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

(1)染色体动力学调控肢体芽中Shh的表达。为了检验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体芽的前部、中间和表达sh的后部。计算出Shh与MFCS 1信号之间的物理距离。结果表明，MFCS - 1信号在表达Shh的后芽细胞中倾向于与Shh信号共定位，而在中间芽细胞中不存在。值得注意的是，Shh-MFCS 1在前肢芽细胞中的距离与后肢芽细胞中的距离相似。考虑到许多多趾突变体中Shh的前位表达，前细胞似乎具有潜在表达Shh的能力。MFCS1和Shh相邻可能表明这种能力。染色体构象捕获实验还显示，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，Shh的表达已经停止。这种染色体结构随肢体发育的变化通过3D-FISH分析在E10.5肢和后期的芽得到证实。(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是Shh的咽特异性远程顺式调控元件。众所周知，Shh在口腔外胚层和咽部内胚层的表达对呼吸道和消化道的发育至关重要。通过对MFCS4嵌套的长达130kb的基因组区域的CNS进行转基因实验，我们发现了两个序列，它们驱动报告基因在牙齿原基、肺和消化道中的表达。这些元件可能作为顺式元件调节嘴、咽、气管和消化道特异性Shh表达。

(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.