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Development of apical ectodermal ridge and changes in subectodermal mesenchyme in mouse limb buds
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PATTERNING OF MYOGENIC CELLS IN THE EARLY CHICK LIMB BUD. M. Solursh, K.L. Jensen, and R.S. Reiter. Department of Biology, University of Iowa, Iowa City, Iowa 52242 The in situ distribution of putative myogenic cells in the limb bud is described by use of indirect immunofluorescence with a monoclonal antibody directed against desmin. At stage 21-24, desminpositive cells are absent from the avascular peripheral zone that extends a relatively constant distance from the limb ectoderm. Positive cells are localized in the central core in regions of the limb bud where venous networks have formed. By stage 26, the desmin-positive cells become shifted to the dorsal and ventral myogenic regions of the limb bud, which now also stain with antibody directed against light meromyosin. In vitro studies were carried out on high density cultures of limb mesenchyme to analyze the effects of the limb ectoderm, the apical ectodermal ridge and vascular endothelial cells on the distribution of myogenic cells. These results support the hypothesis that the early patterning of myogenic cells is influenced by chemotactic and mitogenic stimuli from the already patterned vasculature. (Supported by NIH grants HD05505 and HD18577.)
CELL D E A T H AND P A T T E R N F O R M A T I O N IN MUTANT CHICK EMBRYOS. U.K. Abbott. Dept. of Avian Sciences, UCD, Davis, CA 95616, USA. P a t t e r n m u t a n t s provide a powerful tool for studies of normal morphogenesis. Developmental interactions between chick polydactylous and limbless mutant genes w e r e e x a m i n e d in s t a g e s r a n g i n g f r o m the onset of limb morphogenesis through ossification of b o n e m o d e l s . The mutants employed included the dominant and viable, polydactyly; the autosomal r e c e s s i v e late lethal, e u d i p l o p o d i a ; and the a u t o s o m a l recessive late lethal, l i m b l e s s . P o l y d a c t y l y a l t e r s l i m b m e s e n c h y m e so that a b r o a d e r AER is i n d u c e d and m a i n t a i n e d w h i l e b o t h eudiplopodia and limbless affect responding limb epithelium in opposing fashions. The p a t t e r n and t i m i n g of c e l l d e a t h in the m e s e n c h y m e and e p i t h e l i u m of t h e s e v e r a l types of doubly homozygous embryos permitted a n a l y s i s of the b a s i s of e p i s t a s i s in the limbless combinations and additivity in those involving eudiplopodia and polydactyly.
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EXTRACELLULAR MATRIX MAINTAINS APICAL ECTODERMAL RIDGE IN CULTURE. J.J. Tomasek and J.A. Brier. Department of Anatomy, New York Medical College, Valhalla, N.Y. 10595. Limb outgrowth is dependent upon a reciprocal interaction between apical ectodermal ridge (AER) and underlying mesenchyme. Previous studies have demonstrated that AER cultured in the absence of mesenchyme rapidly dies. In an attempt to determine the role of extracellular matrix (ECM) in limb outgrowth, we have cultured AER on hydrated type I collagen lattices (HCL) and examined the ability of HCL to maintain ridge cells. AER and adjacent nonridge ectoderm were enzymatically isolated from underlying mesenchyme of stages 20-22 duck wing buds and placed onto HCL. Epithelial sheets cultured for 18 or 24 hours were examined microscopically. In contrast to recent studies, we have found that type I collagen is sufficient to maintain viable ridge cells in culture and that they remain morphologically distinguishable from adjacent nonridge ectodermal cells. These results suggest that ECM underlying the AER may play an important role in both its maintenance and ability to induce limb outgrowth.
DEVELOPMENT OF APICAL ECTODERMAL RIDGE AND CHANGES IN SUBECTODERMAL MESENCHYME IN MOUSE LIMB BUDS. A.R.Datu, H.Nakamura and M.Yasuda. Dept. of Anatomy, Hiroshima Univ. Sch. of Med., Kasumi 1-2-3, Minamiku, Hiroshima 734, Japan. To reveal changes in the epithelialmesenchymal interface during limb development, electron microscopic and morphometric studies were done on the cell process meshwork ( C P M ) and extracellular space (ECS) in the subepidermal mesenchyme in mouse forelimb buds between days 9.5 and 12.5 (VP=day 0). At day 9.5, when the apical ectodermal ridge (AER) in the forelimb bud had not appeared yet, no local differences were noted in CPM density and ECS width. However, when the AER was distinct between days 10.5 and 11.5, the CPM beneath the AER became more sparse and the ECS became wider than those beneath the dorsal and ventral non ridge epidermis. With the involution of AER around days 12.0-12.5, the local differences in CPM density and ECS width became less obvious. These findings indicate that lowering of CPM density and widening of the ECS is closely associated with AER development. (Supported by Grant No. 5930003 from the Ministry of Education, Science and Culture, Japan.)
165S
404
PATTERNING OF MYOGENIC CELLS IN THE EARLY CHICK LIMB BUD. M. Solursh, K.L. Jensen, and R.S. Reiter. Department of Biology, University of Iowa, Iowa City, Iowa 52242 The in situ distribution of putative myogenic cells in the limb bud is described by use of indirect immunofluorescence with a monoclonal antibody directed against desmin. At stage 21-24, desminpositive cells are absent from the avascular peripheral zone that extends a relatively constant distance from the limb ectoderm. Positive cells are localized in the central core in regions of the limb bud where venous networks have formed. By stage 26, the desmin-positive cells become shifted to the dorsal and ventral myogenic regions of the limb bud, which now also stain with antibody directed against light meromyosin. In vitro studies were carried out on high density cultures of limb mesenchyme to analyze the effects of the limb ectoderm, the apical ectodermal ridge and vascular endothelial cells on the distribution of myogenic cells. These results support the hypothesis that the early patterning of myogenic cells is influenced by chemotactic and mitogenic stimuli from the already patterned vasculature. (Supported by NIH grants HD05505 and HD18577.)
CELL D E A T H AND P A T T E R N F O R M A T I O N IN MUTANT CHICK EMBRYOS. U.K. Abbott. Dept. of Avian Sciences, UCD, Davis, CA 95616, USA. P a t t e r n m u t a n t s provide a powerful tool for studies of normal morphogenesis. Developmental interactions between chick polydactylous and limbless mutant genes w e r e e x a m i n e d in s t a g e s r a n g i n g f r o m the onset of limb morphogenesis through ossification of b o n e m o d e l s . The mutants employed included the dominant and viable, polydactyly; the autosomal r e c e s s i v e late lethal, e u d i p l o p o d i a ; and the a u t o s o m a l recessive late lethal, l i m b l e s s . P o l y d a c t y l y a l t e r s l i m b m e s e n c h y m e so that a b r o a d e r AER is i n d u c e d and m a i n t a i n e d w h i l e b o t h eudiplopodia and limbless affect responding limb epithelium in opposing fashions. The p a t t e r n and t i m i n g of c e l l d e a t h in the m e s e n c h y m e and e p i t h e l i u m of t h e s e v e r a l types of doubly homozygous embryos permitted a n a l y s i s of the b a s i s of e p i s t a s i s in the limbless combinations and additivity in those involving eudiplopodia and polydactyly.
406
407
EXTRACELLULAR MATRIX MAINTAINS APICAL ECTODERMAL RIDGE IN CULTURE. J.J. Tomasek and J.A. Brier. Department of Anatomy, New York Medical College, Valhalla, N.Y. 10595. Limb outgrowth is dependent upon a reciprocal interaction between apical ectodermal ridge (AER) and underlying mesenchyme. Previous studies have demonstrated that AER cultured in the absence of mesenchyme rapidly dies. In an attempt to determine the role of extracellular matrix (ECM) in limb outgrowth, we have cultured AER on hydrated type I collagen lattices (HCL) and examined the ability of HCL to maintain ridge cells. AER and adjacent nonridge ectoderm were enzymatically isolated from underlying mesenchyme of stages 20-22 duck wing buds and placed onto HCL. Epithelial sheets cultured for 18 or 24 hours were examined microscopically. In contrast to recent studies, we have found that type I collagen is sufficient to maintain viable ridge cells in culture and that they remain morphologically distinguishable from adjacent nonridge ectodermal cells. These results suggest that ECM underlying the AER may play an important role in both its maintenance and ability to induce limb outgrowth.
DEVELOPMENT OF APICAL ECTODERMAL RIDGE AND CHANGES IN SUBECTODERMAL MESENCHYME IN MOUSE LIMB BUDS. A.R.Datu, H.Nakamura and M.Yasuda. Dept. of Anatomy, Hiroshima Univ. Sch. of Med., Kasumi 1-2-3, Minamiku, Hiroshima 734, Japan. To reveal changes in the epithelialmesenchymal interface during limb development, electron microscopic and morphometric studies were done on the cell process meshwork ( C P M ) and extracellular space (ECS) in the subepidermal mesenchyme in mouse forelimb buds between days 9.5 and 12.5 (VP=day 0). At day 9.5, when the apical ectodermal ridge (AER) in the forelimb bud had not appeared yet, no local differences were noted in CPM density and ECS width. However, when the AER was distinct between days 10.5 and 11.5, the CPM beneath the AER became more sparse and the ECS became wider than those beneath the dorsal and ventral non ridge epidermis. With the involution of AER around days 12.0-12.5, the local differences in CPM density and ECS width became less obvious. These findings indicate that lowering of CPM density and widening of the ECS is closely associated with AER development. (Supported by Grant No. 5930003 from the Ministry of Education, Science and Culture, Japan.)
165S