| Project/Area Number |
15390557
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Morphological basic dentistry
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| Research Institution | Tokyo University of Science |
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Principal Investigator |
HOZUMI Nobumichi Tokyo University of Science, Research Institute for Biological Sciences, Professor, 生命科学研究所, 教授 (60051744)
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| Co-Investigator(Kenkyū-buntansha) |
SUZUKI Mitsuhiro Tokyo University of Science, Research Institute for Biological Sciences, Research Associate, 助手 (00321662)
MORIMURA Naoko RIKEN, The Institute for Physical Chemical Research, Researcher, 比較神経発生研究チーム, 研究員 (00349044)
香山 雅子 東京理科大学, 生命科学研究所, 助手 (80318229)
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| Project Period (FY) |
2003 – 2005
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| Project Status |
Completed (Fiscal Year 2005)
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| Budget Amount *help |
¥11,800,000 (Direct Cost: ¥11,800,000)
Fiscal Year 2005: ¥3,000,000 (Direct Cost: ¥3,000,000)
Fiscal Year 2004: ¥4,400,000 (Direct Cost: ¥4,400,000)
Fiscal Year 2003: ¥4,400,000 (Direct Cost: ¥4,400,000)
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| Keywords | Notch / Limb bud micromass culture / SST-REX method / ATDC5 / Chondrogenesis / Ossification / Limb Bud Micromass Culture / DAPT / マウス肢芽 / 間葉系幹細胞 / 細胞凝集 / 内軟骨性骨化 / 遺伝子発現 / 転写因子 / Conditional deletion mutantマウス / Notch1 / 軟骨基質 / 骨芽細胞 |
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| Research Abstract |
In endochondral ossification occurring in longitudinal bone, cartilagineous tissue derived from mesenchymal cells is replaced with bone within ossification centers. Thus, endochondral ossification precedes ossification osteoblasts. Therefore it is prerequisite to elucidate the molecular mechanisms involved in chondrogenesis for better understanding of skeltogenesis. We have shown that Notch signaling stimulates the differentiation of osteoblasts. The current research projects are aimed at elucidation of mechanisms in bone formation regulated by Notch.
Notch1 was localized starting from the mesenchymal condensation stage of embryonic mouse forelimbs. Interestingly, although localization could not be detected in the proliferating chondrocytes, obvious immuno-reactivity indicating its expression was retained in the perichondral region. To assess the effect of Notch activation, we transferred an active form of Notch (NIC) into the cell line by an adenovirus vector. The differentiation of ch
… More
ondrocytes was inhibited. We utilized limb bud micromass culture (LBMC) for further analysis. LBMC is an established model that recapitulates mesenchymal condensation and chondrocyte differentiation. RT-PCR showed that Notch and its related genes were expressed in such cultures at day 1 and day 5, when cell condensation and nodule formation were initiated. Immuno-histochiemical experiments revealed that the expression of Notch1 was initially localized within the nodules and shifted to their peripheral region as the cell differentiation progressed. We disrupted Notch signaling by using a gamma-secretase inhibitor, DAPT, to analyze the function of Notch signaling in the culture system. Blocking Notch signaling by DAPT apparently promoted the initiation of prechondrogenic condensation and fusion of the nodules, and such an effect was reversed by exogenous expression of the Notch cytoplasmic domain. These observations imply that the Notch signal may have an important role in chondrogenic differentiation by negatively regulating the initiation of prechondrogenic condensation and nodule formation.
We constructed a cDNA library from the mRNA fraction derived from a chondrogenic lell line (ATDC5) at the condensation stage by using the signal sequence trap by retrovirus mediated expression (SST-REX) method. We obtained 486 factor (IL-3)-independent clones by screening 5.7 x 10^3 clones. DNA sequencing analysis of the clones identified genes encoding 157 known proteins and 4 novel proteins. A series of the analyses demonstrate that the SST-REX method is a useful experimental system to identify genes involved in the complicated mechanisms of bone formation. Less
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