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

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(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.