Download Developmental regulation of villin gene expression in the epithelial

Development 115, 717-728 (1992)
Printed in Great Britain © The Company of Biologists Limited 1992
717
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
3Laboratoire d’Anatomie Pathologique, Hôpital Sainte-Anne, 1 rue Cabanis, 75674 Paris Cédex 14, France
Cédex 15, France and
*Corresponding author
Summary
The expression of villin, an actin-binding protein and
major structural component of the brush border of
specialized absorptive cells, was studied during mouse
embryogenesis. We show that the ontogeny of villin
expression is limited to the epithelial cell lineages of the
digestive and uro-genital tracts and accounts for the
tissue-specific expression observed in adult mice. This
spatiotemporal pattern of villin expression is distinctive
in sequence, intensity, regional distribution and polarization. During the development of the primitive gut, villin is
faintly and discontinuously expressed in the invaginating
foregut but it is expressed in every cell bordering the
hindgut pocket. Later, villin expression increases along
the developing intestine and concentrates in the brush
border of the epithelium bordering the villi. In gut derivatives, villin is present in liver and pancreas primordia
but only biliary and pancreatic cells maintain a faint
villin expression as observed in adults. In the urogenital
tract, mesonephric tubules are the first mesodermal
derived structures to express villin. This expression is
maintained in the ductuli efferentes, paradidymis and
epoöphoron. Villin then appears in the proximal
metanephric tubules and later increases and concentrates in the brush border of the renal proximal tubular
epithelial cells. Thus villin expression can be considered
as an early marker of the endodermal cell lineage during
the development of the digestive system. Conversely,
during the development of the excretory and genital
system, villin is only expressed after the
mesenchyme/epithelium conversion following the
appearance of tubular structures.
These observations emphasize the multiple levels of
regulation of villin gene activity that occur during mouse
embryogenesis and account for the strict pattern of
tissue-specific expression observed in adults. In the
future, regulatory elements of the villin gene may be used
to target the early expression of oncogenes to the digestive and urogenital tracts of transgenic mice.
Introduction
borders (Bretscher and Weber, 1979) and later shown to be
expressed in several evolutionarily distant animal species.
These observations are the result of numerous investigations
involving immunocytochemical (Reggio et al., 1982;
Dudouet et al., 1987; Figiel et al., 1987; Moll et al., 1987)
and molecular hybridization procedures (Pringault et al.,
1986; Boller et al., 1988). Villin is located in the core of actin
filaments supporting the microvillus membrane forming the
brush border as illustrated by ultrastructural studies
(Dudouet et al., 1987). The key function of villin in the
assembly of the brush border has been suggested by in vitro
experiments showing that villin selfassociates with preformed actin filaments and can sever or bundle actin filaments in a calcium-dependent manner (Glenney et al., 1981;
Matsudaira et al., 1985). In the chick embryo, villin localizes
Most of the tissue-specific proteins involved in highly
specialized cellular structures or metabolic functions can be
used as markers of cell differentiation. However, the corresponding genes are often switched on only at the onset of terminal differentiation and, consequently, their products are
not produced in the immature, determined precursor cells.
Concerning the digestive tract, previous studies have shown
that, in contrast to intestinal hydrolases, villin, a tissuespecific cytoskeletal protein, is already expressed within the
intestinal crypts in the proliferative stem cells (Robine et al.,
1985; Boller et al., 1988). This protein was thus shown to be
an early marker for committed intestinal cells.
Villin was first isolated from chicken intestinal brush
Key words: gut, endoderm differentiation, excretory system,
cytoskeleton.
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R. Maunoury and others
apically and associates with the cytoskeleton of short
microvilli at day 8 (Shibayama et al., 1987). Furthermore, a
recent study in which villin cDNA was transfected in the
CV1 cell line, which does not normally express this protein,
provides evidence that villin is involved in the induction of
microvillus growth, a phenomenon that is associated with the
reorganization of F-actin microfilaments (Friederich et al.,
1989). The elucidation of the primary structure of villin has
shown that villin belongs to a family of closely related Factin-severing proteins including gelsolin in higher eucaryotes, and fragmin and severin in lower eucaryotes (Arpin et
al., 1988; Bazari et al., 1988). Villin displays a large duplicated domain common to other actin-severing proteins and a
specific additional C-terminal domain, conferring the Factin-bundling activity to the molecule (Bretscher and
Weber, 1980; Glenney and Weber, 1981). Elucidation of the
villin gene structure (Pringault et al., 1991) has confirmed
that this protein has evolved in parallel with other severing
proteins, from a common ancestor by duplication and fusion
of specific domains.
The distribution of this protein in normal adult tissues has
been studied in detail demonstrating that villin expression is
restricted to some cell types of the digestive and urogenital
tracts which perform an absorptive role and/or exhibit welldeveloped microvilli at their surfaces (Robine et al., 1985;
Gröne et al., 1986; Osborn et al., 1988; Coudrier et al., 1988;
Elsässer et al., 1991).
In neoplastic tissues, villin expression is retained in most
tumors or cell lines derived from villin-positive epithelia of
the digestive and excretory tracts but has never been
observed in non-epithelial tumors such as sarcoma or lymphoma (Moll et al., 1987; Carboni et al., 1987; West et al.,
1988; Bacchi and Gown, 1991). This pattern of villin synthesis in normal adult tissues and carcinomas has practical
consequences in the differential diagnosis of human tumors.
For instance, as proposed by others for some tissue-specific
cytoskeletal proteins, the immunodetection of villin in
tumors and metastases can guide the pathologist to the
histological origin of carcinomas (Moll et al., 1987; Louvard
et al., 1989). Moreover, the detection of villin in serum of
patients has been proposed for the diagnosis and monitoring
of colorectal cancers (Dudouet et al., 1990).
Studies of the ontogeny of villin gene expression in the
developing mouse embryo should allow us to address a
number of unsolved questions concerning the molecular and
developmental biology of villin. For instance, how is this
tissue-specific regulation taking place? When is villin first
expressed during gut and kidney histogenesis? What is the
sequence of villin expression in the different epithelia that
produce it? How is villin regionally distributed and polarized
within epithelia when they mature? Does villin expression
correlate with defined developmental proccesses? Is there
unexpected transient synthesis of this protein in embryonic
tissues?
We have previously reported that, at the early postimplantation stage of mouse embryogenesis, villin is first detectable
in the primitive endoderm and persists in the extraembryonic
visceral endoderm of the yolk sac, until birth (Maunoury et
al., 1988); this result has been confirmed by Ezzell et al.,
(1989).
The present study extends our investigations to the mouse
embryo itself and illustrates villin synthesis during development of the gut and nephros anlagen and their derivatives. In
these tracts, villin is never found in non-epithelial cells at any
stage of development, a feature that correlates with the pattern of villin expression observed in the corresponding adult
tissues (either normal or neoplastic). Furthermore, villin
appears as an early marker of the primitive and definitive
endodermal cell lineage and as a marker of cells arising after
mesenchyme/epithelium conversion in the developing
kidney.
Materials and methods
Animals, antibodies and immunocytochemical procedures used
have been previously described elsewhere (Maunoury et al.,
1988). In brief, embryos obtained from 129/Sv mice with their
extraembryonic membranes were immediately fixed in a solution of
1% acetic acid in 95% ethanol for 1-2 days at 0°C and embedded in
paraffin. The stage of embryonic development as defined by Theiler
(1972) was determined in this study after examination of histological sections. Immunostaining on five micron thick sections was performed using the ABC procedure (Hsu et al., 1981) as modified by
Maunoury et al., (1988) with the following additional modifications. All incubations and rinses were carried out in phosphatebuffered saline (PBS) for 15 minutes each ; the biotin-labelled secondary antibody and peroxidase-labelled streptavidin were
purchased from Biogenex laboratories. The primary anti-villin antibodies were the rabbit anti-pig villin antibodies affinity purified on
pig villin used in the previous work ; in addition, a polyclonal antimouse villin antibody, affinity purified on mouse villin, has been
recently obtained in our laboratory in order to provide a homologous reagent with high affinity to mouse villin. For this purpose,
using the method described by Bretscher and Weber (1980) with
some modifications, the mucosa from adult mouse intestine were
scraped and snap frozen in liquid nitrogen. A fraction of 2 g of
mucosa was homogenized in 20 ml of buffer containing 5 mM TrisHCl (pH 7.5), 75 mM KCl, 4 mM MgCl2, 1 mM ethylene glycol bis
N, N, N′, N ′-tetraacetic acid (EGTA), 2% Triton X-100, 0.2 mM
adenosine 5′ triphosphate (ATP) and 0.4 mM dithiothreitol (DTT)
in the presence of a cocktail of protease inhibitors. After high-speed
centrifugation (100.000 g for 1 hour) over a sucrose gradient, the
supernatant fraction was dialyzed overnight against KTC buffer (10
mM Tris-HCl, pH 8, 20 mM KCl, 0.2 mM CaCl2, 2.5 mM
β-mercaptoethanol and 1 mM PMSF) and then applied to a
Q-sepharose chromatography column. The proteins were eluted
with a linear gradient of 20-500 mM KCl and dialyzed overnight
against 2 mM Tris-HCl, pH 8, 0.2 mM CaCl2, 0.5 mM β-mercaptoethanol, 0.2 mM ATP and 1 mM PMSF. The dialyzed solution
was applied to the immobilized DNAase I column prepared as
described by Bretscher (1986). Mouse villin was eluted with the
Ca2+ chelating buffer (20 mM Tris-HCl, pH 7.8, 150 mM NaCl, 5
mM EGTA and 1 mM PMSF). 100 µg of pure villin could be
recovered by this method and used either to immunize rabbits as
described by Louvard et al., (1982), or immobilized on activated
Ultrogel beads in order to immunopurify the polyclonal antibodies
obtained. The specificity of the rabbit anti-mouse villin antibodies
has been controlled by immunocytochemistry on mouse adult
tissues sections as well as by immunoreplica.
Negative controls were obtained by replacing the specific
antisera with normal non-immune sera; no labelling was observed,
indicating that the cell procedures and reagents used result in
specific labelling.
Comparative controls were made with a rabbit immunopurified
anti-frog muscle actin (generous gift of Dr A. M. Hill and Dr E.
Karsenti) and anti-Paramecium tubulin previously described
(Adoutte et al., 1985).
Villin in mouse development
Results
The earliest expression of villin in the visceral endoderm
during mouse embryogenesis has been previously reported
(Maunoury et al., 1988) and confirmed by Ezzell et al.,
(1989). Briefly, villin is initially detectable in the early
postimplantation stage (stage 8) in primitive endodermal
cells at the periphery of the egg cylinder and persists in the
extraembryonic visceral endoderm of the yolk sac until birth.
Distal endoderm of the yolk sac is always deprived of villin.
Stage 11
Our present results report yet uncharacterized stages and patterns of villin expression in the embryo itself. Expression
begins at the presomite stage corresponding to formation of
three separate cavities : the amniotic cavity, the exocoelom
and the ectoplacental cleft. In the embryonic area, a faint
villin staining was visible in thin or flattened endodermal
cells (Fig. 1).
Stage 12
A conspicuous characteristic of stage 12 embryos is the
deepening of the anterior intestinal portal region and concomitantly the formation of definitive endoderm. At the
beginning of this stage, only squamous endoderm remnant
cells remain positive for villin, and stipple unstained definitive endodermic cells (Fig. 2). The specificity of this faint
labelling is confirmed in the adjacent section where no staining of these cells is observed with a muscle actin antibody
used as a control. This reagent clearly labels the first heart
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
endoderm (or columnar endoderm) and thin or flattened
embryonic endoderm (or squamous endoderm). The tall columnar
cells contain the largest amounts of villin concentrated in the
apical microvilli, whereas a faint but delineated villin staining is
observed in the squamous thin endodermal cells. ac, amniotic
cavity; eca, ectoplacental cavity; eco, ectoplacental cone; ex,
exocoelom. Bar indicates 200 µm.
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rudiment cells which appear in the mesoderm surrounding
the front end of the embryo (Fig. 2, insert). Later in this stage,
epimyocardium develops rapidly with a very strong expression of muscle actin (Fig. 3, insert). Adjacent to this cellular
mass, in the invaginating foregut, villin remains in a few very
flattened epithelial cells and appears in a few thick cells of
definitive embryonic endoderm (Fig. 3). In the last part of
stage 12, just before the turning of the embryo, villin is discontinously expressed both in thick ventral and thin dorsal
walls of the foregut (Fig. 4A, B, C).
Stage 13
This stage is characterized by the turning of the embryo. The
rotation begins with the head and tail folds ; the mid-trunk
region remains initially in its original position, being bound
to the yolk sac. In the foregut, villin staining is restricted to a
few epithelial cells (Fig. 4D). In contrast, in the newly
formed hindgut, villin is expressed in each thick cell lining
the pocket in a pattern that delineates the apical borders of the
membranes (Fig. 4E).
Stage 14
The turning of the embryo is now complete and the midgut as
well as the vitellin duct are constituted (Fig. 5). Foregut and
hindgut form a continuous tube. In the ring area between
foregut and midgut, liver, gall bladder and pancreas anlagen
emerge and express villin in continuity with the caudal
hindgut, which elongates actively as far as the level of the
posterior neuropore (Fig. 5A-C). In the cranial direction,
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 septum transversum (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).
Stage 17-18
The short oesophagus continues into the large eccentric
stomach anlage. More caudally, in the region of the hepatic
diverticulum, the evaginations of both the ventral pancreas
and the gall bladder primordium are developing.
The mesonephros is now well vascularized. The Wolffian
duct has reached the cloacal wall and the ureteric bud begins
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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 µm.
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 µm.
to develop. Villin first appears in the mesonephric tubules
(Fig. 7) which are more convoluted than at the previous
stage.
Stage 19-20
At this stage, the gut has elongated and intestinal loops are
visible. The stomach is greatly distended and regional differences are visible in its epithelium. The two rudiments of the
pancreas are in contact with each other. Within the liver there
are megakaryocytes indicating the hematopoietic function of
this organ. In the actively developing duodenum, villin is
strongly expressed in the thick pseudostratified epithelium in
contrast with a weak staining in the liver (Fig. 8A). As soon
as the embryo elongates caudally, the hindgut increases in
length and terminates in a dilated blind end : the primitive
cloaca. Villin is present in endodermal cells lining the cloaca
(Fig. 8B) and persists in free cells into the cloacal cavity (Fig.
8B). At this stage, the embryo possesses portions of all three
embryonic excretory systems. Mesonephric tubules are
highly convoluted and are found adjacent to the gonad primordium. Villin is concentrated in the apical cytoplasm of
the monolayered epithelium lining these tubules (Fig. 8C).
The metanephros, still negative for villin, is well delineated
and has many secondary urethric buds.
Stage 22
The gut projects into the wide umbilical hernial sac. All the
mucosal cells lining the intestinal tube from the duodenum to
the rectum stain strongly for villin (Fig. 9A,B). Although
stained with a more moderate intensity, the glandular portion
of the stomach marks the cranial boundary of the villin
expression in the gut (Fig. 9C). Oesophageus as well as the
non-glandular stomach portion are unlabelled for villin. In
derivatives from the hepatopancreatic ring, villin expression
remains moderate and is polarized to the apical side of the
epithelia lining hepatic and pancreatic ducts of various sizes
(Fig. 9A,B).
In the metanephros, villin first appears in the proximal
tubule of the more mature nephrons confined in the pericenter of the organ (Fig. 9C). Villin is concentrated in the apical
cytoplasm of the monolayered epithelial cells which constitute the proximal convoluted tubules. Other nephron regions
such as collecting tubules and glomeruli with well-formed
Bowman’s capsules are unlabelled for villin (Fig. 9D).
Stage 23
The configuration of the intestinal tract and of the urogenital
system shows little change since stage 22, e.g. umbilical
hernia is still present. However, histogenetic changes occur
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
epithelium of the intestinal loops as far as the rectum.
The urogenital system has become separated from the
Villin in mouse development
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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 µm.
rectum during a preceding phase of development and villin is
undetectable in epithelia from bladder, ureter or urethra (no
shown).
The developing kidneys now contain centrally placed
glomeruli, which are more numerous that at day 14.
From day 14 to 15, the mouse thymus undergoes rapid histogenetic and organogenetic changes, disclosing the structure of the adult thymus and lying close to the pericardial
cavity. Each thymic lobe is divided into lobules which are as
yet unsegregated into medullary and cortical zones. Faint
villin-positive cells appear scattered through the entire organ
(Fig. 10A). During the following days until birth, the thymus
enlarges and the density of large villin-positive cells
decreases, by a “dilution effect” among an increasing lymphoid cell population. Under high magnification, villin-positive cells show epithelial and non-lymphoid characteristics
(Fig. 10B). Staining is never very intense but faint and concentrated in, or proximal to, an irregular cytoplasmic membrane. Often some cells are so weakly stained that photographs are unsuccessful. However, this pattern of villin
staining correlates with the currently accepted endodermal
origin of this epithelial cell population (Rugh, 1968). This
observation will be investigated in detail in further studies.
Stage 25
The abdominal cavity has now enlarged, so that the intestinal
loops can reposition since the umbilical hernia is reduced.
The small intestine now has longer, but still rather thick villi
covered by columnar villin-positive epithelium (Fig. 11).
In the liver, blood cell production is increasing and this organ
appears to be more hematopoietic than glycogenogenic with
numerous sinusoid and blood cells. The pancreas has finely
branched, glandular trees, with distinct lumina, and pancreatic islets are budding. A faint villin staining is observed both
in expanding exocrine glandular trees and nascent endocrine
islets (data not shown).
The kidneys still have a large peripheral metanephric
blastema. Near its center, many glomeruli are well developed
and the topography of the corresponding proximal tubules is
conspicuously defined by their selective and intense staining
for villin, which is concentrated in the differentiating brush
border.
At this stage, the anatomy of the mouse is essentially complete.
Stage 28 : postnatal development
INTESTINE
Up to birth, intestinal cells are able to divide along the whole
villus axis. After birth, the crypts are formed and they progressively compartmentalize the predifferentiated cells
which actively proliferate and undergo morphological and
functional maturation from the depth of the crypt to the villus
tip.
Sections of small intestine from the adult mouse cut along
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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 µm.
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 µm.
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 µm.
Villin in mouse development
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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, cloacal membrane;
hb, hindlimb bud; li, liver. Bars : (A) 1 mm, (B, C) 50 µm.
the crypt/villus axis show a labelling of epithelial cells but
not of the villus core. In the immature cells of the crypts a diffuse labelling was seen within the cytoplasm. In cells maturating along the villi, villin concentrates in the brush border
but is absent from mucin granules which appear as clear vesicles in the apex of epithelial cells (not illustrated).
LIVER
The most prominent change during prenatal development of
the liver is an increase in volume of the fetal organ, both in
number and volume of hepatocytes and in the decrease of the
hematopoietic cell compartment. During postnatal weeks,
the hepatic cords disappear and the hepatic parenchyma
becomes progressively lobulated.
In adult mouse liver, the intrahepatic and extrahepatic bile
ducts, and the gall bladder display a faint villin expression in
their epithelia, but bile canaliculi were never labelled (not
shown).
However, when we used villin antibodies under precisely
the same experimental conditions on sections of liver
obtained from adult pig, we have observed a clear delineation
of bile canaliculi as described by Bacchi et al., (1991) in
human normal liver.
PANCREAS
The pancreatic lumen becomes obvious at day 14 and
expands progressively by days 15-16. During the following
prenatal days, however, the luminal spaces diminish and at
birth, as in the adult, the pancreas possesses acini which have
a small lumen. In adult mouse pancreas, only occasional
acinar cells are weakly labelled and even when strongly concentated antibodies are used, villin labelling remains uncertain both for exocrine and endocrine cells. This is in contrast
to positive cells which line the large collecting ducts (not
shown). Those features are reminiscent of the observation
reported in human adult pancreas by Elsässer et al., (1991).
METANEPHROS
The renal blastema remains functional during the first two
postnatal weeks. During this period, the kidney increases in
size and doubles its volume. At 1 month, the kidney attains
the complete maturity of the adult animal. Villin is selectively expressed in the proximal convoluted tubules localized
in the cortex (Fig. 12A). Under high magnification, the now
fully differentiated brush border is associated with a typical
strong staining for villin concentrated at the apical pole of the
cells (Fig. 12B).
MESONEPHROS
Some mesonephric tubules persist and become part of the
duct system of the testis or remain as vestigial structures.
Epigenital tubules form the efferent ductules of the testis or
the epoöphoron in the mesovarium. The paragenital tubules
persist as two vestigial structures : the paradidymis and the
paraoöphoron, respectively in male and female.
In male mesonephric derived structures, we have observed
the villin staining in ductuli efferentes as previously reported
(Robine et al., 1985; Horvat et al., 1990) (not illustrated).
Furthermore, villin is also present both in apical and basolateral membranes of epithelial cells lining the irregular cavities
of the paradidymis (Fig. 12C).
In sections through the mesovarium, the vestigial
mesonephros, which constitutes the epoöphoron, appears as
dispersed villin-positive tubules surrounded by fibrous connective tissue (Fig. 12D). Phase-contrast microscopy or antibodies to Paramecium tubulin show epithelial ciliated cells
intermingled with villin-positive cells. The entire oviduct
and the uterine epithelium cells did not react with the antibodies to villin (not shown).
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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 µm, (D) 50 µm.
Fig. 10. (A) Nominal 15 day embryo, stage 23. (B) Newborn
mice, stage 27. (A) Cross section through the two thymic lobes
(tl). There is not yet clear delimitation between the medulla and
cortex of lobules and faintly villin-positive cells appear scattered
through the entire organ. (B) After birth, the thymus glands have
enlarged rapidly. At high magnification, a single villin-positive
cell appears as a voluminous cell with a large clear nucleus.
Staining is concentrated in the irregular cytoplasmic membrane.
Bars : (A) 100 µm, (B) 10 µm.
THYMUS
With increasing age, the thymic lobes increase in size and
cellularity. Trabeculae penetrate deeper into the lobes and
divide the organ into lobules and the medulla become more
branched and extended.
In postnatal and young adult mice, large villin-positive
cells are localized in thymic medulla and show the same
staining observed in neonatal stage as illustrated in Fig. 10B.
The same results were obtained in the young adult rat. Old
adult mice with regressing thymus were not tested.
Discussion
From the first steps of mouse development to the term of gestation, we report here villin gene expression in epithelial cell
lineages of the digestive and urogenital tracts. The pattern of
villin expression is characteristic in sequence, intensity,
regional distribution and polarization, and accounts for the
tissue-specific expression previously reported in mammalian
adult tissues (Robine et al., 1985).
Concerning early ontogenesis, our previous study
(Maunoury et al., 1988) has demonstrated that villin is first
detected at the early postimplantation stage where it is
restricted to the visceral endodermal cells at the periphery of
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
foregut and hindgut invaginate by day 8-9 of gestation, villin
is expressed in definitive endodermal cells. Each cell from
the hindgut displays a faint and diffuse labelling. In contrast,
only a few epithelial cells of the foregut present faint villin
staining. This might explain the contradictory result obtained
by Ezzell et al. (1989) showing the absence of villin in the
invaginating foregut. The pattern of villin staining (strong
and polarized in the fully differentiated visceral endoderm,
faint and diffuse in the developing hindgut and foregut) is
characteristic of the cell differentiation stage. This difference
allows us to distinguish the primitive from the definitive
endoderm. Immediately after complete turning of the
embryo, a sharp gradient of villin expression is established
along the primitive continuous tube formed by the foregut,
midgut and hindgut. As the hepatic diverticulum emerges by
9 days of gestation, the hepatic endodermal cells maintain
this villin expression. A similar observation has been found
in the pancreas and gall bladder anlagen.
Villin in mouse development
Fig. 11. Nominal 17 day embryo, stage 25. Transverse section
through caudal portion of abdomen. Umbilical hernia is now
withdrawn, the small intestine acquires its villous surface and
shows thick villi covered by strongly villin-positive epithelium. b,
bladder; hl, hindlimb. Bar : 1 mm.
The pattern of villin expression reported in this work in the
case of the developing mouse embryo could be of interest for
phylogenetic studies and comparative ontogenesis. Villin
can be used to identify initial definitive endodermal cells as
well as the emergence of their derivatives. In Xenopus laevis,
villin is a marker for the presumptive intestinal endoderm
and as in the mouse, is not expressed in the blastocyst (M.
Dauça, personal communication), suggesting that the restriction of villin expression to epithelial cell lineage of the digestive tract could be extended to the class of vertebrates. This
marker might be of particular interest to study early histogenesis in other developmental models such as the Zebrafish
for which molecular markers for early organogenesis are
scarce. In contrast, in invertebrates, the presence of villin in
the fertilized sea urchin egg has been recently reported by
Wang and Bonder (1991).
As development proceeds towards organogenesis characterized by complex differentiation events, dynamic and
tissue-specific villin gene regulation takes place. During the
elongation of the primitive tube, villin expression is abolished in the upper part of the gut, whereas it increases without
discontinuity along the entire intestinal part. The glandular
cells of the stomach and the cloacal cells form the boundaries
of this villin expression. Continuation of the intestinal morphogenetic movements leads to villus emergence by 16-18
days and afterwards to crypt formation during the early postnatal period. As this histological terminal differentiation proceeds, villin distribution within the intestinal cells becomes
typical of the adult differentiated status, i.e., diffuse with
moderate apical polarization in immature, proliferative cells
of the crypts and strongly polarized in brush borders of fully
differentiated cells lining the villi of the small intestine. This
is correlated with a 10-fold increase of the villin gene activity
along the crypt villus axis and in differentiating intestinal
cells in culture (Boller et al., 1988; Pringault et al., 1986).
725
During liver formation, a moderate level of villin expression is maintained in cells engaged in duct differentiation 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 compartment 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 implicated 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
and during terminal differentiation of liver parenchymal cells
and acinar cells of the exocrine pancreas, (iii) the enhancing
of expression in differentiated enterocytes. This highlights
similar mechanisms of regulation involved in the genes
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
(Tyner et al., 1990). A functional and structural study of the
villin promoter is currently underway. The comparison of
regulatory elements from genes expressed in the digestive
tract such as the human intestinal alcaline phosphatase gene
(Millan, 1987 ; Henthorn et al., 1988), the rat L-pyruvate
kinase gene (Cognet et al., 1987), the human apolipoprotein
A4 gene (Elshourbagy et al., 1987), the pig neutral aminopeptidase gene (Olsen et al., 1989) and the human fatty acidbinding protein gene (Sweetser et al., 1987) should be useful
for understanding the molecular genetic control of the development of the gastrointestinal tract.
In contrast, during urogenital organogenesis, a very different pattern of villin gene regulation is displayed. Villin is not
detected in the first stages of nephrogenesis leading to
pronephric, mesonephric and metanephric ducts. Villin first
appears in mesonephric tubules at stage 17-18. In adults,
moderate villin expression has been observed in the ductuli
efferentes, paradidymis and epoöphoron, which derive from
some persisting mesonephric tubules. The developing
metanephros remains negative for villin until stage 22, where
it first appears restricted to only the epithelial cells of the
proximal tubules from the more mature nephrons. These
cells are already engaged in their terminal differentiation and
villin is at once concentrated in the apical cytoplasm. Thus,
villin is a specific marker of the proximal tubules in the
developing nephron.
Our data on the distribution of villin at the apical faces of
transporting epithelia show that a high level of villin expression is associated with cells presenting a well-organized
brush border while a low level of villin expression is
observed in cells that do not display such subcellular structure. In this respect, it is worth recalling the morphogenetic
effect of villin when expressed in large amounts inducing the
growth of rudimentary microvilli found at the cell surface of
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 villinpositive 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 µm.
of plasma membrane organization is also associated with the
recruitment of actin from other microfilament structures, a
property that demonstrates the key role of villin in the modu-
lation of actin assembly. The morphogenetic activity of villin
also seems to occur during development and account in part
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 ontogenesis of the mouse is given in Fig. 13. This profile corresponds
to that already observed in adult human and has been shown
to be maintained during carcinogenic processes (Moll et al.,
1987; Carboni et al., 1987; West et al., 1988; Bacchi and
Gown, 1991). Only tumors and cell lines derived from villinpositive epithelia express this protein. However, villin has
been occasionally detected in tumors derived from tissues or
organs devoid of villin in their normal adult state, e.g.
endometrium, ovary and lung. As the present study shows
that the lung anlage, developing oviducts or endometrium
tissue are villin-negative, the unexpected expression in these
few adenocarcinomas cannot be explained by an occasional
reexpression of a fetal phenotype. However, during human
embryogenesis, the time course of development and
ontogeny of villin expression could present some divergences with those of the mouse.
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
normal and neoplasic tissues (Pringault et al., 1986; Boller et
al., 1988; Maunoury et al., 1988).
In consequence, regulatory elements of the villin gene
might be used to drive the expression of heterologous genes
in immature cells of the definitive endodermal lineage, in the
proliferative precursors of the adult intestinal crypts as well
as in differentiated cells from the small and large intestine
mucosa (Pringault, 1990). Murine models reproducing several steps of the progression of human colorectal cancers
could be obtained by targeting the associated oncogenes or
mutated tumor suppressor genes to colonocytes. Moreover,
new cell lines derived from the digestive tract could be established by targeting the SV40 T antigen to the precursors of
intestinal cells mucosa as well as to the biliary cells of the
liver and duct cells of the exocrine pancreas.
In this respect, knowledge of the complete profile of villin
expression during mouse embryogenesis will help to interpret the transgene expression as well as the occurrence of
tumors using mouse models.
We thank Mme J. Regère and Mr J. C. Benichou for the photographic work. We thank Dr R. Kelly for critically reading the manuscript. This study was supported by grants from the Institut
National de la Santé et de la Recherche médicale (no. 86-7008), the
Association pour la Recherche sur le Cancer (no. 6379), the Ligue
Nationale Française contre le Cancer, and the Fondation pour la
Recherche Médicale.
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(Accepted 14 April 1992) .