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)染色体动态调节Shh在肢芽中的表达。为了检查Shh及其远程增强子MFCS1的拓扑关系，我们使用杂交这两个基因组区域的两个探针进行了3D-FISH分析。我们解剖了发育中的肢芽的前部、中间部和表达 Shh 的后部。 Shh 和 MFCS 1 信号之间的物理距离是通过计算计算出来的。结果表明，在表达Shh的后芽细胞中，MFCS 1信号倾向于与Shh信号共定位，但在中间芽细胞中则不然。值得注意的是，前芽细胞中的Shh-MFCS 1 距离显示出与后肢芽细胞中相似的值。考虑到许多多指突变体中前部异位Shh表达，前部细胞似乎具有潜在表达Shh的能力。 MFCS1和Shh的毗邻可能表明了这种能力。染色体构象捕获分析还表明，MFCS1在E10.5肢芽中与Shh的第一个外显子发生物理相互作用，但在后期的芽中则没有相互作用，此时Shh表达已经停止。通过使用 E10.5 肢体和后期的芽进行 3D-FISH 分析，证实了染色体结构随着肢体发育的进展而发生的变化。(2)探索 Shh 表达的新型远程顺式调控元件高度保守的非编码序列 (CNS) ，MFCS4，作为Shh的咽特异性长程顺式调节元件发挥作用。众所周知，Shh 在口腔外胚层和咽内胚层中的表达对于呼吸道和消化道的发育至关重要。通过对 MFCS4 嵌套的 130kb 长基因组区域中的 CNS 进行转基因分析，我们鉴定了两个序列，它们驱动牙齿原基以及肺和消化道中的报告基因表达。这些元件可以作为顺式元件来调节特定于口腔、咽、气管和消化道的Shh表达。