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In vitro segregation of the metanephric nephron
DEVELOPMENTAL
tiIOLOGY
84, 88-95 (1981)
In Vitro Segregation
of the Metanephric
Nephron
PETER EKBLOM,* AARO MIETTINEN,~ ISMO VIRTANEN,* TORSTENWAHLSTR~M,* ANNE DAWNAY,~ AND LAURI SAXEN* *Department
of Pathology
and TDepartment of Bacteriology and Immunology, SDepartment of Nephrology, St. Bartholomew Hospital, Received
June 25,
1980;
accepted
in revised
form
University of Helsinki, London,
Great
October
20,
Helsinki,
Finland,
and
Britain
1980
In vitro segregation of the metanephric nephron was examined using three probes for the main segments: Ruorochrome-conjugated wheat germ agglutinin (WGA) binding to the glomerular epithelial surface, an antiserum against the brush-border antigens (BB) of the proximal tubules, and an antiserum against the Tamm-Horsfall glycoprotein (TH) of the distal tubules. In vivo, these markers appeared sequentially on Days 13 to 15. The same sequence was obtained in experimental recombinants of the metanephric mesenchyme and its inductor. When the inductor was removed after a 24 hr initial transfilter contact with the mesenchyme, segregation was similarly observed after subculture of the isolated mesenchyme. Hence, the sequential, multiphase differentiation of the nephron is initiated during a short induction period.
INTRODUCTION
Tissue Cultures For in vitro transfilter experiments, separated metanephric mesenchymes of 11-day embryos were placed on a Nuclepore filter with the average pore size of 1.0 pm, and fragments of the spinal cord were used as inductors and glued on the lower surface of the filter (Grobstein, 1956, Saxen et al., 1968, Sax& and Lehtonen, 1978). To stop transfilter interaction, the inductor was carefully scraped off after 24 hr, and the mesenthymes were subcultured for up to 6 days. Starting from the second day of culture, tissues were removed at 8 hr intervals, fixed, and examined for the presence of the different markers. The medium consisted of Eagle’s minimum essential medium supplemented with 10% horse serum and antibiotics. Embryo extract was not used.
The developing metanephric nephron has proved a suitable model system for the analysis of multiphasic development and its regulation (Grobstein, 1956, 1967, Saxen et al., 1968). We have recently followed the postinductory in vitro differentiation of the nephron and demonstrated that primary tubules undergo further segregation when the isolated mesenchyme is subcultured. Brush-border (BB) antigens, specific for the proximal tubules, become expressed in such cultures, but many tubular elements are devoid of these antigens in immunofluorescence (Ekblom et al., 1980a). This suggests that these segments of tubules have been geared to another developmental pathway. To test this postulate, we exposed the isolated mesenchyme to an inductor for a short period. When the induction was completed, the inductor was removed, and the maturation of the tubules was followed in vitro. We show that a short induction pulse is followed by a multiphase maturation of the nephron, leading to the segregation of all three main segments, each carrying segment-specific markers.
MATERIAL
AND
Antisera The rabbit anti-rat kidney brush-border (anti-BB) antiserum cross-reacting with mouse kidney has been described earlier (Miettinen and Linder, 1976, Ekblom et al., 1980b). The specimens were analyzed for the presence of Tamm-Horsfall (TH) glycoprotein with the use of two independently prepared antisera. For immunoperoxidase stainings, rabbit anti-TH glycoprotein antiserum described previously was used (Dawnay et al., 1980). For immunofluorescence studies (IFL) and double-staining procedures, an antiserum described below was used. Identical results were obtained with both antisera. The glycoprotein was isolated from human urine by
METHODS
Kidneys Metanephric kidneys of hybrid mouse embryos BALBc X CBA were used. The age of the embryos was determined from the vaginal plug, the appearance of which was designed as Day 0. 0012-1606/81/070088-08~02.00/0 Copyright All rights
Q 1981 by Academic Press, Inc. of reproduction in any form reserved.
88
EKBLOM ET AL.
Metanephric
89
Nephron Segregation
FIG. 1. Sections of 13-day kidneys stained with TRITC-WGA showing an immature glomerulus. The epithelium shows TRITC-WGA binding already at this stage. The cells which form the mesangium and the endothelium are devoid of fluorescence (a). A tangential section through the epithelium shows that fluorescence is localized at the surface of the epithelial cells (b). x800.
the method of Van Dijk et al. (1979). The isolated material was analyzed in polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (Laemmli, 1970), using 8% slab gel. Coomassie blue staining revealed only one polypeptide band with the apparent molecular weight of 94,000 when 100 pg of protein was applied to the gel. In Ouchterlony immunodiffusion, the material gave one precipitation line with the known anti-TH antiserum (Dawnay et al., 1980), but did not react with rabbit antiserum against normal human serum proteins (Behringwerke, Marburg, FRG) or with the anti-BB antiserum. This material was used as TH glycoprotein antigen for the immunization and the preparation of a TH glycoprotein immunoadsorbent. Rabbits were immunized subcutaneously with 0.5 mg of the isolated TH glycoprotein in Freund’s incomplete adjuvant (Difco, Detroit, Mich.). After the third monthly injection, blood samples were taken and tested for antiTH activity by indirect IFL technique. Serum with a l/10,000 titer on mouse kidney was selected for further studies.
gate using TH glycoprotein-Sepharose 4B immunoadsorbant and the elution technique of Hill (1972). The immunologically purified conjugate was diluted in PBS where 10% bovine serum albumin was added. It contained 0.05 mg of IgG in the dilution used. Samples were fixed according to the method of SaintMarie (1962). For double-staining experiments, 200 ~1 of FITC-conjugated anti-BB and 200 ~1 of TRITC-conjugated anti-TH were laid over 6-pm-thick deparaffinized sections either simultaneously or successively. Sections were incubated with the conjugates for 2 hr at +37”C, washed with PBS, and mounted in buffered glycerol. Immunoperoxidase
Staining
The antiserum described previously by Dawnay et al. (1980) was used for indirect immunoperoxidase stainings as described in detail before (Wahlstrom et al., 1979). All appropriate controls as defined by others (DeLellis et al., 1979) were included. Lectin Stainings
Immunofluorescence
Staining
Gammaglobulin fraction was isolated from the antiBB antiserum conjugated with fluorescein isothiocyanate (FITC) and rendered kidney specific as described (Miettinen and Linder, 1976, Ekblom et al., 1980b). The IgG fraction was isolated from the anti-TH antiserum using protein A affinity chromatography (Protein A-Sepharose CL 4B, Pharmacia, Uppsala, Sweden) and conjugated with tetramethylrhodamine isothiocyanate (TRITC; Biological Laboratories, Baltimore, Md.), as described by Goding (1976). Immunologically purified anti-TH antibodies were isolated from the conju-
For lectin binding experiments, tetramethylrhodamine isothiocyanate-coupled wheat germ agglutinin (TRITC-WGA) and fluorescein isothiocyanate-coupled peanut agglutinin (FITC-PNA) were used. These lectins specifically label terminal N-acetyl glucosamine and sialic acid moieties (WGA) and terminal galactosyl residues (PNA) (Bhavanandan and Katlic, 1979, Monsigny et al., 1980). In a preliminary report, we have suggested that lectins may be used to study the surface of the podocytes (Ekblom et al., 1979). Deparaffinized sections were reacted with the WGA conjugate, washed in PBS, and then incubated with the PNA conjugate. In some
90
DEVELOPMENTAL
BIOLOGY
experiments, the deparaffinized sections were first treated with 0.01 U/ml of neuraminidase for 30 min in +37”C (from Vibrio cholerae, Behringwerke) or with hyaluronidase (2 mg/ml, from bovine testis, Serva, Heidelberg, FRG). After the washings, the specimens were embedded in PBS for photography. The lectin binding could be totally inhibited by a preincubation of the lectin conjugates with 0.2M N-acetyl glucos-
VOLUME
84, 1981
amine (WGA) or 0.2 M D-galactose at room temperature.
(PNA)
for 30 min
RESULTS
In Viva Development Induction of the ureter bud leads to a condensation of the mesenchyme. The primary condensates surround
FIG. 2. Sections of 15 and Is-day kidneys double stained with TRITC-WGA and FITC-PNA. Staining with TRITC-WGA becomes more intense at the base of the cells on Day 15, and the epithelial cells move inward (a). In the same section, binding of FITC-PNA is seen only in some tubules (b). X700. After neuraminidase treatment (Nase) FITC-PNA binds to the glomerular epithelial surface (c). Arrow indicates a detached visceral epithelial cell binding the lectin. X700.
EKBLOMETAL.
FIG. 3. A section (TH). The tubules
of a 15-day kidney double stained not expressing the BB antigens
with antisera (a) (arrows)
Metanephric
against the brush-border can be shown to express
the ureteric bud and gradually develop into S-shaped bodies. The upper part will form distal tubules and join the collecting tubule system which develops from the branching ureteric bud. The middle part becomes the proximal tubule, whereas the lower part forms the epithelium of the glomerulus. None of the markers used were detected in 12-day embryos. When 13-day embryonic kidneys were stained with TRITC-WGA, the de-
91
Nephron Segregation
antigens (BB) and the Tamm-Horsfall the TH glycoprotein (b). X750.
glycoprotein
veloping glomerular epithelium showed fluorescence (Fig. 1). When the embryonic kidneys were double stained with the two lectins, TRITC-WGA and FITCPNA, a predominant staining of the glomerular podocyte surfaces was seen with TRITC-WGA (Fig. Ba), whereas FITC-PNA brightly labelled the tubules but not the glomeruli (Fig. 2b). The staining was not altered by the pretreatment of the sections with hyaluronidase.
92
DEVELOPMENTALBIOLOGY
After treatment with neuraminidase, however, a bright staining of glomeruli was seen after the application of FITC-PNA (Fig. 2c), whereas the staining of the tubules remained unaltered. Neuraminidase treatment completely abolished the TRITC-WGA binding to the podocyte surface. Using indirect immunofluorescence, the TH glycoprotein becomes detectable on Day 15 in utero, 1 day after the appearance of the BB antigens. In double staining, where different fluorochromes were used for visualizing the TH glycoprotein and the BB antigens in the same section, the two antigens are invariably found in different segments of the nephron (Figs. 3a, b). The TH glycoprotein is found in smaller tubules, whereas the BB antigens are seen in the tubules connected with the glomerulus. Glomerular structures as well as the macula densa were negative for the two markers. In immunoperoxidase experiments, the TH glycoprotein was also detected first on Day 15 in utero. Subse-
FIG. 4. Sections of a B-day Numerous elongated tubules
VOLUME 84. 1981
quently, more elongated (Fig. 4).
TH-positive
tubules
Transfilter
Continuous
Contact
Combinations,
appear
Metanephric mesenchymes, exposed to the inductor in transfilter cultures, formed glomerular-like bodies on the third day of cultivation, and TRITC-WGA binding was seen in these (Fig. 5a). In double staining sections, no binding of FITC-PNA to these structures could be seen, but the tubules stained intensely (Fig. 5b). Neuraminidase treatment reversed the staining of the glomerular bodies; after enzyme digestion no binding of TRITC-WGA was seen (Fig. 5c), but a bright labeling could be seen with FITC-PNA (Fig. 5d). Metanephric mesenchymes differentiating in vitro expressed the TH glycoprotein after a total culture of 104 hr. Explants fixed prior to this stage did not show this antigen when examined in immunofluroescence or with the immunoperoxidase technique. They showed, however, the BB antigens already after 80 hr in culture.
kidney stained with antiserum against the TH glycoprotein expressing the antigen are seen in the medulla. X150.
and visualized
with
the immunoperoxidase
technique.
93
FIG. 5. Demonstration staining with TRITC-WGA (b). After neuraminidase (d). X660.
of podocytes in an experimental recombinant of metanephric mesenchyme is seen in a cell cluster between tubular structures (a) and FITC-PNA treatment, the TRITC-WGA binding is abolished (c), but now FITC-PNA
TranSfilter Combinations, Short Contact Similar development and identical sequence were obtained when the transfilter contact with the inductor was broken after the initial period of 24 hr and the mesenchyme was then subcultured alone. TRITC-WGA binding could be demonstrated at the end of the third day of culture, the BB antigens at the beginning of the fourth day, and the TH glycoprotein at the beginning of the fifth day. The majority of the tubules expressed the BB antigen, whereas only a few smaller tubules expressed the TH glycoprotein (Fig. 6). DISCUSSION
Differentiation of the mouse metanephric nephron was followed with the use of markers for the three segments of the nephron: the glomerulus, the proximal and distal tubules. These markers were found to appear sequentially during in viva development. All three segments of the nephron could also be obtained as a consequence of a short-term inductive stimulus in vitro. To demonstrate the visceral epithelial surface of the glomerulus, fluorochrome-coupled lectins with a known sugar specificity were used. The coat apparently con-
and the inductor, spinal cord. Intense binds to the tubules in the same section binds to the glomerular-like cell clusters
tains sialic acids and other charged groups (Mohos and Scoza, 1969, Reeves et al., 1978), but the detailed sugar composition has remained unknown. We show that WGA, known to react mainly with sialic acids in biological structures (Bhavanandan and Katlic, 1979, Monsigny et al., 1980), reacted with the surface of the visceral epithelial cells of the glomerulus. When the sections were pretreated with neuraminidase, the glomerular epithelial surface, however, showed an intense fluorescence when stained with PNA, a lectin known to bind to galactose residues. No PNA binding to the glomerular epithelial surface could be found before enzyme treatment. The results suggest that the binding of WGA to the glomerular epithelial surface is mediated by sialic acids, and, furthermore, that the glomerular epithelial surface is rich in galactose masked by peripheral sialic acids. To distinguish between proximal and distal tubules, we used antisera reacting with brush border (BB) of proximal tubules (Miettinen and Linder, 1976, Ekblom et al., 1980b), and with TH glycoprotein found only in distal tubules (Hoyer et al., 19’79, Sikri et al., 1979). The markers characterizing each segment appear sequentially during the in vivo development of the meta-
nephric nephron. The glomerular polyanionic coat ap- became detectable several days after the removal of the inductor. They appeared sequentially with a time inpears on Day 13 of development, and the BB antigens, terval of approximately one cell cycle (i.e., 20 to 24 hr). specific for proximal tubules, become detectable on Day The glomerular polyanionic coat could be demonstrated 14 (Ekblom et al., 1980b). The TH glycoprotein, known on the third day of development, the BB antigens at the to be restricted to distal tubules, appear on Day 15, four onset of the fourth day, and the TH glycoprotein could days after the initial contact with the inducing ureter be detected on Day 5. The time interval and the seand the mesenchymal blastema. This is considerably quence could not be altered in any way by leaving the earlier than perviously reported for other species (Walinductor tissue in contact with the mesenchyme for the lace and Nairn, 1971, Lewis et al., 1972, Hoyer whole culture period. Hence, once initiated by the inet al., 1974). ductor tissue during the first 24-hr period, the develA precise timing of the appearance of the segment opmental program proceeds in the same sequence as markers can be achieved in vitro. Previous transfilter during in vivo development. studies have shown that an initial contact is required The development of the nephron seems to present an between the mesenchymal cells and their inductors example of a multiphase compartmentalization with at et al., 1974, Lehtonen, 1976, Saxen et al., (Wartiovaara least two molecularly distinct levels of differentiation. 1976). The first cells become induced after a 12-hr transThe first change, the appearance of basement memfilter contact. The number of cells subsequently particmesenchyme, ipating in tubule development increases linearly as a brane components in the undifferentiated occurs during the induction period. This is followed by function of time until a maximum response is obtained a period during which all epithelial cells apparently are after 30 hr, after which the inductor can be removed alike and express the basement membrane components (Saxen and Lehtonen, 1978). None of the segment markers were found during this induction period, but they (Ekblom, 1981, Ekblom et al., 1980a). Here we show that
FIG. 6. Sections through a mesenchymal explant induced for 24 hr and subcultured for 96 hr. Figures (a) to (c) demonstrate the in vitro segregation of the nephron into its three main segments. X350. (a) TRITC-WGA binding to the glomerulus-like bodies. (b) Expression of the BB antigens by the proximal tubules. (c) Expression of the TH glycoprotein by the distal tubules.
EKBLOM ET AL.
Metanephric
the completion of basement membrane formation is followed by a terminal differentiation, characterized by the sequential appearance of molecules marking each segment of the nephron. The basement membrane has frequently been suggested to act as a scaffold for the fixation, polarization, and differentiation of epithelial cells (Grobstein, 1967, Bernfield, 1978). Observations on the appearance of the basement membrane proteins in the kidneys have shown that the basement membrane of the distal part of the nephric anlage is formed approximately 24 hr later than that of the proximal tubule (Ekblom, 1981, Ekblom et aZ., 1980a). Thus, one would predict that the distal tubules lag behind the proximal ones in expressing their luminal antigens. This is shown in the present study. Also, in the transfilter in vitro experiments where the precise timing of the events is possible, the TH glycoprotein of the distal tubules was shown to appear 24 hr after the BB antigen of the proximal tubules. Hence, there is both a spatial and temporal correlation between the formation of the basement membrane and the subsequent polarization and differentiation of the cells lining the basement membrane. REFERENCES BERNFIELD, M. R. (1978). The cell periphery in morphogenesis. In “Birth Defects” (J. W. Littlefield and J. DeGrouchy, eds.), pp. lll125. Excerpta Medica, Amsterdam Oxford. BHAVANANDAN, V. P., and KATLIC, A. W. (1979). The interaction of wheat germ agglutinin with sialoglycoproteins. J. BioZ. Chem 254, 4000-4008. DAWNAY, A., MCLEAN, C., and CATTEL, W. R. (1980). The development of radioimmunoassay for Tamm-Horsfall glycoprotein in serum. Biochem. J. 185.679-687. DELELLIS, R. A., STERNBERGER, L. A., MANN, R. B., BANKS, P. M., and NAKANE, B. K. (1979). Immunoperoxidase technique in diagnostic pathology. Amer. J. Clin. Pathol. 71,483-488. EKBLOM, P. (1981). Formation of basement membranes in the embryonic kidney. An immunohistological study. J. CeU BioL (in press). EKBLOM, P., ALITALO, K., VAHERI, A., TIMPL, R., and SAX&N, L. (1980a). Induction of a basement membrane glycoprotein in embryonic kidney: Possible role of laminin in morphogenesis. Proc. Nat. Acad. Sci. USA 77,485-489. EKBLOM, P., MIE~INEN, A., and SAXEN, L. (1980b). Induction of brush border antigens of the proximal tubule in the developing kidney. Develop. Biol. 74, 263-274. EKBLOM, P., MIE~INEN, A., and VIRTANEN, I. (1979). Lectin binding sites in developing mouse metanephros. In “Protides of the biological fluids. Colloquium 27” (H. Peeters, ed.), pp. 463-466. Pergamon Press, Oxford. GODING, J. W. (1976). Conjugation of antibodies with fluorochromes: Modifications to the standard methods. J. ImmunoL Methods 13, 215-226. GROBSTEIN, C. (1956). Transfilter induction of tubules in mouse metanephric mesenchyme. Exp. Cell Res. 10,424~440.
Nephron Segregation
95
GROBSTEIN, C. (1967). Mechanism of organogenetic tissue interaction. Nat. Cancer Inst. Monogr. 26, 279-299. HILL, R. J. (1972). Elution of antibodies from immunoadsorbents: effect of dioxane in promoting release of antibody. J. Immunol. Methods 1, 231-245. HOYER, J. R., RESNICK, J. S., MICHAEL, A. F., and VERNIER, R. L. (1974). Ontogeny of Tamm-Horsfall urinary glycoprotein. Lab. Inwest, 30. 757-761. HOYER, J., SISSON, S. P., and VERNIER, R. L. (1979). Tamm-Horsfall glycoprotein. Ultrastructural immunoperoxidase localization in rat kidney. Lab. Invest. 41, 168-173. LAEMMLI, U. K. (1970). Cleavage of structural proteins during the assembly of head bacteriophage T 4. Nature (London), 227.680-685. LEHTONEN, E. (1976). Transmission of signals in embryonic induction. Med. Biol. 54, 108-128. LEWIS, R. A., SCHWARTZ, R. H., and SCHENK, E. A. (1972). TammHorsfall mucoprotein. II. Ontogenic development. Lab. Invest. 26, 728-730. MIETTINEN, A., and LINDER, E. (1976). Membrane antigens shared by renal proximal tubules and other epithelial associated with absorption and excretion. Clin. Exp. ImmunoL 23.568-577. MOHOS, S. C., and SCOZA, L. (1969). Glomerular sialoprotein. Science 164,1519-1521. MONSIGNY, M., ROCHE, A-C., SENE, C., MAGET-DANA, R., and DELMOITE, F. (1980). Sugar-lectin interactions: How does wheat-germ agglutinin bind sialoglycoconjugates? Eur. J. Biochem. 104, 147153. REEVES, W., CAULFIELD, J. P., and FARQUHAR, M. G. (1978). Differentiation of epithelial foot processes and filtration slits. Sequential appearance of occluding junction, epithelial polyanion, and slit membranes. Lab. Invest. 39.90-100. SAINTE-MARIE, G. (1962). A paraffin embedding technique for studies employing immunofluorescence. J. Histochem. Cytochem. 10, 250256. SAXEN, L., KOSKIMIES, O., LAHTI, A., MIETTINEN, H., RAPOLA, J., and WARTIOVAARA, J. (1968). Differentiation of kidney mesenchyme in an experimental model system. Advan. Morphog. 7,251-293. SAXEN, L., and LEHTONEN, E. (1978). Transfilter induction of kidney tubules as a function of the extent and duration of intercellular contacts. J. EmlnyoL Exp. Morph&. 47,97-109. SAXEN, L., LEHTONEN, E., KARKINEN-J~~SKEL~INEN, M., NORDLING, S., and WARTIOVAARA, J. (1976). Are morphogenetic tissue interactions mediated by transmissible signal substance or through cell contacts? Nature (London) 259, 662-663. SIKRI, K. L., FOSTER, C. L., BLOOMFIELD, F. J., and MARSHALL, R. D. (1979). Localization by immunofluorescence and light- and electronmicroscopic immunoperoxidase techniques of Tamm-Horsfall glycoprotein in adult hamster kidney. Biochem. J. 181.525-532. VAN DIJK, W., LASRHUIS, A.-M., FERWEDA, W. (1979). Preparation and chemical characterization of calf Tamm-Horsfall glycoprotein. B&him. Biophys. Acta 584,121-128. WAHLSTR~M, P., LINDGREN, J., KORHONEN, M., and SEPP~L~, M. (1979). Distinction between endocervical and endometrial adenocarcinoma with immunoperoxidase staining of carcinoembryonic antigens in routine histological specimens. Lancet 2,1159-1160. WALLACE, A. C., and NAIRN, R. C. (1971). Tamm-Horsfall protein in kidneys of human embryos and foreign species. Pathologg 3, 303310. WARTIOVAARA, J., NORDLING, S., LEHTONEN, E., and SAXEN, L. (1974). Transfilter induction of kidney tubules: Correlation with cytoplasmic penetration into Nucleopore filters. J. EmbryoL Ezp. MorphoL 31. 667-686.
tiIOLOGY
84, 88-95 (1981)
In Vitro Segregation
of the Metanephric
Nephron
PETER EKBLOM,* AARO MIETTINEN,~ ISMO VIRTANEN,* TORSTENWAHLSTR~M,* ANNE DAWNAY,~ AND LAURI SAXEN* *Department
of Pathology
and TDepartment of Bacteriology and Immunology, SDepartment of Nephrology, St. Bartholomew Hospital, Received
June 25,
1980;
accepted
in revised
form
University of Helsinki, London,
Great
October
20,
Helsinki,
Finland,
and
Britain
1980
In vitro segregation of the metanephric nephron was examined using three probes for the main segments: Ruorochrome-conjugated wheat germ agglutinin (WGA) binding to the glomerular epithelial surface, an antiserum against the brush-border antigens (BB) of the proximal tubules, and an antiserum against the Tamm-Horsfall glycoprotein (TH) of the distal tubules. In vivo, these markers appeared sequentially on Days 13 to 15. The same sequence was obtained in experimental recombinants of the metanephric mesenchyme and its inductor. When the inductor was removed after a 24 hr initial transfilter contact with the mesenchyme, segregation was similarly observed after subculture of the isolated mesenchyme. Hence, the sequential, multiphase differentiation of the nephron is initiated during a short induction period.
INTRODUCTION
Tissue Cultures For in vitro transfilter experiments, separated metanephric mesenchymes of 11-day embryos were placed on a Nuclepore filter with the average pore size of 1.0 pm, and fragments of the spinal cord were used as inductors and glued on the lower surface of the filter (Grobstein, 1956, Saxen et al., 1968, Sax& and Lehtonen, 1978). To stop transfilter interaction, the inductor was carefully scraped off after 24 hr, and the mesenthymes were subcultured for up to 6 days. Starting from the second day of culture, tissues were removed at 8 hr intervals, fixed, and examined for the presence of the different markers. The medium consisted of Eagle’s minimum essential medium supplemented with 10% horse serum and antibiotics. Embryo extract was not used.
The developing metanephric nephron has proved a suitable model system for the analysis of multiphasic development and its regulation (Grobstein, 1956, 1967, Saxen et al., 1968). We have recently followed the postinductory in vitro differentiation of the nephron and demonstrated that primary tubules undergo further segregation when the isolated mesenchyme is subcultured. Brush-border (BB) antigens, specific for the proximal tubules, become expressed in such cultures, but many tubular elements are devoid of these antigens in immunofluorescence (Ekblom et al., 1980a). This suggests that these segments of tubules have been geared to another developmental pathway. To test this postulate, we exposed the isolated mesenchyme to an inductor for a short period. When the induction was completed, the inductor was removed, and the maturation of the tubules was followed in vitro. We show that a short induction pulse is followed by a multiphase maturation of the nephron, leading to the segregation of all three main segments, each carrying segment-specific markers.
MATERIAL
AND
Antisera The rabbit anti-rat kidney brush-border (anti-BB) antiserum cross-reacting with mouse kidney has been described earlier (Miettinen and Linder, 1976, Ekblom et al., 1980b). The specimens were analyzed for the presence of Tamm-Horsfall (TH) glycoprotein with the use of two independently prepared antisera. For immunoperoxidase stainings, rabbit anti-TH glycoprotein antiserum described previously was used (Dawnay et al., 1980). For immunofluorescence studies (IFL) and double-staining procedures, an antiserum described below was used. Identical results were obtained with both antisera. The glycoprotein was isolated from human urine by
METHODS
Kidneys Metanephric kidneys of hybrid mouse embryos BALBc X CBA were used. The age of the embryos was determined from the vaginal plug, the appearance of which was designed as Day 0. 0012-1606/81/070088-08~02.00/0 Copyright All rights
Q 1981 by Academic Press, Inc. of reproduction in any form reserved.
88
EKBLOM ET AL.
Metanephric
89
Nephron Segregation
FIG. 1. Sections of 13-day kidneys stained with TRITC-WGA showing an immature glomerulus. The epithelium shows TRITC-WGA binding already at this stage. The cells which form the mesangium and the endothelium are devoid of fluorescence (a). A tangential section through the epithelium shows that fluorescence is localized at the surface of the epithelial cells (b). x800.
the method of Van Dijk et al. (1979). The isolated material was analyzed in polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (Laemmli, 1970), using 8% slab gel. Coomassie blue staining revealed only one polypeptide band with the apparent molecular weight of 94,000 when 100 pg of protein was applied to the gel. In Ouchterlony immunodiffusion, the material gave one precipitation line with the known anti-TH antiserum (Dawnay et al., 1980), but did not react with rabbit antiserum against normal human serum proteins (Behringwerke, Marburg, FRG) or with the anti-BB antiserum. This material was used as TH glycoprotein antigen for the immunization and the preparation of a TH glycoprotein immunoadsorbent. Rabbits were immunized subcutaneously with 0.5 mg of the isolated TH glycoprotein in Freund’s incomplete adjuvant (Difco, Detroit, Mich.). After the third monthly injection, blood samples were taken and tested for antiTH activity by indirect IFL technique. Serum with a l/10,000 titer on mouse kidney was selected for further studies.
gate using TH glycoprotein-Sepharose 4B immunoadsorbant and the elution technique of Hill (1972). The immunologically purified conjugate was diluted in PBS where 10% bovine serum albumin was added. It contained 0.05 mg of IgG in the dilution used. Samples were fixed according to the method of SaintMarie (1962). For double-staining experiments, 200 ~1 of FITC-conjugated anti-BB and 200 ~1 of TRITC-conjugated anti-TH were laid over 6-pm-thick deparaffinized sections either simultaneously or successively. Sections were incubated with the conjugates for 2 hr at +37”C, washed with PBS, and mounted in buffered glycerol. Immunoperoxidase
Staining
The antiserum described previously by Dawnay et al. (1980) was used for indirect immunoperoxidase stainings as described in detail before (Wahlstrom et al., 1979). All appropriate controls as defined by others (DeLellis et al., 1979) were included. Lectin Stainings
Immunofluorescence
Staining
Gammaglobulin fraction was isolated from the antiBB antiserum conjugated with fluorescein isothiocyanate (FITC) and rendered kidney specific as described (Miettinen and Linder, 1976, Ekblom et al., 1980b). The IgG fraction was isolated from the anti-TH antiserum using protein A affinity chromatography (Protein A-Sepharose CL 4B, Pharmacia, Uppsala, Sweden) and conjugated with tetramethylrhodamine isothiocyanate (TRITC; Biological Laboratories, Baltimore, Md.), as described by Goding (1976). Immunologically purified anti-TH antibodies were isolated from the conju-
For lectin binding experiments, tetramethylrhodamine isothiocyanate-coupled wheat germ agglutinin (TRITC-WGA) and fluorescein isothiocyanate-coupled peanut agglutinin (FITC-PNA) were used. These lectins specifically label terminal N-acetyl glucosamine and sialic acid moieties (WGA) and terminal galactosyl residues (PNA) (Bhavanandan and Katlic, 1979, Monsigny et al., 1980). In a preliminary report, we have suggested that lectins may be used to study the surface of the podocytes (Ekblom et al., 1979). Deparaffinized sections were reacted with the WGA conjugate, washed in PBS, and then incubated with the PNA conjugate. In some
90
DEVELOPMENTAL
BIOLOGY
experiments, the deparaffinized sections were first treated with 0.01 U/ml of neuraminidase for 30 min in +37”C (from Vibrio cholerae, Behringwerke) or with hyaluronidase (2 mg/ml, from bovine testis, Serva, Heidelberg, FRG). After the washings, the specimens were embedded in PBS for photography. The lectin binding could be totally inhibited by a preincubation of the lectin conjugates with 0.2M N-acetyl glucos-
VOLUME
84, 1981
amine (WGA) or 0.2 M D-galactose at room temperature.
(PNA)
for 30 min
RESULTS
In Viva Development Induction of the ureter bud leads to a condensation of the mesenchyme. The primary condensates surround
FIG. 2. Sections of 15 and Is-day kidneys double stained with TRITC-WGA and FITC-PNA. Staining with TRITC-WGA becomes more intense at the base of the cells on Day 15, and the epithelial cells move inward (a). In the same section, binding of FITC-PNA is seen only in some tubules (b). X700. After neuraminidase treatment (Nase) FITC-PNA binds to the glomerular epithelial surface (c). Arrow indicates a detached visceral epithelial cell binding the lectin. X700.
EKBLOMETAL.
FIG. 3. A section (TH). The tubules
of a 15-day kidney double stained not expressing the BB antigens
with antisera (a) (arrows)
Metanephric
against the brush-border can be shown to express
the ureteric bud and gradually develop into S-shaped bodies. The upper part will form distal tubules and join the collecting tubule system which develops from the branching ureteric bud. The middle part becomes the proximal tubule, whereas the lower part forms the epithelium of the glomerulus. None of the markers used were detected in 12-day embryos. When 13-day embryonic kidneys were stained with TRITC-WGA, the de-
91
Nephron Segregation
antigens (BB) and the Tamm-Horsfall the TH glycoprotein (b). X750.
glycoprotein
veloping glomerular epithelium showed fluorescence (Fig. 1). When the embryonic kidneys were double stained with the two lectins, TRITC-WGA and FITCPNA, a predominant staining of the glomerular podocyte surfaces was seen with TRITC-WGA (Fig. Ba), whereas FITC-PNA brightly labelled the tubules but not the glomeruli (Fig. 2b). The staining was not altered by the pretreatment of the sections with hyaluronidase.
92
DEVELOPMENTALBIOLOGY
After treatment with neuraminidase, however, a bright staining of glomeruli was seen after the application of FITC-PNA (Fig. 2c), whereas the staining of the tubules remained unaltered. Neuraminidase treatment completely abolished the TRITC-WGA binding to the podocyte surface. Using indirect immunofluorescence, the TH glycoprotein becomes detectable on Day 15 in utero, 1 day after the appearance of the BB antigens. In double staining, where different fluorochromes were used for visualizing the TH glycoprotein and the BB antigens in the same section, the two antigens are invariably found in different segments of the nephron (Figs. 3a, b). The TH glycoprotein is found in smaller tubules, whereas the BB antigens are seen in the tubules connected with the glomerulus. Glomerular structures as well as the macula densa were negative for the two markers. In immunoperoxidase experiments, the TH glycoprotein was also detected first on Day 15 in utero. Subse-
FIG. 4. Sections of a B-day Numerous elongated tubules
VOLUME 84. 1981
quently, more elongated (Fig. 4).
TH-positive
tubules
Transfilter
Continuous
Contact
Combinations,
appear
Metanephric mesenchymes, exposed to the inductor in transfilter cultures, formed glomerular-like bodies on the third day of cultivation, and TRITC-WGA binding was seen in these (Fig. 5a). In double staining sections, no binding of FITC-PNA to these structures could be seen, but the tubules stained intensely (Fig. 5b). Neuraminidase treatment reversed the staining of the glomerular bodies; after enzyme digestion no binding of TRITC-WGA was seen (Fig. 5c), but a bright labeling could be seen with FITC-PNA (Fig. 5d). Metanephric mesenchymes differentiating in vitro expressed the TH glycoprotein after a total culture of 104 hr. Explants fixed prior to this stage did not show this antigen when examined in immunofluroescence or with the immunoperoxidase technique. They showed, however, the BB antigens already after 80 hr in culture.
kidney stained with antiserum against the TH glycoprotein expressing the antigen are seen in the medulla. X150.
and visualized
with
the immunoperoxidase
technique.
93
FIG. 5. Demonstration staining with TRITC-WGA (b). After neuraminidase (d). X660.
of podocytes in an experimental recombinant of metanephric mesenchyme is seen in a cell cluster between tubular structures (a) and FITC-PNA treatment, the TRITC-WGA binding is abolished (c), but now FITC-PNA
TranSfilter Combinations, Short Contact Similar development and identical sequence were obtained when the transfilter contact with the inductor was broken after the initial period of 24 hr and the mesenchyme was then subcultured alone. TRITC-WGA binding could be demonstrated at the end of the third day of culture, the BB antigens at the beginning of the fourth day, and the TH glycoprotein at the beginning of the fifth day. The majority of the tubules expressed the BB antigen, whereas only a few smaller tubules expressed the TH glycoprotein (Fig. 6). DISCUSSION
Differentiation of the mouse metanephric nephron was followed with the use of markers for the three segments of the nephron: the glomerulus, the proximal and distal tubules. These markers were found to appear sequentially during in viva development. All three segments of the nephron could also be obtained as a consequence of a short-term inductive stimulus in vitro. To demonstrate the visceral epithelial surface of the glomerulus, fluorochrome-coupled lectins with a known sugar specificity were used. The coat apparently con-
and the inductor, spinal cord. Intense binds to the tubules in the same section binds to the glomerular-like cell clusters
tains sialic acids and other charged groups (Mohos and Scoza, 1969, Reeves et al., 1978), but the detailed sugar composition has remained unknown. We show that WGA, known to react mainly with sialic acids in biological structures (Bhavanandan and Katlic, 1979, Monsigny et al., 1980), reacted with the surface of the visceral epithelial cells of the glomerulus. When the sections were pretreated with neuraminidase, the glomerular epithelial surface, however, showed an intense fluorescence when stained with PNA, a lectin known to bind to galactose residues. No PNA binding to the glomerular epithelial surface could be found before enzyme treatment. The results suggest that the binding of WGA to the glomerular epithelial surface is mediated by sialic acids, and, furthermore, that the glomerular epithelial surface is rich in galactose masked by peripheral sialic acids. To distinguish between proximal and distal tubules, we used antisera reacting with brush border (BB) of proximal tubules (Miettinen and Linder, 1976, Ekblom et al., 1980b), and with TH glycoprotein found only in distal tubules (Hoyer et al., 19’79, Sikri et al., 1979). The markers characterizing each segment appear sequentially during the in vivo development of the meta-
nephric nephron. The glomerular polyanionic coat ap- became detectable several days after the removal of the inductor. They appeared sequentially with a time inpears on Day 13 of development, and the BB antigens, terval of approximately one cell cycle (i.e., 20 to 24 hr). specific for proximal tubules, become detectable on Day The glomerular polyanionic coat could be demonstrated 14 (Ekblom et al., 1980b). The TH glycoprotein, known on the third day of development, the BB antigens at the to be restricted to distal tubules, appear on Day 15, four onset of the fourth day, and the TH glycoprotein could days after the initial contact with the inducing ureter be detected on Day 5. The time interval and the seand the mesenchymal blastema. This is considerably quence could not be altered in any way by leaving the earlier than perviously reported for other species (Walinductor tissue in contact with the mesenchyme for the lace and Nairn, 1971, Lewis et al., 1972, Hoyer whole culture period. Hence, once initiated by the inet al., 1974). ductor tissue during the first 24-hr period, the develA precise timing of the appearance of the segment opmental program proceeds in the same sequence as markers can be achieved in vitro. Previous transfilter during in vivo development. studies have shown that an initial contact is required The development of the nephron seems to present an between the mesenchymal cells and their inductors example of a multiphase compartmentalization with at et al., 1974, Lehtonen, 1976, Saxen et al., (Wartiovaara least two molecularly distinct levels of differentiation. 1976). The first cells become induced after a 12-hr transThe first change, the appearance of basement memfilter contact. The number of cells subsequently particmesenchyme, ipating in tubule development increases linearly as a brane components in the undifferentiated occurs during the induction period. This is followed by function of time until a maximum response is obtained a period during which all epithelial cells apparently are after 30 hr, after which the inductor can be removed alike and express the basement membrane components (Saxen and Lehtonen, 1978). None of the segment markers were found during this induction period, but they (Ekblom, 1981, Ekblom et al., 1980a). Here we show that
FIG. 6. Sections through a mesenchymal explant induced for 24 hr and subcultured for 96 hr. Figures (a) to (c) demonstrate the in vitro segregation of the nephron into its three main segments. X350. (a) TRITC-WGA binding to the glomerulus-like bodies. (b) Expression of the BB antigens by the proximal tubules. (c) Expression of the TH glycoprotein by the distal tubules.
EKBLOM ET AL.
Metanephric
the completion of basement membrane formation is followed by a terminal differentiation, characterized by the sequential appearance of molecules marking each segment of the nephron. The basement membrane has frequently been suggested to act as a scaffold for the fixation, polarization, and differentiation of epithelial cells (Grobstein, 1967, Bernfield, 1978). Observations on the appearance of the basement membrane proteins in the kidneys have shown that the basement membrane of the distal part of the nephric anlage is formed approximately 24 hr later than that of the proximal tubule (Ekblom, 1981, Ekblom et aZ., 1980a). Thus, one would predict that the distal tubules lag behind the proximal ones in expressing their luminal antigens. This is shown in the present study. Also, in the transfilter in vitro experiments where the precise timing of the events is possible, the TH glycoprotein of the distal tubules was shown to appear 24 hr after the BB antigen of the proximal tubules. Hence, there is both a spatial and temporal correlation between the formation of the basement membrane and the subsequent polarization and differentiation of the cells lining the basement membrane. REFERENCES BERNFIELD, M. R. (1978). The cell periphery in morphogenesis. In “Birth Defects” (J. W. Littlefield and J. DeGrouchy, eds.), pp. lll125. Excerpta Medica, Amsterdam Oxford. BHAVANANDAN, V. P., and KATLIC, A. W. (1979). The interaction of wheat germ agglutinin with sialoglycoproteins. J. BioZ. Chem 254, 4000-4008. DAWNAY, A., MCLEAN, C., and CATTEL, W. R. (1980). The development of radioimmunoassay for Tamm-Horsfall glycoprotein in serum. Biochem. J. 185.679-687. DELELLIS, R. A., STERNBERGER, L. A., MANN, R. B., BANKS, P. M., and NAKANE, B. K. (1979). Immunoperoxidase technique in diagnostic pathology. Amer. J. Clin. Pathol. 71,483-488. EKBLOM, P. (1981). Formation of basement membranes in the embryonic kidney. An immunohistological study. J. CeU BioL (in press). EKBLOM, P., ALITALO, K., VAHERI, A., TIMPL, R., and SAX&N, L. (1980a). Induction of a basement membrane glycoprotein in embryonic kidney: Possible role of laminin in morphogenesis. Proc. Nat. Acad. Sci. USA 77,485-489. EKBLOM, P., MIE~INEN, A., and SAXEN, L. (1980b). Induction of brush border antigens of the proximal tubule in the developing kidney. Develop. Biol. 74, 263-274. EKBLOM, P., MIE~INEN, A., and VIRTANEN, I. (1979). Lectin binding sites in developing mouse metanephros. In “Protides of the biological fluids. Colloquium 27” (H. Peeters, ed.), pp. 463-466. Pergamon Press, Oxford. GODING, J. W. (1976). Conjugation of antibodies with fluorochromes: Modifications to the standard methods. J. ImmunoL Methods 13, 215-226. GROBSTEIN, C. (1956). Transfilter induction of tubules in mouse metanephric mesenchyme. Exp. Cell Res. 10,424~440.
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