Figure 3. Cis-regulatory analysis of Hand-like.

(A) Vista alignment depicts sequence conservation between C. intestinalis and C. savignyi in the 5′ intergenic region (Couronne et al., 2003). (B) Diagram of Hand like reporter constructs aligned with (A) and numbered by their distance from the translation start codon. For each construct, relative reporter expression in the TVCs and other lineages is indicated to the right (++++ = nearly all transgenic embryos display denoted reporter staining; +++ = >50% of stained embryos; + = <5% of stained embryos; 0 = no expression). (C–D) Representative Stage 22-Hand-like 2995:+1:lacZ and Hand-like 1100:+1:lacZ embryos, arrows indicate reporter expression in TVCs. (E) Alignment of the highly conserved 179bp distal element, C. int. = Ciona intestinalis, C. sav. = Ciona savignyi, red boxes highlight two conserved Ets1/2 binding sites. (F) Representative embryo showing TVC reporter expression (arrows) driven by the 179bp distal element fused to the Hand-like 296bp basal promoter. (G) Diagram of point mutations, indicated by an X (sequence provided below), introduced into putative Ets1/2 binding sites (red boxes) in the Hand-like -1972-1793:296:lacZ reporter construct (error bars indicate standard deviation, three trials, N>600 for each sample). (H) Representative embryo displaying loss of reporter activity when the two Ets binding motifs are mutated (E1/E2-mut, as diagrammed in G).