FIGURE SUMMARY
Title

Tnni1b-ECR183-d2, an 87 bp cardiac enhancer of zebrafish

Authors
Zhang, Y., Wang, F., Wu, F., Wang, Y., Wang, X., Gui, Y., Li, Q.
Source
Full text @ Peer J.

Diagrams of enhancer activity detection constructs in this study.

(A) Enhancer activity detection construct used for microinjection. (B) Enhancer activity detection construct used for the luciferase reporter assay.

Comparative analysis of the genomic loci of <italic>tnni1b</italic>.

(A) ECR Browser and parameter set between zebrafish and human genomes around the 219 kb genome range encompassing the tnni1b gene and 100 kb regions upstream and downstream of tnni1b. Genes are shown in blue, and the 16 ECRs are shown in the red segment. (B) Overlapping alignment block: sequences of hg19-chr1:201687497-201687890 and zv9-chr6:54883657-54884033.

GFP expression after the injection of zebrafish enhancers.

Lateral views of zebrafish embryos with heart-specific GFP expression after injection with tol2 mRNA and tnni1b-ECR183 (A–C) or tnni1b-ECR183-d2 (D–F), scale bars = 100 µm. The average ratios of heart-specific GFP expression embryos to the total surviving embryos were 10/45 (22%) at 24 hpf, 15/41 (37%) at 48 hpf, and 15/39 (38%) at 72 hpf in the tnni1b-ECR183 group and 7/59 (12%) at 24 hpf, 18/48 (38%) at 48 hpf and 16/43 (37%) at 72 hpf in the tnni1b-ECR183-d2 group. (G) Rate of embryos with GFP expression in different tissues at 48 hpf after injection with tnni1b-ECR183. (H) Rate of embryos with heart-specific GFP expression from transient injections of tnni1b-ECR183 and each truncated enhancer with tol2 transposase at 48 hpf. A t-test was used for statistical analyses between the two groups (G). One-way ANOVA was used to test for homogeneity of variance, and Bonferroni’s test was used to correct for multiple comparisons (H). ** p < 0.01; *** p < 0.001; **** p < 0.0001, n = 3.

Putative TFBS analysis of tnni1b-ECR183 by PROMO and the specific positions of truncated enhancer fragments including tnni1b-ECR183-d1, tnni1b-ECR183-d2, and tnni1b-ECR183-d3.

TFBSs are displayed in fragments of different colors and numbers. The area division of the triangle represents the distribution density of TFBSs.

The heart-specific GFP expression in the stable transgenic zebrafish line <italic>Tg</italic> (tnni1b-ECR183-d2: GFP).

(A–E) Lateral views of zebrafish embryos with heart-specific GFP expression at 24 hpf, 48 hpf, 72 hpf, 96 hpf and 5 dpf. (F) Ventral view of zebrafish embryo with heart-specific GFP expression at 10 dpf. Scale bars = 100 µm.

Heart-specific GFP expression in zebrafish embryos of <italic>Tg</italic> (tnni1b-ECR183-d2: GFP; myl7: mCherry).

(A–F) Lateral views of zebrafish embryos with GFP expression driven by tnni1b-ECR183-d2 (A–C) and those with mCherry expression driven by the promoter of myl7 (D–F). (G–L) Confocal images of ventral views of these embryos with GFP expressed in green (G, J), mCherry expressed in red (H, K) and the overlapping part expressed in yellow (I, L). White line, heart outline; A, atrium; V, ventricle. Scale bars = 100 µm.

Identification of the enhancer activity and analysis of putative TFBSs.

Enhancer activity identification of tnni1b-ECR183 and tnni1b-ECR183-d2 by the luciferase assay in the HEK293 (A) and HL1 cell lines (B). (C) Putative TFBS positions and mutations. Analysis of the enhancer activity of tnni1b-ECR183-d2 after putative TFs were overexpressed (D) or after NKX2.5 (E) and JUN (F) binding sites were mutated. One-way ANOVA was used to test for homogeneity of variance, and Bonferroni’s test was used to correct for multiple comparisons (A, B, D). A t-test was performed for statistical analyses between two groups (E, F). * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001, n = 3.

Functional analysis of human enhancers.

(A–F) Lateral views of zebrafish embryos with GFP expression after injection with tol2 mRNA and tnni1b-ECR183-h179. (G–I) Lateral views of zebrafish embryos with GFP expression after injection with tol2 mRNA and tnni1b-ECR183-h84. The arrowheads indicate GFP expression in the skeletal muscles (A, B, D, F, G, H, I), craniofacial muscles (B, C, D, F) and hearts (E, F), scale bars = 100 µm. Rate of embryos with GFP expression in different tissues after injection with tnni1b-ECR183-h179 (J). Enhancer activity identification of tnni1b-ECR183-h179 and tnni1b-ECR183-h84 in HEK293 cell lines (K). Analysis of the enhancer activity of tnni1b-ECR183-h179 (L) and tnni1b-ECR183-h84 (M) after putative TFs were overexpressed. One-way ANOVA was used to test for homogeneity of variance, and Bonferroni’s test was used to correct for multiple comparisons. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001, n = 3.

Acknowledgments
This image is the copyrighted work of the attributed author or publisher, and ZFIN has permission only to display this image to its users. Additional permissions should be obtained from the applicable author or publisher of the image. Full text @ Peer J.