2000 Fiscal Year Final Research Report Summary
Developmental and molecular cell biological study of the mechanisms of limb differentiation
| Project/Area Number |
10470004
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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 |
General anatomy (including Histology/Embryology)
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| Research Institution | KYOTO UNIVERSITY |
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Principal Investigator |
SHIOTA Kohei Kyoto Univ., Anatomy, Professor, 医学研究科, 教授 (80109529)
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| Co-Investigator(Kenkyū-buntansha) |
MORIMOTO Takeshi Kyoto Univ., Anatomy, Instructor, 医学研究科, 助手 (00311746)
TAKIGAWA Toshiya Kyoto Univ., Anatomy, Instructor, 医学研究科, 助手 (90263095)
ISHIBASHI Makoto Kyoto Univ., Anatomy, Lecturer, 医学研究科, 講師 (30232341)
MIURA Takashi Kyoto Univ., Anatomy, Instructor, 医学研究科, 助手 (10324617)
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| Project Period (FY) |
1998 – 2000
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| Keywords | Limb bud / Programmed cell death / Vascular remodelling / Matrix metalloproteinase / Limb anomalies / Mouse fetus |
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| Research Abstract |
We have reported that so-called "programmed cell death" (PCD) in the interdigital tissue of the fetal mouse limb is apoptosis with DNA fragmentation and that the regional heterogeneity and temporal sequence of cell death in the developing limb play essential roles in patterning the limb. However, the cells which undergo apoptosiss were not identified nor was the process of how the ECM is disrupted during digit separation. We examined the remodeling of the microvascular network in the interdigital tissue of mouse fetuses and found that cell death occurs in PECAM1/C031-positive endothelial cells. Whole-mount in situ hybridization and Northern blot analysis revealed that gelatinase/MMP2 was highly expressed in the areas where vascular remodeling was occurring. In the limb of Hammertoe mutant (Hm/Hm) mouse fetuses in which interdigital webbing persists, neither apoptosis nor the regression of microvascular plexus in the interdigital tissue were observed to occur normally. Our study has provided the first evidence that apoptotic cell death occurs in endothelial cells of the interdigital microvascular plexus and that vascular remodeling and gelatinase/MMP2 expression may play essential roles in the separation of mammalian digits.
Next, to analyze the mechanisms of chondrogenic pattern formation in the developing limb, we utilized a micromass culture system of limb mesenchymal cells, and examined the chondrogenic pattern formation in vitro. The periodic nature of limb chondrogenesis was retained in micromass culture. We developed a novel method to automatically measure the periodicity of the pattern and demonstrated that the reaction-diffusion model is the most plausible for explaining the periodic chondrogenic pattern formation. We also demonstrated that TGFβ2 acts as an "activator"-like molecule in chondrogenic pattern formation and is possibly responsible for the cell sorting phenomenon.
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