Download Localization of the Wilms` tumour protein WT1 in avian embryos

Cell Tissue Res (2001) 303:173–186
DOI 10.1007/s004410000307
REGULAR ARTICLE
R. Carmona · M. González-Iriarte
J.M. Pérez-Pomares · R. Muñoz-Chápuli
Localization of the Wilms’ tumour protein WT1 in avian embryos
Received: 3 August 2000 / Accepted: 9 October 2000 / Published online: 19 December 2000
© Springer-Verlag 2000
Abstract The Wilms’ tumour suppressor gene WT1 encodes a zinc-finger transcription factor which is essential
for the development of kidney, gonads, spleen and adrenals. WT1-null embryos lack all of these viscerae and
they also show a thin ventricular myocardium and unexpectedly die from cardiac failure between 13 and 15 days
post coitum. We studied the localization of the WT1 protein in chick and quail embryos between stages HH18
and HH35. In early embryos, WT1 protein was located
in specific areas of the coelomic mesothelium adjacent to
the nephric ducts, the myocardium or the primordia of
the endodermal organs (gut, liver and lungs). These mesothelial areas also showed localized expression of Slug,
a zinc-finger transcription factor involved in epithelialmesenchymal transitions. WT1+ mesenchymal cells were
always found below the immunoreactive mesothelial areas, either forming a narrow band on the surface of the
endodermal organs (gut, liver and lungs) or migrating
throughout the mesodermal organs (mesonephros, metanephros, gonads, spleen and heart). In the developing
heart, the invasion of WT1+ cells started at stage HH26,
and all the ventricular myocardium was pervaded by
these cells, presumably derived from the epicardium, at
HH30. We suggest that WT1 is not required for the epithelial-mesenchymal transition of the coelomic mesothelium, but it might be a marker of the mesothelial-derived
cells, where this protein would be acting as a repressor
of the differentiation.
This work was supported by grants PM98-0219 and 1FD97-0693
(Ministerio de Educación y Cultura, Spain). Mauricio González is
the recipient of a fellowship from Ministerio de Educación y Cultura
R. Carmona · M. González-Iriarte · J.M. Pérez-Pomares
R. Muñoz-Chápuli (✉)
Department of Animal Biology, Faculty of Science,
University of Málaga, 29071 Málaga, Spain
e-mail: [email protected]
Tel.: +34-952131853, Fax: +34-952132000
J.M. Pérez-Pomares
Department of Anatomy and Cell Biology,
Medical University of South Carolina, Charleston, 29425 SC,
USA
Keywords Wilms’ tumour · WT1 · Mesothelium · Chick
embryo · Quail embryo
Introduction
The Wilms’ tumour gene codes for a zinc-finger transcription factor which has been involved in many normal
and pathological processes. The 10-exon WT1 gene contains two alternately spliced regions, thus encoding four
distinct protein isoforms (Haber et al. 1991). WT1 is essential for the development of the kidney and gonads
(Kreidberg et al. 1993), adrenals (Moore et al. 1999) and
spleen (Herzer et al. 1999). Mice homozygous for a
WT1 deletion lack these organs but they unexpectedly
die from cardiac failure between 13 and 15 days of gestation (Kreidberg et al. 1993), suggesting a role for WT1
in cardiac development.
Mutated forms of WT1 have been related to congenital abnormalities such as a chromosome 11p deletion
syndrome known as the WAGR syndrome (Wilms’ tumor, aniridia, genitourinary anomaly and mental retardation) (Brown et al. 1992; Haber and Housman 1992), the
Denys-Drash syndrome (Baird et al. 1992; Pelletier et al.
1991a) as well as several kinds of acute myeloid leukemias (King-Underwood et al. 1996; Bergmann et al.
1997).
It was initially thought that the functions of WT1
were specifically related to the normal differentiation of
the kidneys and gonads (Pritchard-Jones et al. 1990).
However, in recent years, a large number of in vitro reporter assays have involved WT1 in a wide spectrum of
fundamental cell processes, including control of proliferation and differentiation mainly through transcriptional
repression of genes of growth factors, their receptors,
and other transcription factors (reviewed in Little et al.
1999; Davies et al. 1999). However, it is possible that
most genes with WT1-responsive promoters are not regulated in vivo by WT1, and the sometimes conflicting results obtained might depend upon the experimental conditions (Reddy et al. 1995; Reddy and Licht 1996). Thus,
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the physiological functions of WT1 during the embryonic development remain uncertain.
Besides its functions as a transcription factor, WT1
has also been involved in RNA metabolism. In fact, the
WT1 isoform lacking the KTS (lysine-serine-threonine)
insertion binds RNA and shows a speckled pattern of
distribution which is thought to colocalize with spliceosomal proteins (Larsson et al. 1995; Kennedy et al. 1996;
Bardeesy and Pelletier 1998).
During normal development WT1 is expressed in a
dynamic and tissue-specific pattern in a specific population of neurones in the neural tube and in several mesoderm-derived tissues, namely in mesothelia (coelomic
epithelia), derivatives of the intermediate mesoderm
such as the meso- and metanephros, gonads and adrenals
(Pritchard-Jones 1990; Pelletier et al. 1991b; Armstrong
et al. 1993; Rackley et al. 1993), and the limbs (Moore et
al. 1998). These papers show localization of WT1
mRNA through in situ hybridization in mammalian embryos, but only limited data are available about the presence of WT1 protein during embryonic development
(Charles et al. 1997). On the other hand, the presence of
this important transcription factor has not yet been studied in the avian embryo, a very important system for descriptive and experimental embryology. It is important to
emphasize that data about the precise temporal and spatial localization of a transcription factor in embryonic
tissues where well-characterized developmental events
occur, can provide good insights into their possible functions.
Our aim was to study the localization of the WT1 protein by immunohistochemistry in embryos of chick and
quail, in order to test several hypotheses proposed about
the normal functions of the protein, such as its involvement in processes of transition between mesenchyme
and epithelium and vice versa. With this purpose we
have checked the presence, in the embryonic areas where
WT1 is expressed, of Slug, another zinc-finger transcription factor which is involved in the epithelial-mesenchymal transition (Nieto et al. 1994; Duband et al. 1995;
Savagner et al. 1997; Carmona et al. 2000).
Materials and methods
The animals used in our research program were handled in compliance with the international guidelines for animal care and welfare. Chick and quail eggs were kept in a rocking incubator at
38°C. The avian embryos were staged according to the Hamburger
and Hamilton (1951) stages of chick development.
The spatial and temporal immunoreactive pattern of WT1 was
studied in a sample consisting of 16 embryos of quail (Coturnix
coturnix japonica), which were collected at stages HH18–HH28,
and 8 embryos of chick (Gallus gallus), collected at stages
HH18–HH35. Mouse embryos, 11.5 days post coitum, were used
as positive controls.
For WT1 immunohistochemistry, the embryos were excised
and cryoprotected in 10%, 20% and 30% sucrose solutions, where
they were kept at 4°C until they sunk. Then, the embryos were
embedded in OCT and snap frozen in liquid-nitrogen-cooled isopentane. The frozen embryos were sectioned in a cryostate, and
14-µm sections were collected on poly-L-lysine-coated slides and
fixed for 10 min in 1:1 methanol acetone at –20°C. The sections
were then rehydrated in TRIS-phosphate-buffered saline (TPBS)
and the endogenous peroxidase activity was quenched by incubation for 30 min with 3% hydrogen peroxide in TPBS. After washing, non-specific binding sites were saturated for 30 min with 16%
sheep serum, 1% bovine serum albumin and 0.5% Triton X-100 in
TPBS (SBT). Endogenous biotin was blocked with the avidinbiotin blocking kit (Vector, Burlingame, CA). The slides were then
incubated overnight at 4°C in polyclonal anti-human WT1 diluted
1:500 in SBT (0.4 µg IgG/ml). Control slides were incubated with
the antibody preadsorbed for 1 h with the immunogen (4 µg/ml) or
in SBT containing non-immune rabbit IgG. Then, the slides were
washed in TPBS (3×5 min), incubated for 1 h at room temperature
in biotin-conjugated anti-rabbit goat IgG (Sigma) diluted 1:100 in
SBT, washed again and incubated for 1 h in avidin-peroxidase
complex (Sigma) diluted 1:150 in TPBS. After washing, peroxidase activity was developed with Sigma Fast 3,3’-diaminobenzidine (DAB) tablets according to the supplier’s instructions.
A second set of embryos were fixed in 4% paraformaldehyde
in TRIS-phosphate-buffered saline (TPBS) for 1 h. After fixation,
the embryos were washed, dehydrated in an ethanolic series finishing in butanol, and embedded in paraffin; 10-µm sections were
then obtained with a Leitz microtome and collected on poly-Llysine-coated slides. The sections were dewaxed in xylene, hydrated in an ethanolic series and washed in TPBS. Then, the sections
were boiled in a microwave oven for 10 min in 10 mM citric acid
buffer (pH 6.0) to recover antigenicity. Immunostaining was performed as described above. However, this method gave inconsistent results in both avian and mouse embryos, since a number of
sections showed a strong non-specific staining consisting of dark
spots within most cell nuclei throughout the embryo. Particularly,
mitotic cells showed a distinct non-specific staining. A number of
sections, however, were stained in the same fashion as the frozen
embryos.
The staining pattern of WT1 was studied by laser confocal microscopy in monolayers of epicardial cells obtained by culture of
chick proepicardia on collagen gels (Bernanke and Markwald
1982). The immunostaining was performed as described, but diluting 1:100 the primary antibody, and substituting the avidin-peroxidase by avidin-TRITC conjugate (Sigma)
For the immunolocalization of Slug, the procedure was similar
except for the fixation of the excised embryos, which was performed in 4% paraformaldehyde in TRIS-phosphate-buffered saline (TPBS) for 30 min. After fixation, the embryos were washed,
cryoprotected, snap frozen and sectioned. The sections were postfixed in 4% paraformaldehyde in TPBS for 15 min, and washed 3
times in TPBS for 15 min before further processing.
The affinity-purified anti-WT1 polyclonal antibody (sc-192,
Santa Cruz Biotechnologies) was developed by immunizing rabbits against the 19-carboxy-terminus amino acids of the human
WT1 protein. The antibody has been used to immunolocalize WT1
in paraffin-embedded sections of mice embryos (Toyooka et al.
1998; Lee et al. 1999) and in cell culture (English and Licht 1999;
Little et al. 1999).
The anti-chick Slug monoclonal antibody (clone 62.1E6) was
obtained from the Developmental Studies Hybridoma Bank. It has
been used for the immunodetection of Slug protein in premigratory neural crest cells (Liem et al. 1995, 1997) and in the developing
avian heart (Carmona et al. 2000; Romano and Runyan 1999).
For histological purposes, some chick embryos were fixed in
1% paraformaldehyde, 1% glutaraldehyde in PBS, and washed
and postfixed in 1% OsO4 for 90 min. After washing, the embryos
were dehydrated in an ethanolic series finishing in acetone and
embedded in Araldite 502. Semithin (0.5–1 µm) sections were obtained with a Reichert UMO-2 ultramicrotome and stained with
toluidine blue.
For the detection of WT1 protein in Western blots, embryo
chick hearts (stages HH29 and HH39) were homogenized in 1 ml
Tyrode’s solution containing protease inhibitors (0.5 µg/ml pepstatin, 1.0 µg/ml leupeptin, 0.1 mM phenylmethylsulphonylfluoride).
The suspensions were centrifuged at 8000 g for 15 min in a microcentrifuge (BHG-Hermle, Gosheim, Germany). Protein content in
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the homogenate was determined by the Bradford technique. Appropriate volumes of Laemmli’s sample buffer were added to each
fraction to a final concentration of 1 µg protein/µl. Proteins were
separated on 12% polyacrylamide gels loaded with 15 µl/lane. After electrophoresis, proteins were transferred to a nitrocellulose
membrane (Bio-rad) using a semidry transfer cell (Bio-rad transBlot SD). The blots were treated with blocking solution (20 mM
TRIS, 0.9% NaCl, 10% non-fat milk) and then reacted with a
1:500 dilution of anti-WT1. Specific antigen-antibody reactions
were visualized with a commercial immunoassay kit protocol
(ECL Plus detection system, Amersham).
Results
Specificity of the immunostaining
The immunostaining obtained with the anti-human WT1
antibody in the frozen chick and quail embryos closely
matched both the published pattern of expression of WT1
mRNA in mouse embryos and the immunostaining of
11.5-dpc mouse embryos used as positive controls. Indeed,
the carboxyl-terminus domain of WT1 is very well conserved among vertebrates (Kent et al. 1995). The carboxylterminal 19-amino-acid sequence which was used to raise
the antiserum differs by a single amino acid between human and mouse, and there are only two mismatches between mouse and Xenopus. Unfortunately, the corresponding chick WT1 sequence is not available in the databases,
since the published clone lacks the C-terminal 25 amino
acids (accession number X85731; Kent et al. 1995).
However, the known chick sequence at the carboxy
terminus is virtually identical between mouse and chick
Fig. 1 Western blot analysis of heart extracts of chick embryos,
stages HH29 and HH39. The anti-WT1 polyclonal antibody shows
a single reactive band of approximately 42 kDa
(one mismatch in 106 amino acids). Thus, we assume
that the human epitope against which the antiserum was
raised must be very well conserved in the chick and quail
protein.
On the other hand, Western blot analysis of HH29 and
HH39 chick embryo heart extracts revealed a single
band, 42 kDa (Fig. 1), which corresponds to the predicted molecular weight of the chick WT1 protein (assuming
a most probable length of 417 amino acids). The C-19
antibody gives three main bands in human cell extracts,
due to the existence of different isoforms originated
through alternative splicing and alternative translational
start sites (supplier’s data). However, neither alternative
splicing of exon 5 nor the presence of an N-terminal
polyproline run occurs in chick (Kent et al. 1995; Little
et al. 1999). Thus, a single main band would be the expected result in Western blots performed on chick cell
extract. Finally, preabsorption of the antiserum with the
immunogen abolished the immunoreactivity (Fig. 5F).
All these data strongly support the specificity of the immunostaining obtained.
Subcellular staining pattern
In both chick and quail embryos, the immunoperoxidase
staining was nuclear and diffuse, although more intense
in quail than in chick. In quail, but not in chick, a paler
circular area was evident within the stained nuclei
(Fig. 3A, C). This coincides with the presence, in the
quail nuclei, of a characteristic mass of heterochromatin.
Fig. 2 Monolayer of chick epicardial cells grown on a collagen
gel, immunostained with the anti-WT1 antibody and observed
with a laser confocal microscope. The staining pattern shows a
few large domains within the nuclei. Scale bar 2 µm
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Confocal immunofluorescence images of a cultured
chick epicardial monolayer onto a collagen gel showed a
more detailed staining pattern consisting of a few, intensely stained nuclear domains (Fig. 2).
HH18–19
We will describe first the immunostaining in the mesothelium, then the labelling of the submesothelial mesenchymal cells and, finally, the labelling of the other cells
of the embryo.
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A strong staining was found in the proepicardium, the
early epicardium, and the mesothelium lining the gut, the
liver primordium, the allantois and the nephrogenic ridges (Figs. 3A, B, 4, 5A, B). The strongest immunoreactivity was found in the mesothelial areas closer to the nephric duct. However, the dorsal mesenterium and the parietal mesothelium were not stained. The boundary between the positive and negative mesothelium was very
well delimited in the lateral limit of the nephrogenic
ridges (Fig. 3A).
Most mesenchymal cells within the proepicardial and
subepicardial matrix were WT1+ (Fig. 5B). Immunoreactive cells were also abundant in the nephrogenic ridges,
around the nephric duct and forming a broad band between the ducts and the dorsal aorta (Fig. 3A, B). A thin
layer of WT1+ mesenchymal cells was also observed immediately below the coelomic mesothelium of the gut
and liver primordium (Fig. 5B). Mesenchymal cells were
never observed in areas of the coelomic wall not covered
by WT1+ mesothelial cells.
HH20–22
▲
Strongly stained mesothelial cells covered the proepicardium and the primitive epicardium, as well as the nephrogenic ridges, especially in the proximity of the nephric
ducts. WT1+ mesothelial cells were also evident covering the liver and in some areas of the dorsal mesenterium
(Fig. 6C, D). These immunoreactive areas showed morphological evidence of epithelial-mesenchymal transition
in histological sections (Fig. 6A, B). The mesothelium
covering the emerging lung buds was not stained by
Fig. 4A–F WT1 immunostaining in some organs of avian embryos. A Quail embryo, HH24. Transverse section. The WT1-immunoreactive cells form a narrow band in the outer areas of the endodermal organs, oesophagus (OE), lung buds (LB) and liver (LI).
However, WT1+ cells surround the mesonephric ducts (arrowheads) and the dorsal aorta (AO), and arrive at the areas where the
metanephros will differentiate in later stages (M). B Chick embryo, HH30. Transverse section. The left gonad is filled with
WT1+ cells and lined by an immunoreactive mesothelium (arrow).
C Chick embryo, HH30. Transverse section. The large amount of
WT1+ cells in the spleen (SP) contrasts with the small number of
immunoreactive cells in the proventriculus (PV), where there is a
thin layer of submesothelial cells (arrow) and a few cells sparse in
the inner layer (arrowheads). D Chick embryo, HH35. Transverse
section of the oesophagus (OE). A layer of submesothelial cells is
WT1+, and some immunoreactive cells seem to be migrating toward inner areas (arrows). E, F Quail and chick embryos, HH26
and HH30, respectively. Transverse sections of the spinal cord, at
a thoracic level. WT1+ cells can be seen in the mantle layer (ML)
between the ependyma (EPN) and the ventral horn of the grey
matter (VH). The number of cells increase between HH26 and
HH30, and their location shifts to a more ventral level (RP roof
plate). Scale bars 90 µm (A), 29 µm (B), 22 µm (C), 38 µm (E),
30 µm (D, F), respectively
▲
Fig. 3A–E WT1 immunolocalization in the mesonephros and developing metanephros. A Quail embryo, HH18. Transverse section. WT1+ cells can be seen in the coelomic mesothelium covering the mesonephric ducts (MD) and around the ducts, being more
abundant in the medial area (arrow). Note the faint immunoreactivity of some cells dorsal to the mesonephric ducts. The limit between the WT1+ and the WT1– mesothelial cells is very sharp (arrowhead). The early allantoid bud (AL) also shows mesothelial
and submesothelial WT1+ cells (DM dorsal mesenterium). B Quail
embryo, HH19. Frontotransverse section. A number of WT1+ cells
medial to the mesonephric ducts (MD) are arranged in vesicles
(arrowheads). Other cells, however, are located in the ventrolateral aortic wall (small arrow). Note the strong immunoreactivity of
the coelomic mesothelium closer to the mesonephric duct (large
arrow). C Quail embryo, HH24. Transverse section. The mesonephric tubules are WT1–, but they are surrounded by immunoreactive cells, which also extend to the dorsal areas where the metanephros will differentiate in later stages (M). A WT1-immunoreactive mesonephric vesicle is shown (MV). The strong immunoreactivity of the mesothelium adjacent to the mesonephric duct
(MD) contrasts with the faint staining of the dorsal mesentery
(DM) at this level. D Chick embryo, HH30. Transverse section.
The mesonephric tubules are not stained, but the glomeruli (G)
keep the WT1 immunoreactivity. The metanephric mesenchyme
(MM) is WT1+. E Chick embryo, HH30. Transverse section. In the
lateral part of the mesonephros (MN), the müllerian duct (MU) is
surrounded by WT1+ mesenchymal cells and covered by immunoreactive mesothelial cells (BW lateral body wall). Scale bars
22 µm (A), 28 µm (B, E), 32 µm (C), 30 µm (D)
HH22. Scattered areas of the parietal (somatic) mesothelium were immunoreactive. However, the extraembryonic coelomic mesothelia showed a strong immunoreactivity.
A narrow band of WT1+ mesenchymal cells was
found in the submesothelial layer of the liver, gut, parietal pericardium and dorsal mesentery. However, immunoreactive mesenchymal cells were very abundant and
reached deep levels in the proepicardium, subepicardium
and nephrogenic ridges. In the anterior part of the trunk,
ahead of the level of the mesonephros, abundant WT1immunoreactive cells filled all the space between the
coelom, the dorsal mesentery, the aorta, the sympathetic
ganglia and the ventral limit of the somites (Fig. 6D). In
more posterior areas, WT1+ mesenchymal cells were
very abundant in the mesonephric ridges as well as in the
lateral and ventrolateral areas of the dorsal aorta and surrounding the postcardinal veins. The most dorsal limit of
the immunoreactive cells was again the ventral end of
the somite and the sympathetic ganglia.
Some strongly stained cells were arranged in vesicles
of circular section, which probably constituted the precursors of the glomerular cells of the mesonephric tubules, as demonstrated by comparison with histological
sections (Fig. 6A, C, E). In frontal sections, the existence
of a posteroanterior gradient was evident in the organization of these vesicles which seemed to lose the WT1 immunoreactivity coinciding with the acquisition of the
epithelial features. Clusters of WT1-negative cells were
observed inside these vesicles. In the quail embryos
these inner cells expressed the QH1 antigen, suggesting
a vascular fate (Fig. 6E). The differentiated mesonephric
tubules were always WT1 negative.
In all the cases of WT1+ mesenchymal cells (either a
narrow layer of submesothelial cells or large areas filled
with immunoreactive cells), we observed a gradient of
immunostaining, the cells closer to the coelom always
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Fig. 4A–F Legend see page 177
179
Fig. 5A–F WT1 immunostaining in the heart of avian embryos.
A Quail embryo, HH18. The proepicardial villi (arrow) and the
early epicardium (arrowhead), which covers the atrioventricular
groove (AV), are immunoreactive. Note the lack of immunoreactivity in the myocardium (MY) and the endocardial cushion mesenchyme (EC). B Quail embryo, HH19. Transverse section. All
the proepicardial (small arrows), epicardial (EP) and subepicardial
cells (arrowhead) are intensely WT1+. Note the immunoreactivity
of the mesothelium covering the liver primordium (LI) (arrow).
C Quail embryo, HH26. Transverse section. Immunoreactive cells
(arrows) are detected in the compact layer of the myocardium
(MY), but not in the trabeculate myocardium (TR) (SE subepicardi-
um, PC parietal pericardium). D Quail embryo, HH27. Transverse
section. Most epicardial (EP) and subepicardial (SE) cells are immunoreactive, but WT1+ cells are scarce in the atrial myocardium
(AM). E Chick embryo, HH30. Transverse section. The ventricular
myocardium is infiltrated by WT1+ cells, which sometimes are arranged in rows and occupy the clefts between the myocardiocytes
(arrows) (SE subepicardium, EC atrioventricular endocardial
cushion). F Quail embryo, HH28. Transverse section. Control section incubated with the primary antibody preadsorbed with the immunogen (A atrium, V ventricle, OE oesophagus, LB lung buds).
Scale bars 36 µm (A, E), 27 µm (B), 37 µm (C), 32 µm (D),
260 µm (F)
Fig. 6A–E WT1 immunostaining in the aorta-gonad-mesonephros
region. A, B Quail embryo, HH20. Transverse semithin section.
The areas where WT1 immunoreactivity is detected (compare with
C) show morphological evidence of epithelial-mesenchymal transition, including cell overriding and basal cytoplasmic processes
(arrows). Note the morphological changes of the mesothelium adjacent to the mesonephric duct (MD). The WT1– mesothelial cells
of the dorsal mesentery (DM) show a more typical epithelial morphology (arrowhead in B). Note the connection (arrowhead in A)
of the vascular lumen of the glomerulus (G) with the dorsal aorta
(AO). A developing glomerulus is shown by the red arrowhead
(CV postcardinal vein, SV subcardinal vein). C Quail embryo,
HH22. Note the immunoreactivity of the flexure of the dorsal mesentery (DM), where immunoreactive cells seem to be migrating
towards inner areas, and also in the mesothelium (the limit of the
immunoreactive cells is shown by the arrowhead) and developing
glomerulus (G). Other abbreviations as in A. D Quail embryo,
HH21. Periaortic area anterior to the mesonephros. The distribution of the immunoreactive cells is similar to that observed at
more posterior levels, including an increase in the immunoreactivity close to the lateroventral aortic walls (arrowheads) and cardinal veins (CV). Note the presence of WT1+ cells close to the aortic
wall (arrows), where clusters of haematopoietic stem cells are differentiating in this stage (large arrow). WT1+ mesenchymal cells
(M) fill the area between the lateral wall of the aorta, the myotome
(MT) and the sympathetic ganglia (DM dorsal mesentery). E Quail
embryo, HH21. Double immunostaining of WT1 (immunoperoxidase) and the vascular antigen QH1 (immunofluorescence) superimposed through digital image processing. Before a vascular lumen appears in the developing glomeruli (G), a cluster of QH1+
cells can be seen in its cavity, and a connection with the aorta
(AO) is evident (C coelom, CV postcardinal vein). Scale bars
36 µm (A, B), 26 µm (C), 32 µm (D), 24 µm (E)
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Fig. 7 Quail embryos, HH22 (B, F), HH24 (A, C, D), and HH26
(E). Comparison between the staining patterns obtained with antibodies against WT1 (A, C, E) and Slug (B, D, E) in the laryngotracheal groove (A, B), lungs (C, D) and liver (E, F). The Slugimmunoreactive cells are present throughout these areas, but the
WT1+ cells remain at the surface. However, note the images which
suggest migration of the outer cells inside these organs (arrows).
The mesothelium is stained with both antibodies (EN endodermal
tissue, LB lung buds, LI liver). Scale bars 43 µm (A, B), 54 µm
(C), 46 µm (D, E), 41 µm (F)
um, concretely in the ventricle (Fig. 5C). These cells appear in clefts between the myocardial cells. On the other
hand, in lungs, gut and liver, the WT1+ cells remain in a
thin submesothelial layer (Figs. 4A, 7C, E).
In the HH26 embryos a new domain of WT1+ cells
appear in the spinal cord. A few immunoreactive cells
(8–16 cells per section on each side) can be seen in the
lateral areas, slightly dorsal to the middle level (Fig. 4E).
being more stained (Fig. 6C; see also Fig. 3A, B for a
earlier stage). An exception to this rule were the nephrogenic cells arranged in the mesonephric vesicles, which
showed a strong immunoreactivity.
HH27–35
HH24–26
The immunostaining pattern of the mesothelial cells is
similar to that of the previous stages, but new areas of
immunoreactive mesothelial cells have appeared around
the oesophagus, lining the lung buds (Figs. 4A, 7C), and
in a well-defined area posterior to the connection of the
cardiac outflow tract, close to the laryngotracheal groove
and ventral to the aortic sac (Fig. 7A). The labelling of
the parietal and visceral mesothelia is more generalized
than in the previous stages, but the intensity of the labelling is still stronger in specific areas characterized by the
proximity of endodermal-derived organs, myocardium
and nephric ducts (Figs. 3C, 7E).
WT1+ mesenchymal cells form a crescent-shaped area
around the dorsal aorta, extending dorsally to the level of
the sympathetic ganglia (Fig. 4A), and they are very
abundant in the mesonephros and subepicardium
(Figs. 3C, 5C). In the embryos of stage HH26, WT1+
cells can be seen for the first time within the myocardi-
The mesothelium covering the heart, trachea, lungs, liver, oesophagus, stomach, gonads, spleen, mesenteries
and mesonephros is immunoreactive, although the staining has decreased in the parietal mesothelium. A narrow
band of submesothelial immunoreactive cells is evident
in the oesophagus, stomach, lungs and liver, where some
cells seem to be migrating into the inner core of WT1–
cells (Fig. 4D). Immunoreactive mesenchymal cells can
be seen throughout the metanephros, gonads and spleen
(Figs. 3D, 4B, C, respectively), dorsal mesocardium and
mesenteries (especially the ventral mesentery of the liver). Most of the mesonephros shows no WT1+ cells, except for the glomeruli (Fig. 3D). WT1+ cells are very
abundant around the müllerian ducts (Fig. 3E). Most
cells at the subepicardium are WT1+ (Fig. 5D), while
many immunoreactive cells can be seen within the ventricular myocardium, sometimes forming rows between
the myocardiocytes (Fig. 5E). However, WT1+ cells are
very scarce in the atrial myocardium (Fig. 5D). The periaortic WT1+ cells have become less abundant in the anterior part of the trunk, although small groups of cells are
detected ventrally to the sympathetic ganglia and lateral
to the vertebrae. The cells in the spinal cord have increased in number and they are located at a more ventral
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position than in the previous stages, in the mantle, between the ependymal layer and the ventral horns of the
medulla (Fig. 4F).
Slug immunolocalization in the coelomic wall
The mesothelial cells lining the nephrogenic ridges, mesenteries, developing gut, allantois, lungs, liver and heart
were Slug immunoreactive, showing a pattern very similar to that described for the WT1+ mesothelial cells, i.e.
an increase in immunoreactivity in the proximity of
some organs such as nephric ducts, lung buds, liver primordium or myocardium (Fig. 7B, D, F). All these organs showed abundant Slug+ mesenchymal cells. However, unlike the WT1+ mesenchymal cells, Slug+ mesenchymal cells were observed throughout the primordia of
the endodermal organs such as lungs and liver (Fig. 7D,
F), not remaining restricted to the submesothelial layer.
Another difference was observed in the heart; most subepicardial cells were Slug+, but Slug-immunoreactive
cells were not observed infiltrating within the ventricular
myocardium. In the genitourinary system, Slug+ mesenchymal cells were very abundant in the developing gonads, but very scarce in the mesonephros (not shown).
Discussion
The normal roles played by the WT1 protein in the embryonic development have been the object of much discussion. A better knowledge of these roles is made difficult by the multiple functions which WT1 has been
shown to perform in a number of in vitro reporter assays
as commented upon in the “Introduction”. WT1 has been
reported to inhibit cell growth but also to be necessary
for proliferation of leukemic cell lines (Algar et al.
1996). Some experimental evidence shows that WT1 is
able to induce apoptosis but also to protect from apoptosis acting as a survival factor. The promoters of a number of genes have been shown to be either activated and
repressed by WT1, and a further role in splicing has also
been proposed (reviewed in Little et al. 1999).
These disparate and sometimes contradictory results
can partially be explained by the existence of different
isoforms of the protein, generated by alternative splicing,
RNA editing and alternative translational start sites
(Davies et al. 1999). However, we are far from explaining the precise functions performed by WT1 during the
development.
In order to interpret our results, we will start from two
important points: first, WT1 has been involved in phenotypic shifts between mesenchyme and epithelium as well
as between epithelium and mesenchyme (Moore et al.
1999). Second, the phenotype of the WT1-null embryos
has shown its requirement for the development of kidney, gonads, spleen and adrenals (Kreidberg et al. 1993;
Moore et al. 1999), although the lack of WT1 causes a
lethal failure in the development of the myocardium, a
tissue which does not express WT1 (Moore et al. 1999).
We will try to integrate all these data with our own observations about the sites of localization of WT1 protein
during the embryonic development in order to suggest a
hypothesis about their normal physiological functions.
The first point to be mentioned is the close relationship between the localization of WT1 in the mesothelium
and submesothelial cells. The protein was localized in
definite areas of the coelomic mesothelium and in the
layer of the cells immediately below, but never in one of
these layers alone, with the exception of the very early
epicardium, which is directly attached to the myocardium, without a subepicardial layer. On the other hand, until stage HH24, the presence of WT1 was always related
to the proximity of one of these tissues: nephric ducts,
endoderm-derived organ primordia or myocardium. In
these cases, the limit between the immunoreactive and
not immunoreactive mesothelial areas was clearly delimited. After stage HH24, WT1 was found throughout the
coelomic mesothelium, but the strongest and most persistent immunoreactivity of the mesothelial cells always
coincided with the proximity of the mentioned tissues or
other tissues developed later, such as the müllerian ducts.
Interestingly, a significant correlation was found in
the mesothelial cells between the presence of WT1 and
Slug, another zinc-finger transcription factor. Slug and
WT1 immunoreactivities were strong in the same mesothelial areas (epicardium, liver, lungs and nephrogenic
ridges) and in the same developmental stages. However,
in spite of the coincidence in the mesothelial cells, interesting differences were observed in the pattern of immunoreactivity of the underlying mesenchyme. Thus, in the
endoderm-derived organs, WT1+ cells were only seen in
the outermost layer, while Slug+ cells were observed
throughout organ primordia such as lungs or liver. In the
heart, Slug+ and WT1+ cells were very abundant in the
subepicardium, where vascular and connective tissue
was forming, but only WT1+ cells invaded the myocardium. The developing gonads were also widely invaded by
Slug+- and WT1+-immunoreactive cells, but there was a
coincident downregulation of both Slug and WT1 in the
differentiated mesonephros. Furthermore, in other systems of epithelial-mesenchymal transition where Slug is
expressed, such as the endocardial cushions of the heart
(Carmona et al. 2000), WT1+ cells were not observed in
the stages studied.
Most papers dealing with the developmental function
of WT1 have stressed the coincidence between its expression in the developing kidney (where a transformation of the nephrogenic mesenchyme into epithelium occurs) and in the coelomic epithelium. Thus, it has been
frequently suggested that WT1 might be involved in the
formation of the mesothelium by aggregation of mesenchymal cells. This idea can be traced back to the paper
by Pritchard-Jones et al. (1990), although the flaws of
this hypothesis have been shown (Armstrong et al.
1993). Basically, these authors rightly remark that the
early coelomic epithelium (and many other embryonic
epithelia) forms without expression of WT1. A more re-
183
cent contribution suggests instead that this transcription
factor may enable cells to flip between mesenchymal and
epithelial cell states (Moore et al. 1999). We think that
there are powerful reasons to believe that WT1 is specifically expressed or upregulated in areas of transformation of mesothelial cells into mesenchyme. These reasons are:
1. No WT1 expression is detected in most of the early
coelomic mesothelium of the avian embryos, as already noted by Armstrong et al. (1993). WT1 protein
is first localized in very precise mesothelial areas, always coinciding with the proximity of specific tissues
which might be playing an inductive role (nephric
ducts, müllerian ducts, endodermal derivatives and
myocardium).
2. In most of the mesothelial areas where WT1 is expressed or upregulated, an epithelial to mesenchymal
transition has been described, but not the reverse, for
example, in the proepicardium (Pérez-Pomares et al.
1997) and the epicardium (Pérez Pomares et al. 1997,
1998; Dettman et al. 1998, Vrancken Peeters et al.
1999). The presence of WT1 in epicardial-derived
cells of mouse embryos has been remarked upon by
Moore et al. (1999). The contribution of mesothelial
cells to the mesenchyme of other developing organs
where WT1 is widely expressed, such as mesonephros, gonads and adrenal cortex, was well known for
the older anatomists. Indeed, a large number of papers
in the classical anatomical literature deal with this developmental event (reviewed in Gruenwald 1942).
Unfortunately, these important observations seem to
have been neglected in the most recent literature. For
example, it is a well-known fact that the cells which
form the adrenal cortex arise from the peritoneal epithelium of the mesonephric ridges (Bellairs and
Osmond 1998). Sertoli cells of the testicle are also
mesothelial derivatives (Bellairs and Osmond 1998;
Karl and Capel 1998) and express WT1 even in adults
(Mundlos et al. 1993). We have shown elsewhere evidence of a migration of mesothelial-derived cells to
the paraaortic areas of avian embryos (Pérez-Pomares
et al. 1999), where WT1+ cells are very abundant according to our observations (see Fig. 6D).
3. It has been claimed that WT1 expression in the mesoand metanephric mesenchyme is related to their ability to transform into epithelium. However, we have
shown that WT1 immunoreactivity persists in the
paravertebral metanephric mesenchyme, dorsal to the
mesonephros, for a long time without signs of differentiation. We have also shown that WT1 immunoreactivity suddenly disappears, coinciding with the differentiation of the mesonephric mesenchyme into epithelial structures. Finally, it is important to note that
the main site of WT1 expression in the adult kidney,
the podocytes, is not entirely epithelial, showing an
intermediate phenotype between epithelium and mesenchyme (Davies 1996). Sertoli cells, which express
WT1 throughout adult life as stated above, also show
mesenchymal characteristics. These observations
would argue against a significant role of WT1 in the
acquisition of a fully differentiated epithelial phenotype, although other findings have strongly supported
this role.
4. The temporal and spatial expression of WT1 in the
coelomic mesothelium significantly correlates with
the presence of Slug, a transcription factor which
plays a key role in the epithelial-mesenchymal transition (Duband et al. 1995; Savagner et al. 1997). Indeed, Slug inactivation in avian embryos impairs the
epithelial-mesenchymal transformation of the neuroepithelium into the neural crest cells (Nieto et al.
1994). Snail, the probable functional homologue of
Slug in mammals (Sefton et al. 1998), has been
shown to downregulate E-cadherin expression, thus
contributing to the phenotypic shift between epithelium and mesenchyme (Batlle et al. 2000; Cano et al.
2000).
In conclusion, we think that there are reasons to believe
that WT1 is present in those mesothelial cells fated to
transform to mesenchyme or, at least, which are able to
achieve such a transformation. We also think that the
submesothelial WT1+ cells represent, probably, a population of undifferentiated mesothelial-derived cells, a point
of view already adopted by Moore et al. (1999) in relation to the epicardial-derived cells.
However, we think that WT1 is not required for the
process of epithelial-mesenchymal transition. In fact, the
main embryonic processes of epithelial-mesenchymal
transition, such as gastrulation, neural crest or endocardial cushion formation progress without WT1 expression
and they are normal in WT1-null embryos. On the other
hand, WT1-null embryos apparently show mesothelialderived mesenchyme, for example, in the subepicardium
(Kreidberg et al. 1993), although the number of subepicardial mesenchymal cells is greatly reduced (Moore et
al. 1999).
We think that a hypothetical function of WT1 which
would be consistent with the available observations is to
maintain the mesothelial-derived mesenchyme in an undifferentiated state. Moore et al. (1999) had already suggested that WT1 enables cells to flip between mesenchymal and epithelial cell states. We suggest that in some organs, especially those derived from endoderm, these cells
would soon lose their WT1 protein, and would subsequently migrate to inner zones and differentiate, probably
into fibroblasts and smooth muscle cells. It would explain
the narrow submesothelial band of WT1+ cells. However,
in other organs such as gonads, spleen, adrenal cortex,
kidneys and heart (i.e. in the mesodermal-derived viscerae), mesothelial-derived cells are apparently able to migrate into inner areas keeping their WT1 protein. It does
not mean, however, that WT1 cannot also perform alternative functions related to differentiation or cell survival, depending on the developmental or cellular context.
Interestingly, all the mesodermal organs invaded by
WT1+ cells, but not the endoderm-derived viscerae, are
184
severely affected or even absent in WT1-deficient mouse
embryos (Kreidberg et al. 1993; Moore et al. 1999). This
can be explained in the context of our hypothesis if we
assume that the mesothelial-derived cells which should
normally contribute to the primary sex cords, corticoadrenal tissue or nephric blastema, prematurely differentiate in the absence of WT1, possibly into fibroblasts. In
the case of the kidney, the lack of an inducing blastema
would cause agenesia of the ureteric bud, whose absence
could, in turn, induce apoptosis in mesenchymal cells
(Davies 1996).
In the case of the heart, our hypothesis probably also
accounts for the cardiac defects in WT1-null embryos.
The undifferentiated WT1+ epicardial-derived cells
which invade the myocardium might play a signalling
role which would be lost in WT1-deficient embryos by
premature differentiation. It is important to remark that
the epicardium produces retinoids (Moss et al. 1998),
and these molecules are essential for the differentiation
of the ventricular myocardium (Kubalak and Sucov
1999). Since retinoids are hydrophobic, it is conceivable
that the massive migration of WT1+ epicardial-derived
cells inside the myocardium is essential for the delivery
of retinoids to the inner layers of the ventricle. In the
WT1-null embryos, epicardial-derived cells would normally form, but they would differentiate prematurely and
would stop producing retinoids. Thus, the ventricular
myocardium would be defective, resulting in cardiac
failure. It might be significant that the RXRα-null mice
embryos show the same cardiac phenotype as the WT1null embryos, as already noted by Kubalak and Sucov
(1999). Both types of embryos show thin ventricular
walls and die between 14.5 and 15.5 dpc from cardiac
failure (Sucov et al. 1994; Dyson et al. 1995). In the
RXRα-null embryos, the lack of the retinoid receptor
causes an anomalous persistence of the atrial-specific
MLC2a myosin isoform, either by premature differentiation of the ventricular myocardium or by the acquisition
of an atrial phenotype in the ventricle (Dyson et al.
1995). Thus, a prediction of our hypothesis is the anomalous persistence of MLC2a isoform in the ventricle of
WT1-null embryos.
Although there is a close relationship between the
presence of WT1 protein and the coelomic wall, we have
observed immunoreactive cells in the spinal cord, an observation which has also been made in mouse embryos
(Armstrong et al. 1993; Moore et al. 1998). In this regard, it might be significant that the Drosophila zinc-finger protein Klumpfuss, which shows sequence similarities to vertebrate WT1, is expressed in a subset of neuronal precursors, where it is involved in the determination
of the cell fate (Yang et al. 1997). On the other hand, we
have not observed the presence of WT1 protein in other
locations where evidence of WT1 gene expression has
been reported, such as the fourth ventricle of the brain or
the limb (Armstrong et al. 1993; Moore et al. 1998).
A final question is the relationship between WT1+
cells and the origin of haematopoietic cells. Two facts
are relevant in this regard: (1) WT1 is expressed by early
haematopoietic progenitors in humans (Baird and
Simmons 1997) and (2) we have shown that WT1+ cells
are very abundant in the AGM (aorta-gonad-mesonephros) region, where the definitive haematopoietic stem
cells differentiate in the avian and mammalian embryos
(Cormier et al. 1988; Pardanaud et al. 1996). We have
localized WT1+ cells very close to the lateroventral aortic wall, a site reached by mesothelial-derived cells
(Pérez Pomares et al. 1999), where haematopoietic stem
cells are being incorporated into the blood stream
(Cormier et al. 1988; Tavian et al. 1996). It is tempting
to connect all these observations. If WT1+ cells differentiate into haematopoietic progenitors in the AGM region,
and we assume that WT1 is a marker of mesothelialderived cells, it would imply that these cells can give rise
to haematopoietic stem cells. This idea is not new. In fact
we have proposed a model about the differentiation of
haemangioblasts from pluripotential mesothelial-derived
cells, a model supported by a number of observations
and also by phylogenetic considerations (Muñoz-Chapuli
et al. 1999). For example, it is interesting to note that in
many invertebrates, such as the echinoderms, blood cells
derive from the coelomic epithelium (Vanden Bossche
and Jangoux 1976).
In conclusion, we suggest that one of the possible
functions of WT1 may be related to the transient acquisition of an undifferentiated, pluripotential state by mesothelial-derived cells. It is important to note that the coelomic mesothelium of the embryo has been regarded as
mesoderm arranged as a flat epithelium, capable of differentiation along the same lineages that the mesoderm
normally displays during embryogenesis (Donna et al.
1991; Colas et al. 2000). From the evolutionary point of
view, the dedifferentiation of mesothelial-derived cells
would serve at least two aims: first to regain the epithelial state in order to give rise to a new system of cavities
lined with a mesodermal epithelium, with excretory
functions; and, second, to keep the mesenchymal state in
order to provide the endoderm-derived organs (lungs,
liver, gut) of connective and muscular tissue. From this
point of view, it might be possible to understand some of
the serious and disparate consequences of WT1 malfunction, such as renal tumour with aberrant differentiation,
particularly muscle, but also occasionally bone or cartilage (Davies et al. 1999).
Acknowledgements The monoclonal antibodies anti-Slug and
QH1 were obtained from the Developmental Studies Hybridoma
Bank maintained by the Department of Pharmacology and Molecular Sciences, John Hopkins University School of Medicine, Baltimore, MD 21205, and the Department of Biological Sciences,
University of Iowa, Iowa City, IA 52242, under contract NO1HD-2-3144 from the National Institute of Child Health and Human Development (NICHD). The authors sincerely thank Amelia
Aranega, Jorge Domínguez, Jesús Santamaría, Gerardo Atencia
and María José Aranda for their help.
185
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