Figure 5. Combinations of cis-regulatory elements with distinct chromatin accessibility profiles are required for Ebf transcription in pharyngeal-muscle precursors.
(A) A 12 kb region of the scaffold L24 displaying expression profiles of RNA-seq and chromatin accessibility profiles of ATAC-seq (normalized tag count) in the Ebf locus. sgRNAs used to target ATAC-seq peaks are shown in red; intronic antisense riboprobes are shown in orange (B) Schematic representation showing sequential opening of cis-regulatory elements required for Ebf activation in pharyngeal muscle founder cells, and maintenance by auto-regulation in committed precursor. (C) Schematic representation of Ebf cis-regulatory elements targeted for CRISPR/Cas9-mediated deletions. Shapes represent binding sites located in the regulatory elements and differentially accessible over time. (D) Proportions of larva halves showing the indicated Ebf transcription patterns, in indicated experimental conditions; all the treatments were significant versus Tyrosinase (Fisher exact test, p < 0.001). (E) Endogenous expression of Ebf visualized by in situ (green) in TyrosinaseCRISPR and upon CRISPR/Cas9-induced deletion of ATAC-seq peaks as indicated, at stage 25 (E) and 27 (F) based on Hotta et al. (2007). For stage 25, an anti-sense riboprobe for the full length cDNA was used, whereas for stage 27 an intronic anti-sense riboprobe targeting the first three introns of Ebf transcript (orange lines) as previously used in Wang et al. (2013). 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.
Figure 5—figure supplement 1. Combinations of cis-regulatory elements with distinct chromatin accessibility profiles are required for Tbx1/10 transcription in pharyngeal-muscle precursors.
(A) An 11 kb region on chromosome seven displaying expression profiles of RNA-seq and chromatin accessibility profiles ATAC-seq normalized tag count in Tbx1/10 locus. (B) STVC-specific enhancer (T12) (Razy-Krajka et al., 2018) driven in vivo reporter expression (green) in ASMFs and SHPs at stage 26 (Hotta et al., 2007). Nuclei of B7.5 lineage cells are labelled by Mesp>H2B::mCherry (red). Scale bar, 20 μm. T12 enhancer tested alone or fused to the intronic element (T12+KhC7.914). Statistical significance of the difference in reporter expression was tested using a Fisher exact test (p < 0.001) (C) Motif scores in each experimentally validated peak in the Tbx1/10 locus. Only the highest match score is shown for each motif. (D) Sequence alignment of Tbx1/10 enhancer (T12) between Ciona robusta/Ciona savignyi. Conserved blocks in the orange boxes with putative Forkhead binding sites. In blue is highlighted the single guide RNA (sgRNA#2) used to target CRISPR/Cas9 system, with the PAM domain in red; the point mutations induced in two conserved putative Forkhead binding sites (Fox1 and Fox2) are in bold and red after the asterisks. (E) Proportion of larvae expressing both GFP and mCherry in the STVC progeny when co-electroporated wild-type and mutant Tbx1/10 reporters lacking the indicated putative Forkhead binding sites and Mesp>H2B::mCherry in comparison to the control. (F) Proportions of larvae halves showing GFP expressed in the ASMFs and SHPs in embryos electroporated with Mesp>Cas9 along with single guide RNAs targeting Tyrosinase (controlCRISPR) as well as Foxf (FoxfCRISPR). (G) Schematic representation of regulatory elements in Tbx1/10 locus as displayed in ATAC-seq profiles targeted for CRISPR/Cas9-mediated deletions. (H) Proportions of larvae halves showing the indicated Tbx1/10 transcription patterns, in indicated experimental conditions (Fisher exact test, p < 0.001). (I) Endogenous expression of Tbx1/10 visualized by in situ (green) in TyrosinaseCRISPR (left panel) and upon CRISPR/Cas9-induced deletion of TVC-specific peaks (right panel) at stage 25 according to Hotta et al. (2007). Nuclei of B7.5 lineage cells are labelled by Mesp>nls::LacZ and revealed with an anti beta-galactosidase antibody (red). Nuclei of B7.5 lineage cells are labelled by Mesp>nls::LacZ and revealed with an anti beta-galactosidase antibody (red). Scale bar = 10 µm. Experiment performed in biological replicates. Total numbers of individual halves scored per condition are shown in 'n='. All the treatments were significant versus Control (Tyrosinase) (Fisher exact test, p < 0.001).
Figure 5—figure supplement 2. Ebf regulatory regions showing differentially accessibility over time contain distinct binding motifs.
(A) Motif scores in each experimentally validated peak in the Ebf locus. Only the highest match score is shown for each motif. (B) Endogenous expression of Ebf visualized by in situ (green) in TyrosinaseCRISPR and upon CRISPR/Cas9-induced deletions of TVC-specific peaks at stage 24 according to Hotta et al. (2007). Nuclei of B7.5 lineage cells are labelled by Mesp>nls::LacZ and revealed with an anti beta-galactosidase antibody (red). Nuclei of B7.5 lineage cells are labelled by Mesp>nls::LacZ and revealed with an anti beta-galactosidase antibody (red). Scale bar = 10 µm. (C) Proportions of larvae halves showing the indicated Ebf transcription patterns, in indicated experimental conditions. Experiment performed in biological replicates. All the treatments were significant versus Control (Tyrosinase) (Fisher exact test, p < 0.001).
Figure 5—figure supplement 3. CRISPR/Cas9-mediated deletions on individual accessible elements upstream of Ebf caused phenotypic impact on pharyngeal muscle precursors morphogenesis.
(A) Schematic representation of Ebf cis-regulatory elements targeted for CRISPR/Cas9-mediated deletions; the shapes of the distinct cis-regulatory elements are as in Figure 5C. (B) Proportions of larva halves showing GFP-driven STVC-specific enhancer of Tbx1/10 in indicated experimental conditions; all the treatments were significant versus Tyrosinase (Fisher exact test, p < 0.001; ‘n’ is the total number of individual halves scored per condition.). (C) Example of an embryo at 28 hpf showing GFP expression only in the ASM (solid arrowhead) and SHP (arrow) but not in the FHP (open arrowheads), where Tbx1/10 enhancer is not active (TyrosinaseCRISPR, first panel on the left). Targeted deletions in KhL24.37 peak induced ASMP cell migration defects. B7.5 lineage cells are labelled by Mesp>LacZ. Scale bar = 10 µm.
Figure 5—figure supplement 4. Intronic and distal enhancer accessibility in the Tbx1/10 locus tested by dCas9-KRAB.
(A) ASM-specific enhancer of Ebf (Ebf-3348/–178) (Wang et al., 2013) driven in vivo reporter expression (green) in embryos at stage 27 (Hotta et al., 2007) electroporated with Mesp>dCas9 KRAB along with single guide RNAs targeting Tyrosinase (control, left panel) as well as intronic (KhC7.914) and distal (KhC7.909) of Tbx1/10 locus (right panel) as in Figure 5—figure supplement 1G. Nuclei of B7.5 lineage cells are labelled by Mesp>H2B::mCherry (red). White asterisk indicated central nervous system (CNS). Scale bar, 50 μm. (B) Proportions of larvae halves showing GFP expressed in the ASMFs in indicated experimental conditions (Fisher exact test, p < 0.001). (C) Double in situ hybridization of Ebf (green) and Tbx1/10 (red) on embryos at stage 27 electroporated with Mesp>dCas9 KRAB along with single guide RNAs targeting Tyrosinase (control, left panel) as well as intronic (KhC7.914) and distal (KhC7.909) of Tbx1/10 locus. White asterisk indicated central nervous system (CNS). Scale bar, 20 μm. (D) Proportions of larvae halves expressing both Tbx1/10 and Ebf in the ASMFs in indicated experimental conditions. Experiment performed in biological replicates. Total numbers of individual halves scored per condition are shown in 'n='. All the treatments were significant versus Control (Tyrosinase) (Fisher exact test, p < 0.001).