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Figure 3
Constructs carrying well-characterised CREs from the PAX6 locus (PAX6-7CE3, hindbrain enhancer, and PAX6-SIMO, lens enhancer). (A) Confocal images of 96hpf F1 embryos derived from founder lines injected with the cassettes indicated above each image panel. Top two panels show dye-swap experiment (eGFP and mCherry reporters swapped between the two CREs) with a neutral sequence (–, no insulator activity) between the two CRE-reporter cassettes. eGFP and mCherry expression is observed in both lens and hindbrain indicating complete crosstalk between 7CE3 and SIMO CREs. Bottom panel: inclusion of three copies of the well-characterised chicken β-globin 5′HS4 (3XcHS4) insulator restricts the activities of each enhancer to their respective specific domains. Scale bars = 100 μm. (B) Average of mean fluorescence intensities ratios (G/C: eGFP/mCherry; C/G: mCherry/eGFP) in the lens and hindbrain at 72 and 120 hr post fertilisation (hpf) in F1 embryos derived from founders bearing constructs without (–) or with 1×, 2× or 3× insulator sequences. Each bar indicates average of ratios of mean fluorescence intensities from at least five independent images of embryos bearing the assay construct indicated (n ≥ 5, error bars indicate standard deviation). A highly significant difference in fluorescence intensity ratios (computed by two-tailed Student’s t-test) was observed between embryos at the same stage of development harbouring constructs with no insulator (–) and those with three copies of the insulator (3xI). This demonstrates that fluorescence is progressively restricted to the tissue where the associated CRE is active as the number of copies of the insulator increases. Raw data used for plotting the graphs are provided in Figure 3—source data 1. L, Lens; H, hindbrain, ****p<0.0001, ***p<0.001, **p<0.01.
Quantitative assessment of tissue-specific enhancer activity and effect of insulation on crosstalk between cis-regulatory elements (CREs) in dual-CRE dual-reporter constructs.