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Immunocytochemical demonstration of collagen IV, fibronectin and laminin in phylum platyhelminthes
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EPITHELIAL INTERACTIONS WITH MESENCHYMAL MATRICES INVOLVE A CELL SURFACE PROTEOGLYCAN. hi, Jalkanen, K. Hayaslli. S. Saunders and M. Bernfield. University of Turku, SF-20520 Turku, Finland, UMDNJ-Rutgers Medical School, Piscataway, NJ 08854, USA and Stanford University, CA 94305, USA. Cultured mouse mammary epithelial cells (NMuMG) bear a cell surface proteoglycan (PG) consisting of at least a membraneassociated domain and an ectodomaln, which i) contains both heparan and chondroitin sulfate glycosaminoglycan chains, ii) binds with high affinity Type I, III and V collagen fibrils and C-terminal heparin binding domain of fibronectin, and iii) is recognized by monoclonal antibody 281-2. On NMuMG cell cultures the PG is sequestered at basolateral surfaces and colocalizes with filamentous framework of F-actin, suggesting that when the PG is cross-linked by matrix ligand, it can interact with cytoskeletal elements of epithelial cells. The PG localizes to epithelial cell-cell and cell-matrix interphases in vivo and its appearance is also developmentally regulated: i) squamous and transitional epithelia stain intensely over their entire surfaces, but the most superficial and differentiated cells fail to stain, ii) cuboidal and columnar epithelia stain moderately and only at their basolateral surfaces, but many secretory alveolar cells fail to stain. The ectodomain can also be shed (proliferation or rounding up of NMuMG cells) by cleavage from its membraneassociated domain. Therefore, i) by its association with the cytoskeleton and matrix, the PG could transmit changes in the matrix into changes in epithelial behavior, and ii) by shedding of ectodomain the epithelial cells can loosen their association with the matrix or with other cells. These both occur during normal epithelial development and the invasion of carcinomas.
PRODUCTION AND CELLULAR SOURCE OF EXTRACELLULARMATRIX PROTEINS IN HYBRID INTESTINES. M. Kedin~er~ P. Simon-Assman% F. Bouziges and K. Raffen. Unit4 61 INSERM, Strasbourg, France. The production and deposition of extracellular matrix proteins and the cellular origin of type-IV collagen have been analyzed immunocytochemically* in cocultured or transplanted intestinal epithelial-mesenchymal cell associations. In a first model, intestinal endodermal cells cultured on top of confluent monolayers of intestinal mesenchymal or of skin fibroblastic cells underwent a gradual differentiation**. We could show that I) all interstitial matrix and basement membrane (BM) proteins were deposit within the fibroblastic layer over the whole culture period, 2) contacts between the epithelial cells and the fibroblastic cell populations, whatever their organ of origin were required for the production of the BM, 3) the formation of the BM was progressive as assessed by the shift of a spot-like labeling to a continuous linear pattern at the epithelial/mesenchymal interface, and paralleled epithelial cell differentiation. In a second model, chick-rat epithelial/mesenchymal hybrids developed as intracoelomic grafts, were used; and the immunocytochemical detection of type-IV collagen was performed with species-specific antibodies. We could show that anti-chick antibodies stained the epithelial/mesenchymal interface in chick mesenchyme/ rat endoderm recombinants while anti-ma~alian antibodies stained the inverse associations. These data emphasize the role of tissue interactions in BM formation and show that the mesenchymal compartment is the main endogenous source of type IV collagen. * Antibodies were provided by Drs. Von'D~r Mark and Timpl (Max Planck InstitOt for Biochemiej MOnich). ~* Kedinger et al. Cell Differentiation, in press.
59 MEROSIN, A TISSUE-SPECIFIC BASEMENT MEMBRANE PROTEIN IS EXPRESSED LATE IN NERVE AND MUSCLE DEVELOPMENT. I . L e i v o l , 2 and E.Engva111. 1La 3 o l l a Cancer Research F o u n d a t i o n , La 3 o l l a , CA, and 2Department o f P a t h o l o g y , U n i v e r s i t y of H e l s i n k i , F i n l a n d .
6@ ~ r ~ 1 % h - ~ . i ( ] B Of (X)11~m~l IV, II~II1 ~ l~ll-ill ill I~lylul P l y . Paula Lindroos and Maria J. Still. Inst. of Biology, Abo Akademi~ 20500 Abo~ Finland.
Cross-reactivity betweenhtm~n and invertebrateEC~ components suggests that most of thesemoleculesare highly conservedthrough evolution. Very l i t t l e is
A novel tissue-specific basement membrane p r o t e i n a s s o c i a t e d w i t h basement membranes o f 5chwann c e l l s , s t r i a t e d muscle and t r o p h o b l a s t has been i d e n t i f i e d using monoclonal a n t i b o d i e s . In a n t i b o d y a f f i n i t y chromatography of p r o t e o l y t i c d i g e s t s o f human p l a c e n t a t h e s e a n t i b o d i e s bound a 65 kD p o l y p e p t i d e which was immunologically distinct from t y p e IV c o l l a g e n , l a m i n i n and f i b r o n e c t i n . The 65 kD p o l y p e p t i d e p r e s u m a b l y r e p r e s e n t s a fragment of a novel basement m e m b r a n e - a s s o c i a t e d p r o t e i n named m e r o s i n . In d e v e l o p i n g mouse t i s s u e s m e r o s i n was f i r s t detected i n t r u n k and h i n d l i m b s k e l e t a l muscles and s c i a t i c n e r v e s a t t h e newborn s t a g e . The r e s t r i c t e d t i s s u e distribution and l a t e d e v e l o p m e n t a l appearance o f m e r o s i n suggest t h a t t h e p r o t e i n has a t i s s u e - s p e c i f i c f u n c t i o n associated with a high l e v e l of d i f f e r e n t i a t i o n .
~nown about ECM in phylum Platyhelminthes, which reIa'esents the lowest Bilateria. In this study antibodies ~ I n s t human fibronectin, laminin and collagen IV gave positive reactions in the following platyhelminths: Polycells nigra (Turbellaria) Digh~llobothrium dendriticum (Cestoda) and Bunodera luciopercae (Digenea). The positive reactions were detected by inmlunocytochemical PAP-staining and immunofluoreseenee methods. In all species, positive staining of collagen IV, fibronectin and isminin was intense in the subepithelial basal l~mxlna. Intm/noreactivity a/so occurred around muscles and as a very thin netwoPk in the parenchyma. The distribution of collagen IV, l ~ i n and fibronectin, respectively, was very similar, indicating that laminin and fibronectin are co-existing as linking molecules to collagen IV. Due to the small size of these ~ s , ~-immtu~cytochemlstry will be needed to clarify this statement. The results iudicate, however, that in the ability to synthesize collagen IV, fibronectin and laminin, phylum Platyhelmlnthes does not represent an evolutionsry "dead end".
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EPITHELIAL INTERACTIONS WITH MESENCHYMAL MATRICES INVOLVE A CELL SURFACE PROTEOGLYCAN. hi, Jalkanen, K. Hayaslli. S. Saunders and M. Bernfield. University of Turku, SF-20520 Turku, Finland, UMDNJ-Rutgers Medical School, Piscataway, NJ 08854, USA and Stanford University, CA 94305, USA. Cultured mouse mammary epithelial cells (NMuMG) bear a cell surface proteoglycan (PG) consisting of at least a membraneassociated domain and an ectodomaln, which i) contains both heparan and chondroitin sulfate glycosaminoglycan chains, ii) binds with high affinity Type I, III and V collagen fibrils and C-terminal heparin binding domain of fibronectin, and iii) is recognized by monoclonal antibody 281-2. On NMuMG cell cultures the PG is sequestered at basolateral surfaces and colocalizes with filamentous framework of F-actin, suggesting that when the PG is cross-linked by matrix ligand, it can interact with cytoskeletal elements of epithelial cells. The PG localizes to epithelial cell-cell and cell-matrix interphases in vivo and its appearance is also developmentally regulated: i) squamous and transitional epithelia stain intensely over their entire surfaces, but the most superficial and differentiated cells fail to stain, ii) cuboidal and columnar epithelia stain moderately and only at their basolateral surfaces, but many secretory alveolar cells fail to stain. The ectodomain can also be shed (proliferation or rounding up of NMuMG cells) by cleavage from its membraneassociated domain. Therefore, i) by its association with the cytoskeleton and matrix, the PG could transmit changes in the matrix into changes in epithelial behavior, and ii) by shedding of ectodomain the epithelial cells can loosen their association with the matrix or with other cells. These both occur during normal epithelial development and the invasion of carcinomas.
PRODUCTION AND CELLULAR SOURCE OF EXTRACELLULARMATRIX PROTEINS IN HYBRID INTESTINES. M. Kedin~er~ P. Simon-Assman% F. Bouziges and K. Raffen. Unit4 61 INSERM, Strasbourg, France. The production and deposition of extracellular matrix proteins and the cellular origin of type-IV collagen have been analyzed immunocytochemically* in cocultured or transplanted intestinal epithelial-mesenchymal cell associations. In a first model, intestinal endodermal cells cultured on top of confluent monolayers of intestinal mesenchymal or of skin fibroblastic cells underwent a gradual differentiation**. We could show that I) all interstitial matrix and basement membrane (BM) proteins were deposit within the fibroblastic layer over the whole culture period, 2) contacts between the epithelial cells and the fibroblastic cell populations, whatever their organ of origin were required for the production of the BM, 3) the formation of the BM was progressive as assessed by the shift of a spot-like labeling to a continuous linear pattern at the epithelial/mesenchymal interface, and paralleled epithelial cell differentiation. In a second model, chick-rat epithelial/mesenchymal hybrids developed as intracoelomic grafts, were used; and the immunocytochemical detection of type-IV collagen was performed with species-specific antibodies. We could show that anti-chick antibodies stained the epithelial/mesenchymal interface in chick mesenchyme/ rat endoderm recombinants while anti-ma~alian antibodies stained the inverse associations. These data emphasize the role of tissue interactions in BM formation and show that the mesenchymal compartment is the main endogenous source of type IV collagen. * Antibodies were provided by Drs. Von'D~r Mark and Timpl (Max Planck InstitOt for Biochemiej MOnich). ~* Kedinger et al. Cell Differentiation, in press.
59 MEROSIN, A TISSUE-SPECIFIC BASEMENT MEMBRANE PROTEIN IS EXPRESSED LATE IN NERVE AND MUSCLE DEVELOPMENT. I . L e i v o l , 2 and E.Engva111. 1La 3 o l l a Cancer Research F o u n d a t i o n , La 3 o l l a , CA, and 2Department o f P a t h o l o g y , U n i v e r s i t y of H e l s i n k i , F i n l a n d .
6@ ~ r ~ 1 % h - ~ . i ( ] B Of (X)11~m~l IV, II~II1 ~ l~ll-ill ill I~lylul P l y . Paula Lindroos and Maria J. Still. Inst. of Biology, Abo Akademi~ 20500 Abo~ Finland.
Cross-reactivity betweenhtm~n and invertebrateEC~ components suggests that most of thesemoleculesare highly conservedthrough evolution. Very l i t t l e is
A novel tissue-specific basement membrane p r o t e i n a s s o c i a t e d w i t h basement membranes o f 5chwann c e l l s , s t r i a t e d muscle and t r o p h o b l a s t has been i d e n t i f i e d using monoclonal a n t i b o d i e s . In a n t i b o d y a f f i n i t y chromatography of p r o t e o l y t i c d i g e s t s o f human p l a c e n t a t h e s e a n t i b o d i e s bound a 65 kD p o l y p e p t i d e which was immunologically distinct from t y p e IV c o l l a g e n , l a m i n i n and f i b r o n e c t i n . The 65 kD p o l y p e p t i d e p r e s u m a b l y r e p r e s e n t s a fragment of a novel basement m e m b r a n e - a s s o c i a t e d p r o t e i n named m e r o s i n . In d e v e l o p i n g mouse t i s s u e s m e r o s i n was f i r s t detected i n t r u n k and h i n d l i m b s k e l e t a l muscles and s c i a t i c n e r v e s a t t h e newborn s t a g e . The r e s t r i c t e d t i s s u e distribution and l a t e d e v e l o p m e n t a l appearance o f m e r o s i n suggest t h a t t h e p r o t e i n has a t i s s u e - s p e c i f i c f u n c t i o n associated with a high l e v e l of d i f f e r e n t i a t i o n .
~nown about ECM in phylum Platyhelminthes, which reIa'esents the lowest Bilateria. In this study antibodies ~ I n s t human fibronectin, laminin and collagen IV gave positive reactions in the following platyhelminths: Polycells nigra (Turbellaria) Digh~llobothrium dendriticum (Cestoda) and Bunodera luciopercae (Digenea). The positive reactions were detected by inmlunocytochemical PAP-staining and immunofluoreseenee methods. In all species, positive staining of collagen IV, fibronectin and isminin was intense in the subepithelial basal l~mxlna. Intm/noreactivity a/so occurred around muscles and as a very thin netwoPk in the parenchyma. The distribution of collagen IV, l ~ i n and fibronectin, respectively, was very similar, indicating that laminin and fibronectin are co-existing as linking molecules to collagen IV. Due to the small size of these ~ s , ~-immtu~cytochemlstry will be needed to clarify this statement. The results iudicate, however, that in the ability to synthesize collagen IV, fibronectin and laminin, phylum Platyhelmlnthes does not represent an evolutionsry "dead end".
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