phospholipid-binding protein, different cytokeratin protein, ENDO A, in both par- might be bound to the membrane (Janmey & Matsu- ietal and visceral endoderm cells (Boiler & Kemler, daira, 1988) and, as an actin filament-severing protein, 1983). These antibodies have been useful in monitoring can accelerate actin polymerization (Matsudaira & the in vitro 'transdifferentiation' of the visceral endo- Janmey, 1988). The two activities suggest a membrane- derm into parietal endoderm (Casanova & Grabel, associated actin-nucleating role for villin. Our immuno- 1988). In contrast to alpha-fetoprotein or the cytokera- electron micrographs of 8-5 day visceral endoderm tin proteins, villin is a marker for the entire visceral microvilli (see Fig. 6) provide evidence for this postu- endoderm. In this context, it would be interesting to lated membrane association of villin during the early examine whether villin expression is down-regulated stages of microvillus assembly. A similar mechanism for during visceral endoderm transdifferentiation. microvilli assembly may operate in the sea urchin egg. 418 R. M. Ezzell, M. M. Chafel and P. T. Matsudaira

FERTILIZATION VIII IN F1VBBH Casanova for helpful suggestions, and the members of Dr (days after) Rudolph Jaenisch's laboratory, especially Ruth Curry and r-T-0 - present In ooeyte 2-cell -•- Doris Grotkopp, for providing expertise and mice. This investigation was supported by grants POl CA44704 and ROl 4-2 DK35306 from the National Institutes of Health. present In embryonic Implantation -^> appearsln - -4 1 and extraembryonlc primitive endodorm tissues References gastrulatlon -&• - -6 . _s apical localization In _^ ALICEA, H. A. & MOOSEKER, M. S. (1988). Characterization of closure ol gut••• visceral endoderm villin from the intestinal brush border of the rat, and comparative - -10 appears In gut -•- _ aplc«l localization In ' viscsral endoderm analysis with avian villin. Cell Motil. Cytoskel. 9, 60-72. ALTMAN, L. G., SCHNEIDER, B. G. & PAPERMASTER, D. S. (1984). - -12 apical tocaBzallon _^.~ Rapid embedding of tissues in lowicryl K4M for immunoelectron In gut microscopy. J. Histochem. Cytochem. 32, 1217-1223. mlcrovDII • -14 appear In gut- . apical tocaBzallon BARAK, L. S., YOCUM, R. R. & WEBB, W. W. (1981). In vivo In gut •-16 staining of cytoskeletal actin by autointernalization of nontoxic concentrations of nitrobenzoxadiazole-phallacidin. J. Cell Biol. - -18 89, 368-372. BOLLER, K. & KEMLER, R. (1983). In vitro differentiation of 1-1-20 embryonal carcinoma cells characterized by monoclonal BIRTH antibodies against embryonal cell markers. Cold Spring Harbor Conf. Cell Proliferation. 10, 39-49. Fig. 9. Time course of villin and fimbrin expression in the BRETSCHER, A., OSBORN, M., WEHLAND, J. & WEBER, K. (1981). developing mouse, relating the appearance and distribution Villin associates with specific microfilamentous structures as seen by immunofluorescence microscopy on tissue sections and cells of these proteins to the differentiation of the visceral microinjected with villin. Expl Cell Res. 135, 213-219. endoderm and the intestinal epithelium. Villin redistributes BRETSCHER, A. & WEBER, K. (1980). Fimbrin, a new microfilament- to the apical surface 2-3 days after it appears in the associated protein present in microvilli and other cell surfaces. /. primitive endoderm and the gut; 2-3 days later fimbrin is Cell Biol, 86, 335-340. localized to the apical surface in both of these tissues. CASANOVA, J. E. & GRABEL, L. B. (1988). The role of cell interactions in the differentiation of teratocarcinoma-derived parietal and visceral endoderm. Devi Biol. 129, 124-139. At fertilization, the egg microvilli rapidly elongate COCKROFT, D. L. & GARDNER, R. L. (1987). Clonal analysis of the through a process mediated by polymerization of pre- developmental potential of 6th and 7th day visceral endoderm existing actin (reviewed by Schatten, 1982). In examin- cells in the mouse. Development 101, 143-155. DRENCKHAHN, D., HOFMANN, H. D. & MANNHERZ, H. G. (1983). ing the cortex of the unfertilized sea urchin egg, Henson Evidence for the association of villin with core filaments and & Begg (1988) observed short actin filaments woven rootlets of intestinal epithelial microvilli. Cell Tissue Res. 228, into a tight network, which they propose may provide 409-414. nucleation sites for actin polymerization during micro- DZIADEK, M. (1978). Modulation of alphafetoprotein synthesis in villi growth. the early postimplantation embryo. J. Embryol. exp. Morph. 46, 135-146. In the second stage of microvillar biogenesis, fimbrin DZIADEK, M. & ADAMSON, E. (1978). Localization and synthesis of becomes concentrated at the membrane 2-3 days after alphafetoprotein in postimplantation mouse embryos. villin. The later accumulation of fimbrin may result J. Embryol. exp. Morph. 43, 289-313. from changes in actin filament concentration at the GARDNER, R. L. (1983). Origin and differentiation of extraembryonic tissues in the mouse. Int. Rev. exp. Path. 24, membrane. In the chicken gut, there is a dramatic 63-133. increase in microvilli length late in development. Stid- GARDNER, R. L. (1984). An in situ cell marker for clonal analysis of will & Burgess (1986) have reported a shift in the development of the extraembryonic endoderm in the mouse. J. monomeric to F-actin ratio from 3:7 to 1:1 just prior to Embryol. exp. Morph. 80, 251-288. GLENNEY, J., KAULFUS, P., MATSUDAIRA, P. T. & WEBER, K. this increase in microvilli length. In the undifferentiated (1981). F-actin binding and bundling properties of fimbrin, a cell, the actin concentration may be suboptimal for major cytoskeletal protein of the microvillus core filaments. /. bundle formation. However, as more filaments become biol. Chem. 256, 9283-9288. associated with the membrane, a critical threshold for HENSON, J. H. & BEGG, D. A. (1988). Filamentous actin bundle formation could be reached and actin filaments organization in the unfertilized sea urchin egg cortex. Devi Biol. 127, 338-348. would become cross-linked. In examining the distri- HOCAN, B. L. M. & NEWMAN, R. (1984). A scanning electron bution of actin during development of the chick duo- microscope study of the extraembryonic endoderm of the 8th-day denum, Noda and Mitsui (1988) report that actin mouse embryo. Differen. 26, 138-143. changes from being concentrated in areas involved in ISHIKAWA, T., YAGYU, K. & SEGUCHI, H. (1986). A scanning electron microscopic study of the surface morphology of visceral formation of the previllous ridge to the apical surface endoderm and ectoderm in postimplantation mouse embryos. J. during microvilli assembly. Such a scheme might ex- Electron Miscrosc. 35, 185-194. plain why fimbrin concentration at the membrane lags JANMEY, P. & MATSUDAIRA, P. T. (1988). Functional comparison of behind that of villin, but increases before the formation villin and gelsolin: effects of Ca2+, KC1, and of microvilli in the developing gut is complete. 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