| Project/Area Number |
10480209
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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 |
Developmental biology
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| Research Institution | Nara Institute of Science and Technology |
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Principal Investigator |
OGURA Toshihiko Nara Institute of Science and Technology, Graduate School of Biological Sciences, Associate Professor, バイオサイエンス研究科, 助教授 (60273851)
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| Project Period (FY) |
1998 – 1999
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| Project Status |
Completed (Fiscal Year 1999)
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| Budget Amount *help |
¥9,600,000 (Direct Cost: ¥9,600,000)
Fiscal Year 1999: ¥3,800,000 (Direct Cost: ¥3,800,000)
Fiscal Year 1998: ¥5,800,000 (Direct Cost: ¥5,800,000)
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| Keywords | limb bud / Alx4 / Strong's luxoid / preaxial polydactyly / Shh / Gli3 / 前後軸 / ソニックヘッジホグ(Shh) / レチノイン酸 / 多指症 |
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| Research Abstract |
To study the possible roles of the anterior portion of limb bud during pattern formation along its anteroposterior axis, we cloned several candidate genes which are expressed exclusively in the anterior portion of chicken limb buds. In our initial analysis, we found that one clone (Alx4) shows the highest homology to the Drosophila paired -type transcription factor aristaless. Whole mount in situ hybridization analysis revealed that this gene is expressed exclusively in the anterior part of limb buds from very early stages of development.
To investigate the relationship between Alx4 and Sonic hedgehog (Shh), precise expression patterns were further analyzed. Alx4 was found to be expressed from very early stages while Shh seems to be expressed in the posterior portion where Alx4 is not expressed. Hence Alx4 expression precedes to that of Shh. This indicates that Alx4 might negatively regulate Shh at the early stages of limb development. This also indicates that mutation of Alx 4 could ca
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use disruption of this negative regulation, which in turn could cause ectopic induction of Shh at the anterior portion of limb buds and the preaxial polydactyly. Our mapping analysis of mouse Alx4 revealed that this gene is located at the position of Strong's luxoid (1st : mouse preaxial polydactyly mutant).
To investigate further, we cloned Alx4 cDNA from 1stィイD1JィエD1 mutant strain. Sequence analysis identified 16 bp deletion in the Alx4 homeodomain, which causes premature termination of Alx4. Since Strong's luxoid mutants show ectopic expression of Shh at the anterior part of limb buds, these lines of evidence strongly suggest that the negative loop between Alx4 and Shh plays an important role during establishment of anterior-posterior axis of limb.
Chicken embryos provide an ideal system to analyze this kind of genetic hierarchy. Thus we confirmed this negative loop in chicken embryos by implanting Shh-soaked beads in the anterior portion of limb where normally Alx4 is expressed. Shh-soaked beads rapidly downregulated Alx4 while another anterior marker Gli3 was suppressed slowly. This indicates that negative feedback loop between Alx4 and Shh plays a critical role in establishing the anterior-posterior polarity of limb buds and that Gli3, which was also shown to act in an independent pathway of Alx4, is involved in the maintenance of anterior-posterior polarity at later stages.
Taken together, our data indicate that Alx4 is one of critical factors involved in the establishment of anterior-posterior axis of limb buds. Our data also indicate that disruption of the negative Alx4 feedback loop causes preaxial polydactyly phenotype. Less
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