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Differential expression of laminin A and B1 chains in the developing mouse kidney
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247
DYNAMICS
OF THE EXTRACELLULR MATRIX (ECM) DURING MOEPHOGENESIS MARKSON Y. WEISS D.W. DOLJANSKI F. HEBREW UNIVEEITY - HADASSAH MEDICAL SCHOOL, JERUSALEM, ISRAEL. Turnover of ECM and cell-ECM interactions were studled in cartilaginous limb-bone rudiments growing in organ culture. Feaora frca 6 d a y - o l d c h i c k embryos w e r e p u l s e - l a b e l e d w i t h s S S - e u l f a t e o r l 4 C - g l u c o s s m i n e and c h a s e d f o r s e v e r a l d a y s . Growth r a t e s o f t h e r u d i m e n t s were s i m i l a r to t h o s e o f femora i n v i v o . ECM components e x h i b i t e d rapid turnover; their m e t a b o l i c f a t e was t o be r e l e a s e d i n t o t h e c u l t u r e medium as m a c r o m o l e c u l e s . When t h e rudiment s h a f t s were c u t h o r i z o n t a l l y , each femur-half exhibited normal growth, morphogenesis,and ECM turnover for at least 5 days. Heating to 46.5°C for 1 hour killed the rudiments'cells,leaving the ECH. intact. When the cut surfaces of a heated and a living half femur were brought into apposition in organ culture,the latter,as expected,grewnormally, incorporated thymldlne, and synthesieed ECM. The appoeed c e l l - f r e e half femur behaved sime/larly in all these regards. In contrast, when t h e r e m a i n i n g h a l f o f t h e h e a t e d femur was c u l t u r e d s e p a r a t e l y , t h e r e was no growth0 th~midine incorporation, or ECM component s y n t h e s i s . I t is s u g g e s t e d t h a t c h o n d r o b l a s t s t r a n s l o c a t e from t h e l i v i n g half-rudiment to t h e a p p o s e d , h e a t - i n a c t i v a t e d one.
DIFFERENTIAL EXPRESSION OF LAMININ A AND B1 CHAINS IN THE DEVELOPING MOUSE KIDNEY. M. Dziadek* and R. Timpl, Centre for Early Human Development, Monash University, Clayton Victoria, Australia. Polyclonal antibodies which react with laminin A and B1 chain epitopes were used in immunofluoreseence studies to localize these chains in basement membranes (BMs) of mouse kidney during development. Antibodies against the B1 chain react with all BMs of the adult kidney. Antibodies against the A chain react with proximal but not distal tubule BMs, with mesangial but not capillary regions of the glomerular BM, with Bowman's capsule, but not with blood vessel BMs. Enzyme treatments (proteases, hyaluronidase, heparatinase), denaturation (I.0-6.0M GuHCI), or periodate pretreatment failed to reveal A chain staining in the negative BMs$ In embryonic (13.5d) and neonatal (2-9d) kidneys all tubular and glomerular BMs as well as mesenchymal tissue were stained with B1 chain antibodies. Staining with A chain antibodies was restricted to BMs around tubules in the embryonic kidney, while developing glomeruli and mesenchymal tissue were unstained. Tubular BMs which did not react with A chain antibodies were first observed in the neonatal period (2d). These studies demonstrate that differential localization of laminin A and BI chains in kidney BMs is already established during early development of this organ.
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Laminin isoforms in the developing kidney
EXPRESSION OF F I B R O N E C T I N ( F N ) I N EYE TISSUES AFTER LENS REMOVAL AND RETINA DETACHMENT IN N E W T . E . N . G r i g o r y a n , * A.E.Dolnikova~V.M.Belkin. Institute of D e v e l o p m e n t a l BiologyTAcad.Sci.; I n s t i t u t e of B i o l o g i c a l and Medical Chemistry,Acad.Med. Sci.,Moscow,USSR.
M. Ekblom, L. Fecker, G. Mugrauer, G. Klein, L. Sorokin, E. Illgen, S. Conzelmann and P. Ekblom Friedrich-Miescher-Laborat orium der Max-Planck-Gesellschaft, D-7400 Tt~bingen, FRG l.auninin, a basement membrane glycoprotein, is essential for several differentiation events in the embryo. The first isolated laminin isoform contains three polypeptide chains (A, B1 and B2), each encoded by a different gene. We studied laminin B1, B2 and A chain mRNA expression in the developing mouse kidney. Mesenchymal cells from this tissue can be induced to differentiate into epithelium in vitro. Northern blotting showed that the mesenchyme began to express B1 and B2 chain mRNA as soon as it was induced to differentiate, but it did not express A chain mRNA until conversion of mesenchyme to a polarized epithelium started. No evidence for truncated forms of laminin A chains could be obtained: with probes detecting coding regions either near the 3' or 5' ends, only 10 kb transcripts were seen. In situ hybridization of tissue sections using the 3' cDNA clone and immunofluorescence using monoclonal antibodies showed that the A chain mRNA and polypeptide were expressed exclusivelyin developing epithelia. However, endtohelial cells of the developing kidney expressed neither the A chain mRNA nor the polypeptide, although these cells are polarized and produce a basement membrane containing B chain polypeptides. The studies show that variant forms of laminin with no A chain are present not only in embryonic mesenchyme but also in several basement membranes.
Appearance and distribution of F N d u r i n g cell p r o l i f e r a t i o n and transdi fferentiation i n n e w t o p e r a t e d eyes were investigated using indirect immu nofluorescence and radioautography.Af ter lens removal and retina detachm e n t i n t e n s i v e FN e x p r e s s i o n in a r e a s of c e l l s m i g r a t i o n a n d p r o l i f e r a t i o n (iris i n n e r l a y e r , g r o w t h z o n e s a n d re t i n a a n l a g e at t h e eye p e r i p h e r y ) w a s observed.FN e x p r e s s i o n in B r u c h ' s m e mbrane correlated with cell behavior in c o r r e s p o n d i n g r e t i n a l p i g m e n t epithelium(RPE) a r e a , b e i n g l o w e r i n zones of R P E c e l l s t r a n s d i f f e r e n t i a t i o n and much higher in zones retaining t h e i r i n i t i a l c e l l t y p e . T h e l a t t e r we re a l s o c h a r a c t e r i z e d by more intensi ve F N e x p r e s s i o n i n a p i c a l c e l l m e m b r a n e w h e r e a s in t r a n s d i f f e r e n t i a t i n g RPE cells the diffuse FN distribution in t h e c e l l s u r f a c e w a s o b s e r v e d . T h e r o l e of F N in t r a n s d i f f e r e n t i a t i o n of R P E c e l l s a n d in r e g e n e r a t i o n of t h e n e w t eye t i s s u e s is d i c u s s e d .
$78
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DYNAMICS
OF THE EXTRACELLULR MATRIX (ECM) DURING MOEPHOGENESIS MARKSON Y. WEISS D.W. DOLJANSKI F. HEBREW UNIVEEITY - HADASSAH MEDICAL SCHOOL, JERUSALEM, ISRAEL. Turnover of ECM and cell-ECM interactions were studled in cartilaginous limb-bone rudiments growing in organ culture. Feaora frca 6 d a y - o l d c h i c k embryos w e r e p u l s e - l a b e l e d w i t h s S S - e u l f a t e o r l 4 C - g l u c o s s m i n e and c h a s e d f o r s e v e r a l d a y s . Growth r a t e s o f t h e r u d i m e n t s were s i m i l a r to t h o s e o f femora i n v i v o . ECM components e x h i b i t e d rapid turnover; their m e t a b o l i c f a t e was t o be r e l e a s e d i n t o t h e c u l t u r e medium as m a c r o m o l e c u l e s . When t h e rudiment s h a f t s were c u t h o r i z o n t a l l y , each femur-half exhibited normal growth, morphogenesis,and ECM turnover for at least 5 days. Heating to 46.5°C for 1 hour killed the rudiments'cells,leaving the ECH. intact. When the cut surfaces of a heated and a living half femur were brought into apposition in organ culture,the latter,as expected,grewnormally, incorporated thymldlne, and synthesieed ECM. The appoeed c e l l - f r e e half femur behaved sime/larly in all these regards. In contrast, when t h e r e m a i n i n g h a l f o f t h e h e a t e d femur was c u l t u r e d s e p a r a t e l y , t h e r e was no growth0 th~midine incorporation, or ECM component s y n t h e s i s . I t is s u g g e s t e d t h a t c h o n d r o b l a s t s t r a n s l o c a t e from t h e l i v i n g half-rudiment to t h e a p p o s e d , h e a t - i n a c t i v a t e d one.
DIFFERENTIAL EXPRESSION OF LAMININ A AND B1 CHAINS IN THE DEVELOPING MOUSE KIDNEY. M. Dziadek* and R. Timpl, Centre for Early Human Development, Monash University, Clayton Victoria, Australia. Polyclonal antibodies which react with laminin A and B1 chain epitopes were used in immunofluoreseence studies to localize these chains in basement membranes (BMs) of mouse kidney during development. Antibodies against the B1 chain react with all BMs of the adult kidney. Antibodies against the A chain react with proximal but not distal tubule BMs, with mesangial but not capillary regions of the glomerular BM, with Bowman's capsule, but not with blood vessel BMs. Enzyme treatments (proteases, hyaluronidase, heparatinase), denaturation (I.0-6.0M GuHCI), or periodate pretreatment failed to reveal A chain staining in the negative BMs$ In embryonic (13.5d) and neonatal (2-9d) kidneys all tubular and glomerular BMs as well as mesenchymal tissue were stained with B1 chain antibodies. Staining with A chain antibodies was restricted to BMs around tubules in the embryonic kidney, while developing glomeruli and mesenchymal tissue were unstained. Tubular BMs which did not react with A chain antibodies were first observed in the neonatal period (2d). These studies demonstrate that differential localization of laminin A and BI chains in kidney BMs is already established during early development of this organ.
248
249
Laminin isoforms in the developing kidney
EXPRESSION OF F I B R O N E C T I N ( F N ) I N EYE TISSUES AFTER LENS REMOVAL AND RETINA DETACHMENT IN N E W T . E . N . G r i g o r y a n , * A.E.Dolnikova~V.M.Belkin. Institute of D e v e l o p m e n t a l BiologyTAcad.Sci.; I n s t i t u t e of B i o l o g i c a l and Medical Chemistry,Acad.Med. Sci.,Moscow,USSR.
M. Ekblom, L. Fecker, G. Mugrauer, G. Klein, L. Sorokin, E. Illgen, S. Conzelmann and P. Ekblom Friedrich-Miescher-Laborat orium der Max-Planck-Gesellschaft, D-7400 Tt~bingen, FRG l.auninin, a basement membrane glycoprotein, is essential for several differentiation events in the embryo. The first isolated laminin isoform contains three polypeptide chains (A, B1 and B2), each encoded by a different gene. We studied laminin B1, B2 and A chain mRNA expression in the developing mouse kidney. Mesenchymal cells from this tissue can be induced to differentiate into epithelium in vitro. Northern blotting showed that the mesenchyme began to express B1 and B2 chain mRNA as soon as it was induced to differentiate, but it did not express A chain mRNA until conversion of mesenchyme to a polarized epithelium started. No evidence for truncated forms of laminin A chains could be obtained: with probes detecting coding regions either near the 3' or 5' ends, only 10 kb transcripts were seen. In situ hybridization of tissue sections using the 3' cDNA clone and immunofluorescence using monoclonal antibodies showed that the A chain mRNA and polypeptide were expressed exclusivelyin developing epithelia. However, endtohelial cells of the developing kidney expressed neither the A chain mRNA nor the polypeptide, although these cells are polarized and produce a basement membrane containing B chain polypeptides. The studies show that variant forms of laminin with no A chain are present not only in embryonic mesenchyme but also in several basement membranes.
Appearance and distribution of F N d u r i n g cell p r o l i f e r a t i o n and transdi fferentiation i n n e w t o p e r a t e d eyes were investigated using indirect immu nofluorescence and radioautography.Af ter lens removal and retina detachm e n t i n t e n s i v e FN e x p r e s s i o n in a r e a s of c e l l s m i g r a t i o n a n d p r o l i f e r a t i o n (iris i n n e r l a y e r , g r o w t h z o n e s a n d re t i n a a n l a g e at t h e eye p e r i p h e r y ) w a s observed.FN e x p r e s s i o n in B r u c h ' s m e mbrane correlated with cell behavior in c o r r e s p o n d i n g r e t i n a l p i g m e n t epithelium(RPE) a r e a , b e i n g l o w e r i n zones of R P E c e l l s t r a n s d i f f e r e n t i a t i o n and much higher in zones retaining t h e i r i n i t i a l c e l l t y p e . T h e l a t t e r we re a l s o c h a r a c t e r i z e d by more intensi ve F N e x p r e s s i o n i n a p i c a l c e l l m e m b r a n e w h e r e a s in t r a n s d i f f e r e n t i a t i n g RPE cells the diffuse FN distribution in t h e c e l l s u r f a c e w a s o b s e r v e d . T h e r o l e of F N in t r a n s d i f f e r e n t i a t i o n of R P E c e l l s a n d in r e g e n e r a t i o n of t h e n e w t eye t i s s u e s is d i c u s s e d .
$78