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The role of extracellular matrix components in the pathfinding of embryonic retinal axons
250 THE ROLE OF E X T R A C E L L U L A R MATRIX COMPONENTS IN THE P A T H F I N D I N G OF EMBRYONIC RETINAL AXONS W. Halfter, Friedrich Miescher-Institut, P.O. Box 2543, 4002 Basel, Switzerland During retina morphogenesis, optic axons grow out from their parent ganglion cells and navigate toward the optic disc. A detailed analysis of the orientation of newly formed processes shows that the initial growth of nerve fibers is directed from the very beginning. D i s t u r b a n c e of the basal surface of the retina causes aberrant fiber tracts suggesting that the information for directed fiber growth is located at this site of the tissue. By a mechanical cleavage of the retina, the basal surface of the retina, which represents the environment of axons in vivo, can be isolated and used as a substratum for axonal growth in vitro. A similar procedure allows the isolation of the basal lamina of the pigment e p i t h e l i u m for similar assays. C o m p a r a t i v e studies of the in vivo-derived matrices with collagen, fibronectin or laminin shows that the basal lamina preparations are far superior in promoting neurite growth than any other substrates, however, the basal laminae had no effect on the orientation of axonal growth. Antisera against the retina, pigment epithelium, and glomerular basal lamina disturb the normal neurite extension on basal laminae by causing very strong neurite fasciculation or by inhibiting the linear extension of fibers from the explant. The same experiments, however, also showed that the antisera did not alter the principal direction of fiber growth from the explant. The antisera experiments show that extracellular matrix components from basal laminae are crucial for the rapid and linear neurite extension, but not for the directionality of fiber growth.
251 Development: o f e D N A probel for rethta c o g n / n , z h e embryoudLc cbXck r e t / a a - a p e c / f / c ceiL1, r e e o g n 4 t 4 n u prot:e/n. R.E. liensuan and A.S.M. g r / a h n a g a o , DeparCment: o f B£olugy, B o s t o n U n i v e r s i t y , B o s t o n , M A 02215 USA. Messenger KNA was p r e p a r e d f r o m day e m b r y o n i c c h i c k u e u r a i r e f i n e r.issue and used to make cD N A. Two expression libraries were constructed: in Zambda G T I I and IJtmbda Zap II v e c t o r s . These were screened w i t h t h e sperAf£c p o l y g o n a l ant&serum a g a i n s t reCtne,$~pngin using both alkaline phoephntaae-and *'J[I]c o n j u g a t e d s e c o n d a r y enrAbody. Five c l o n e s r e s u l t e d from m u l t i p l e s c r e e n i n g and t h e s e are b e i n g invest~4Jaced f o r t h e i r r e t i n a c o g n i n eperAJ~cicy. The a m o u n t of c o g n i n m It N A 4. b e i n g £nvesr.tgated at d i f f e r e n t stages of development of the £ntoet retina and £n d h s s o c t a t e d r e l i n e c e l l s at d / f f e r e n t s t a g e s o f n e u r o n a l c o i l differenCiaCton i n sight
c~d~ure.
252
253
Expression of the laminin A and B chains in chimeric and cultured embryonic kidneys
The role of f i b r o n e c t i n and laminin in the migration of the W o l f f i a n duct of avian embryos. M. Jacob, B. Christ, H.J. Jacob, I. Flam~e, S. Britsch, R.E. P o e l m a n n
K. Holm-Sainin 1 . L. Risteli 2, and H. Sariola3
IDepartment of Pathology, University of Helsinki, Finland. 2 Department of Medical Biochemistry, University of Oulu, Finland.3Department of Paediatric Pathology, University Central Hospital of Helsinki, Finland The expression of laminin in embryonic kidneys growing in chicken chorioallantoic membranes or in vitro w a s
The role of the two e x t r a c e l l u l a r m a t r i x glycoproteins fibronectin and laminin in the migration of the tip of the W o l f f i a n duct is examined by m i c r o i n j e c t i o n of synthetic peptides. The amino acid sequence RGD is found to be the m a j o r part of the c e l l - b i n d i n g domain of f i b r o n e c t i n which is r e c o g n i z e d by a receptor of the integrin family. Synthetic peptides containing this active sequence are able to c o m p e t i t i v e l y inhibit the a t t a c h m e n t of cells to fibronectin. In 2-day old chick embryos the p e n t a p e p t i d e GRGDS leads to a c o m p a c t i o n of the m e s e n c h y m a l cells at the tip of the Wolffian duct and inhibits their normal migration. The p e n t a p e p t i d e YIGSR m e d i a t e s the attachment of laminin to a 67-KDa receptor on the cell surface. A f t e r m i c r o i n j e c t i o n of this peptide cells at the tip of the duct anlage are found to m i g r a t e far lateral over the somatopleure. Cell b r i d g e s with the somites are also observed. The control peptide SHLVE has no d e t e c t a b l e biological activity. These data supported by i n v e s t i g a t i o n s with antibodies may provide insights into the mechanisms of cell- e x t r a c e l l u l a r matrix interactions.
followed with mouse-specific, affinity-purified antibodies against the laminin A and B chains. In mousechicken chimeric kidneys the vasculaturc originates from chicken, and the nephric tubules from mouse. In these chimeras the laminin B chain is expressed in the undifferentiated mesenchyme, but laminin A chain appears first with tubular differentiation. Deposition of laminin chains was also studied in wholemount preparations of cultured embryonic kidneys. Peter Ekblom and his colleques have earlier shown that, during induction of kidney tubules, laminia is expressed as small dots, and later,during tubular differentiation, a linear basement membrane pattern is established. We show that in the induced mcsenchyme the dots are composed of the laminin B chain, which also forms spikes that are attached to the tips of the ureter bud. Similar spiked pattern of the basement membrane can be seen between the Wolffian duct and the surrounding muscle. Spikes, which are not seen in adult kidneys,may represent one of the earliest stages in the formation of the epithelial basement membrane and direct induced mesenchymal cells for the subsequent fusion of the ureter bud and the secretory tubules.
$79
251 Development: o f e D N A probel for rethta c o g n / n , z h e embryoudLc cbXck r e t / a a - a p e c / f / c ceiL1, r e e o g n 4 t 4 n u prot:e/n. R.E. liensuan and A.S.M. g r / a h n a g a o , DeparCment: o f B£olugy, B o s t o n U n i v e r s i t y , B o s t o n , M A 02215 USA. Messenger KNA was p r e p a r e d f r o m day e m b r y o n i c c h i c k u e u r a i r e f i n e r.issue and used to make cD N A. Two expression libraries were constructed: in Zambda G T I I and IJtmbda Zap II v e c t o r s . These were screened w i t h t h e sperAf£c p o l y g o n a l ant&serum a g a i n s t reCtne,$~pngin using both alkaline phoephntaae-and *'J[I]c o n j u g a t e d s e c o n d a r y enrAbody. Five c l o n e s r e s u l t e d from m u l t i p l e s c r e e n i n g and t h e s e are b e i n g invest~4Jaced f o r t h e i r r e t i n a c o g n i n eperAJ~cicy. The a m o u n t of c o g n i n m It N A 4. b e i n g £nvesr.tgated at d i f f e r e n t stages of development of the £ntoet retina and £n d h s s o c t a t e d r e l i n e c e l l s at d / f f e r e n t s t a g e s o f n e u r o n a l c o i l differenCiaCton i n sight
c~d~ure.
252
253
Expression of the laminin A and B chains in chimeric and cultured embryonic kidneys
The role of f i b r o n e c t i n and laminin in the migration of the W o l f f i a n duct of avian embryos. M. Jacob, B. Christ, H.J. Jacob, I. Flam~e, S. Britsch, R.E. P o e l m a n n
K. Holm-Sainin 1 . L. Risteli 2, and H. Sariola3
IDepartment of Pathology, University of Helsinki, Finland. 2 Department of Medical Biochemistry, University of Oulu, Finland.3Department of Paediatric Pathology, University Central Hospital of Helsinki, Finland The expression of laminin in embryonic kidneys growing in chicken chorioallantoic membranes or in vitro w a s
The role of the two e x t r a c e l l u l a r m a t r i x glycoproteins fibronectin and laminin in the migration of the tip of the W o l f f i a n duct is examined by m i c r o i n j e c t i o n of synthetic peptides. The amino acid sequence RGD is found to be the m a j o r part of the c e l l - b i n d i n g domain of f i b r o n e c t i n which is r e c o g n i z e d by a receptor of the integrin family. Synthetic peptides containing this active sequence are able to c o m p e t i t i v e l y inhibit the a t t a c h m e n t of cells to fibronectin. In 2-day old chick embryos the p e n t a p e p t i d e GRGDS leads to a c o m p a c t i o n of the m e s e n c h y m a l cells at the tip of the Wolffian duct and inhibits their normal migration. The p e n t a p e p t i d e YIGSR m e d i a t e s the attachment of laminin to a 67-KDa receptor on the cell surface. A f t e r m i c r o i n j e c t i o n of this peptide cells at the tip of the duct anlage are found to m i g r a t e far lateral over the somatopleure. Cell b r i d g e s with the somites are also observed. The control peptide SHLVE has no d e t e c t a b l e biological activity. These data supported by i n v e s t i g a t i o n s with antibodies may provide insights into the mechanisms of cell- e x t r a c e l l u l a r matrix interactions.
followed with mouse-specific, affinity-purified antibodies against the laminin A and B chains. In mousechicken chimeric kidneys the vasculaturc originates from chicken, and the nephric tubules from mouse. In these chimeras the laminin B chain is expressed in the undifferentiated mesenchyme, but laminin A chain appears first with tubular differentiation. Deposition of laminin chains was also studied in wholemount preparations of cultured embryonic kidneys. Peter Ekblom and his colleques have earlier shown that, during induction of kidney tubules, laminia is expressed as small dots, and later,during tubular differentiation, a linear basement membrane pattern is established. We show that in the induced mcsenchyme the dots are composed of the laminin B chain, which also forms spikes that are attached to the tips of the ureter bud. Similar spiked pattern of the basement membrane can be seen between the Wolffian duct and the surrounding muscle. Spikes, which are not seen in adult kidneys,may represent one of the earliest stages in the formation of the epithelial basement membrane and direct induced mesenchymal cells for the subsequent fusion of the ureter bud and the secretory tubules.
$79