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. 2019 Nov 20;8:e49921. doi: 10.7554/eLife.49921

Figure 6. Multiple copies of a weak Ebf enhancer drive ectopic reporter gene expression.

Figure 6.

(A) Schematic representation of cardiopharyngeal lineage cells at Stage 27 (Hotta et al., 2007); First heart precursors (FHPs, red and open arrowheads), second heart precursors (SHPs, orange and arrows), inner ASM precursors and derivatives (iASMPs, violet and solid arrowhead), outer ASM precursors and derivatives (oASMPs, dark blue and solid arrowhead) (see (Razy-Krajka et al., 2014); black bars link sister cells. (B) Lineage tracing in individual larvae expressing single (1x) and multiple copy (3x) of Ebf cis-regulatory element, KhL24.37, driving H2B::mCherry (red) in cardiopharynegal progenitors at stage 27; B7.5 lineage is marked with Mesp>H2B::GFP (green). The single copy of KhL24.37 element drives H2B::mCherry reporter expression specifically in the ASMPs (upper left panel, in white); three copies of KhL24.37 (3x L24.37) drives expression in ASMPs and induces ectopic expression SHPs (lower left panel, in white). Experiment performed in biological replicate. Scale bar = 30 µm. (C) Proportions of embryos expressing H2B::mCherry in indicated cell-type progenitors by the indicated cis-regulaory elements. The ‘full length’ upstream region encompassing all combined enhancers with endogenous spacing (Ebf-full length −3348 /- 178) (Wang et al., 2013) as well as the concatemer of KhL24.37, .36, .35, and .34 elements, lacking endogenous spacer sequences were used as controls. Statistical analysis using a Fisher exact test showed all comparisons with either control to be significant (p < 0.01); ‘n’ is the total number of individual halves scored per condition. (D) Schematic representation of cardiopharyngeal lineage cells at Stage 24 (Hotta et al., 2007); First heart precursors (FHPs, red and open arrowheads), secondary TVC (arrows). (E) Expression of GFP visualized by in situ hybridization on embryos at Stage 24 electroporated with single, multi copies and full-length of Ebf cis-regulatory element. Nuclei of B7.5 lineage cells are labelled by Mesp>nls::LacZ and revealed with an anti beta-galactosidase antibody (red). Mesp-driven hCD4::mCherry accumulates at the cell membrane as revealed by anti mCherry antibody (Blue). Scale bar = 10 µm. (F) Proportions of embryos showing GFP-driven by the indicated Ebf cis-regulatory element (Fisher exact test, p < 0.01).