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. 2009 Sep 15;23(18):2166–2178. doi: 10.1101/gad.1842409

Figure 2.

Figure 2.

An essential CArG box in the miR-143/145 upstream enhancer. (A) Summary of transgenic constructs used to delineate the miR-143/145 enhancer is shown. Evolutionary conservation of the genomic region upstream of pre-miR-143 is shown at the top. Numbers of F0 transgenic embryos showing cardiac expression at E12.5 per total transgenic embryos obtained are indicated in the right column. A single mutation of the CArG in Reporter A abolished expression of the lacZ transgene in the embryonic heart and adult aorta. Embryonic heart expression was assayed in F0 and stable mouse lines, whereas adult aorta expression was assayed in two independent stable mouse lines. The 1.5-kb enhancer construct (Reporter B) contains only the two evolutionarily conserved regions and is sufficient to drive lacZ expression in the embryonic heart and adult aorta. Mutation of the CArG box in Reporter B abolished lacZ expression in the embryonic heart and adult aorta as determined in two stable mouse lines. (B) Transgenic mice were generated with a lacZ reporter linked to 5.5 kb of genomic DNA upstream of the miR-143/145 gene (Reporter A in A). The embryo in panel e was cleared to allow visualization of internal structures. Panel f is a high magnification of the embryo in panel e to show lacZ expression in SMCs lining the dorsal aorta (ao) and intersomitic arteries (isa). (Panel g) Adult aorta. (Panel h) Subdermal vasculature. (Panel i) Transverse section showing lacZ staining in an artery in skeletal muscle. (Panel j) Bladder. (ao) Aorta; (cc) cardiac crescent; (ht) heart. (C) Histological sections of Reporter B transgenic mice at various embryonic stages. (Bars: panel a, 100 μm; panels b,c, 200 μm. (a) Common atrial chamber; (bc) bulbus cordis; (la) left atrium; (lv) left ventricle; (ra) right atrium; (rv) right ventricle; (v) common ventricular chamber. (D) Histological sections of transgenic mice with wild-type genomic regions or the same regions with mutations in the CArG box at the indicated ages. Note that the CArG box mutation abolishes lacZ expression in the heart and vasculature. (a) Common atrial chamber; (ao) aorta; (bc) bulbus cordis; (la) left atrium; (lv) left ventricle; (ra) right atrium; (rv) right ventricle; (v) common ventricular chamber. Bars: panels a,b,g,h, 100 μm; panels c–f, 200 μm.