The apical surface of transporting epithelia is specially modified to absorb nutrients efficiently by amplifying its surface area as microvilli. Each microvillus is supported by an underlying core of bundled actin filaments. Villin and fimbrin are two actin-binding proteins that bundle actin filaments in the intestine and kidney brush border epithelium. To better understand their function in the assembly of the cytoskeleton during epithelial differentiation, we examined the pattern of villin and fimbrin expression in the developing mouse using immunofluorescence and immunoelectron microscopy. Villin is first detected at day 5 in the primitive endoderm of the postimplantation embryo and is later restricted to the visceral endoderm. By day 8-5, villin becomes redistributed to the apical surface in the visceral endoderm, appearing in the gut at day 10 and concentrating in the apical cytoplasm of the differentiating intestinal epithelium 2–3 days later. In contrast, fimbrin is found in the oocyte and in all tissues of the early embryo. In both the visceral endoderm and gut epithelium, fimbrin concentrates at the apical surface 2–3 days after villin; this redistribution occurs when the visceral endoderm microvilli first contain organized microfilament bundles and when microvilli first begin to appear in the gut. These results suggest a common mechanism of assembly of the absorptive surface of two different tissues in the embryo and identify villin as a useful marker for the visceral endoderm.

In absorptive epithelium, microvilli promote efficient uptake of nutrients by increasing the apical surface area. The actin cytoskeleton plays a structural role in maintaining surface area by stabilizing these fingerlike projections. In the intestine and kidney proximal tubule brush border, actin filaments in the microvillus core are organized by two proteins, villin and fimbrin (Mooseker, 1985). Villin is a member of a family of actin-severing proteins that regulate the length and assembly of actin filaments in a Ca2+-dependent manner (Stossel et al. 1985; Pollard & Cooper, 1986; Matsudaira & Janmey, 1988): at submicromolar levels of Ca2+, villin cross-links actin filaments into bundles; at Ca2+ concentrations above 1 μm, villin binds to and caps the barbed, fast-assembly end of the filament; above 10 gm Ca2+, villin severs actin filaments. Not all microvilli contain villin. Villin is mainly found in the microvilli of intestinal and kidney brush borders (Bretscher et al. 1981; Drenckhahn et al. 1983; Rodman et al. 1986) and in the duct-lining cells of the pancreas, liver and epididymis (Robine et al. 1985).

Fimbrin is thought to be the primary actin filament cross-linker of the microvillus core because, in vitro, fimbrin cross-links actin filaments into uniformly polarized bundles structurally similar to those observed within microvilli (Glenney et al. 1981; Matsudaira et al. 1983). Unlike villin, fimbrin is present in a variety of nonbrush-border-containing cell types (Bretscher & Weber, 1980). It is found in highly ordered actin-containing structures, such as microvilli, microspikes and stereocilia, as well as in less organized actin network in membrane ruffles.

Since villin and fimbrin play an important role in the structural organization of actin filaments in brush border microvilli, determining the spatial and temporal distribution of these two actin-binding proteins during development is important in understanding microvilli assembly and the role of actin-associated proteins in epithelial morphogenesis. Shibayama et al. (1987) recently examined the appearance of villin and fimbrin in the developing chicken and found that both proteins are present in the gut at the earliest stage examined (day 7 of incubation). However, villin and fimbrin accumulated asynchronously in the apical cytoplasm of the differentiating epithelium: villin displayed concentrated apical staining by day 8, whereas fimbrin became apically concentrated at day 10. Shibayama et al. (1987) proposed that the early appearance and differential apical accumulation of villin and fimbrin in the gut play a role in microvillus assembly and growth during development.

Using immunocytochemistry at the light and electron microscopic levels, we have extended these observations in the mouse embryo to determine how early in mammalian development villin and fimbrin appear and to correlate the distribution of these proteins with the assembly of the microvillus cytoskeleton. We find that villin is sequentially expressed in two tissues that have similar absorptive functions: first, in the visceral endoderm (an extraembryonic tissue derived from the primitive endoderm) and then in the intestine (derived from the fetal or definitive endoderm). Our results confirm the recent report by Maunoury et al. (1988) describing villin distribution and expression during endoderm development in the mouse. The pattern of villin expression in the visceral endoderm and the gut correlates with lineage tracer analysis showing that the visceral endoderm does not contribute to the definitive gut of the embryo (reviewed by Rossant, 1986). In contrast, fimbrin is present throughout development, beginning with the primary oocyte. Fimbrin becomes concentrated after villin in the apical cytoplasm of the visceral endoderm, when the micro villi are straight and microfilaments are organized into bundles, and in the developing gut during the appearance of microvilli.

Mice and embryos

Inbred 8- to 12-week-old FVB strain mice were used for this study. The age of the embryos was determined by designating midday on the day the vaginal plug was found as day 0-5 of gestation. Embryos younger than day 5 were obtained by superovulating mice by intraperitoneal injection of pregnant mare serum gonadotropin (5i.u.) followed 48 h later by human chorionic gonadotropin (5i.u.). Later stage embryos were obtained from mated nonhormone-primed females.

Villin and fimbrin were purified from chicken intestinal epithelial cells using the methods of Matsudaira et al. (1985) and Glenney et al. (1981). Antisera reactive with villin (R200.2) and fimbrin (R163.3) were prepared by subcutaneous injection of villin headpiece (Matsudaira et al. 1985) or whole fimbrin, respectively, into rabbits using standard procedures. The mouse monoclonal anti-actin antibody C4, prepared against chicken gizzard actin (Lessard, 1988), was a gift from Drs James Lessard and Nancy Sawtell (Children’s Hospital Research Foundation, Cincinatti, OH).

Affinity-purified antibodies were prepared by first precipitating immunoglobulins from antisera with 45 % ammonium sulfate, dialyzing against PBS and incubating with Immobilon membranes (Millipore Corp.) to which were absorbed elec-trophoretically purified villin or fimbrin. Bound antibodies were eluted with 0T M-glycine-HCl (pH2·7), quickly adjusted to pH 7·5 with lm-sodium phosphate (pH 8·5), and dialysed against PBS containing 0-02 % NaN3.

The specificity of the antibodies for their respective antigens was assessed using immunoblots. Adult chicken and mouse intestinal epithelial cells, isolated using the procedure of Matsudaira & Burgess (1979), and visceral yolk sacs manually dissected from 10·5-day mouse embryos were prepared for electrophoresis by sonicating for 10s in 50 vol. of a sample buffer consisting of 2% SDS, 20% glycerol, and 0·2M-dithiothreitol in 50 mm-Tris-glycine buffer (pH6·8), boiled for 5 min, and centrifuged for 10 min at 14000g to remove insoluble material. Samples were electrophoresed on 5 %-15 % SDS-polyacrylamide minigels (Matsudaira & Burgess, 1978), and then electrophoretically transferred onto 0-22 pm pore size nitrocellulose (NC) paper (Schleicher & Schuell, Inc., Keene, NH) for 2h at 0·5 A in a transfer buffer consisting of 10mm-3-(cyclohexylamino)-l-propanesulfonic acid (pH 11) and 10% methanol. After washing in Trisbuffered saline, pH 7-4 (TBS) with 0-1% Tween-20 (three times, 15 min each), nonspecific protein binding was blocked by incubating the NC paper in a blocking solution consisting of 5 % bovine serum albumin (BSA) and 2 % nonfat dry milk in TBS for 2h at 37°C. The NC paper was incubated in affinity-purified antibodies (2izg ml-1 in blocking solution containing 0·2% NP-40) overnight at 4°C and washed three times (15 min each) in TBS with 0T % NP-40. The NC paper was incubated again in blocking solution for 1 h at 37 °C prior to incubating in ,25I-Protein A (Amersham Corp.) in blocking buffer containing 0·2 % NP-40. After washing three times in TBS with 0-1 % NP-40, the NC paper was dried and exposed to X-ray film.

Fluorescence microscopy

Ovaries, embryos and intestines were fixed for 6h on ice in 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 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 (in an isopentane-liquid N2 bath) in OCT compound (Miles, Naperville, IL) for cryostat sectioning. Frozen sections (4—6 μm thick) were mounted on polylysine-coated microscope slides and extracted with acetone for 2 min at —20°C prior to staining with antibodies. The sections were not allowed to dry during the extraction and staining procedure.

For antibody staining, the sections were first covered with a blocking solution (3% BSA in PBS-EGTA), incubated for 1h at 37°C, and rinsed in PBS. The sections were then incubated for 1h at 37 °C in affinity-purified antibodies (20 μg ml−1 in blocking solution) to either villin or fimbrin or with normal rabbit 1gG, washed three times (15 min each) in PBS-EGTA in staining dishes using magnetic stirring, and incubated for 1h at 37°C in fluorescein-conjugated donkey anti-rabbit 1gG (Jackson Immunoresearch) diluted 1:100 in blocking solution. Slides were washed in PBS-EGTA and a drop of l mg ml−1 p-phenylenediamine (SIGMA) in a 9:1 mixture of glycerol and PBS-EGTA was added to each section before application of a coverslip and sealing with clear nail polish.

For localization of filamentous (F-) actin, acetone-extracted sections were stained for 30 min at ambient temperature with rhodamine-phalloidin (Molecular Probes, Junction City, OR) diluted 1:20 in PBS-EGTA, and washed in PBS-EGTA. All slides were examined with a Zeiss Axiophot microscope and photographed using Kodak TMAX 400 film developed at 1000 ASA with Kodak TMAX developer.

Electron microscopy and ultrastructural immunocytochemistry

For ultrastructural studies, early embryos and intestines dissected from later-stage embryos were fixed for 2–16 h in 3 % glutaraldehyde, 1·5 % paraformaldehyde and 1·5 % acrolein in 0-lm-sodium cacodylate (pH7·2). Specimens were rinsed three times in 0·1 m-sodium cacodylate (pH 7·2) for 1 h, and postfixed in 2% OsO4 in 01m-sodium cacodylate (pH 7-2) on ice. After rinsing four times (15 min each) in cold distilled water, specimens were stained for 1h at 4 °C in 1 % aqueous uranyl acetate, rinsed again in cold distilled water and dehydrated through a graded ethanol series. Dehydration was continued in a 1:1 mixture of propylene oxide and ethanol for 15 min, followed by two changes of 100% propylene oxide for 15 min each. Specimens were embedded in a Polybed 812-Araldite mixture (Polysciences, Inc.) by first infiltrating in a 3:1 mixture of propylene oxide and resin for 1 h, a 1:1 mixture for 2 h, and finally a 100% resin mixture for 12–24 h. Resin was polymerized at 50°C for 3–4 days.

To determine areas to be examined in the electron microscope, 1μm thick sections were cut with glass knives from manually trimmed blocks, stained with 1% toluidine blue in 1 % sodium borate for 4 min at 70°C, rinsed in distilled water, and examined by light microscopy. The blocks were then retrimmed, and ultrathin (silver) sections were cut with a diamond knife. Sections collected on 200-mesh grids were stained with 1 % aqueous uranyl acetate, rinsed in distilled water, and stained with 0·31 % lead citrate using the procedure of Venable & Coggeshall (1965).

For ultrastructural immunocytochemistry, embryos were fixed for 16 h at 4°C in a periodate-lysine-paraformaldehyde fixative (pH6·9) as described by McLean & Nakane (1974), rinsed for 30 min in PBS-EGTA, dehydrated in a graded series of dimethylformamide, 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 sections were mounted on 200-mesh Formvar-coated nickel grids, rehydrated for 10 min with PBS-EGTA, incubated for Ih at ambient temperature in the blocking solution used for immunofluorescence staining (see above) and stained overnight at 4°C with 40 μg ml−1 affinity-purified rabbit anti-villin or anti-fimbrin antibody, or with 100 μg ml−1 anti-actin mouse monoclonal 1gG 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 1gG (for villin and fimbrin) or goat anti-mouse 1gG (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 10min, rinsed in distilled water, and dried. All specimens were examined in a Philips 410 LS electron microscope.

Specificity of antibodies to villin and fimbrin

The specificity of the affinity-purified antibodies used in these studies was ascertained on immunoblots of SDS-solubilized adult chicken and mouse intestinal epithelial cells and visceral yolk sac from day 10-5 mouse embryos (Fig. 1). Antibodies to chicken villin and fimbrin were cross-reactive with a single band of the appropriate molecular mass on immunoblots of electrophoretically transferred proteins. Villin has a molecular mass of 95 kd in chickens and 93 kd in mouse, whereas we find that fimbrin is 68 kd in both the chicken and mouse intestine and in mouse 10-5 day visceral yolk sac. In the rat, villin has a molecular mass of 91 kd (Alicea & Mooseker, 1988).

Fig. 1.

Specificity of antibodies for chicken and mouse villin and fimbrin on immunoblots. Samples were electrophoresed on 5 %-15 % SDS-polyacrylamide gels, transferred to NC paper and incubated with affinity-purified antibodies to chicken villin (left panel) and chicken fimbrin (right panel). Lanes 1, adult chicken intestinal epithelial cells; lanes 2, adult mouse intestinal epithelial cells; lanes 3, visceral yolk sac from 10 ·5 day mouse embryo. Villin is 95 kd in chicken and 93 kd in mouse, whereas fimbrin is 68 kd in both chicken and mouse.

Fig. 1.

Specificity of antibodies for chicken and mouse villin and fimbrin on immunoblots. Samples were electrophoresed on 5 %-15 % SDS-polyacrylamide gels, transferred to NC paper and incubated with affinity-purified antibodies to chicken villin (left panel) and chicken fimbrin (right panel). Lanes 1, adult chicken intestinal epithelial cells; lanes 2, adult mouse intestinal epithelial cells; lanes 3, visceral yolk sac from 10 ·5 day mouse embryo. Villin is 95 kd in chicken and 93 kd in mouse, whereas fimbrin is 68 kd in both chicken and mouse.

Localization of villin and fimbrin in the early embryo

The distribution of villin during early development was determined by immunocytochemical staining of frozen sections of embryos. Villin was first detected soon after implantation at day 5 as a diffuse cytoplasmic staining of the primitive endoderm, a layer of cells lining the blastocoelic surface of the inner cell mass (Fig. 2A and 2B). Villin was present in the primitive endoderm cells bordering the ‘ventral’ surface of the inner cell mass and not in the primitive endoderm cells along the lateral sides of the inner cell mass. By day 6, the ventral primitive endoderm cells had differentiated into the visceral endoderm, which continued to express villin (Fig. 2C). In contrast, primitive endoderm cells along the lateral sides of the inner cell mass become motile and migrate along the inner surface of the trophecto-derm; there they differentiate into the parietal endoderm, which contains no detectable villin. By 6-5 days, the inner cell mass had developed into an elongated, cylinder-shaped embryo (the embryonic ectoderm),surrounded by visceral endoderm. Villin was found in the visceral endoderm, but not in the underlying embryonic and extraembryonic ectoderm (Fig. 2D and 2E).

Fig. 2.

Early-stage embryos stained with anti-villin antibodies examined with immunofluorescence (A, C and D) and phasecontrast 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 μm.

Fig. 2.

Early-stage embryos stained with anti-villin antibodies examined with immunofluorescence (A, C and D) and phasecontrast 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 μm.

As seen in both immunofluorescence of embryos from 7 to 9 days of development (Fig. 3) and immuno blots of day 10·5 visceral yolk sac (see Fig. 1), villin continued to be expressed in the visceral endoderm as it differentiated into the epithelial lining of the visceral yolk sac. During this period, the embryonic endoderm, which is contiguous to the visceral yolk sac, begins to fold into the gut. Examination of 8·5 day embryos shows villin to be present in the visceral yolk sac but absent in the invaginating gut (Fig. 3B). The abrupt decrease in villin staining marks a boundary between the visceral endoderm and the differentiating embryonic endoderm.

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 μm for A (left and right) and B (left), and 50pm for B and C (right).

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 μm for A (left and right) and B (left), and 50pm for B and C (right).

At day 6·5 villin was concentrated in the apical cytoplasm of the visceral endoderm and, by day 8-5, the apical localization became more prominent (compare Figs 2D and 3B, right panel). This same distribution pattern is seen for villin in the epithelial lining of the adult gut (see Fig. 7). Staining of the visceral yolk sac with rhodamine-phalloidin, a compound that selectively binds F-actin (Barak et al. 1981), reveals a corresponding accumulation of F-actin in the apical surface in the visceral endoderm (Fig. 3C).

Fimbrin was expressed throughout development, beginning with the preovulatory oocyte (Figs 4A-5D). Examination of sections of ovaries stained with anti-fimbrin antibodies showed bright fluorescence in oocytes at all stages of growth. In primary oocytes, fimbrin shows a punctate distribution in the cytoplasm and a thin, submembranous concentration. In later-stage oocytes, fimbrin exhibits a diffuse cytoplasmic distribution localized to the cell cortex. Staining of oocytes with rhodamine-phalloidin revealed a similar cortical concentration of F-actin (not shown); suggesting that fimbrin is associated with submembranous actin filaments and may be involved in bundling actin filaments in the microvilli covering the surface of the mouse oocyte (Wassarman & Josefowicz, 1978). Follicle cells surrounding the oocyte and adjacent ovarian tissues also stain with anti-fimbrin antibodies, but the fluorescence intensity is weak and diffuse. Neither oocytes nor ovarian tissues stained with anti-villin antibodies.

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 (ICM) 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, 20 μm for A-D, 50 μm for E-H.

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 (ICM) 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, 20 μm for A-D, 50 μm for E-H.

Fig. 5.

Electron microscopie 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 μm.

Fig. 5.

Electron microscopie 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 μm.

After implantation at day 5, fimbrin was found in all embryonic and extraembryonic tissues of the embryo (Fig. 4E). At 8·5 days diffuse cytoplasmic fimbrin staining was found in the epithelial lining of the 8·5 day foregut (Fig. 4F), which does not express villin until ∼2 days later in development (see Fig. 7). At the same time, fimbrin becomes redistributed to the apical cytoplasm of the visceral endoderm. This localization to the apical surface became more prominent by day 10·5 (compare Fig. 4G and 4H).

Ultrastructural localization of villin and fimbrin during differentiation of the visceral endoderm

At the ultrastructural level, the apical surface of the visceral endoderm changed in morphology during development (Fig. 5). At day 6·5, the visceral endoderm was covered by long, bulbous projections that resembled microvilli, morphology similar to that observed by Hogan & Newman (1984) and Ishikawa et al. (1986) using the scanning electron microscope to examine the surface of the mouse visceral endoderm. By day 8·5, these structures were more compact and contained loose arrays of filaments. At day 10·5 and day 12·5, the microvilli were straight and microfilament bundles were visible.

To investigate further the distribution of villin, fimbrin and actin in the visceral endoderm, Lowicryl-embedded sections of the visceral endoderm were stained with antibodies to villin, fimbrin or actin, followed by gold-labeled secondary antibodies. Fig. 6 shows the ultrastructural distribution of villin, fimbrin and actin in the microvilli and apical cytoplasm of 8·5 day and 10·5 day visceral endoderm. Although diffusely distributed in the apical cytoplasm, all three proteins were also associated with the cytoplasmic surface of the microvillar membranes in 8·5 day visceral endoderm. By day 10·5, these proteins were concentrated in the microvillus core. The change in distribution of villin,fimbrin and actin correlates with the appearance of rigid microvilli containing filament bundles (see Fig. 5C). A similar organization of villin and fimbrin was found in the kidney proximal tubule brush border (Rodman et al. 1986). Control experiments showed that when pre-immune primary antibody was used, labeling of sections with gold particles was diffuse and minimal (not shown).

Fig. 6.

Ultrastructural localization of villin (A), fimbrin (B), and actin (C) in the visceral endoderm microvilli from an 8·5 day (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 μm.

Fig. 6.

Ultrastructural localization of villin (A), fimbrin (B), and actin (C) in the visceral endoderm microvilli from an 8·5 day (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 μm.

Localization of villin and fimbrin in the developing intestine

The pattern of villin and fimbrin distribution in the developing intestine was similar to that seen in the visceral endoderm. Villin was first detected in the intestinal epithelium at day 10, after the embryonic endoderm had formed a tube and closed off from the visceral endoderm (Fig. 7). No villin was seen in the underlying mesenchyme or connective tissues. The villin-staining pattern in the intestinal cells at this stage of development was mottled and was localized to the cell boundaries. Fimbrin was more evenly distributed throughout the cell but was slightly more concentrated along the basal surface of the epithelium (Fig. 7A). By day 12·5, the intestine had become eliptical in cross section. During this time, villin became concentrated at the apical or luminal surface of the intestine. Fimbrin remained distributed in the cytoplasm and did not display apical localization until day 15·5, 2–3 days after the redistribution of villin. The distribution of villin and fimbrin at this stage of development resembles its localization in the adult gut (Fig. 7D and 7d).

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 comer of (c) points to smooth muscle surrounding gut, which stains with anti-fimbrin antibodies. Bars, 50 μm.

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 comer of (c) points to smooth muscle surrounding gut, which stains with anti-fimbrin antibodies. Bars, 50 μm.

Ultrastructural morphology of the developing mouse intestine

Before day 16 of development, the apical surface of the intestinal epithelium was sparsely populated by microvilli, and those present were short and protruded from the cell at various angles (Fig. 8A and 8B). By day 16·5, there were numerous microvilli, each containing a bundle of microfilaments (Fig. 8C); however, compared with the microvilli of the adult brush border (Fig. 8D), the embryonic microvilli were short and their actin bundles did not extend into the apical cytoplasm. There also was no network of filaments or well-defined junctional complexes that delineated the terminal web. The apical accumulation of villin and fimbrin in the intestinal epithelium before the appearance of microvilli (see Fig. 7) may account for the compact morphology of the microvilli when they first appear in the gut.

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 pm.

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 pm.

Our results demonstrate that the microvillar core proteins villin and fimbrin are differentially localized during mouse development but display identical patterns of accumulation in both the extraembryonic visceral endoderm and the embryonic intestinal epithelium. Fimbrin is present in the preovulatory oocyte and is detected throughout development in various embryonic tissues.

In contrast, villin is first detected in the primitive endoderm before gastrulation and later in the intestinal epithelium after the gut has sealed into a tube. As these tissues differentiate, villin accumulates at the apical surface of the cells within 2-3 days after it first appears, and fimbrin redistributes to the apical surface 2-3 days later. A similar asynchrony in the redistribution of villin and fimbrin was observed by Shibayama et al. (1987) studying the chicken gut. The expression of villin during mouse development has also been examined by Maunoury et al. (1988). They also observe that villin first appears in the visceral endoderm followed later by the gut; and immunoblot and Northern blot analysis of extraembryonic and embryonic tissues revealed, respectively, a single polypeptide of 93 kd and an mRNA of 3·4 kb in length. Fig. 9 summarizes our findings and relates the appearance and distribution of villin and fimbrin to the differentiation of the visceral endoderm and the gut. Our results provide information about functional relationships between villin-containing cells derived from embryonic and extraembryonic lineages, and suggest a mechanism for the assembly of the microvillus cytoskeleton.

Fig. 9.

Time course of villin and fimbrin expression in the developing mouse, relating the appearance and distribution of these proteins to the differentiation of the visceral endoderm and the intestinal epithelium. Villin redistributes to the apical surface 2 – 3 days after it appears in the primitive endoderm and the gut; 2 – 3 days later fimbrin is localized to the apical surface in both of these tissues.

Fig. 9.

Time course of villin and fimbrin expression in the developing mouse, relating the appearance and distribution of these proteins to the differentiation of the visceral endoderm and the intestinal epithelium. Villin redistributes to the apical surface 2 – 3 days after it appears in the primitive endoderm and the gut; 2 – 3 days later fimbrin is localized to the apical surface in both of these tissues.

Villin is a marker of the visceral endoderm

The inner cell mass gives rise to the embryonic tissues, including the gut endoderm, whereas the surrounding primitive endoderm gives rise to two extraembryonic cell types: the visceral endoderm, which remains associated with the differentiating inner cell mass; and the parietal endoderm, which colonizes the inner surface of the trophectoderm (Gardner, 1983, 1984; Rossant, 1986). When Cockroft & Gardner (1987) examined the developmental potential of the visceral endoderm by injecting genetically marked visceral endoderm cells into blastocysts, they found the injected cells had differentiated into visceral and parietal endoderm but did not become part of the embryo. Lawson et al. (1986) and Lawson & Pederson (1987), injecting various cells of the postimplantation embryo with horseradish peroxidase, showed that the definitive endoderm arises from the embryonic ectoderm and that the visceral endoderm contributes few, if any, cells to the gut. The presence of villin in the visceral endoderm and its absence in the undifferentiated embryonic gut (see Fig. 3) is consistent with data showing that the visceral endoderm does not contribute cells to the fetal gut.

Our studies, and those of Maunoury et al. (1988), show that villin is a valuable marker for the visceral endoderm and can be used in conjunction with other markers to investigate the differentiation of extraembryonic tissues in the mouse embryo. For example, alpha-fetoprotein is detected in visceral endoderm cells that are in contact with the embryonic, but not the extraembryonic, ectoderm (Dziadek, 1978; Dziadek & Adamson, 1978). In comparison, ENDO C, a member of the cytokeratin family of intermediate filament proteins, is detected by the monoclonal antibody TROMA-3 only in parietal endoderm cells, whereas a second monoclonal antibody, TROMA-1, recognizes a different cytokeratin protein, ENDO A, in both parietal and visceral endoderm cells (Boiler & Kemler, 1983). These antibodies have been useful in monitoring the in vitro ‘transdifferentiation’ of the visceral endoderm into parietal endoderm (Casanova & Grabe!, 1988). In contrast to alpha-fetoprotein or the cytokeratin proteins, villin is a marker for the entire visceral endoderm. In this context, it would be interesting to examine whether villin expression is down-regulated during visceral endoderm transdifferentiation.

Role of villin and fimbrin in microvillus assembly

The identical sequence in the accumulation of villin and fimbrin at the apical surface in the visceral endoderm and in the intestinal epithelium (see Fig. 9) suggests that the two tissues employ a common mechanism for assembling the microvillar cytoskeleton during development. As discussed by Shibayama et al. (1987) for the chicken, this mechanism may first involve the growth of actin filaments from membrane nucleation sites and the cross-linking of the newly assembled filaments into bundles. This scheme is consistent with the order of appearance and actin-binding activities of villin and fimbrin. Villin, as a phospholipid-binding protein, might be bound to the membrane (Janmey & Matsudaira, 1988) and, as an actin filament-severing protein, can accelerate actin polymerization (Matsudaira & Janmey, 1988). The two activities suggest a membrane-associated actin-nucleating role for villin. Our immunoelectron micrographs of 8 · 5 day visceral endoderm microvilli (see Fig. 6) provide evidence for this postulated membrane association of villin during the early stages of microvillus assembly. A similar mechanism for microvilli assembly may operate in the sea urchin egg.

At fertilization, the egg microvilli rapidly elongate through a process mediated by polymerization of preexisting actin (reviewed by Schatten, 1982). In examining the cortex of the unfertilized sea urchin egg, Henson & Begg (1988) observed short actin filaments woven into a tight network, which they propose may provide nucleation sites for actin polymerization during microvilli growth.

In the second stage of microvillar biogenesis, fimbrin becomes concentrated at the membrane 2 –3 days after villin. The later accumulation of fimbrin may result from changes in actin filament concentration at the membrane. In the chicken gut, there is a dramatic increase in microvilli length late in development. Stid-will & Burgess (1986) have reported a shift in the monomeric to F-actin ratio from 3:7 to 1:1 just prior to this increase in microvilli length. In the undifferentiated cell, the actin concentration may be suboptimal for bundle formation. However, as more filaments become associated with the membrane, a critical threshold for bundle formation could be reached and actin filaments would become cross-linked. In examining the distribution of actin during development of the chick duodenum, Noda and Mitsui (1988) report that actin changes from being concentrated in areas involved in formation of the previllous ridge to the apical surface during microvilli assembly. Such a scheme might explain why fimbrin concentration at the membrane lags behind that of villin, but increases before the formation of microvilli in the developing gut is complete. Thus, the differences in localization may be due to different functions of villin and fimbrin.

We thank Drs Roger Pederson, Rudolph Jaenisch and Jim Casanova for helpful suggestions, and the members of Dr Rudolph Jaenisch’s laboratory, especially Ruth Curry and Doris Grotkopp, for providing expertise and mice. This investigation was supported by grants PO1 CA44704 and RO1 DK35306 from the National Institutes of Health.

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We apologize to the authors for the delay in publication of this paper due to problems in transmission to the Cambridge office.