DEV01344 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. Genomic and transcriptional start site mapping and preliminary transgenic analysis. A. Primer extension and S1 nuclease digestion products obtained using chick H+H stage 36 skeletal muscle (skel m.) or heart polyA+ RNA are shown to the left beside sequence gel primed with primer extension oligonucleotide (nt +242-219). These reveal a strong common start site at –163 from the ATG translational start site for Nkx-2.5 along with several minor downstream initiation sites. Transcriptional start site (arrowhead) and genomic sequence immediately surrounding the initiation site (boxed) are shown vertically at immediate right of the sequence gel, and in the context of 500 bp of 5¢ flanking region upstream of the translational start codon shaded in gray at lower right of the genomic sequence. B. Schematic map of an approximately 27 kb region of chick Nkx-2.5 representative of 20 overlapping EMBL4 Gallus gallus genomic clones (Clontech) obtained by screening with full length cNkx-2.5 cDNA sequences (Schultheiss et al., 1995). Chick activating regions or CARs are shown as colored bars or ovals, reflecting the extent of their characterization (see subsequent figures). Transcriptional start site is shown as arrowhead, and exons represented as black boxes. Coding regions and exon are shown amplified above genomic map; promoter proximal region shown in A is represented as yellow oval. Homeodomain/NK region is boxed in grey in exon 2, with approximate intron/exon lengths shown above in kb. C. Schematic representations of hsp68-lacZ reporter constructs used in preliminary transgenic screening are shown below a map of their corresponding cNkx-2.5 genomic sequences in B. Arrows on reporter constructs indicate both endogenous Nkx-2.5 (CAR2) and heterologous hsp68 promoters. D through K: Representative whole-mount x-gal stained transgenic embryos bearing transient CAR1 (D,E) or stable CAR2 (F-H) and CAR3 (I-K) hsp reporter genes, harvested at embryonic stages indicated at lower left. Shown are both whole embryos (D-G, I, J) and close-ups of E10.5 hearts (H, k). Results for CAR2 and CAR3 are representative of 2/3 stable lines; results for CAR1 are representative of 4/4 E7.5 and 4/11 E10.5 transient transgenic embryos. B: BamH1; H: HincII; K: KpnI; BA: branchial arches; cc: cardiac crescent; Ht: heart; OFT: outflow tract; LV: left ventricle; RV: right ventricle; LA: left atrium; RA: right atrium; St: stomach.

• Supplemental Figure 2 -

  Fig. S2. CAR analysis with endogenous chick Nkx-2.5 promoter. A. Map of cNkx-2.5 genomic regions and CAR enhancers, shown as in Fig. 1. Gray overlines indicate genomic regions that failed to drive transgenic expression in the heart (data not shown). B. Constructs combining various CAR enhancers constructed using a minimal Nkx2.5-lacZ promoter reporter (Construct N1) contains lacZ coding regions fused in frame with the N-terminal cNkx-2.5 coding region, and approximately 2kb of 5¢ flanking sequence (nt –2046 to +1; yellow bar) excluding a conserved enhancer in CAR2 (red oval). Construct N1 shows no lacZ expression at E 7.5 and E 10.5 (data not shown). Construct N2 contains 2.4 kb of 5¢ flanking region (orange bar) that includes the conserved CAR2 enhancer (red oval) (Liberatore et al., 2002) as well as the endogenous cNkx-2.5 promoter. Construct N3 includes the 2kb CAR3 (nt +976 to +3047; green bar) from the 3¢ flanking region fused to the minimal cNkx-2.5 promoter (N1). Construct N4 combines both CAR2 and CAR3 with the endogenous promoter in their endogenous positions and orientations. Construct N5 fuses a 6kb region of distal 5¢ flanking region containing the partially characterized CAR1 to the Nkx2.5-lacZ (N1) construct. Construct N6 combines CARs 1, 2 and 3 in abbreviated fashion as shown, in their endogenous orientations and positions relative to the transcriptional start site. c-O) Representative lacZ expression patterns from Nkx2.5-lacZ transgenes. c-e: N2; f-h: N3; i,j: N4; k,l: N5; m-O: N6. Embryonic stages are shown at bottom left of each photo. Whole embryos (c, d, f, g, I, k-m) and close-ups of E10.5 hearts (e, h, j, n) are shown. Sectioned heart shown in O is from embryo diplayed in N. Ratio of transient embryos (N1-3, 5,6) or stable lines (N4) displaying these expression patterns are as follow: N1: 10/10 transients; N2: 7/8 transients; N3: 7/9 transients; N4: 6/8 transients and 2/3 stable lines; N5: 6/6 transients; N6: 7/9 transients. Abbreviations: AVV: atrioventricular valve; En: endocardium; Ep: epicardium; PC: pericardium, and as in Supplementary Fig. 1.