Fig. 3

Characterization of the C. inflata FoxF TVC enhancer. a mVISTA alignments depict sequence conservation between C. robusta and C. savignyi and between C. robusta and C. inflata for the FoxF gene and 5′ intergenic region (LAGAN alignment, conservation across 100 bp window, conservation > 70% highlighted). There is increased conservation associated with the FoxF coding region (orange) and conserved TVC enhancer (purple). b ClustalW alignment of the 183 bp conserved TVC enhancer with Ets1/2 (red), ATTA (blue), and TGTT (orange)-binding motifs highlighted. Dark-shaded-binding motifs were required for reporter expression and boxed-binding motifs exhibited no functionality. C. robusta FoxF-binding motif knockout data come from Beh et al. and Woznica et al. [41, 58]. c–e Representative embryos showing the activity of Coinfl.FoxF −2622 GFP reporter constructs in C. inflata and C. robusta (arrows indicate expression in TVCs, and scale bar is 50 μm). f Representative C. inflata mid-tailbud stage embryo displaying expression of Coinf.FoxF in TVCs (arrow) and epidermis. g Effect of Ets1/2 and ATTA-binding motif knockouts (Δ) on reporter expression driven by the C. inflata 146 bp minimal TVC enhancer fused to a 255 bp basal promoter (Coinfl.FoxF −547/−401::−255). Names of binding motifs correspond to the names in b. LacZ reporter constructs are diagramed on the left with X indicating a binding motif knockout. The graph depicts %TVC expression in C. inflata (number of trials ≥ 2, total N ≥ 150, and error bars indicate standard deviation). Significance relative to Coinfl.FoxF −547/−401::−255 was determined with a Student’s t test, p < 0.05 indicated by *