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Biochemical and ultrastructural studies on vitamin A induced proximalization of limb regeneration in axolotl
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177 THE CELLULAR BASIS OF RETINOID-INDUCED ALTERATIONS IN PATTERN FORMATION. M.Maden and S.Keeble. National Institute f o r Medical Research, London, NW7 IAA, U.K. During normal regeneration of the axolotl limb only those elements removed are replaced. But after retinoid treatment proximodistal duplications appear, result -ing in the regeneration of complete l i m ~ from distal amputation levels. It seems that retinoids change the positional information of limb cells and respecify them in a proximal direction. The cellular changes induced by retinoids include the induction of cartilage matrix breakdown, the induction of mucopolysaccharide production in the epidermis, the inhibition of cell division, the stimulation of fibronectin production and other extracellular matrix changes. Each of these effects has been tested for its role in changing pattern formation and these experiments will be described. Further work to determine the cellular location and mode of action of retinoids in blastemal cells will be described. These include the characteristics of cytoplasmic binding proteins, whether there are cell surface receptors and whether retinoids enter the nucleus.
LI~ ~ IN THE ~ NHBRYO: OF SURGICAL ~ C ~ E S . K. ~meoka, N. Wan~k and S.V. Bryant. Developmental Biology Center, University of California, Irvine, CA 92717. Techniques for in s i t u microsurgical manipulation of the mouse embryo have been developed. All operations were performed on outbred Swiss Webster mice. Previously described in utero surgical techniques were used for experimentation on the mouse fetus of 14 days of gestation (plug day = day I) and older. However, we found these techniques to be ineffective for work on embryos of earlier stages of development due to the fragility of the embryo and the continuous contractions of the uterus. We have found that development of embryos will continue normally after the musc,]sr layers of the uterus have been cut away, leaving the placenta in tact. Fmbryos are thus exposed to the coelemic cavity and development continues exo utero. Embryos of day 10 of gestation and older develop normally following this procedure in virtually 100%o of the cases. Without the hinderance of the uterus we have been able to successfully perform surgical operations on these early developmental stages and have started investigations on the regulative ability of the developing mouse limb bud. This work was supported by a gift from Monsanto
Compsny.
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178 BIOCHEMICAL AND ULTRASTRUCTURAL STUDIES ON V I T A M I N A INDUCED PROXIMALIZATION OF L I M B R E G E N E R A T I O N IN A X O L O T L . K . K . S h a r m a and H.J. Anton. Zoologisches Institut Univ. K g l n , 5 K S l n 41 W e s t G e r m a n y . In 3 5 m m s i z e a x o l o t l s 15 d a y s treatment w i t h 75 I U / m l V i t a m i n A palmitate after fore-arm amputation r e s u l t e d in p r o x i m a l i z a t i o n of regeneration.Vitamin A decreased 15% m e a n D N A c o n t e n t a n d 20% Sphase cell population of b l a s t e m a l mesenchyme on 1 0 t h d a y . P r o t e i n s were separated on 15% SDS p o l y a c rylamide gels.Amount of p r o t e i n s in ] 6 . 5 , 4 1 . 6 a n d 78 Kd b a n d s of blastema epidermis,mesenchyme and adjacent stump decreased a f t e r 15 d a y s t r e a t m e n t . In m e s e n c h y m e of treated blastema 1 2 . 4 a n d 13.9 Kd b a n d s w e r e f a i n t a n d 12 a n d 38 Kd bands were absent.These c e l l s showed small Golgi apparatus less r o u g h ER a n d i n t e r c e l l u l a r fibrils and more nuclear pores than controls.To conclude,Vitamin A alters synthetic machinery of b l a s t e m a and adjacent stump cells during proximalization of r e g e n e r a t i o n .
A MODEL FOR THE ROLE OF INTERFACIAL TENSIONS WITHIN CELL SHEETS IN THE EARLY MORPHOGENETIC EVENTS OF AMPHIBIAN DEVELOPMENT. J. E. Mittenthal, Department of
Anatomical Sciences, University of Illinois, Urbana, IL 61801, U.S.A. A model which treats an epithelium as a fluid elastic shell predicts the morphogenetlc effects of differential adhesion within the shell. Inferences from this model are designated with *. The late blastula contains an animalvegetal cohesion gradient, with high cohesion near the animal pole (Holtfreter). *This gradient can produce epiboly. Epiboly generates an adhesive disparity at the blastopore site. *This disparity initiates involution, bringing marginal zone mesoderm in contact with the under-slde of the gradient. Involution proceeds as the mesoderm migrates up this gradient. *The gradient in mesoderm and ectoderm elongates the embryo. Dorsal mesoderm induces overlying ectoderm to increase its cohesion; *this initiates thickening of the neural plate. Thus each movement in the sequence tends to resolve an instability, but generates a new instability which drives the next phase of movement.
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177 THE CELLULAR BASIS OF RETINOID-INDUCED ALTERATIONS IN PATTERN FORMATION. M.Maden and S.Keeble. National Institute f o r Medical Research, London, NW7 IAA, U.K. During normal regeneration of the axolotl limb only those elements removed are replaced. But after retinoid treatment proximodistal duplications appear, result -ing in the regeneration of complete l i m ~ from distal amputation levels. It seems that retinoids change the positional information of limb cells and respecify them in a proximal direction. The cellular changes induced by retinoids include the induction of cartilage matrix breakdown, the induction of mucopolysaccharide production in the epidermis, the inhibition of cell division, the stimulation of fibronectin production and other extracellular matrix changes. Each of these effects has been tested for its role in changing pattern formation and these experiments will be described. Further work to determine the cellular location and mode of action of retinoids in blastemal cells will be described. These include the characteristics of cytoplasmic binding proteins, whether there are cell surface receptors and whether retinoids enter the nucleus.
LI~ ~ IN THE ~ NHBRYO: OF SURGICAL ~ C ~ E S . K. ~meoka, N. Wan~k and S.V. Bryant. Developmental Biology Center, University of California, Irvine, CA 92717. Techniques for in s i t u microsurgical manipulation of the mouse embryo have been developed. All operations were performed on outbred Swiss Webster mice. Previously described in utero surgical techniques were used for experimentation on the mouse fetus of 14 days of gestation (plug day = day I) and older. However, we found these techniques to be ineffective for work on embryos of earlier stages of development due to the fragility of the embryo and the continuous contractions of the uterus. We have found that development of embryos will continue normally after the musc,]sr layers of the uterus have been cut away, leaving the placenta in tact. Fmbryos are thus exposed to the coelemic cavity and development continues exo utero. Embryos of day 10 of gestation and older develop normally following this procedure in virtually 100%o of the cases. Without the hinderance of the uterus we have been able to successfully perform surgical operations on these early developmental stages and have started investigations on the regulative ability of the developing mouse limb bud. This work was supported by a gift from Monsanto
Compsny.
179
178 BIOCHEMICAL AND ULTRASTRUCTURAL STUDIES ON V I T A M I N A INDUCED PROXIMALIZATION OF L I M B R E G E N E R A T I O N IN A X O L O T L . K . K . S h a r m a and H.J. Anton. Zoologisches Institut Univ. K g l n , 5 K S l n 41 W e s t G e r m a n y . In 3 5 m m s i z e a x o l o t l s 15 d a y s treatment w i t h 75 I U / m l V i t a m i n A palmitate after fore-arm amputation r e s u l t e d in p r o x i m a l i z a t i o n of regeneration.Vitamin A decreased 15% m e a n D N A c o n t e n t a n d 20% Sphase cell population of b l a s t e m a l mesenchyme on 1 0 t h d a y . P r o t e i n s were separated on 15% SDS p o l y a c rylamide gels.Amount of p r o t e i n s in ] 6 . 5 , 4 1 . 6 a n d 78 Kd b a n d s of blastema epidermis,mesenchyme and adjacent stump decreased a f t e r 15 d a y s t r e a t m e n t . In m e s e n c h y m e of treated blastema 1 2 . 4 a n d 13.9 Kd b a n d s w e r e f a i n t a n d 12 a n d 38 Kd bands were absent.These c e l l s showed small Golgi apparatus less r o u g h ER a n d i n t e r c e l l u l a r fibrils and more nuclear pores than controls.To conclude,Vitamin A alters synthetic machinery of b l a s t e m a and adjacent stump cells during proximalization of r e g e n e r a t i o n .
A MODEL FOR THE ROLE OF INTERFACIAL TENSIONS WITHIN CELL SHEETS IN THE EARLY MORPHOGENETIC EVENTS OF AMPHIBIAN DEVELOPMENT. J. E. Mittenthal, Department of
Anatomical Sciences, University of Illinois, Urbana, IL 61801, U.S.A. A model which treats an epithelium as a fluid elastic shell predicts the morphogenetlc effects of differential adhesion within the shell. Inferences from this model are designated with *. The late blastula contains an animalvegetal cohesion gradient, with high cohesion near the animal pole (Holtfreter). *This gradient can produce epiboly. Epiboly generates an adhesive disparity at the blastopore site. *This disparity initiates involution, bringing marginal zone mesoderm in contact with the under-slde of the gradient. Involution proceeds as the mesoderm migrates up this gradient. *The gradient in mesoderm and ectoderm elongates the embryo. Dorsal mesoderm induces overlying ectoderm to increase its cohesion; *this initiates thickening of the neural plate. Thus each movement in the sequence tends to resolve an instability, but generates a new instability which drives the next phase of movement.
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