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Fig. 1. BMP response and in vivo transgene expression maps to CAR3 enhancer. (A) Genomic flanking regions of chick Nkx2.5 containing CAR enhancers as shown were assayed for BMP response in P19 cells in the context of both hsp68-lacZ and Nkx2.5-luciferase reporters (Nkx2.5-lux). Only the CAR3 enhancer is BMP responsive. (B) Deletion mapping of BMP response and transgenic expression for 3 kb 3' genomic flanking region of chick Nkx2.5which contains CAR3, using hsp68-lacZ promoter/reporter. Nucleotide positions of 5' and 3' ends of deleted flanking regions are given relative to transcriptional start site of chick Nkx2.5. Extent of the genomic fragments yielding positive reporter lacZ expression in F0 transient transgenic mouse embryos are shown by black bars (C3-2, C3-4, C3-6, C3-8 and C3-10); negative reciprocal fragments are represented as gray bars (C3-1, C3-3, C3-5, C3-7 and C3-9). BMP response of these hsp-lacZ-CAR3 reporters is shown to immediate right of their representations, expressed as normalized fold induction by BMP. (C-L) Representative X-gal stained E7.5 F0 transient transgenic mice embryos injected with C3 reporters C3-1 to C3-10. (M) BMP response of 2 kb CAR3 (fragment C3-2) and 200 bp BMPRE (fragment C3-10) enhancers linked to Nkx2.5-lux reporters (Nkx2.5-lux-CAR3 and Nkx2.5-lux-BMPRE, respectively). Extents of CAR3 and BMPRE enhancers and nucleotide positions relative to transcriptional start site are shown as above. BMP response in P19 cell assay is shown to right as fold induction above basal. (N-S) Representative X-gal stained F0 transient transgenic mice embryos injected with Nkx2.5-lacZ-CAR3 (N-Q) and Nkx-lacZ-BMPRE (R,S). Embryonic stages are shown at bottom left of pictures. Frequency of transient or stable (C3-2) transgene expression in cardiac region (cardiac crescent): C3-1, 0/20 embryos; C3-2, 2/3 lines; C3-3, 0/18 embryos; C3-4, 4/5 embryos; C3-5, 1/19 embryos; C3-6, 1/1 embryos; C3-7, 1/12 embryos; C3-8, 12/12 embryos; C3-9, 0/5 embryos; C3-10, 12/16 embryos. CC, cardiac crescent; LS, lateral somite; OFT, outflow tract; AL, allantois; BA, branchial arches; St, stomach; RV, right ventricle; H, heart; LV, left ventricle.