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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, 174 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 175 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 176 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. 177 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 178 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) 181 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 182 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). 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