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Immunochemical and biochemical studies of epithelial initiation of cementogenesis
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413 DISTRIBUTION OF HYALURONATE IN PALATAL SHELVES VISUALIZED BY COMPUTER-ASSISTED DIFFERENCE PICTURES. L. Brink ley and _J. Morris-Wiman. Dept. Anat. & Cell Biol., Med Sch., U. Mich., Ann Arbor, MI 48109 Hyaluronate (HA) in tissue sections can only be positively identified by its susceptibility to digestion by Strept Omyces hyaluronidase combined with Alcian blue staining. Previously, HA localization required a visual comparison of two adjacent serial sections, a control and an enzyme-digested. We have used computer algorithms which permit the images of the control and enzyme-digested sections to be registered with each other, one image subtracted from the other and the resulting difference picture displayed. The distribution of HA along the rostralcaudal axis of mouse secondary palatal shelves was followed during the course of shelf closure. Prior to closure the central portion of the posterior twothirds of the shelf is HA-rich. During elevation the shelves expand and the HA pattern becomes much more dispersed. This Js consistent with the hypothesis that shelf closure is partially driven by the expansion of an HA gel-fiber networks Supported by NIH grants DE02774 and 5-KO4-DE00104 to LLB. 415
IM}~NOCHEMICAL AND BIOCHEMICAL STUDIES OF EPITHELIAL INITIATION OF CEMENTOGENESIS. P.Bringas,Jr., M.Y.Hsu, C.Bessem, T.F. Jaskoll and H.C.Slavkin. Lab. Develop. Biol., School of Dentistry, University of Southern California, Los Angeles, California 90089-0191. Following crown formation and enamel extracellular matrix (ECM) deposition, epithelial tissue thereafter appears to regulate root formation of tooth organs. The present study was designed to determine functions of epithelial root sheath tissue in the initiation of cementum ECM which covers root surfaces. Using mouse molar tooth organs, we compared in situ and in vitro development using immunochemical, biochemical and morphological assays. Using metabolic labeling with 3H-tryptophan or 35S-methionine, fractionation of labeled proteins with 2-dimensional SDS gel electrophoresis, subsequent Western transfer or fluorography, and indirect inmlunofluorescence, we observed epithelial root sheath to produce enamel-like proteins upon root dentine (ECM). Epithelial products may recruit ectomesenchyme cells to complete cementum ECM formation. Supported by research grants DE-02848 and DE-06425 (USPHS), 414 INHIBITION OF LIMB CHONDROGENESIS BY A VERATRUM-ALKALOID:TEMPORAL SPECIFICITY IN VIVO AND IN VITRO. M.A. Campbell, K.S. Brown, J.Hassell, E.Horigan_n, & R.F.Keeler. Lab. Devel. Biol. & Anomalies, NIDR, NIH, Bethesda, MD & USDA, Logan, UT Jervine, a steroidal alkaloid derived from plants of the genus Veratrum, produces characteristic malformations. Defects were restricted to structures which are initially formed in cartilage. Peak sensitivity of specific anlagen occurred prior to condensation of the prechondrogenic mesenchyme. The temporal specificity of sensitivity was further studied by using limb bud cells from mouse embryos, which undergo chondrogenesis in vitro. Prior to differentiation, exposure of cultures to jervine suppressed subsequent accumulation of cartilage-specific macromolecules. Treatment after differentiation had no such effect. These studies indicate that jervine specifically blocks an early event in the differentiation of mesenchyme to cartilage. It is proposed that as a temporally-specific disruptor of normal development, jervine could be used to identify specific premorphogenic cell populations.
LECTIN RECEPTOR SITES AND ENDOGEN O U S L E C T I N S IN T H E C H I C K E M B R Y O . POSSIBLE RELATIONSHIPS WITH GERM CELL MIGRATION. E.Didier, P.Didier D.Bayle and N.Fargeix. Laboratoire de Biologie Animale. Universit~ de Clermont II.BP 45.63170 Aubi~re (France) . Glycoconjugates from axial extracellular matrices (ECM) h a v e been investigated via cytochemical and biochemical methods. The mesenterial and gonadal ECMs bind fluorescent and/or peroxidase-conjugated WGA (Wheat germ agglutinin) a n d B E A ( B o l e t u s e d u l i s a g glutinin) which indicates that they display glucosaminyl and lactosaminyl residues. Whereas no dif f e r e n c e is o b s e r v e d at t h e l e v e l of g o n a d s , d i s s y m e t r i c a l labelling is s e e n o n t h e m e s e n t e r i a l E C M at s t a g e 2 3 - 2 5 , w h i c h c o u l d be c o r r e lated to the left-right asymetry of g e r m c e l l d i s t r i b u t i o n . In a d d i t i o n , c r u d e e x t r a c t s of 4-5 d a y embryos contain soluble and membrane agglutinins specific for galactose, lactose, glucose and amine s u g a r s .
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413 DISTRIBUTION OF HYALURONATE IN PALATAL SHELVES VISUALIZED BY COMPUTER-ASSISTED DIFFERENCE PICTURES. L. Brink ley and _J. Morris-Wiman. Dept. Anat. & Cell Biol., Med Sch., U. Mich., Ann Arbor, MI 48109 Hyaluronate (HA) in tissue sections can only be positively identified by its susceptibility to digestion by Strept Omyces hyaluronidase combined with Alcian blue staining. Previously, HA localization required a visual comparison of two adjacent serial sections, a control and an enzyme-digested. We have used computer algorithms which permit the images of the control and enzyme-digested sections to be registered with each other, one image subtracted from the other and the resulting difference picture displayed. The distribution of HA along the rostralcaudal axis of mouse secondary palatal shelves was followed during the course of shelf closure. Prior to closure the central portion of the posterior twothirds of the shelf is HA-rich. During elevation the shelves expand and the HA pattern becomes much more dispersed. This Js consistent with the hypothesis that shelf closure is partially driven by the expansion of an HA gel-fiber networks Supported by NIH grants DE02774 and 5-KO4-DE00104 to LLB. 415
IM}~NOCHEMICAL AND BIOCHEMICAL STUDIES OF EPITHELIAL INITIATION OF CEMENTOGENESIS. P.Bringas,Jr., M.Y.Hsu, C.Bessem, T.F. Jaskoll and H.C.Slavkin. Lab. Develop. Biol., School of Dentistry, University of Southern California, Los Angeles, California 90089-0191. Following crown formation and enamel extracellular matrix (ECM) deposition, epithelial tissue thereafter appears to regulate root formation of tooth organs. The present study was designed to determine functions of epithelial root sheath tissue in the initiation of cementum ECM which covers root surfaces. Using mouse molar tooth organs, we compared in situ and in vitro development using immunochemical, biochemical and morphological assays. Using metabolic labeling with 3H-tryptophan or 35S-methionine, fractionation of labeled proteins with 2-dimensional SDS gel electrophoresis, subsequent Western transfer or fluorography, and indirect inmlunofluorescence, we observed epithelial root sheath to produce enamel-like proteins upon root dentine (ECM). Epithelial products may recruit ectomesenchyme cells to complete cementum ECM formation. Supported by research grants DE-02848 and DE-06425 (USPHS), 414 INHIBITION OF LIMB CHONDROGENESIS BY A VERATRUM-ALKALOID:TEMPORAL SPECIFICITY IN VIVO AND IN VITRO. M.A. Campbell, K.S. Brown, J.Hassell, E.Horigan_n, & R.F.Keeler. Lab. Devel. Biol. & Anomalies, NIDR, NIH, Bethesda, MD & USDA, Logan, UT Jervine, a steroidal alkaloid derived from plants of the genus Veratrum, produces characteristic malformations. Defects were restricted to structures which are initially formed in cartilage. Peak sensitivity of specific anlagen occurred prior to condensation of the prechondrogenic mesenchyme. The temporal specificity of sensitivity was further studied by using limb bud cells from mouse embryos, which undergo chondrogenesis in vitro. Prior to differentiation, exposure of cultures to jervine suppressed subsequent accumulation of cartilage-specific macromolecules. Treatment after differentiation had no such effect. These studies indicate that jervine specifically blocks an early event in the differentiation of mesenchyme to cartilage. It is proposed that as a temporally-specific disruptor of normal development, jervine could be used to identify specific premorphogenic cell populations.
LECTIN RECEPTOR SITES AND ENDOGEN O U S L E C T I N S IN T H E C H I C K E M B R Y O . POSSIBLE RELATIONSHIPS WITH GERM CELL MIGRATION. E.Didier, P.Didier D.Bayle and N.Fargeix. Laboratoire de Biologie Animale. Universit~ de Clermont II.BP 45.63170 Aubi~re (France) . Glycoconjugates from axial extracellular matrices (ECM) h a v e been investigated via cytochemical and biochemical methods. The mesenterial and gonadal ECMs bind fluorescent and/or peroxidase-conjugated WGA (Wheat germ agglutinin) a n d B E A ( B o l e t u s e d u l i s a g glutinin) which indicates that they display glucosaminyl and lactosaminyl residues. Whereas no dif f e r e n c e is o b s e r v e d at t h e l e v e l of g o n a d s , d i s s y m e t r i c a l labelling is s e e n o n t h e m e s e n t e r i a l E C M at s t a g e 2 3 - 2 5 , w h i c h c o u l d be c o r r e lated to the left-right asymetry of g e r m c e l l d i s t r i b u t i o n . In a d d i t i o n , c r u d e e x t r a c t s of 4-5 d a y embryos contain soluble and membrane agglutinins specific for galactose, lactose, glucose and amine s u g a r s .
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