Abstract The Wilms’ tumour suppressor gene WT1 en-codes a zinc-finger transcription factor which is essentialfor the development of kidney, gonads, spleen and adre-nals. WT1-null embryos lack all of these viscerae andthey also show a thin ventricular myocardium and unex-pectedly die from cardiac failure between 13 and 15 dayspost coitum. We studied the localization of the WT1 pro-tein in chick and quail embryos between stages HH18and HH35. In early embryos, WT1 protein was locatedin specific areas of the coelomic mesothelium adjacent tothe nephric ducts, the myocardium or the primordia ofthe endodermal organs (gut, liver and lungs). These me-sothelial areas also showed localized expression of Slug,a zinc-finger transcription factor involved in epithelial-mesenchymal transitions. WT1+ mesenchymal cells werealways found below the immunoreactive mesothelial ar-eas, either forming a narrow band on the surface of theendodermal organs (gut, liver and lungs) or migratingthroughout the mesodermal organs (mesonephros, meta-nephros, gonads, spleen and heart). In the developingheart, the invasion of WT1+ cells started at stage HH26,and all the ventricular myocardium was pervaded bythese cells, presumably derived from the epicardium, atHH30. We suggest that WT1 is not required for the epi-thelial-mesenchymal transition of the coelomic mesothe-lium, but it might be a marker of the mesothelial-derivedcells, where this protein would be acting as a repressorof the differentiation.

Keywords Wilms’ tumour · WT1 · Mesothelium · Chickembryo · Quail embryo

Introduction

The Wilms’ tumour gene codes for a zinc-finger tran-scription factor which has been involved in many normaland pathological processes. The 10-exon WT1 gene con-tains two alternately spliced regions, thus encoding fourdistinct protein isoforms (Haber et al. 1991). WT1 is es-sential for the development of the kidney and gonads(Kreidberg et al. 1993), adrenals (Moore et al. 1999) andspleen (Herzer et al. 1999). Mice homozygous for aWT1 deletion lack these organs but they unexpectedlydie from cardiac failure between 13 and 15 days of ges-tation (Kreidberg et al. 1993), suggesting a role for WT1in cardiac development.

Mutated forms of WT1 have been related to congeni-tal abnormalities such as a chromosome 11p deletionsyndrome known as the WAGR syndrome (Wilms’ tu-mor, aniridia, genitourinary anomaly and mental retarda-tion) (Brown et al. 1992; Haber and Housman 1992), theDenys-Drash syndrome (Baird et al. 1992; Pelletier et al.1991a) as well as several kinds of acute myeloid leuke-mias (King-Underwood et al. 1996; Bergmann et al.1997).

It was initially thought that the functions of WT1were specifically related to the normal differentiation ofthe kidneys and gonads (Pritchard-Jones et al. 1990).However, in recent years, a large number of in vitro re-porter assays have involved WT1 in a wide spectrum offundamental cell processes, including control of prolifer-ation and differentiation mainly through transcriptionalrepression 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 thatmost genes with WT1-responsive promoters are not reg-ulated in vivo by WT1, and the sometimes conflicting re-sults obtained might depend upon the experimental con-ditions (Reddy et al. 1995; Reddy and Licht 1996). Thus,

This work was supported by grants PM98-0219 and 1FD97-0693(Ministerio de Educación y Cultura, Spain). Mauricio González isthe recipient of a fellowship from Ministerio de Educación y Cultura

R. Carmona · M. González-Iriarte · J.M. Pérez-PomaresR. Muñoz-Chápuli (✉ )Department of Animal Biology, Faculty of Science, University of Málaga, 29071 Málaga, Spaine-mail: chapuli@uma.esTel.: +34-952131853, Fax: +34-952132000

J.M. Pérez-PomaresDepartment of Anatomy and Cell Biology, Medical University of South Carolina, Charleston, 29425 SC,USA

Cell Tissue Res (2001) 303:173–186DOI 10.1007/s004410000307

R E G U L A R A R T I C L E

R. Carmona · M. González-IriarteJ.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

the physiological functions of WT1 during the embryon-ic development remain uncertain.

Besides its functions as a transcription factor, WT1has also been involved in RNA metabolism. In fact, theWT1 isoform lacking the KTS (lysine-serine-threonine)insertion binds RNA and shows a speckled pattern ofdistribution which is thought to colocalize with spliceo-somal proteins (Larsson et al. 1995; Kennedy et al. 1996;Bardeesy and Pelletier 1998).

During normal development WT1 is expressed in adynamic and tissue-specific pattern in a specific popula-tion of neurones in the neural tube and in several meso-derm-derived tissues, namely in mesothelia (coelomicepithelia), derivatives of the intermediate mesodermsuch as the meso- and metanephros, gonads and adrenals(Pritchard-Jones 1990; Pelletier et al. 1991b; Armstronget al. 1993; Rackley et al. 1993), and the limbs (Moore etal. 1998). These papers show localization of WT1mRNA through in situ hybridization in mammalian em-bryos, but only limited data are available about the pres-ence of WT1 protein during embryonic development(Charles et al. 1997). On the other hand, the presence ofthis important transcription factor has not yet been stud-ied in the avian embryo, a very important system for de-scriptive and experimental embryology. It is important toemphasize that data about the precise temporal and spa-tial localization of a transcription factor in embryonictissues where well-characterized developmental eventsoccur, can provide good insights into their possible func-tions.

Our aim was to study the localization of the WT1 pro-tein by immunohistochemistry in embryos of chick andquail, in order to test several hypotheses proposed aboutthe normal functions of the protein, such as its involve-ment in processes of transition between mesenchymeand epithelium and vice versa. With this purpose wehave checked the presence, in the embryonic areas whereWT1 is expressed, of Slug, another zinc-finger transcrip-tion factor which is involved in the epithelial-mesenchy-mal 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 com-pliance with the international guidelines for animal care and wel-fare. Chick and quail eggs were kept in a rocking incubator at38°C. The avian embryos were staged according to the Hamburgerand Hamilton (1951) stages of chick development.

The spatial and temporal immunoreactive pattern of WT1 wasstudied in a sample consisting of 16 embryos of quail (Coturnixcoturnix japonica), which were collected at stages HH18–HH28,and 8 embryos of chick (Gallus gallus), collected at stagesHH18–HH35. Mouse embryos, 11.5 days post coitum, were usedas positive controls.

For WT1 immunohistochemistry, the embryos were excisedand cryoprotected in 10%, 20% and 30% sucrose solutions, wherethey were kept at 4°C until they sunk. Then, the embryos wereembedded in OCT and snap frozen in liquid-nitrogen-cooled iso-pentane. The frozen embryos were sectioned in a cryostate, and14-µm sections were collected on poly-L-lysine-coated slides and

fixed for 10 min in 1:1 methanol acetone at –20°C. The sectionswere then rehydrated in TRIS-phosphate-buffered saline (TPBS)and the endogenous peroxidase activity was quenched by incuba-tion for 30 min with 3% hydrogen peroxide in TPBS. After wash-ing, non-specific binding sites were saturated for 30 min with 16%sheep serum, 1% bovine serum albumin and 0.5% Triton X-100 inTPBS (SBT). Endogenous biotin was blocked with the avidin-biotin blocking kit (Vector, Burlingame, CA). The slides were thenincubated overnight at 4°C in polyclonal anti-human WT1 diluted1:500 in SBT (0.4 µg IgG/ml). Control slides were incubated withthe antibody preadsorbed for 1 h with the immunogen (4 µg/ml) orin SBT containing non-immune rabbit IgG. Then, the slides werewashed in TPBS (3×5 min), incubated for 1 h at room temperaturein biotin-conjugated anti-rabbit goat IgG (Sigma) diluted 1:100 inSBT, washed again and incubated for 1 h in avidin-peroxidasecomplex (Sigma) diluted 1:150 in TPBS. After washing, peroxi-dase activity was developed with Sigma Fast 3,3’-diaminobenzidi-ne (DAB) tablets according to the supplier’s instructions.

A second set of embryos were fixed in 4% paraformaldehydein TRIS-phosphate-buffered saline (TPBS) for 1 h. After fixation,the embryos were washed, dehydrated in an ethanolic series fin-ishing in butanol, and embedded in paraffin; 10-µm sections werethen obtained with a Leitz microtome and collected on poly-L-lysine-coated slides. The sections were dewaxed in xylene, hydrat-ed in an ethanolic series and washed in TPBS. Then, the sectionswere boiled in a microwave oven for 10 min in 10 mM citric acidbuffer (pH 6.0) to recover antigenicity. Immunostaining was per-formed as described above. However, this method gave inconsis-tent results in both avian and mouse embryos, since a number ofsections showed a strong non-specific staining consisting of darkspots within most cell nuclei throughout the embryo. Particularly,mitotic cells showed a distinct non-specific staining. A number ofsections, however, were stained in the same fashion as the frozenembryos.

The staining pattern of WT1 was studied by laser confocal mi-croscopy in monolayers of epicardial cells obtained by culture ofchick proepicardia on collagen gels (Bernanke and Markwald1982). The immunostaining was performed as described, but dilut-ing 1:100 the primary antibody, and substituting the avidin-peroxi-dase by avidin-TRITC conjugate (Sigma)

For the immunolocalization of Slug, the procedure was similarexcept for the fixation of the excised embryos, which was per-formed in 4% paraformaldehyde in TRIS-phosphate-buffered sa-line (TPBS) for 30 min. After fixation, the embryos were washed,cryoprotected, snap frozen and sectioned. The sections were post-fixed in 4% paraformaldehyde in TPBS for 15 min, and washed 3times in TPBS for 15 min before further processing.

The affinity-purified anti-WT1 polyclonal antibody (sc-192,Santa Cruz Biotechnologies) was developed by immunizing rab-bits against the 19-carboxy-terminus amino acids of the humanWT1 protein. The antibody has been used to immunolocalize WT1in 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) wasobtained from the Developmental Studies Hybridoma Bank. It hasbeen used for the immunodetection of Slug protein in premigrato-ry neural crest cells (Liem et al. 1995, 1997) and in the developingavian heart (Carmona et al. 2000; Romano and Runyan 1999).

For histological purposes, some chick embryos were fixed in1% paraformaldehyde, 1% glutaraldehyde in PBS, and washedand postfixed in 1% OsO4 for 90 min. After washing, the embryoswere dehydrated in an ethanolic series finishing in acetone andembedded in Araldite 502. Semithin (0.5–1 µm) sections were ob-tained with a Reichert UMO-2 ultramicrotome and stained withtoluidine blue.

For the detection of WT1 protein in Western blots, embryochick hearts (stages HH29 and HH39) were homogenized in 1 mlTyrode’s solution containing protease inhibitors (0.5 µg/ml pepsta-tin, 1.0 µg/ml leupeptin, 0.1 mM phenylmethylsulphonylfluoride).The suspensions were centrifuged at 8000 g for 15 min in a micro-centrifuge (BHG-Hermle, Gosheim, Germany). Protein content in

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the homogenate was determined by the Bradford technique. Ap-propriate volumes of Laemmli’s sample buffer were added to eachfraction to a final concentration of 1 µg protein/µl. Proteins wereseparated on 12% polyacrylamide gels loaded with 15 µl/lane. Af-ter electrophoresis, proteins were transferred to a nitrocellulosemembrane (Bio-rad) using a semidry transfer cell (Bio-rad trans-Blot SD). The blots were treated with blocking solution (20 mMTRIS, 0.9% NaCl, 10% non-fat milk) and then reacted with a1:500 dilution of anti-WT1. Specific antigen-antibody reactionswere visualized with a commercial immunoassay kit protocol(ECL Plus detection system, Amersham).

Results

Specificity of the immunostaining

The immunostaining obtained with the anti-human WT1antibody in the frozen chick and quail embryos closelymatched both the published pattern of expression of WT1mRNA in mouse embryos and the immunostaining of11.5-dpc mouse embryos used as positive controls. Indeed,the carboxyl-terminus domain of WT1 is very well con-served among vertebrates (Kent et al. 1995). The carboxyl-terminal 19-amino-acid sequence which was used to raisethe antiserum differs by a single amino acid between hu-man and mouse, and there are only two mismatches be-tween mouse and Xenopus. Unfortunately, the correspond-ing chick WT1 sequence is not available in the databases,since the published clone lacks the C-terminal 25 aminoacids (accession number X85731; Kent et al. 1995).

However, the known chick sequence at the carboxyterminus is virtually identical between mouse and chick

(one mismatch in 106 amino acids). Thus, we assumethat the human epitope against which the antiserum wasraised must be very well conserved in the chick and quailprotein.

On the other hand, Western blot analysis of HH29 andHH39 chick embryo heart extracts revealed a singleband, 42 kDa (Fig. 1), which corresponds to the predict-ed molecular weight of the chick WT1 protein (assuminga most probable length of 417 amino acids). The C-19antibody gives three main bands in human cell extracts,due to the existence of different isoforms originatedthrough alternative splicing and alternative translationalstart sites (supplier’s data). However, neither alternativesplicing of exon 5 nor the presence of an N-terminalpolyproline run occurs in chick (Kent et al. 1995; Littleet al. 1999). Thus, a single main band would be the ex-pected result in Western blots performed on chick cellextract. Finally, preabsorption of the antiserum with theimmunogen abolished the immunoreactivity (Fig. 5F).All these data strongly support the specificity of the im-munostaining obtained.

Subcellular staining pattern

In both chick and quail embryos, the immunoperoxidasestaining was nuclear and diffuse, although more intensein quail than in chick. In quail, but not in chick, a palercircular area was evident within the stained nuclei(Fig. 3A, C). This coincides with the presence, in thequail nuclei, of a characteristic mass of heterochromatin.

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Fig. 1 Western blot analysis of heart extracts of chick embryos,stages HH29 and HH39. The anti-WT1 polyclonal antibody showsa single reactive band of approximately 42 kDa

Fig. 2 Monolayer of chick epicardial cells grown on a collagengel, immunostained with the anti-WT1 antibody and observedwith a laser confocal microscope. The staining pattern shows afew large domains within the nuclei. Scale bar 2 µm

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Confocal immunofluorescence images of a culturedchick epicardial monolayer onto a collagen gel showed amore detailed staining pattern consisting of a few, in-tensely stained nuclear domains (Fig. 2).

HH18–19

We will describe first the immunostaining in the meso-thelium, then the labelling of the submesothelial mesen-chymal cells and, finally, the labelling of the other cellsof the embryo.

A strong staining was found in the proepicardium, theearly epicardium, and the mesothelium lining the gut, theliver primordium, the allantois and the nephrogenic ridg-es (Figs. 3A, B, 4, 5A, B). The strongest immunoreactiv-ity was found in the mesothelial areas closer to the neph-ric duct. However, the dorsal mesenterium and the pari-etal mesothelium were not stained. The boundary be-tween the positive and negative mesothelium was verywell delimited in the lateral limit of the nephrogenicridges (Fig. 3A).

Most mesenchymal cells within the proepicardial andsubepicardial matrix were WT1+ (Fig. 5B). Immunoreac-tive cells were also abundant in the nephrogenic ridges,around the nephric duct and forming a broad band be-tween the ducts and the dorsal aorta (Fig. 3A, B). A thinlayer of WT1+ mesenchymal cells was also observed im-mediately below the coelomic mesothelium of the gutand liver primordium (Fig. 5B). Mesenchymal cells werenever observed in areas of the coelomic wall not coveredby WT1+ mesothelial cells.

HH20–22

Strongly stained mesothelial cells covered the proepicar-dium and the primitive epicardium, as well as the neph-rogenic ridges, especially in the proximity of the nephricducts. WT1+ mesothelial cells were also evident cover-ing the liver and in some areas of the dorsal mesenterium(Fig. 6C, D). These immunoreactive areas showed mor-phological evidence of epithelial-mesenchymal transitionin histological sections (Fig. 6A, B). The mesotheliumcovering the emerging lung buds was not stained by

HH22. Scattered areas of the parietal (somatic) mesothe-lium were immunoreactive. However, the extraembryon-ic coelomic mesothelia showed a strong immunoreactivi-ty.

A narrow band of WT1+ mesenchymal cells wasfound in the submesothelial layer of the liver, gut, pari-etal pericardium and dorsal mesentery. However, immu-noreactive mesenchymal cells were very abundant andreached deep levels in the proepicardium, subepicardiumand nephrogenic ridges. In the anterior part of the trunk,ahead of the level of the mesonephros, abundant WT1-immunoreactive cells filled all the space between thecoelom, the dorsal mesentery, the aorta, the sympatheticganglia and the ventral limit of the somites (Fig. 6D). Inmore posterior areas, WT1+ mesenchymal cells werevery abundant in the mesonephric ridges as well as in thelateral and ventrolateral areas of the dorsal aorta and sur-rounding the postcardinal veins. The most dorsal limit ofthe immunoreactive cells was again the ventral end ofthe somite and the sympathetic ganglia.

Some strongly stained cells were arranged in vesiclesof circular section, which probably constituted the pre-cursors of the glomerular cells of the mesonephric tu-bules, as demonstrated by comparison with histologicalsections (Fig. 6A, C, E). In frontal sections, the existenceof a posteroanterior gradient was evident in the organiza-tion of these vesicles which seemed to lose the WT1 im-munoreactivity coinciding with the acquisition of theepithelial features. Clusters of WT1-negative cells wereobserved inside these vesicles. In the quail embryosthese inner cells expressed the QH1 antigen, suggestinga vascular fate (Fig. 6E). The differentiated mesonephrictubules were always WT1 negative.

In all the cases of WT1+ mesenchymal cells (either anarrow layer of submesothelial cells or large areas filledwith immunoreactive cells), we observed a gradient ofimmunostaining, the cells closer to the coelom always

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Fig. 3A–E WT1 immunolocalization in the mesonephros and de-veloping metanephros. A Quail embryo, HH18. Transverse sec-tion. WT1+ cells can be seen in the coelomic mesothelium cover-ing the mesonephric ducts (MD) and around the ducts, being moreabundant in the medial area (arrow). Note the faint immunoreac-tivity of some cells dorsal to the mesonephric ducts. The limit be-tween the WT1+ and the WT1– mesothelial cells is very sharp (ar-rowhead). The early allantoid bud (AL) also shows mesothelialand submesothelial WT1+ cells (DM dorsal mesenterium). B Quailembryo, HH19. Frontotransverse section. A number of WT1+ cellsmedial to the mesonephric ducts (MD) are arranged in vesicles(arrowheads). Other cells, however, are located in the ventrolater-al aortic wall (small arrow). Note the strong immunoreactivity ofthe coelomic mesothelium closer to the mesonephric duct (largearrow). C Quail embryo, HH24. Transverse section. The meso-nephric tubules are WT1–, but they are surrounded by immunore-active cells, which also extend to the dorsal areas where the meta-nephros will differentiate in later stages (M). A WT1-immunore-active mesonephric vesicle is shown (MV). The strong immunore-activity 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 thelateral part of the mesonephros (MN), the müllerian duct (MU) issurrounded by WT1+ mesenchymal cells and covered by immuno-reactive mesothelial cells (BW lateral body wall). Scale bars22 µm (A), 28 µm (B, E), 32 µm (C), 30 µm (D)

Fig. 4A–F WT1 immunostaining in some organs of avian embry-os. A Quail embryo, HH24. Transverse section. The WT1-immu-noreactive cells form a narrow band in the outer areas of the endo-dermal organs, oesophagus (OE), lung buds (LB) and liver (LI).However, WT1+ cells surround the mesonephric ducts (arrow-heads) and the dorsal aorta (AO), and arrive at the areas where themetanephros will differentiate in later stages (M). B Chick em-bryo, HH30. Transverse section. The left gonad is filled withWT1+ cells and lined by an immunoreactive mesothelium (arrow).C Chick embryo, HH30. Transverse section. The large amount ofWT1+ cells in the spleen (SP) contrasts with the small number ofimmunoreactive cells in the proventriculus (PV), where there is athin layer of submesothelial cells (arrow) and a few cells sparse inthe inner layer (arrowheads). D Chick embryo, HH35. Transversesection of the oesophagus (OE). A layer of submesothelial cells isWT1+, and some immunoreactive cells seem to be migrating to-ward inner areas (arrows). E, F Quail and chick embryos, HH26and HH30, respectively. Transverse sections of the spinal cord, ata thoracic level. WT1+ cells can be seen in the mantle layer (ML)between the ependyma (EPN) and the ventral horn of the greymatter (VH). The number of cells increase between HH26 andHH30, and their location shifts to a more ventral level (RP roofplate). Scale bars 90 µm (A), 29 µm (B), 22 µm (C), 38 µm (E),30 µm (D, F), respectively

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Fig. 4A–F Legend see page 177

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Fig. 5A–F WT1 immunostaining in the heart of avian embryos.A Quail embryo, HH18. The proepicardial villi (arrow) and theearly epicardium (arrowhead), which covers the atrioventriculargroove (AV), are immunoreactive. Note the lack of immunoreac-tivity in the myocardium (MY) and the endocardial cushion mes-enchyme (EC). B Quail embryo, HH19. Transverse section. Allthe proepicardial (small arrows), epicardial (EP) and subepicardialcells (arrowhead) are intensely WT1+. Note the immunoreactivityof 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. Transversesection. Most epicardial (EP) and subepicardial (SE) cells are im-munoreactive, but WT1+ cells are scarce in the atrial myocardium(AM). E Chick embryo, HH30. Transverse section. The ventricularmyocardium is infiltrated by WT1+ cells, which sometimes are ar-ranged in rows and occupy the clefts between the myocardiocytes(arrows) (SE subepicardium, EC atrioventricular endocardialcushion). F Quail embryo, HH28. Transverse section. Control sec-tion incubated with the primary antibody preadsorbed with the im-munogen (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-mesonephrosregion. A, B Quail embryo, HH20. Transverse semithin section.The areas where WT1 immunoreactivity is detected (compare withC) show morphological evidence of epithelial-mesenchymal tran-sition, including cell overriding and basal cytoplasmic processes(arrows). Note the morphological changes of the mesothelium ad-jacent to the mesonephric duct (MD). The WT1– mesothelial cellsof the dorsal mesentery (DM) show a more typical epithelial mor-phology (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 me-sentery (DM), where immunoreactive cells seem to be migratingtowards inner areas, and also in the mesothelium (the limit of theimmunoreactive cells is shown by the arrowhead) and developingglomerulus (G). Other abbreviations as in A. D Quail embryo,

HH21. Periaortic area anterior to the mesonephros. The distribu-tion of the immunoreactive cells is similar to that observed atmore posterior levels, including an increase in the immunoreactiv-ity close to the lateroventral aortic walls (arrowheads) and cardi-nal veins (CV). Note the presence of WT1+ cells close to the aorticwall (arrows), where clusters of haematopoietic stem cells are dif-ferentiating 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 Quailembryo, HH21. Double immunostaining of WT1 (immunoperoxi-dase) and the vascular antigen QH1 (immunofluorescence) super-imposed through digital image processing. Before a vascular lu-men 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 bars36 µm (A, B), 26 µm (C), 32 µm (D), 24 µm (E)

being more stained (Fig. 6C; see also Fig. 3A, B for aearlier stage). An exception to this rule were the nephro-genic cells arranged in the mesonephric vesicles, whichshowed a strong immunoreactivity.

HH24–26

The immunostaining pattern of the mesothelial cells issimilar to that of the previous stages, but new areas ofimmunoreactive mesothelial cells have appeared aroundthe oesophagus, lining the lung buds (Figs. 4A, 7C), andin a well-defined area posterior to the connection of thecardiac outflow tract, close to the laryngotracheal grooveand ventral to the aortic sac (Fig. 7A). The labelling ofthe parietal and visceral mesothelia is more generalizedthan in the previous stages, but the intensity of the label-ling is still stronger in specific areas characterized by theproximity of endodermal-derived organs, myocardiumand nephric ducts (Figs. 3C, 7E).

WT1+ mesenchymal cells form a crescent-shaped areaaround the dorsal aorta, extending dorsally to the level ofthe sympathetic ganglia (Fig. 4A), and they are veryabundant 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-

um, concretely in the ventricle (Fig. 5C). These cells ap-pear in clefts between the myocardial cells. On the otherhand, in lungs, gut and liver, the WT1+ cells remain in athin submesothelial layer (Figs. 4A, 7C, E).

In the HH26 embryos a new domain of WT1+ cellsappear in the spinal cord. A few immunoreactive cells(8–16 cells per section on each side) can be seen in thelateral areas, slightly dorsal to the middle level (Fig. 4E).

HH27–35

The mesothelium covering the heart, trachea, lungs, liv-er, oesophagus, stomach, gonads, spleen, mesenteriesand mesonephros is immunoreactive, although the stain-ing has decreased in the parietal mesothelium. A narrowband of submesothelial immunoreactive cells is evidentin the oesophagus, stomach, lungs and liver, where somecells seem to be migrating into the inner core of WT1–

cells (Fig. 4D). Immunoreactive mesenchymal cells canbe seen throughout the metanephros, gonads and spleen(Figs. 3D, 4B, C, respectively), dorsal mesocardium andmesenteries (especially the ventral mesentery of the liv-er). Most of the mesonephros shows no WT1+ cells, ex-cept for the glomeruli (Fig. 3D). WT1+ cells are veryabundant around the müllerian ducts (Fig. 3E). Mostcells at the subepicardium are WT1+ (Fig. 5D), whilemany immunoreactive cells can be seen within the ven-tricular myocardium, sometimes forming rows betweenthe myocardiocytes (Fig. 5E). However, WT1+ cells arevery scarce in the atrial myocardium (Fig. 5D). The peri-aortic WT1+ cells have become less abundant in the an-terior part of the trunk, although small groups of cells aredetected ventrally to the sympathetic ganglia and lateralto the vertebrae. The cells in the spinal cord have in-creased in number and they are located at a more ventral

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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 anti-bodies against WT1 (A, C, E) and Slug (B, D, E) in the laryngo-tracheal groove (A, B), lungs (C, D) and liver (E, F). The Slug-immunoreactive cells are present throughout these areas, but theWT1+ cells remain at the surface. However, note the images whichsuggest migration of the outer cells inside these organs (arrows).The mesothelium is stained with both antibodies (EN endodermaltissue, LB lung buds, LI liver). Scale bars 43 µm (A, B), 54 µm(C), 46 µm (D, E), 41 µm (F)

position than in the previous stages, in the mantle, be-tween the ependymal layer and the ventral horns of themedulla (Fig. 4F).

Slug immunolocalization in the coelomic wall

The mesothelial cells lining the nephrogenic ridges, me-senteries, developing gut, allantois, lungs, liver and heartwere Slug immunoreactive, showing a pattern very simi-lar to that described for the WT1+ mesothelial cells, i.e.an increase in immunoreactivity in the proximity ofsome organs such as nephric ducts, lung buds, liver pri-mordium or myocardium (Fig. 7B, D, F). All these or-gans showed abundant Slug+ mesenchymal cells. How-ever, unlike the WT1+ mesenchymal cells, Slug+ mesen-chymal cells were observed throughout the primordia ofthe 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 sub-epicardial cells were Slug+, but Slug-immunoreactivecells were not observed infiltrating within the ventricularmyocardium. In the genitourinary system, Slug+ mesen-chymal cells were very abundant in the developing go-nads, but very scarce in the mesonephros (not shown).

Discussion

The normal roles played by the WT1 protein in the em-bryonic development have been the object of much dis-cussion. A better knowledge of these roles is made diffi-cult by the multiple functions which WT1 has beenshown to perform in a number of in vitro reporter assaysas commented upon in the “Introduction”. WT1 has beenreported to inhibit cell growth but also to be necessaryfor proliferation of leukemic cell lines (Algar et al.1996). Some experimental evidence shows that WT1 isable to induce apoptosis but also to protect from apopto-sis acting as a survival factor. The promoters of a num-ber of genes have been shown to be either activated andrepressed by WT1, and a further role in splicing has alsobeen proposed (reviewed in Little et al. 1999).

These disparate and sometimes contradictory resultscan partially be explained by the existence of differentisoforms of the protein, generated by alternative splicing,RNA editing and alternative translational start sites (Davies et al. 1999). However, we are far from explain-ing the precise functions performed by WT1 during thedevelopment.

In order to interpret our results, we will start from twoimportant points: first, WT1 has been involved in pheno-typic shifts between mesenchyme and epithelium as wellas between epithelium and mesenchyme (Moore et al.1999). Second, the phenotype of the WT1-null embryoshas shown its requirement for the development of kid-ney, gonads, spleen and adrenals (Kreidberg et al. 1993;Moore et al. 1999), although the lack of WT1 causes alethal 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 ob-servations about the sites of localization of WT1 proteinduring the embryonic development in order to suggest ahypothesis about their normal physiological functions.

The first point to be mentioned is the close relation-ship between the localization of WT1 in the mesotheliumand submesothelial cells. The protein was localized indefinite areas of the coelomic mesothelium and in thelayer of the cells immediately below, but never in one ofthese layers alone, with the exception of the very earlyepicardium, which is directly attached to the myocardi-um, without a subepicardial layer. On the other hand, un-til stage HH24, the presence of WT1 was always relatedto the proximity of one of these tissues: nephric ducts,endoderm-derived organ primordia or myocardium. Inthese cases, the limit between the immunoreactive andnot immunoreactive mesothelial areas was clearly delim-ited. After stage HH24, WT1 was found throughout thecoelomic mesothelium, but the strongest and most per-sistent immunoreactivity of the mesothelial cells alwayscoincided with the proximity of the mentioned tissues orother tissues developed later, such as the müllerian ducts.

Interestingly, a significant correlation was found inthe mesothelial cells between the presence of WT1 andSlug, another zinc-finger transcription factor. Slug andWT1 immunoreactivities were strong in the same meso-thelial areas (epicardium, liver, lungs and nephrogenicridges) and in the same developmental stages. However,in spite of the coincidence in the mesothelial cells, inter-esting differences were observed in the pattern of immu-noreactivity of the underlying mesenchyme. Thus, in theendoderm-derived organs, WT1+ cells were only seen inthe outermost layer, while Slug+ cells were observedthroughout organ primordia such as lungs or liver. In theheart, Slug+ and WT1+ cells were very abundant in thesubepicardium, where vascular and connective tissuewas forming, but only WT1+ cells invaded the myocardi-um. The developing gonads were also widely invaded bySlug+- and WT1+-immunoreactive cells, but there was acoincident downregulation of both Slug and WT1 in thedifferentiated mesonephros. Furthermore, in other sys-tems of epithelial-mesenchymal transition where Slug isexpressed, such as the endocardial cushions of the heart(Carmona et al. 2000), WT1+ cells were not observed inthe stages studied.

Most papers dealing with the developmental functionof WT1 have stressed the coincidence between its ex-pression in the developing kidney (where a transforma-tion of the nephrogenic mesenchyme into epithelium oc-curs) and in the coelomic epithelium. Thus, it has beenfrequently suggested that WT1 might be involved in theformation of the mesothelium by aggregation of mesen-chymal cells. This idea can be traced back to the paperby Pritchard-Jones et al. (1990), although the flaws ofthis hypothesis have been shown (Armstrong et al.1993). Basically, these authors rightly remark that theearly coelomic epithelium (and many other embryonicepithelia) forms without expression of WT1. A more re-

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cent contribution suggests instead that this transcriptionfactor may enable cells to flip between mesenchymal andepithelial cell states (Moore et al. 1999). We think thatthere are powerful reasons to believe that WT1 is specif-ically expressed or upregulated in areas of transforma-tion of mesothelial cells into mesenchyme. These rea-sons are:

1. No WT1 expression is detected in most of the earlycoelomic mesothelium of the avian embryos, as al-ready noted by Armstrong et al. (1993). WT1 proteinis first localized in very precise mesothelial areas, al-ways coinciding with the proximity of specific tissueswhich might be playing an inductive role (nephricducts, müllerian ducts, endodermal derivatives andmyocardium).

2. In most of the mesothelial areas where WT1 is ex-pressed or upregulated, an epithelial to mesenchymaltransition has been described, but not the reverse, forexample, 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-derivedcells of mouse embryos has been remarked upon byMoore et al. (1999). The contribution of mesothelialcells to the mesenchyme of other developing organswhere WT1 is widely expressed, such as mesoneph-ros, gonads and adrenal cortex, was well known forthe older anatomists. Indeed, a large number of papersin the classical anatomical literature deal with this de-velopmental event (reviewed in Gruenwald 1942).Unfortunately, these important observations seem tohave been neglected in the most recent literature. Forexample, it is a well-known fact that the cells whichform the adrenal cortex arise from the peritoneal epi-thelium of the mesonephric ridges (Bellairs and Osmond 1998). Sertoli cells of the testicle are alsomesothelial derivatives (Bellairs and Osmond 1998;Karl and Capel 1998) and express WT1 even in adults(Mundlos et al. 1993). We have shown elsewhere evi-dence of a migration of mesothelial-derived cells tothe paraaortic areas of avian embryos (Pérez-Pomareset al. 1999), where WT1+ cells are very abundant ac-cording to our observations (see Fig. 6D).

3. It has been claimed that WT1 expression in the meso-and metanephric mesenchyme is related to their abili-ty to transform into epithelium. However, we haveshown that WT1 immunoreactivity persists in theparavertebral metanephric mesenchyme, dorsal to themesonephros, for a long time without signs of differ-entiation. We have also shown that WT1 immunore-activity suddenly disappears, coinciding with the dif-ferentiation of the mesonephric mesenchyme into epi-thelial structures. Finally, it is important to note thatthe main site of WT1 expression in the adult kidney,the podocytes, is not entirely epithelial, showing anintermediate phenotype between epithelium and mes-enchyme (Davies 1996). Sertoli cells, which expressWT1 throughout adult life as stated above, also show

mesenchymal characteristics. These observationswould argue against a significant role of WT1 in theacquisition of a fully differentiated epithelial pheno-type, although other findings have strongly supportedthis role.

4. The temporal and spatial expression of WT1 in thecoelomic mesothelium significantly correlates withthe presence of Slug, a transcription factor whichplays a key role in the epithelial-mesenchymal transi-tion (Duband et al. 1995; Savagner et al. 1997). In-deed, Slug inactivation in avian embryos impairs theepithelial-mesenchymal transformation of the neuro-epithelium into the neural crest cells (Nieto et al.1994). Snail, the probable functional homologue ofSlug in mammals (Sefton et al. 1998), has beenshown to downregulate E-cadherin expression, thuscontributing to the phenotypic shift between epitheli-um and mesenchyme (Batlle et al. 2000; Cano et al.2000).

In conclusion, we think that there are reasons to believethat WT1 is present in those mesothelial cells fated totransform to mesenchyme or, at least, which are able toachieve such a transformation. We also think that thesubmesothelial WT1+ cells represent, probably, a popula-tion of undifferentiated mesothelial-derived cells, a pointof view already adopted by Moore et al. (1999) in rela-tion to the epicardial-derived cells.

However, we think that WT1 is not required for theprocess of epithelial-mesenchymal transition. In fact, themain embryonic processes of epithelial-mesenchymaltransition, such as gastrulation, neural crest or endocardi-al cushion formation progress without WT1 expressionand they are normal in WT1-null embryos. On the otherhand, WT1-null embryos apparently show mesothelial-derived mesenchyme, for example, in the subepicardium(Kreidberg et al. 1993), although the number of subepi-cardial mesenchymal cells is greatly reduced (Moore etal. 1999).

We think that a hypothetical function of WT1 whichwould be consistent with the available observations is tomaintain the mesothelial-derived mesenchyme in an un-differentiated state. Moore et al. (1999) had already sug-gested that WT1 enables cells to flip between mesenchy-mal and epithelial cell states. We suggest that in some or-gans, especially those derived from endoderm, these cellswould soon lose their WT1 protein, and would subse-quently migrate to inner zones and differentiate, probablyinto fibroblasts and smooth muscle cells. It would explainthe 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 vis-cerae), mesothelial-derived cells are apparently able to mi-grate into inner areas keeping their WT1 protein. It doesnot mean, however, that WT1 cannot also perform alterna-tive functions related to differentiation or cell survival, de-pending on the developmental or cellular context.

Interestingly, all the mesodermal organs invaded byWT1+ cells, but not the endoderm-derived viscerae, are

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severely affected or even absent in WT1-deficient mouseembryos (Kreidberg et al. 1993; Moore et al. 1999). Thiscan be explained in the context of our hypothesis if weassume that the mesothelial-derived cells which shouldnormally contribute to the primary sex cords, corticoad-renal tissue or nephric blastema, prematurely differenti-ate in the absence of WT1, possibly into fibroblasts. Inthe case of the kidney, the lack of an inducing blastemawould cause agenesia of the ureteric bud, whose absencecould, in turn, induce apoptosis in mesenchymal cells(Davies 1996).

In the case of the heart, our hypothesis probably alsoaccounts for the cardiac defects in WT1-null embryos.The undifferentiated WT1+ epicardial-derived cellswhich invade the myocardium might play a signallingrole which would be lost in WT1-deficient embryos bypremature differentiation. It is important to remark thatthe epicardium produces retinoids (Moss et al. 1998),and these molecules are essential for the differentiationof the ventricular myocardium (Kubalak and Sucov1999). Since retinoids are hydrophobic, it is conceivablethat the massive migration of WT1+ epicardial-derivedcells inside the myocardium is essential for the deliveryof retinoids to the inner layers of the ventricle. In theWT1-null embryos, epicardial-derived cells would nor-mally form, but they would differentiate prematurely andwould stop producing retinoids. Thus, the ventricularmyocardium would be defective, resulting in cardiacfailure. It might be significant that the RXRα-null miceembryos show the same cardiac phenotype as the WT1-null embryos, as already noted by Kubalak and Sucov(1999). Both types of embryos show thin ventricularwalls and die between 14.5 and 15.5 dpc from cardiacfailure (Sucov et al. 1994; Dyson et al. 1995). In theRXRα-null embryos, the lack of the retinoid receptorcauses an anomalous persistence of the atrial-specificMLC2a myosin isoform, either by premature differentia-tion of the ventricular myocardium or by the acquisitionof an atrial phenotype in the ventricle (Dyson et al.1995). Thus, a prediction of our hypothesis is the anoma-lous persistence of MLC2a isoform in the ventricle ofWT1-null embryos.

Although there is a close relationship between thepresence of WT1 protein and the coelomic wall, we haveobserved immunoreactive cells in the spinal cord, an ob-servation which has also been made in mouse embryos(Armstrong et al. 1993; Moore et al. 1998). In this re-gard, it might be significant that the Drosophila zinc-fin-ger protein Klumpfuss, which shows sequence similari-ties to vertebrate WT1, is expressed in a subset of neuro-nal precursors, where it is involved in the determinationof the cell fate (Yang et al. 1997). On the other hand, wehave not observed the presence of WT1 protein in otherlocations where evidence of WT1 gene expression hasbeen reported, such as the fourth ventricle of the brain orthe 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 factsare 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+ cellsare very abundant in the AGM (aorta-gonad-mesoneph-ros) region, where the definitive haematopoietic stemcells differentiate in the avian and mammalian embryos(Cormier et al. 1988; Pardanaud et al. 1996). We havelocalized WT1+ cells very close to the lateroventral aor-tic wall, a site reached by mesothelial-derived cells(Pérez Pomares et al. 1999), where haematopoietic stemcells are being incorporated into the blood stream(Cormier et al. 1988; Tavian et al. 1996). It is temptingto connect all these observations. If WT1+ cells differen-tiate into haematopoietic progenitors in the AGM region,and we assume that WT1 is a marker of mesothelial-derived cells, it would imply that these cells can give riseto haematopoietic stem cells. This idea is not new. In factwe have proposed a model about the differentiation ofhaemangioblasts from pluripotential mesothelial-derivedcells, a model supported by a number of observationsand also by phylogenetic considerations (Muñoz-Chapuliet al. 1999). For example, it is interesting to note that inmany invertebrates, such as the echinoderms, blood cellsderive from the coelomic epithelium (Vanden Bosscheand Jangoux 1976).

In conclusion, we suggest that one of the possiblefunctions of WT1 may be related to the transient acquisi-tion of an undifferentiated, pluripotential state by meso-thelial-derived cells. It is important to note that the coe-lomic mesothelium of the embryo has been regarded asmesoderm arranged as a flat epithelium, capable of dif-ferentiation along the same lineages that the mesodermnormally displays during embryogenesis (Donna et al.1991; Colas et al. 2000). From the evolutionary point ofview, the dedifferentiation of mesothelial-derived cellswould serve at least two aims: first to regain the epithe-lial state in order to give rise to a new system of cavitieslined with a mesodermal epithelium, with excretoryfunctions; and, second, to keep the mesenchymal state inorder to provide the endoderm-derived organs (lungs,liver, gut) of connective and muscular tissue. From thispoint of view, it might be possible to understand some ofthe serious and disparate consequences of WT1 malfunc-tion, such as renal tumour with aberrant differentiation,particularly muscle, but also occasionally bone or carti-lage (Davies et al. 1999).

Acknowledgements The monoclonal antibodies anti-Slug andQH1 were obtained from the Developmental Studies HybridomaBank maintained by the Department of Pharmacology and Molec-ular Sciences, John Hopkins University School of Medicine, Balti-more, MD 21205, and the Department of Biological Sciences,University of Iowa, Iowa City, IA 52242, under contract NO1-HD-2-3144 from the National Institute of Child Health and Hu-man Development (NICHD). The authors sincerely thank AmeliaAranega, Jorge Domínguez, Jesús Santamaría, Gerardo Atenciaand María José Aranda for their help.

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