Differential expression of the laminin A and B chains in chimeric kidneys

Cell Differentiation, 24 (1988) 223-228 Elsevier ScientificPublishers Ireland, Ltd.

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Differential expression of the laminin A and B chains in chimeric kidneys K. H o l m 1, L. Risteli 2 and H. Sariola 1 1 Department of Pathology, University ofHelsinki, Helsinki, Finland and 2 Department of Medical Biochemistry, University of Oulu, Oulu, Finland

(Accepted 2 May 1988)

The expression of laminin in embryonic kidneys growing in ovo is followed with mouse-specific, affinity-purified antibodies against the laminin A and B chains. In mouse kidneys growing on the chicken chorioallantoic membrane, the epithelium and nephrogenic mesenchyme are derived from mouse and the vasculature from chicken cborioallantoic vessels. Hence, with the mouse-specific antibodies, it is possible to analyze the deposition of laminin chains by the nephrogenic tissue, because iuminin derived from the chicken vasculature remains unstained. In these chimeras, only the iaminin B chain, but not the A chain, is expressed in the undifferentiated nephrogenic mesenchyme. The basement membrane around the ureter bud is labeled by the antibodies against both laminin A and B chains. In the mesenchyme, the laminin A chain appears when the mesenchyme converts into tubules. The results suggest that the iaminin A and B chains are synthesized differentially in the embryonic nephrogenic tissue. Kidney differentiation; Laminin; Laminin chains; Inductive tissue interaction

Introduction Laminin is a basement-specific glycoprotein with a molecular size of about 900 kDa (Timpl et al., 1979). It is composed of two B chains (B1 and B2) and one A chain that are covalently linked together with sulphur bridges (Timpl et al., 1979; Cooper et al., 1981; Risteli and Timpl, 1981; Hogan et al., 1985). Laminin promotes axonal growth, cell attachment and migration, and it is involved in the metastasis of malignant cells (re-

Correspondence address: Harmu Sariola, Ph.D., Department of Pathology, Universityof Helsinki, Haartmaninkatu 3, SF-00290 Helsinki, Finland.

views: Liotta, 1985; Timpl and Dziadek, 1986). In the embryonic kidney, laminin has been found in the induced mesenchymal cell condensates that later form kidney tubules, and in the basement membranes of vessels, nephric-tubules and glomeruli (Ekblom et al., 1980). Experiments with chimeric kidneys have shown that in the tubular basement membranes, larninin (as well as type IV collagen) is derived from the epithelial cells but, in the glomerular basement membrane, has a dual origin (Sariola et al., 1984). In the present study we have analyzed the pattern of laminin in the mesenchyme and epithelium of embryonic kidneys. As most cell types of the metanephros seem to express laminin, chimeric kidneys between mouse and chicken, and

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224 mouse-specific antibodies were chosen for the study. By these tools, it is possible to follow the deposition of the laminin chains by the murine nephrogenic tissue without visualizing laminin derived from the vasculature.

Material and Methods

Tissue cultures and chorioallantoic grafts Murine l l - d a y embryonic ( C B A / N M R I ) metanephric kidneys were microdissected, pipetted on 7-day chicken chorioallantoic membranes (CAMs) and grafted for 10 days. Then the grafts were harvested, fixed and processed for immunohistology. For details, see Sariola et al. (1983). Immunohistology and antibodies The kidney grafts were frozen in solid CO2, cut with cryostat at 6 #m, and stained by the biotinavidin peroxidase method (Dakopatts a/s, Denmark) as described earlier (Sariola et al., 1983). When the primary antibody was omitted, no staining was seen. Laminin was prepared from the transplantable EHS-tumor grown in mice as described (Timpl et al., 1979). Its subunit chains A and B (containing both B1 and B2) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in 6.25% gels, the bands were cut out and the gel slices used for immunizing rabbits (Cooper et al., 1981). After the primary injection, two boosters were given. For use in immunohistochemistry, antibodies that could recognize their antigenic determinants in native laminin were purified by affinity chromatography on intact laminin. The antisera were first passed over columns of immobilized mouse type IV collagen and bovine type III pN-collagen to remove any cross-reacting antibodies (Furthmayr, 1982). Final purification was carried out on laminin, coupled with Sepharose 4B; the antisera were chromatographed on this column twice and the eluates combined. The ability of the antibodies to bind native laminin was tested with immunoprecipitation of 3H-proline-labeled laminin, synthesized by PYS-2 cells. The final concentration of the antibody against the A chain was 0.19

mg/ml specific IgG and against the B chain, 0.20 mg/ml specific IgG. The antibodies against laminin A and B chain were diluted at 1:30 for immunohistochemistry and at 1:300 for immunoblotting. Commercial polyclonal antibodies against laminin (Bethesda Research Laboratories (BRL), MD, U.S.A.) were used at 1 : 200 for immunoperoxidase and at 1 : 8 0 0 for immunoblotting. Secondary antibodies in immunoperoxidase staining and immunoblotting were biotinylated a-rabbit immunoglobulins (Dakopatts) and they were diluted at 1 : 300.

Gel electrophoresis and immunoblotting SDS-PAGE was carried out according to the method of Laemmli (1970) and immunoblotting according to Towbin et al. (1979). Briefly, native laminin (Collaborative Research, MA, U.S.A., concentration 0.1 mg/ml) was run electrophoretically with Mini Protean II Dual slab cell system in 5% gel. Proteins were transferred to cationized nylon membrane (Zeta-Probe) with Mini TransBlot transfer cell system. The electrophoresis equipment was from Bio-Rad (Richmond, CA, U.S.A.). Total protein detection was carried out with a Biotin-Blot kit (Bio-Rad). Immunostaining with chain-specific laminin A and B antibodies and BRL a-laminin was accomplished with the avidin-biotin peroxidase method. Results

The chain specificity of the laminin antibodies The antibodies against the A chain and the B chain were immunoblotted against native laminin run under reduced conditions in the SDS-PAGE (Fig. 1) and were shown to react specifically with the A and B chains, respectively. The commercial antibody against laminin reacted with both chains. In immunoperoxidase stainings of mouse and chicken tissues, the chain-specific antibodies turned out to be mouse-specific, whereas the commercial antibody against laminin reacted with both chicken and mouse (Fig. 2). Laminin expression in interspecies chimeric kidneys When transplanted on chicken CAMs, undifferentiated mouse kidneys become vascularized by

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the chorioallantoic vessels (Ekblom et al., 1982). In these chimeras, the laminin A chain is expressed in the basement membrane of the ureter bud and of the secretory nephrons, but not in the undifferentiated mesenchyme. In the mesenchymal condensates that represent prospective nephric tubules, few dots of the laminin A chain are found. The laminin B chain is found in abundance in all cell types of the embryonic kidney: in the uninduced and induced mesenchyme, secretory tubules and ureter bud. In the uninduced mesenchyme, the B chain has a diffuse staining pattern. With condensation of the mesenchyme, laminin B becomes organized in a strong dotted pattern and, with epithelial polarization of tubules, is finally layered in a sheet typical of a basement membrane. Hence, the laminin chains codistribute in the basement membranes, but only the laminin B chain seems to be expressed in the undifferentiated mesenchyme, whereas the laminin A chain appears with tubular differentiation (Fig. 3).

Discussion

The interspecies chimeric kidney grafts provide a possibility of analyzing the source of the extracellular matrix molecules with species-specific antibodies (Sariola, 1985). By mouse-specific anti-

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Fig. 3. Expression of the laminin chains in chimeric mouse/chicken kidneys. (a) The A-chain-specific antibody against laminin labels only the basement membranes around the ureter bud (ub) and secretory tubules (t), but not the mesenchyme (m). Few dots of reactivity are seen in the condensed mesenchymal areas. The chicken chorioallantoic membrane (cam) and vessels remain unstained. (b) The B-chain-specific antibody reacts with all nephrogenic cell types. Note the abundant intracytoplasmic staining of the antibody. (c) With the B-chain-specific antibody, the chorioallantoic membrane remains unlabeled. (d) The BRL a-laminin antibody reacts with the chimeric kidney and with blood vessels (v) in the chorioallantoic membrane. Biotin-avidin peroxidase staining of frozen sections, magnifications a and b × 320, c and d × 160.

b o d i e s a g a i n s t the l a m i n i n A a n d B chains, it is n o w s h o w n that the B c h a i n is e x p r e s s e d in the u n d i f f e r e n t i a t e d m e s e n c h y m e , w h e r e a s the A c h a i n d o e s not a p p e a r until the t u b u l a r d i f f e r e n t i a t i o n o f the m e s e n c h y m e . H o w e v e r , b o t h c h a i n s are c o n s t a n t l y p r e s e n t in the b a s e m e n t m e m b r a n e o f the u r e t e r b u d a n d of the s e c r e t o r y tubules. L a m i n i n e x p r e s s i o n in the e m b r y o n i c k i d n e y has p r e v i o u s l y b e e n a n a l y z e d in several i m m u n o -

h i s t o c h e m i c a l r e p o r t s . H o w e v e r , the results have b e e n c o n f l i c t i n g . A l l a u t h o r s agree t h a t l a m i n i n is f o u n d a r o u n d the e p i t h e l i a l a n d v a s c u l a r cells ( E k b l o m et al., 1980; S a r i o l a et al., 1983; B o n a d i o et al., 1984), b u t t h e r e is a c o n t r a d i c t i o n w h e t h e r (Sax6n a n d L e h t o n e n , 1987) or n o t ( E k b l o m et al., 1980) lan'finin is e x p r e s s e d in the u n d i f f e r e n t i a t e d n e p h r o g e n i c m e s e n c h y m a l cells• Since t h e y a r e n o w s h o w n to e x p r e s s o n l y the B chair., the pre-

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sent study suggests that the controversial results may be explained by the different reactivity of the antibodies to the chains of laminin. When native laminin has been used as immunogen, the laminin A chain is only partly exposed, and strong immunoresponse is obtained only against the B chain. This problem can be settled when the purified chains of laminin are used as immunogens or when monoclonal antibodies are produced. With the chain-specific antibodies we show that the expression of the laminin A chain becomes apparent with tubular differentiation of the nephrogenic mesenchyme. Although we cannot exclude the possibility that an undetectable amount of the A chain could be synthesized by mesenchymal cells, the present results suggest that there may be a variation in the ratio of laminin chains synthesized by the mesenchyme during differentiation. Asynchronous synthesis of the laminin chains has been found in early embryos (Cooper and MacQueen, 1983). Being abundantly expressed in the uninduced and induced mesenchyme, laminin B may have a specific function in mesenchymal condensation, which is the first sign of differentiation into nephric tubules. The laminin B chain may serve as an adhesive molecule creating circumstances in which the epithelial polarization and transmission of the inductive signals become possible.

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Induction of basement membrane glycoprotein in embryonic kidney: possible role of larninin in morphogenesis. Proc. Natl. Acad. Sci. USA 77, 485-489 (1980). Ekblom, P., H. Sariola, M. Karkinen-J~i~iskel~inen and L. Saxrn: The origin of the glomerular endothelium. Cell Differ. 11, 35-39 (1982). Furthmayr, H.: Immunization procedures, isolation by affinity chromatography and serological and immunochemical characterization of collagen specific antibodies. In: Immunochemistry of the Extracellular Matrix, Vol. I, ed. H. Furthmayr (CRC Press, Boca Raton) pp. 143-170 (1982). Hogan, B.,-D. Barlow, N. Green, R. EUiott, J. McVey, G. Patel, M. Kurkinen and A. Cooper: Laminin: towards the structure of the protein and its genes. In: Basement Membranes, ed. S. Shibata (Elsevier Science Publishers, Amsterdam) pp. 147-154 (1985). Laemmli, U.K.: Cleavage of structure proteins during the assembly of the head of bacteriophage T 4. Nature 227: 680-685 (1970). Liotta, L.: Mechanisms of cancer invasion and metastasis. In: Important Advances in Oncology. Part I: Basic Research, eds. V. DeVita, S. Hellman and S. Rosenberg, (J.B. Lippincott Company, Philadelphia) pp. 28-41 (1985). Risteli, L. and R. Timpl: Isolation and characterization of pepsin fragments of laminin from human placental and renal basement membranes. Biochem. J. 193, 749-755

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