Characterization of the C. inflata Hand-like TVC enhancer. a Minimization of the C. inflata Hand-like (HL) upstream genomic fragment to test two predicted enhancers. LacZ reporter constructs are diagramed on the left. The graph depicts %TVC expression in C. robusta (number of trials ≥ 2, total N ≥ 75, and error bars indicate standard deviation). Significance relative to Coinfl.HL −1737 was determined with a Student t test (p < 0.001 indicated by ***). The second predicted enhancer is both necessary and sufficient for reporter expression in the TVCs. b–e Representative embryos showing the expression of LacZ reporter constructs that contain the second predicted enhancer (Coinfl.HL −1048) or lack the second predicted enhancer (Coinfl.HL −899) in both C. robusta and C. inflata (arrows indicate expression in TVCs, and scale bar is 50 μm). f Effect of Ets1/2 and ATTA-binding motif knockouts (Δ) on the expression of a C. inflata Hand-like::LacZ reporter construct containing a 1048 bp upstream genomic fragment (Coinfl.HL −1048). Names of binding motifs correspond to the names in panel B. LacZ reporter constructs are diagramed on the left with X indicating a binding motif knockout. The graph depicts %TVC expression in C. robusta (number of trials ≥ 2, total N ≥ 25, and error bars indicate standard deviation). Significance relative to Coinfl.HL −1048 was determined with a Student t test (p < 0.01 indicated by ** and p < .001 indicated by ***). g Comparison of Hand-like TVC enhancer structure in C. robusta and C. inflata. Darkly shaded binding motifs were required for reporter expression. Lightly shaded binding motifs exhibited ‘limited” functionality as assessed by mutagenesis of multiple sites in the minimal Cirobu.FoxF enhancer  or by a non-significant reduction in reporter expression following mutagenesis (this study). Boxed binding motifs exhibited no functionality. C. robusta Hand-like binding motif knockout data comes from Woznica et al.