Fig. 5

Characterization of the C. inflata Mesp founder lineage enhancer. a Minimization of the C. inflata Mesp 5′ intergenic region to identify the B7.5 founder lineage enhancer. LacZ reporter constructs are diagramed on the left. The graphs depict % founder lineage (TVC + ATM) expression or % founder lineage + primary tail muscle lineage expression in C. robusta and C. inflata (number of trials ≥ 2, total N ≥ 25, and error bars indicate standard deviation). Significance relative to Coinfl.Mesp −651 was determined with a Student t test (p < 0.01 indicated by ** and p < 0.001 indicated by ***). b Representative C. robusta embryo showing founder lineage-specific expression of Coinfl.Mesp −866 (arrows indicate TVCs and ATMs, and scale bar is 50 μm). c Representative C. robusta embryo showing the founder lineage and primary tail muscle lineage expression for Coinfl.Mesp −576. d Representative C. robusta embryo showing the lack of founder lineage expression for Coinfl.Mesp −421. e Representative C. inflata embryo showing the founder lineage-specific expression for Coinfl.Mesp −866. f Effect of TBX6 and LHX3 binding motif knockouts (Δ) on the expression of the C. inflata Mesp founder cell enhancer. Binding motifs designated as shown in g. LacZ reporter constructs are diagramed on the left with an X indicating a binding motif knockout. The graphs depict % founder lineage expression in C. robusta and C. inflata (number of trials ≥ 2, total N ≥ 75, and error bars indicate standard deviation). Significance relative to Coinfl.Mesp −576 or the minimal −576/−421 enhancer was determined with a Student t test (p < 0.05 indicated by * and p < 0.01 indicated by **). g Structure of the C. inflata Mesp founder cell enhancer. Darkly shaded green TBX6 motifs were required for reporter expression, and lightly shaded pink LHX binding motifs exhibited some functionality, as determined by mutagenesis of multiple motifs. There is no conservation of functional binding motifs in the aligned upstream genomic region of C. robusta. h Representative C. robusta embryo showing the founder lineage and primary tail muscle lineage expression for Coinfl.Mesp −576/−421::−138. i–k Representative C. robusta embryos showing lack of reporter expression for i Coinfl.Mesp ΔT1, and j Coinfl.Mesp ΔL1,2,3,4 and k reporter expression in the primary tail muscle lineage, but not the founder lineage for Coinfl.Mesp ΔL4,5,6