Development 115, 717-728 (1992) 717 Printed in Great Britain © The Company of Biologists Limited 1992

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

ROGER MAUNOURY1, SYLVIE ROBINE2,*, ERIC PRINGAULT2, NADINE LÉONARD3, JEAN ALFRED GAILLARD2 and DANIEL LOUVARD2

1Institut Cochin de Génetique Moléculaire, 22 rue Méchain, 75014 Paris, France 2Institut Pasteur, Departement de Biologie Moléculaire, URA-CNRS 1149, 25 rue du Docteur Roux, 75724 Paris Cédex 15, France and 3Laboratoire d’Anatomie Pathologique, Hôpital Sainte-Anne, 1 rue Cabanis, 75674 Paris Cédex 14, France *Corresponding author

Summary

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

Introduction borders (Bretscher and Weber, 1979) and later shown to be expressed in several evolutionarily distant animal species. Most of the tissue-specific involved in highly These observations are the result of numerous investigations specialized cellular structures or metabolic functions can be involving immunocytochemical (Reggio et al., 1982; used as markers of cell differentiation. However, the corre- Dudouet et al., 1987; Figiel et al., 1987; Moll et al., 1987) sponding genes are often switched on only at the onset of ter- and molecular hybridization procedures (Pringault et al., minal differentiation and, consequently, their products are 1986; Boller et al., 1988). Villin is located in the core of actin not produced in the immature, determined precursor cells. filaments supporting the membrane forming the Concerning the digestive tract, previous studies have shown brush border as illustrated by ultrastructural studies that, in contrast to intestinal hydrolases, villin, a tissue- (Dudouet et al., 1987). The key function of villin in the specific cytoskeletal protein, is already expressed within the assembly of the brush border has been suggested by in vitro intestinal crypts in the proliferative stem cells (Robine et al., experiments showing that villin selfassociates with pre- 1985; Boller et al., 1988). This protein was thus shown to be formed actin filaments and can sever or bundle actin fila- an early marker for committed intestinal cells. ments in a calcium-dependent manner (Glenney et al., 1981; Villin was first isolated from chicken intestinal brush Matsudaira et al., 1985). In the chick embryo, villin localizes 718 R. Maunoury and others apically and associates with the cytoskeleton of short embryo itself and illustrates villin synthesis during develop- microvilli at day 8 (Shibayama et al., 1987). Furthermore, a ment of the gut and nephros anlagen and their derivatives. In recent study in which villin cDNA was transfected in the these tracts, villin is never found in non-epithelial cells at any CV1 cell line, which does not normally express this protein, stage of development, a feature that correlates with the pat- provides evidence that villin is involved in the induction of tern of villin expression observed in the corresponding adult microvillus growth, a phenomenon that is associated with the tissues (either normal or neoplastic). Furthermore, villin reorganization of F-actin microfilaments (Friederich et al., appears as an early marker of the primitive and definitive 1989). The elucidation of the primary structure of villin has endodermal cell lineage and as a marker of cells arising after shown that villin belongs to a family of closely related F- mesenchyme/epithelium conversion in the developing actin-severing proteins including in higher eucary- kidney. otes, and fragmin and severin in lower eucaryotes (Arpin et al., 1988; Bazari et al., 1988). Villin displays a large dupli- Materials and methods cated domain common to other actin-severing proteins and a specific additional C-terminal domain, conferring the F- Animals, antibodies and immunocytochemical procedures used actin-bundling activity to the molecule (Bretscher and have been previously described elsewhere (Maunoury et al., Weber, 1980; Glenney and Weber, 1981). Elucidation of the 1988). In brief, embryos obtained from 129/Sv mice with their villin gene structure (Pringault et al., 1991) has confirmed extraembryonic membranes were immediately fixed in a solution of that this protein has evolved in parallel with other severing 1% acetic acid in 95% ethanol for 1-2 days at 0°C and embedded in proteins, from a common ancestor by duplication and fusion paraffin. The stage of embryonic development as defined by Theiler (1972) was determined in this study after examination of histologi- of specific domains. cal sections. Immunostaining on five micron thick sections was per- The distribution of this protein in normal adult tissues has formed using the ABC procedure (Hsu et al., 1981) as modified by been studied in detail demonstrating that villin expression is Maunoury et al., (1988) with the following additional modifica- restricted to some cell types of the digestive and urogenital tions. All incubations and rinses were carried out in phosphate- tracts which perform an absorptive role and/or exhibit well- buffered saline (PBS) for 15 minutes each ; the biotin-labelled sec- developed microvilli at their surfaces (Robine et al., 1985; ondary antibody and peroxidase-labelled streptavidin were Gröne et al., 1986; Osborn et al., 1988; Coudrier et al., 1988; purchased from Biogenex laboratories. The primary anti-villin anti- Elsässer et al., 1991). bodies were the rabbit anti-pig villin antibodies affinity purified on In neoplastic tissues, villin expression is retained in most pig villin used in the previous work ; in addition, a polyclonal anti- tumors or cell lines derived from villin-positive epithelia of mouse villin antibody, affinity purified on mouse villin, has been recently obtained in our laboratory in order to provide a homolo- the digestive and excretory tracts but has never been gous reagent with high affinity to mouse villin. For this purpose, observed in non-epithelial tumors such as sarcoma or lym- using the method described by Bretscher and Weber (1980) with phoma (Moll et al., 1987; Carboni et al., 1987; West et al., some modifications, the mucosa from adult mouse intestine were 1988; Bacchi and Gown, 1991). This pattern of villin synthe- scraped and snap frozen in liquid nitrogen. A fraction of 2 g of sis in normal adult tissues and carcinomas has practical mucosa was homogenized in 20 ml of buffer containing 5 mM Tris- consequences in the differential diagnosis of human tumors. HCl (pH 7.5), 75 mM KCl, 4 mM MgCl2, 1 mM ethylene glycol bis For instance, as proposed by others for some tissue-specific N, N, N¢, N¢-tetraacetic acid (EGTA), 2% Triton X-100, 0.2 mM cytoskeletal proteins, the immunodetection of villin in adenosine 5¢triphosphate (ATP) and 0.4 mM dithiothreitol (DTT) tumors and metastases can guide the pathologist to the in the presence of a cocktail of protease inhibitors. After high-speed histological origin of carcinomas (Moll et al., 1987; Louvard centrifugation (100.000 g for 1 hour) over a sucrose gradient, the et al., 1989). Moreover, the detection of villin in serum of supernatant fraction was dialyzed overnight against KTC buffer (10 mM Tris-HCl, pH 8, 20 mM KCl, 0.2 mM CaCl2, 2.5 mM patients has been proposed for the diagnosis and monitoring b-mercaptoethanol and 1 mM PMSF) and then applied to a of colorectal cancers (Dudouet et al., 1990). Q-sepharose chromatography column. The proteins were eluted Studies of the ontogeny of villin gene expression in the with a linear gradient of 20-500 mM KCl and dialyzed overnight developing mouse embryo should allow us to address a against 2 mM Tris-HCl, pH 8, 0.2 mM CaCl2, 0.5 mM b-mercap- number of unsolved questions concerning the molecular and toethanol, 0.2 mM ATP and 1 mM PMSF. The dialyzed solution developmental biology of villin. For instance, how is this was applied to the immobilized DNAase I column prepared as tissue-specific regulation taking place? When is villin first described by Bretscher (1986). Mouse villin was eluted with the 2+ expressed during gut and kidney histogenesis? What is the Ca chelating buffer (20 mM Tris-HCl, pH 7.8, 150 mM NaCl, 5 sequence of villin expression in the different epithelia that mM EGTA and 1 mM PMSF). 100 mg of pure villin could be produce it? How is villin regionally distributed and polarized recovered by this method and used either to immunize rabbits as described by Louvard et al., (1982), or immobilized on activated within epithelia when they mature? Does villin expression Ultrogel beads in order to immunopurify the polyclonal antibodies correlate with defined developmental proccesses? Is there obtained. The specificity of the rabbit anti-mouse villin antibodies unexpected transient synthesis of this protein in embryonic has been controlled by immunocytochemistry on mouse adult tissues? tissues sections as well as by immunoreplica. We have previously reported that, at the early postimplan- Negative controls were obtained by replacing the specific tation stage of mouse embryogenesis, villin is first detectable antisera with normal non-immune sera; no labelling was observed, in the primitive endoderm and persists in the extraembryonic indicating that the cell procedures and reagents used result in visceral endoderm of the yolk sac, until birth (Maunoury et specific labelling. Comparative controls were made with a rabbit immunopurified al., 1988); this result has been confirmed by Ezzell et al., anti-frog muscle actin (generous gift of Dr A. M. Hill and Dr E. (1989). Karsenti) and anti-Paramecium tubulin previously described The present study extends our investigations to the mouse (Adoutte et al., 1985). Villin in mouse development 719

Results rudiment cells which appear in the mesoderm surrounding the front end of the embryo (Fig. 2, insert). Later in this stage, The earliest expression of villin in the visceral endoderm epimyocardium develops rapidly with a very strong expres- during mouse embryogenesis has been previously reported sion of muscle actin (Fig. 3, insert). Adjacent to this cellular (Maunoury et al., 1988) and confirmed by Ezzell et al., mass, in the invaginating foregut, villin remains in a few very (1989). Briefly, villin is initially detectable in the early flattened epithelial cells and appears in a few thick cells of postimplantation stage (stage 8) in primitive endodermal definitive embryonic endoderm (Fig. 3). In the last part of cells at the periphery of the egg cylinder and persists in the stage 12, just before the turning of the embryo, villin is dis- extraembryonic visceral endoderm of the yolk sac until birth. continously expressed both in thick ventral and thin dorsal Distal endoderm of the yolk sac is always deprived of villin. walls of the foregut (Fig. 4A, B, C).

Stage 11 Stage 13 Our present results report yet uncharacterized stages and pat- This stage is characterized by the turning of the embryo. The terns of villin expression in the embryo itself. Expression rotation begins with the head and tail folds ; the mid-trunk begins at the presomite stage corresponding to formation of region remains initially in its original position, being bound three separate cavities : the amniotic cavity, the exocoelom to the yolk sac. In the foregut, villin staining is restricted to a and the ectoplacental cleft. In the embryonic area, a faint few epithelial cells (Fig. 4D). In contrast, in the newly villin staining was visible in thin or flattened endodermal formed hindgut, villin is expressed in each thick cell lining cells (Fig. 1). the pocket in a pattern that delineates the apical borders of the membranes (Fig. 4E). Stage 12 A conspicuous characteristic of stage 12 embryos is the Stage 14 deepening of the anterior intestinal portal region and con- The turning of the embryo is now complete and the as comitantly the formation of definitive endoderm. At the well as the vitellin duct are constituted (Fig. 5). Foregut and beginning of this stage, only squamous endoderm remnant hindgut form a continuous tube. In the ring area between cells remain positive for villin, and stipple unstained defini- foregut and midgut, liver, gall bladder and pancreas anlagen tive endodermic cells (Fig. 2). The specificity of this faint emerge and express villin in continuity with the caudal labelling is confirmed in the adjacent section where no stain- hindgut, which elongates actively as far as the level of the ing of these cells is observed with a muscle actin antibody posterior neuropore (Fig. 5A-C). In the cranial direction, used as a control. This reagent clearly labels the first heart only a short distance exists between the hepatic diverticulum and the lung rudiment. The stomach has not yet been formed and its presumptive area bounds the cranial limit for villin staining. For the first time, the mesonephric duct is apparent and is in continuity with the pronephric duct. At this and later stages of embryonic development, pro- and mesonephric ducts are unlabelled for villin.

Stage 15-16 The most conspicuous change at this stage is the formation of the lung anlage; however, villin is not observed in the early lung rudiment (not illustrated) nor later when this organ develops. Within the hepatoduodenal field, the hepatic cords which are weakly stained for villin start to invade the mesenchymal tissue of the (Fig. 6). Mesonephric tubules are well-formed and present a typical S-shaped pattern; no staining can yet be observed in this tissue (not illustrated). Fig. 1. Nominal 8 day embryo, presomite development, stage 11.5. Midfrontal section through cranial end of the notochord (arrow). Two arrowheads limit the visceral extraembryonic thick Stage 17-18 endoderm (or columnar endoderm) and thin or flattened embryonic endoderm (or squamous endoderm). The tall columnar The short oesophagus continues into the large eccentric cells contain the largest amounts of villin concentrated in the stomach anlage. More caudally, in the region of the hepatic apical microvilli, whereas a faint but delineated villin staining is diverticulum, the evaginations of both the ventral pancreas observed in the squamous thin endodermal cells. ac, amniotic and the gall bladder primordium are developing. cavity; eca, ectoplacental cavity; eco, ectoplacental cone; ex, The mesonephros is now well vascularized. The Wolffian exocoelom. Bar indicates 200 mm. duct has reached the cloacal wall and the ureteric bud begins 720 R. Maunoury and others

Fig. 2. Nominal 8 day embryo, first somite development, stage 12. Two consecutive sagittal sections stained with villin or actin antibodies. Villlin staining is observed in the remnant squamous endodermal cells, still present in number in the caudal part of the embryo. In the region of the foregut (arrow), the labelling is discontinuous and the definitive endodermal cells are yet unlabelled. Actin staining (insert) reveals the first heart rudiment cells in the cardiogenic area (arrow). al, allantois. Bar indicates 200 mm. Fig. 3. Nominal 8 day embryo, 2-3 somites, stage 12. Two consecutive sagittal sections at the level of the foregut pocket (f) stained with villin and actin antibodies. At this stage of development, in the invaginating foregut, scarce very flattened epithelial cells (arrows) but also a few thick cell (arrowhead) express villin. In the companion section, epimyocardium appears sharply delineated by staining strongly for actin (insert). ac, amniotic cavity; ex, excoelom; p, pericardial coelom. Bar indicates 100 mm. to develop. Villin first appears in the mesonephric tubules mucosal cells lining the intestinal tube from the duodenum to (Fig. 7) which are more convoluted than at the previous the rectum stain strongly for villin (Fig. 9A,B). Although stage. stained with a more moderate intensity, the glandular portion of the stomach marks the cranial boundary of the villin Stage 19-20 expression in the gut (Fig. 9C). Oesophageus as well as the non-glandular stomach portion are unlabelled for villin. In At this stage, the gut has elongated and intestinal loops are derivatives from the hepatopancreatic ring, villin expression visible. The stomach is greatly distended and regional differ- remains moderate and is polarized to the apical side of the ences are visible in its epithelium. The two rudiments of the epithelia lining hepatic and pancreatic ducts of various sizes pancreas are in contact with each other. Within the liver there (Fig. 9A,B). are megakaryocytes indicating the hematopoietic function of In the metanephros, villin first appears in the proximal this organ. In the actively developing duodenum, villin is tubule of the more mature nephrons confined in the pericen- strongly expressed in the thick pseudostratified epithelium in contrast with a weak staining in the liver (Fig. 8A). As soon ter of the organ (Fig. 9C). Villin is concentrated in the apical as the embryo elongates caudally, the hindgut increases in cytoplasm of the monolayered epithelial cells which consti- length and terminates in a dilated blind end : the primitive tute the proximal convoluted tubules. Other nephron regions . Villin is present in endodermal cells lining the cloaca such as collecting tubules and glomeruli with well-formed (Fig. 8B) and persists in free cells into the cloacal cavity (Fig. Bowman’s capsules are unlabelled for villin (Fig. 9D). 8B). At this stage, the embryo possesses portions of all three embryonic excretory systems. Mesonephric tubules are Stage 23 highly convoluted and are found adjacent to the gonad pri- mordium. Villin is concentrated in the apical cytoplasm of The configuration of the intestinal tract and of the urogenital the monolayered epithelium lining these tubules (Fig. 8C). system shows little change since stage 22, e.g. umbilical The metanephros, still negative for villin, is well delineated hernia is still present. However, histogenetic changes occur and has many secondary urethric buds. both in the small intestine where numerous and relatively thick villi develop, and in the large intestine where crypts are forming. Villin is intensely expressed in the proliferative Stage 22 epithelium of the intestinal loops as far as the rectum. The gut projects into the wide umbilical hernial sac. All the The urogenital system has become separated from the Villin in mouse development 721

Fig. 4. Turning of the embryo. Nominal 8 day embryo, 6 somites, stage 12. (A, B and C) Transverse sections, slightly oblique, to neural groove. The rotation has not yet begun, but an axial symmetry exists as, a straight line between anterior and posterior neural groove can be drawn (double arrow) (A, darkfield). In foregut, the floor is thickened while the dorsal epithelium remains thin. Villin is discontinuously expressed both in thick and thin walls of the foregut. In serial sections, apical concentration of villin in microvilli is prominent in cells facing the entire heart level (B and C). Nominal 8.5 day, 9-10 somites, stage 13. Both cranial and caudal parts of these embryos have started their rotations, coinciding with the midgut formation. (D) In foregut, villin staining is restricted to the scarce tall epithelial cells showing dense microvilli (arrow). (E) At the level of the hindgut villin is expressed in all the thick cells bordering the pocket just formed and delimited by the arrowheads. The staining delineates the apical portions of the epithelial hindgut cell membranes. al, allantois; *, foregut; h, heart; oa, omphalomesenteric artery. Bars, 100 mm. rectum during a preceding phase of development and villin is The small intestine now has longer, but still rather thick villi undetectable in epithelia from bladder, ureter or urethra (no covered by columnar villin-positive epithelium (Fig. 11). shown). In the liver, blood cell production is increasing and this organ The developing kidneys now contain centrally placed appears to be more hematopoietic than glycogenogenic with glomeruli, which are more numerous that at day 14. numerous sinusoid and blood cells. The pancreas has finely From day 14 to 15, the mouse thymus undergoes rapid his- branched, glandular trees, with distinct lumina, and pancre- togenetic and organogenetic changes, disclosing the struc- atic islets are budding. A faint villin staining is observed both ture of the adult thymus and lying close to the pericardial in expanding exocrine glandular trees and nascent endocrine cavity. Each thymic lobe is divided into lobules which are as islets (data not shown). yet unsegregated into medullary and cortical zones. Faint The kidneys still have a large peripheral metanephric villin-positive cells appear scattered through the entire organ blastema. Near its center, many glomeruli are well developed (Fig. 10A). During the following days until birth, the thymus and the topography of the corresponding proximal tubules is enlarges and the density of large villin-positive cells conspicuously defined by their selective and intense staining decreases, by a “dilution effect” among an increasing lym- for villin, which is concentrated in the differentiating brush phoid cell population. Under high magnification, villin-posi- border. tive cells show epithelial and non-lymphoid characteristics At this stage, the of the mouse is essentially com- (Fig. 10B). Staining is never very intense but faint and con- plete. centrated in, or proximal to, an irregular cytoplasmic mem- brane. Often some cells are so weakly stained that pho- tographs are unsuccessful. However, this pattern of villin Stage 28 : postnatal development staining correlates with the currently accepted endodermal INTESTINE origin of this epithelial cell population (Rugh, 1968). This Up to birth, intestinal cells are able to divide along the whole observation will be investigated in detail in further studies. villus axis. After birth, the crypts are formed and they pro- gressively compartmentalize the predifferentiated cells which actively proliferate and undergo morphological and Stage 25 functional maturation from the depth of the crypt to the villus The abdominal cavity has now enlarged, so that the intestinal tip. loops can reposition since the umbilical hernia is reduced. Sections of small intestine from the adult mouse cut along 722 R. Maunoury and others

Fig. 5. Nominal 9 day embryo, 13-14 somites, stage 14. Turning of the embryo is complete. Midgut and vitellin duct are well formed. (A) Sagittal section through area junction precisely between foregut and midgut. The ventral endoderm in the anterior intestinal portal region projects into the loose mesenchyme of the septum transversum (st) and forms a diverticulum (arrow) which with the dorsal endoderm constitutes the hepatopancreatic ring. Pseudostratified epithelium of this region express non-polarised villin. (B) Section showing the boundary between the primitive endoderm of the vitellin duct and the definitive endoderm of the midgut (arrows). Notice the distinct villin localization which is polarized to the apex of the vitellin duct epithelial cells while newly formed midgut cells present a more diffuse staining. (C) In hindgut caudal part, the staining for villin extends as far as the level of posterior neuropore. ao, aorta; b, blood island; ca, caudal artery; h, hindgut; star, intestinal portal; m, midgut; nt, neural tube; pn, posterior neuropore; st, septum transversum. Bars : (A, B, C) 100 mm.

Fig. 6. Nominal 10 day embryo, stage 16. Sagittal paramedian section through the hepato-pancreatic ring. A slight staining for villin is observed in hepatic cords (arrows) which are sprouting from the cranial region of the hepatic diverticulum and invading the loose mesenchyme of the septum transversum (st). Contrary to the cranial portion, the caudal portion of the hepatic diverticulum (arrowhead) projects no outgrowths in surrounding mesenchyme. Villin is intensely expressed in presumptive stomach (*) and duodenum areae (d) but neither detected in lung bud. Compare with Fig. 5A and note the narrowing of the duodenal portion. Bar : 100 mm. Fig. 7. Nominal 11 day embryo, stage 18. Section through four nephric units (arrows) in mesonephros showing tubules light-stained for villin. a, aorta; vcp, vein cardinalis posterior; c, coelom. Bar : 100 mm. Villin in mouse development 723

Fig. 8. Nominal 12 day embryo, stage 20. (A) Transverse section at the abdominal level show continuous villin expression in gut from stomach (s) through intestinal loops up to cloaca (arrow). (B) The cloacal cavity is delimited by pseudostratified epithelium and contains free cells positive for villin. (C) By 12 days the embryo displays pieces of each of the three embryonic excretory systems. At this stage, in transverse section just dorsal to the gonad primordium (g), only mesonephric tubules present a clear localized villin staining, (arrow). More posterior to the gonad, the cuboidal epithelium of the mesonephric Wolffian duct is negative (arrowhead). cm, ; hb, hindlimb bud; li, liver. Bars : (A) 1 mm, (B, C) 50 mm.

shown). Those features are reminiscent of the observation the crypt/villus axis show a labelling of epithelial cells but reported in human adult pancreas by Elsässer et al., (1991). not of the villus core. In the immature cells of the crypts a dif- fuse labelling was seen within the cytoplasm. In cells matu- METANEPHROS rating along the villi, villin concentrates in the brush border The renal blastema remains functional during the first two but is absent from mucin granules which appear as clear vesi- postnatal weeks. During this period, the kidney increases in cles in the apex of epithelial cells (not illustrated). size and doubles its volume. At 1 month, the kidney attains the complete maturity of the adult animal. Villin is selec- LIVER tively expressed in the proximal convoluted tubules localized The most prominent change during prenatal development of in the cortex (Fig. 12A). Under high magnification, the now the liver is an increase in volume of the fetal organ, both in fully differentiated brush border is associated with a typical number and volume of hepatocytes and in the decrease of the strong staining for villin concentrated at the apical pole of the hematopoietic cell compartment. During postnatal weeks, cells (Fig. 12B). the hepatic cords disappear and the hepatic parenchyma becomes progressively lobulated. MESONEPHROS In adult mouse liver, the intrahepatic and extrahepatic bile Some mesonephric tubules persist and become part of the ducts, and the gall bladder display a faint villin expression in duct system of the testis or remain as vestigial structures. their epithelia, but bile canaliculi were never labelled (not Epigenital tubules form the efferent ductules of the testis or shown). the epoöphoron in the mesovarium. The paragenital tubules However, when we used villin antibodies under precisely persist as two vestigial structures : the paradidymis and the the same experimental conditions on sections of liver paraoöphoron, respectively in male and female. obtained from adult pig, we have observed a clear delineation In male mesonephric derived structures, we have observed of bile canaliculi as described by Bacchi et al., (1991) in the villin staining in ductuli efferentes as previously reported human normal liver. (Robine et al., 1985; Horvat et al., 1990) (not illustrated). Furthermore, villin is also present both in apical and basolat- PANCREAS eral membranes of epithelial cells lining the irregular cavities The pancreatic lumen becomes obvious at day 14 and of the paradidymis (Fig. 12C). expands progressively by days 15-16. During the following In sections through the mesovarium, the vestigial prenatal days, however, the luminal spaces diminish and at mesonephros, which constitutes the epoöphoron, appears as birth, as in the adult, the pancreas possesses acini which have dispersed villin-positive tubules surrounded by fibrous con- a small lumen. In adult mouse pancreas, only occasional nective tissue (Fig. 12D). Phase-contrast microscopy or anti- acinar cells are weakly labelled and even when strongly con- bodies to Paramecium tubulin show epithelial ciliated cells centated antibodies are used, villin labelling remains uncer- intermingled with villin-positive cells. The entire oviduct tain both for exocrine and endocrine cells. This is in contrast and the uterine epithelium cells did not react with the anti- to positive cells which line the large collecting ducts (not bodies to villin (not shown). 724 R. Maunoury and others

Fig. 9. Nominal 14 day embryo, stage 22. (A) Transverse section showing the opening of the common bile duct (cbd) in the duodenum (d) through the duodenal papilla. Note the various levels of staining intensity for villin : strongest for duodenum, medium for duodenal papilla and weakly for common bile duct, hepatic ducts (hd) and intrahepatic ducts arising out of the lumina structures bordering the portal vein (arrows). (B) Same male embryo as in A. A more caudal section showing through the pancreas (p), the hindgut (hg) and the urogenital septum (us). In acini and pancreatic ductules, moderate expression of villin is concentrated in apical side. All serial sections between A and B covering the entire intestinal loops show strong staining for villin in the gut epithelium. In the same sections, genital and urinary ducts : Wolffian (stars), Müller (in center between the two stars) and ureter (u) are constantly negative for villin as illustrated in B. (C) A midsagittal section through the glandular portion of the stomach (st) showing moderate villin expression. Oesophagus (star) as the non glandular portion of the stomach, here not visible, are negative for villin. In metanephros (m) note the first expression of villin appearing in the proximal tubule issued from the more mature nephroi localized at the pericenter of the organ. (D) High magnification of the developing metanephros. At the urinary pole, the villin staining begins in the cells that constitute the origin of the proximal convoluted tubule, i.e., a funnel-shaped structure (arrow). The latter is in continuity with a thin tubule (double arrow) which becomes curved and then transforms in a thick tubule (triple arrow). Villin is continuously expressed as far as the tubule coils up in a new curve, and then delineating the junction area with distal tubule (star) communicating itself with a collecting tubule. At this stage of nephrogenic development, the juxtaglomerular apparatus appears and macula densa, negative for villin, is visible as a flattened tube at the vascular pole level (arrowhead). a, adrenal gland; pv, portal vein; li, liver; s, spleen. Bars : (A, B, C) 100 mm, (D) 50 mm.

Fig. 10. (A) Nominal 15 day embryo, stage 23. (B) Newborn regional distribution and polarization, and accounts for the mice, stage 27. (A) Cross section through the two thymic lobes tissue-specific expression previously reported in mammalian (tl). There is not yet clear delimitation between the medulla and adult tissues (Robine et al., 1985). cortex of lobules and faintly villin-positive cells appear scattered Concerning early ontogenesis, our previous study through the entire organ. (B) After birth, the thymus glands have enlarged rapidly. At high magnification, a single villin-positive (Maunoury et al., 1988) has demonstrated that villin is first cell appears as a voluminous cell with a large clear nucleus. detected at the early postimplantation stage where it is Staining is concentrated in the irregular cytoplasmic membrane. restricted to the visceral endodermal cells at the periphery of Bars : (A) 100 mm, (B) 10 mm. the egg cylinder. In this extraembryonic differentiated tissue, villin is located at the apex of the cells. The present report extends this study to the embryo proper. As soon as the THYMUS foregut and hindgut invaginate by day 8-9 of gestation, villin With increasing age, the thymic lobes increase in size and is expressed in definitive endodermal cells. Each cell from cellularity. Trabeculae penetrate deeper into the lobes and the hindgut displays a faint and diffuse labelling. In contrast, divide the organ into lobules and the medulla become more only a few epithelial cells of the foregut present faint villin branched and extended. staining. This might explain the contradictory result obtained In postnatal and young adult mice, large villin-positive by Ezzell et al. (1989) showing the absence of villin in the cells are localized in thymic medulla and show the same invaginating foregut. The pattern of villin staining (strong staining observed in neonatal stage as illustrated in Fig. 10B. and polarized in the fully differentiated visceral endoderm, The same results were obtained in the young adult rat. Old faint and diffuse in the developing hindgut and foregut) is adult mice with regressing thymus were not tested. characteristic of the cell differentiation stage. This difference allows us to distinguish the primitive from the definitive endoderm. Immediately after complete turning of the Discussion embryo, a sharp gradient of villin expression is established along the primitive continuous tube formed by the foregut, From the first steps of mouse development to the term of ges- midgut and hindgut. As the hepatic diverticulum emerges by tation, we report here villin gene expression in epithelial cell 9 days of gestation, the hepatic endodermal cells maintain lineages of the digestive and urogenital tracts. The pattern of this villin expression. A similar observation has been found villin expression is characteristic in sequence, intensity, in the pancreas and gall bladder anlagen. Villin in mouse development 725

During liver formation, a moderate level of villin ex- pression is maintained in cells engaged in duct differentia- tion of the biliary tree. In contrast to that observed in the hepatic cords, villin appears polarized to the apical pole of the epithelial cells forming the intrahepatic and extrahepatic ductal structures. In cells differentiating into hepatocytes, moderate villin expression persists only as long as hepatic cord structures exist, i.e. until the hematopoietic cell com- partment decreases in neonatal life. Villin is undetectable in adult mouse parenchymal hepatocyte. Thus, a differential regulation of villin gene expression arises in these two hepatic epithelia which derive from common precursor cells. A similar observation was found in the developing pancreas : villin gene expression is down-regulated in acinar cells but continues to be active in duct cells. Multiple levels of villin gene regulation are thus impli- cated in the histogenesis of the digestive tract : (i) the switch on from the outset of primitive and definitive endodermal cells, (ii) the silencing in the upper part of the primitive gut Fig. 11. Nominal 17 day embryo, stage 25. Transverse section and during terminal differentiation of liver parenchymal cells through caudal portion of abdomen. Umbilical hernia is now and acinar cells of the exocrine pancreas, (iii) the enhancing withdrawn, the small intestine acquires its villous surface and of expression in differentiated enterocytes. This highlights shows thick villi covered by strongly villin-positive epithelium. b, similar mechanisms of regulation involved in the genes bladder; hl, hindlimb. Bar : 1 mm. encoding for villin and alpha-feto-protein. For instance, the latter is also expressed in visceral endoderm, fetal gut and fetal liver but restricted in adults to enteroendocrine cells The pattern of villin expression reported in this work in the (Tyner et al., 1990). A functional and structural study of the case of the developing mouse embryo could be of interest for villin promoter is currently underway. The comparison of phylogenetic studies and comparative ontogenesis. Villin regulatory elements from genes expressed in the digestive can be used to identify initial definitive endodermal cells as tract such as the human intestinal alcaline phosphatase gene well as the emergence of their derivatives. In Xenopus laevis, (Millan, 1987 ; Henthorn et al., 1988), the rat L-pyruvate villin is a marker for the presumptive intestinal endoderm kinase gene (Cognet et al., 1987), the human apolipoprotein and as in the mouse, is not expressed in the blastocyst (M. A4 gene (Elshourbagy et al., 1987), the pig neutral amino- Dauça, personal communication), suggesting that the restric- peptidase gene (Olsen et al., 1989) and the human fatty acid- tion of villin expression to epithelial cell lineage of the diges- binding protein gene (Sweetser et al., 1987) should be useful tive tract could be extended to the class of vertebrates. This for understanding the molecular genetic control of the devel- marker might be of particular interest to study early histo- opment of the gastrointestinal tract. genesis in other developmental models such as the Zebrafish In contrast, during urogenital organogenesis, a very differ- for which molecular markers for early organogenesis are ent pattern of villin gene regulation is displayed. Villin is not scarce. In contrast, in invertebrates, the presence of villin in detected in the first stages of nephrogenesis leading to the fertilized sea urchin egg has been recently reported by pronephric, mesonephric and metanephric ducts. Villin first Wang and Bonder (1991). appears in mesonephric tubules at stage 17-18. In adults, As development proceeds towards organogenesis charac- moderate villin expression has been observed in the ductuli terized by complex differentiation events, dynamic and efferentes, paradidymis and epoöphoron, which derive from tissue-specific villin gene regulation takes place. During the some persisting mesonephric tubules. The developing elongation of the primitive tube, villin expression is abol- metanephros remains negative for villin until stage 22, where ished in the upper part of the gut, whereas it increases without it first appears restricted to only the epithelial cells of the discontinuity along the entire intestinal part. The glandular proximal tubules from the more mature nephrons. These cells of the stomach and the cloacal cells form the boundaries cells are already engaged in their terminal differentiation and of this villin expression. Continuation of the intestinal mor- villin is at once concentrated in the apical cytoplasm. Thus, phogenetic movements leads to villus emergence by 16-18 villin is a specific marker of the proximal tubules in the days and afterwards to crypt formation during the early post- developing nephron. natal period. As this histological terminal differentiation pro- Our data on the distribution of villin at the apical faces of ceeds, villin distribution within the intestinal cells becomes transporting epithelia show that a high level of villin ex- typical of the adult differentiated status, i.e., diffuse with pression is associated with cells presenting a well-organized moderate apical polarization in immature, proliferative cells brush border while a low level of villin expression is of the crypts and strongly polarized in brush borders of fully observed in cells that do not display such subcellular struc- differentiated cells lining the villi of the small intestine. This ture. In this respect, it is worth recalling the morphogenetic is correlated with a 10-fold increase of the villin gene activity effect of villin when expressed in large amounts inducing the along the crypt villus axis and in differentiating intestinal growth of rudimentary microvilli found at the cell surface of cells in culture (Boller et al., 1988; Pringault et al., 1986). transfected fibroblasts (Friederich et al., 1989). This change 726 R. Maunoury and others

Fig. 12. Villin localization in tissues of the adult mouse. (A-B) Kidney. (A) In a longitudinal section of the entire organ, a strong villin staining delineates the kidney cortex (c). Kidney medulla (m), papilla (p), fat and vessels of the hilus (h) as adrenal gland (a) were negative. (B) High magnification of the same preparation as A. Villin is concentrated in the dense brush border of the proximal convoluted tubules. In the center of the picture, a section through a renal corpuscule shows the urinary pole (arrowhead) with villin- positive prismatic cells in continuity with those of the corresponding proximal tubule. On the opposite side (arrow), the vascular pole elements are negative for villin. (C-D) Evidence of villin in two vestigial mesonephrotic structures in male and female adult mouse : the paradidymis and the epoöphoron. (C) The entire paradidymis appears as a small structure of about 0.2 mm in diameter, surrounded by fibrous connective tissue (ct). In the center, irregular communicating cavities are delimited by a continuous epithelium uniformly stained while the adjacent epididymis duct wall (top right) is negative. (D) Mesonephric remnants so-called the epoöphoron persist as dispersed tubular structures surrounded by fibrous connective tissue. Villin immunoreactivity is seen in epithelial cells lining the tubules. Phase contrast (insert) shows that the epithelial ciliated cells (arrows) were negative for villin. Bars : (A) 1 mm, (B, C, D) 50 mm. of plasma membrane organization is also associated with the lation of actin assembly. The morphogenetic activity of villin recruitment of actin from other microfilament structures, a also seems to occur during development and account in part property that demonstrates the key role of villin in the modu- for the brush border assembly. Villin in mouse development 727

Fig. 13. General view of villin expression during ontogenesis of the mouse.

A general view of villin gene expression during ontogen- could be obtained by targeting the associated oncogenes or esis of the mouse is given in Fig. 13. This profile corresponds mutated tumor suppressor genes to colonocytes. Moreover, to that already observed in adult human and has been shown new cell lines derived from the digestive tract could be estab- to be maintained during carcinogenic processes (Moll et al., lished by targeting the SV40 T antigen to the precursors of 1987; Carboni et al., 1987; West et al., 1988; Bacchi and intestinal cells mucosa as well as to the biliary cells of the Gown, 1991). Only tumors and cell lines derived from villin- liver and duct cells of the exocrine pancreas. positive epithelia express this protein. However, villin has In this respect, knowledge of the complete profile of villin been occasionally detected in tumors derived from tissues or expression during mouse embryogenesis will help to inter- organs devoid of villin in their normal adult state, e.g. pret the transgene expression as well as the occurrence of endometrium, ovary and lung. As the present study shows tumors using mouse models. that the lung anlage, developing oviducts or endometrium tissue are villin-negative, the unexpected expression in these We thank Mme J. Regère and Mr J. C. Benichou for the photo- few adenocarcinomas cannot be explained by an occasional graphic work. We thank Dr R. Kelly for critically reading the man- uscript. This study was supported by grants from the Institut reexpression of a fetal phenotype. However, during human National de la Santé et de la Recherche médicale (no. 86-7008), the embryogenesis, the time course of development and Association pour la Recherche sur le Cancer (no. 6379), the Ligue ontogeny of villin expression could present some diver- Nationale Française contre le Cancer, and the Fondation pour la gences with those of the mouse. Recherche Médicale. Previous studies have suggested that the regulation of the villin synthesis was controlled at the transcriptional level in embryonic primitive and definitive endoderm, and in adult References normal and neoplasic tissues (Pringault et al., 1986; Boller et al., 1988; Maunoury et al., 1988). Adoutte, A., Claisse, M., Maunoury, R. and Beisson, J. (1985). Tubulin evolution : ciliate-specific epitopes are conserved in the ciliary tubulin In consequence, regulatory elements of the villin gene of metazoa. J. Mol. Evol. 22, 220-229. might be used to drive the expression of heterologous genes Arpin, M., Pringault, E., Finidori, J., Garcia, A., Jeltsch, J-M., in immature cells of the definitive endodermal lineage, in the Vandekerckhove, J. and Louvard, D. (1988). Sequence of human proliferative precursors of the adult intestinal crypts as well villin : a large duplicated domain homologous with other actin-severing as in differentiated cells from the small and large intestine proteins and a large unique small carboxy-terminal domain related to villin specificity. J. Cell Biol. 107, 1759-1766. mucosa (Pringault, 1990). Murine models reproducing sev- Bacchi, C. E. and Gown, A. M. (1991). Distribution and pattern of eral steps of the progression of human colorectal cancers expression of villin, a gastrointestinal-associated cytoskeletal protein, in 728 R. Maunoury and others

human carcinomas : A study employing paraffin-embedded tissue. Lab. Horvat, B., Osborn, M. and Damjanov, I. (1990). Expression of villin in Invest. 64, 418-424. the mouse oviduct and the seminiferous ducts. Histochemistry 93, 661- Bazari, W. L., Matsudaira, P., Wallek, M., Smeal, T., Jakes, R. and 663. Ahmed, Y. (1988). Villin sequence and peptide map identify six Hsu, S. M., Raine, L. and Fanger, H. (1981). Use of avidin-biotin- homologous domains. Proc. Natl. Acad. Sci. USA 85, 4986-4990. peroxidase complex (ABC) in immunoperoxydase techniques : A Boller, K., Arpin, M., Pringault, E., Mangeat, P. and Reggio, H. comparison between ABC and unlabeled antibody (PAP) procedures. (1988). Differential distribution of villin and villin mRNA in mouse J. Histochem. Cytochem. 29, 577-580. intestinal epithelial cells. Differentiation 39, 51-57. Louvard, D., Reggio, H. and Warren, G. (1982). Antibodies to the golgi Bretscher, A. and Weber, K. (1979). Villin : the major microfilament- complex and the rough endoplasmic reticulum. J. Cell Biol. 92, 92-107. associated protein of the intestinal microvillus. Proc. Natl. Acad. Sci. Louvard, D., Arpin, M., Coudrier E., Dudouet, B., Finidori, J., USA 76, 2321-2325. Garcia, A., Huet, C., Maunoury, R., Pringault, E., Robine, S. and Bretscher, A. and Weber, K. (1980). Villin is a major protein of the Sahuquillo-Merino, C. (1989). Use of villin for the histopathological microvillus cytoskeleton which binds both G and F actin in a calcium- and serological diagnosis of digestive cancers. In Cytoskeletal Proteins dependent manner. Cell 20, 839-847. in Tumor Diagnosis. Current Communications in Molecular Biology, Bretscher, A. (1986). Purification of the intestinal microvillus CSH 201-213. cytoskeletal proteins villin, fimbrin, and ezrin. In Methods in Matsudaira, P., Jakes, R. and Walker, J. E. (1985). A gelsolin-like Enzymology (ed. R.B. Vallee), vol. 134, pp. 24-37. Orlando, Florida : Ca2+ dependent actin-binding domain in villin. Nature 315, 248-250. Academic Press. Maunoury, R., Robine, S., Pringault, E., Huet, C., Guénet, J. L., Carboni, J. M., Howe, C. L., West, A. B., Barwick, K. W., Mooseker, Gaillard, J. A. and Louvard, D. (1988). Villin expression in the M. S. and Morrow, J. S. (1987). Characterization of intestinal brush visceral endoderm and in the gut anlage during early mouse border cytoskeletal proteins of normal and neoplastic human epithelial embryogenesis. EMBO J. 7, 3321-3329. cells : a comparison with the avian brush border. Am. J. Pathol. 129, Millan, J. L. (1987). Promoter structure of the human intestinal alkaline 589-600. phosphatase gene. Nucl. Acids Res. 15, 10599. Cognet, M., Lone, Y. C., Vaulont, S., Kahn, A. and Marie, J. (1987). Moll, R., Robine, S., Dudouet, B. and Louvard, D. (1987). Villin: a Structure of the rat L-type pyruvate kinase gene. J. Mol. Biol. 196, 11- cytoskeletal protein and a differentiation marker expressed in some 25. human adenocarcinomas. Virchows Arch. B 54, 155-169. Coudrier, E., Kerjaschki, D. and Louvard, D. (1988). Cytoskeleton Olsen, J., Sjöström, H. and Noren, O. (1989). Cloning of the pig organization and submembranous interactions in intestinal and renal aminopeptidase N gene. FEBS Letters 251, 275-281. brush borders. Kidney International 34, 309-320. Osborn, M., Mazzoleni, G., Santini, D., Marrano, D., Martinelli, G. Dudouet, B., Robine, S., Huet, C., Sahuquillo-Merino, C., Blair, L., and Weber, K. (1988). Villin, intestinal brush border hydrolases and Coudrier, E. and Louvard, D. (1987). Changes in villin synthesis and keratin polypeptides in intestinal metaplasia and gastric cancer ; an subcellular distribution during intestinal differentiation of HT29-18 immunohistologic study emphasizing the different degrees of intestinal clones. J. Cell Biol. 105, 365-369. and gastric differentiation in signet ring cell carcinomas. Virchows Dudouet, B., Jacob, L., Beuzeboc, P., Magdelenat, H., Robine, S., Arch. A 413, 303-312. Chapuis, Y., Christoforov, B. Cremer, G. A., Pouillard, P., Pringault, E., Arpin, M., Garcia, A., Finidori, J. and Louvard, D. Bonnichon, P., Pinon, F., Salmon, R. J., Pointereau-Bellanger, A., (1986). A human villin cDNA clone to investigate the differentiation of Bellanger, J., Maunoury, M. T. and Louvard, D. (1990). Presence of intestinal and kidney cells in vivo and in culture. EMBO J. 5, 3119- villin, a tissue-specific cytoskeletal protein, in sera of patients and an 3124. initial clinical evaluation of its value for the diagnosis and follow-up of Pringault, E. (1990). Regulation d’un gène marqueur de la différenciation colorectal cancers. Cancer Research 50, 438-443. des cellules intestinales : la villine. Annales de l’Inst. Pasteur 2, 120- Elsässer, H. P., Klöppel, G., Mannherz, H. G., Flocke, K., and Kern, 126. H. F. (1991). Immunohistochemical demonstration of villin in the Pringault, E., Robine, S. and Louvard, D. (1991). Structure of the normal human pancreas and in chronic pancreatitis. Histochemistry 95, human villin gene. Proc. Natl. Acad. Sci. U.S.A. 88, 10811-10815. 383-390. Reggio, H. E., Coudrier, E. and Louvard, D. (1982). Surface and Elshourbagy, N. A., Walker, D. W., Paik, Y-K., Boguski, M. S., cytoplasmic domains in polarized epithelial cells. In Membranes in Freeman, M., Gordon, J. I. and Taylor, J. M. (1987). Structure and Growth and Development. pp. 89-105. New York : Alan R. Liss, Inc. expression of the human apolipoprotein A-IV gene. J. Biol. Chem. 262, Robine, S., Huet, C., Moll, R., Sahuquillo-Merino, C., Coudrier, E., 7973-7981. Zweibaum, A. and Louvard, D. (1985). Can villin be used to identify Ezzell, R. M., Chafel, M. M. and Matsudaira, P. T. (1989). Differential malignant and undifferentiated normal digestive epithelial cells ? Proc. localization of villin and fimbrin during development of the mouse Natl. Acad. Sci. USA 82, 8488-8492. visceral endoderm and intestinal epithelium. Development 106, 407- Rugh, R. (1968). The Mouse : Its Reproduction and Development. 419. Minneapolis, MN : Burgess Publishing Company. Figiel, A., Schilt, J., Dudouet, B., Robine, S. and Dauça, M. (1987). A 2 Shibayama, T., Carboni, J. M. and Mooseker, M. S. (1987). Assembly dimensional gel analysis of proteins expressed during the of the intestinal brush border : appearance and redistribution of differentiation of the amphibian intestinal epithelium with special microvillar core proteins in developing chick enterocytes. J. Cell Biol. emphasis on villin expression and localization. Differentiation 36, 116- 105, 335-344. 124. Sweetser, D.,A., Birkenmeier, E. H., Klisak, I. J., Zollman, S., Friederich, E., Huet, C., Arpin, M. and Louvard, D. (1989). Villin Sparkes, R. S., Mohandas, T., Lusis, A. J. and Gordon, J. I. (1987). induces microvilli growth and actin redistribution in transfected The human and rodent intestinal fatty acid binding protein genes. J. fibroblasts. Cell 59, 461-475. Biol. Chem. 262, 16060-16071. Glenney, Jr., J. R. and Weber, K. (1981). Calcium control of Theiler, K. (1972). The House Mouse. Berlin : Springer Verlag. microfilaments : uncoupling of the F-actin-severing and -bunding Tyner, A. L., Godbout, R., Compton, R. S. and Tilghman, S. M. activity of villin by limited proteolysis in vitro. Proc. Natl. Acad. Sci. (1990). The ontogeny of a-fetoprotein gene expression in the mouse U.S.A. 78, 2810-2814. gastrointestinal tract. J. Cell Biol. 110, 915-927. Glenney, Jr., J. R., Geisler, N., Kaulfus, P. and Weber, K. (1981). Wang, F-S. and Bonder, E. M. (1991). Sea urchin egg villin : Demonstration of at least two different actin-binding sites in villin, a identification of villin in a non-epithelial cell from an invertebrate calcium-regulated modulator of F-actin organization. J. Biol. Chem. species. J. Cell Science 100, 61-71. 256, 8156-8161. West, A. B., Isaac, C. A., Carboni, J. M., Morrow, J. S., Mooseker, M. Gröne, H-J., Weber, K., Helmchen, U. and Osborn, M. (1986). Villin - S. and Barwick, K. W. (1988). Localization of villin, a cytoskeletal a marker of brush border differentiation and cellular origin in human protein specific to microvilli, in human ileum and colon and in colonic renal cell carcinomas. Am. J. Pathol. 124, 294-302. neoplasms. Gastroenterology 94, 343-352. Henthorn, P. S., Raducha, M., Kadesch, T., Weiss, M. J. and Harris, H. (1988). Sequence and characterization of the human intestinal alkaline phosphatase gene. J. Biol. Chem. 263, 12011-12019. (Accepted 14 April 1992).