Novel signaling center in anterior portion of limb buds

肢芽前部的新型信号中心

• 批准号: 10480209
• 负责人: OGURA Toshihiko
• 金额: $ 6.14万
• 依托单位: Nara Institute of Science and Technology
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1998
• 资助国家: 日本
• 起止时间: 1998 至 1999
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10480209/
• 关键词: 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

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 … More 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

为了研究鸡肢芽前部在其前后轴线模式形成过程中的可能作用，我们克隆了几个仅在鸡肢芽前部表达的候选基因。在我们的初步分析中，我们发现一个克隆(ALX4)与果蝇配对型转录因子aristess具有最高的同源性。全套原位杂交分析表明，该基因仅在发育早期的肢芽前部表达。为了研究ALX4与Sonic Hedgehog(Shh)的关系，进一步分析了其精确的表达模式。发现ALX4在早期就有表达，而Shh似乎在ALX4不表达的后部表达。因此，ALX4的表达先于Shh。这表明ALX4可能在肢体发育的早期阶段负向调节Shh。这也表明，ALX4的突变可能导致…更多的使用破坏这一负调控，这反过来可能导致在肢芽的前部异位诱导Shh和轴前多指。我们对小鼠alx4基因的定位分析表明，该基因位于Strong‘s Luxid(第1个：小鼠轴前多指突变体)的位置，为了进一步研究，我们从第1个ィイD1JィエD1突变株中克隆了alx4基因。序列分析表明，ALX4同源结构域存在16个碱基缺失，导致ALX4提前终止。由于Strong的豪华突变体显示Shh在肢芽的前部异位表达，这些证据有力地表明ALX4和Shh之间的负环在肢体前后轴的建立中起着重要作用。鸡胚胎为分析这种遗传等级提供了一个理想的系统。因此，我们通过在正常表达ALX4的肢体前部植入Shh浸泡的珠子，在鸡胚中证实了这一负环。Shh浸泡的珠子迅速下调ALX4的表达，而另一个前标记Gli3被缓慢抑制。这表明ALX4和Shh之间的负反馈环在建立肢芽的前后极性中起着关键作用，Gli3也被证明在ALX4的独立通路中起作用，参与后期的前后极性的维持。我们的数据还表明，ALX4负反馈环的中断会导致轴前多指表型。较少

项目成果

M. Takahashi et al.: "The role of Alx4 in the establishment of auteroposterior polarity during vertebrate limb development"Development. 125. 4417-4425 (1998)

M. Takahashi等人：“Alx4在脊椎动物肢体发育过程中前后极性建立中的作用”开发。

Motohisa Takahashi, Koji Tamura, Dirk Buscher, Hiroshi Masuya, Sayuri Yonei-Tamura, Ken Matsumoto, Mayumi Naitoh-Matsuo, Jun Takeuchi, Keiko Ogura, Toshihiko Shiroishi, Toshihiko Ogura, Juan Carlos Izpisua Belmonte: "The role of Alx4 in the establishment

Motohisa Takahashi、Koji Tamura、Dirk Buscher、Hiroshi Masuya、Sayuri Yonei-Tamura、Ken Matsumoto、Mayumi Naitoh-Matsuo、Jun Takeuchi、Keiko Ogura、Toshihiko Shiroishi、Toshihiko Ogura、Juan Carlos Izpisua Belmonte：“Alx4 在建立中的作用

M.Takahashi et al.: "The role of Alx4 in the establishmeut of anteroposterior polarity during vertebrate ilwb developwent"Developwent. 125. 4417-4425 (1998)

M.Takahashi 等人：“Alx4 在脊椎动物 ilwb 发育过程中前后极性建立中的作用”。

M.Takahashi et al.: "The role of Alx4 in the establishment of auteroposterior polarity during verfebrate limb development." Development.125. 4417-4425 (1998)

M.Takahashi 等人：“Alx4 在脊椎动物肢体发育过程中前后极性建立中的作用。”