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

Figure 7. Multimer of one weak Ebf enhancer drives ectopic pharyngeal muscle fate specification.

Figure 7.

(A) Targeted expression of an Ebf cDNA by three copies of KhL24.37 element induces expression of endogenous Ebf in four cells compared to the control where Ebf is detected only in the two ASMPs. Proportions of embryos expressing Ebf in ASMPs only or in ASMPs and SHPs in the indicated conditions. The single KhL24.37 cis-regulatory was used as control. (B) Expression of Ebf visualized by in situ hybridization on embryos at Stage 26 electroporated with single or three copies KhL24.37 element driving Ebf cDNA. Intron-specific probes show nascent Ebf transcripts in ASMPs. 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. (C) Proportions of embryos showing GFP-driven STVC-specific enhancer of Tbx1/10 (Fisher exact test, p < 0.01). The ‘full length’ upstream element encompassing all combined enhancers with endogenous spacing (Ebf-full length −3348 / -178) as well as the ‘full length’ without endogenous spacing (KhL24.37, .36, .35, .34) and the single copy KhL24.37 element driving Ebf were used as a positive controls. (D) Example of an embryo at 28 hpf showing GFP expression only in the ASMP (solid arrowhead) and SHP (arrow) but not in the FHP (open arrowheads), where Tbx1/10 enhancer is not active. Targeted expression of Ebf cDNA by three copies of KhL24.37 element induces cells that normally form the second heart lineage to migrate alongside the pharyngeal muscle. Nuclei of B7.5 lineage cells are labelled by Mesp>H2B::mCherry. Scale bar = 15 µm. (E) Proposed role of combinatorial logics in fostering both precision and spatial and temporal accuracy of cell fate choices. The Ebf-full length, with or without endogenous spacers, fosters spatial/temporal accurate and precise cell fate choice, whereas the single copy of KhL24.37 (1x L24.37) gives spatiotemporally accurate reporter expression but is likely insufficient to induce a precise ASM fate. Multiple copies of KhL24.37 cis-regulatory element rescue a high reporter activity that reflects precise pharyngeal fate at the expense of spatial and temporal accuracy. The shapes of the distinct cis-regulatory elements are as in Figure 5C. Statistical analysis using a Fisher exact test (p < 0.01); ‘n’ is the total number of individual halves scored per condition.