1986 Fiscal Year Final Research Report Summary
Extracellular Matrix Macromolecules of Developing Limbs
| Project/Area Number |
59570651
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
Orthopaedic surgery
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| Research Institution | Institute for Developmental Research |
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Principal Investigator |
OOHIRA Atsuhiko Institute for Developmental Research, その他, 研究員 (20101074)
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| Co-Investigator(Kenkyū-buntansha) |
MATSUI Fumiko Institute for Developmental Research, 発生学部, 研究助手
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| Project Period (FY) |
1984 – 1986
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| Keywords | Limb anomalies / Developing limbs / Rat limb buds / Chondrogenesis / Extracellular matrix / Proteoglycans / Chondroitin sulfate |
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| Research Abstract |
We have analyzed proteoglycans quantitatively as well as qualitatively in the pathological cartilaginous or osseous tissues excised from patients with various congenital skeletal disorders, especially limb anomalies, and found in most cases abnormalities in the amount and/or structure of proteoglycans. Our observations support the general idea that extracellular matrix macromolecules including proteoglycans are involved in cell differentiation and morphogenesis of skeletal tissues. To clarify the role of proteoglycans in limb morphogenesis, we isolated and characterized proteoglycans synthesized by developing limb buds of the rat.
Limb buds, dissected from rat fetuses on day 13, 14 or 15 of gestation, were incubated in a culture medium containing [ <^(35)S> ]sulfate for 3 hr. [ <^(35)S> ]labeled proteoglycans were solubilized in 4 M guanidine HCl containing protease inhibitors and a detergent, 0.8% MEGA-9. A chondroitin sulfate proteoglycan and a heparan sulfate proteoglycan were isolat
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ed from day-13 limb buds. In addition, single chain heparan sulfate was also isolated. Our microscopic examination revealed that mesenchymal-cell condensation to form the primordia of the limb skeleton became observable at this stage. Cartilage-type proteoglycan could be detected for the first time on day 14, and the amount increased thereafter. This finding indicates that chondrogenesis in rat limbs begins on day 14 of gestation.
Polysaccharide preparation obtained from day-13 limb buds by proteolysis was separated by two-dimensional electrophoresis on cellulose acetate film into four Alcian blue-stained spots; hyaluronate, heparan sulfate, chondroitin sulfate and an unknown component. Interestingly, the unknown component was not found in polysaccharide preparations obtained either from the brain or tail of rat fetuses at the same developmental stages. Although characterization of this component is not satisfactory at this moment mainly owing to the small amount available, the component could be involved in limb morphogenesis. Less
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