Limb bud-specific Shh enhancer that is 1Mb apart from Shh coding sequence

肢芽特异性 Shh 增强子，距 Shh 编码序列 1Mb

基本信息

批准号：
17370003
负责人：
SHIROISHI Toshihiko
金额：
$ 9.34万
依托单位：
National Institute of Genetics
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2005
资助国家：
日本
起止时间：
2005 至 2006
项目状态：
已结题

项目摘要

(1)Chromosomal dynamism regulates Shh expression in limb buds.To examine topological relationship of Shh and its remote enhancer, MFCS1, we performed 3D-FISH analysis using two probes hybridizing these two genome regions. We dissected anterior, intermediate and Shh-expressing posterior portions of developing limb buds. Physical distance between Shh and MFCS 1 signals was computationally calculated. The result revealed that the MFCS 1 signal tends to colocalize with the Shh signal in the Shh-expressing posterior bud cells but not in the intermediate bud cells. Notably, the Shh-MFCS 1 distance in the anterior bud cells showed similar value to that in the posterior limb bud cells. Considering an anterior ectopic Shh expression in many polydactylous mutants, the anterior cell appears to have a competence to express Shh potentially. Adjoining of MFCS1 and Shh may indicate this competence.The chromosome conformation capture assay also revealed that MFCS1 physically interacts with first exon of Shh in the E10.5 limb buds but not in the buds at the later stage, in which Shh expression already ceased. This change in chromosomal structure as limb development progresses was confirmed by 3D-FISH analysis using buds at E10.5 limb and the later stage.(2)Exploration of novel remote cis-regulatroy element for Shh expressionA highly conserved noncoding-sequence (CNS), MFCS4, functions as a pharynx-specific long-range cis-regulatory element of Shh. It is well known that expression of Shh in the oral ectoderm and pharyngeal endoderm is essential for development of the respiratory and digestive tracts. By transgenic assay for CNS in the 130kb-long genomic region nested to MFCS4, we identified two sequences, which drive reporter gene expression in the tooth primordium and in the lung and digestive tract. These elements may serve cis-elements to regulate Shh expression specific to mouth, pharynx, trachea and digestive tract.

（1）染色体动力学调节肢体芽中的SHH表达。为了检查SHH及其远程增强子MFCS1的拓扑关系，我们使用两个探针杂交这两个基因组区域进行了3D-FISH分析。我们解剖了发育中的肢体的前，中间和表达SHH的后部。计算SHH和MFCS 1信号之间的物理距离。结果表明，MFCS 1信号倾向于与表达SHH的后芽细胞中的SHH信号共定位，而在中间芽细胞中则不会将其共定位。值得注意的是，前芽细胞中的SHH-MFCS 1距离显示的值与后肢芽细胞中的值相似。考虑到许多多肌突变体中的前异位SHH表达，前细胞似乎具有潜在表达SHH的能力。 MFCS1和SHH的毗邻可能表明这种能力。染色体构象捕获测定法还表明，MFCS1在E10.5肢体芽中与SHH的第一个外显子进行物理相互作用，但在后期的芽中不在芽中，SHH表达已经停止。 This change in chromosomal structure as limb development progresses was confirmed by 3D-FISH analysis using buds at E10.5 limb and the later stage.(2)Exploration of novel remote cis-regulatroy element for Shh expressionA highly conserved noncoding-sequence (CNS), MFCS4, functions as a pharynx-specific long-range cis-regulatory element of Shh.众所周知，在口腔外胚层和咽内胚层中SHH的表达对于呼吸道和消化道的发展至关重要。通过在嵌套在MFCS4身上的130kb长基因组区域中CNS的转基因测定，我们鉴定出了两个序列，它们驱动了牙齿原始中的报告基因表达，在牙齿和肺和消化道中。这些元素可以使用顺式元素来调节特定于口腔，咽，气管和消化道的SHH表达。