FIGURE SUMMARY
Title

Decoding an Organ Regeneration Switch by Dissecting Cardiac Regeneration Enhancers

Authors
Begeman, I.J., Shin, K., Osorio-Méndez, D., Kurth, A., Lee, N., Chamberlain, T.J., Pelegri, F.J., Kang, J.
Source
Full text @ Development

Regeneration-dependent activity of cardiac LEN (cLEN). (A) Schematic of lepb regeneration enhancer (LEN) containing two tissue-specific regeneration modules. A transgene construct examined for cLEN activity consists of 317 bp of cLEN, lepb 2 kb upstream promoter (P2) and EGFP reporter sequences. (B) Schematic of cLEN annotated with predicted TF binding sites. (C-F) Zebrafish strain and experimental strategy undertaken to ablate CMs (C) and images of uninjured and ablated hearts in larvae (D), juveniles (E) and adults (F). (G-I) Dynamic cLEN activity during zebrafish heart regeneration. (G) Experimental design to determine cLEN activity during heart regeneration. (H) Quantification of EGFP expression intensity normalized to images of wild-type uninjured hearts at the wound area. The data are presented as the mean±s.d. Numbers of animals are shown in Table S3. *P<0.05; **P<0.01; N.S., not significant. One-way ANOVA with Tukey's post-hoc test. (I) Images of cardiac sections of uninjured and regenerating hearts. Scale bars: 100 µm in E″,F″,I; 50 µm in D′.

Molecular dissection of cLEN to identify the fragment containing injury-responsive elements. (A) Transgene constructs examined for regeneration-dependent expression in response to cardiac injury. Endocardial expression results are summarized on the right. EC, endocardial cell. Uninj. and Reg. correspond to uninjured and 3 dpa regenerating hearts, respectively. (B) Images of sections of 3 dpa hearts from transgenic fish carrying cLEN fragments. Numbers of animals are shown in Table S3. The arrows indicate endocardial EGFP induction. Note that the P2 promoter directs basal expression in CMs in response to injury (see Fig. S3; Kang et al., 2016). MHC, myosin heavy chain, a CM marker. (C) Transgenic constructs used to examine enhancer activity in the F0 mosaic injured hearts. Two AP-1 binding sites were mutated in cLENAP-1m (black circles). AFNEGx3 is a synthetic cardiac regeneration enhancer consisting of three tandem copies of the AP-1-FOX-NFAT-ETS-GATA motifs. (D) Schematic of enhancer assays in the F0 mosaic hearts. (E) Images of injured hearts of larvae injected with P2, cLEN, cLENAP-1m and AFNEGx3. Note that larvae carrying transgenic constructs were selected by mCherry expression in the lens, without a cardiac EGFP signal. The arrows indicate injury-responsive EGFP induction in the injured hearts. Numbers at the bottom corners indicate the total ratio of embryos showing the corresponding expression pattern. Scale bars: 100 µm in B; 50 µm in E.

Active repression of cLEN establishes spatiotemporal gene expression in uninjured and regenerating hearts. (A) Transgene constructs examined for reporter gene expression. Endocardial expression results are summarized on the right. EC, endocardial cell. Uninj. and Reg. correspond to uninjured and 3 dpa regenerating hearts, respectively. (B) Images of sections of transgenic fish carrying cLEN and cLENΔ47. Top, uninjured hearts. Middle, remote zone of 3 dpa hearts. Bottom, border zone of 3 dpa hearts. The arrows indicate endocardial EGFP expression. (C-E) Top, schematic of uninjured and 3 dpa hearts. Area of quantification is marked in blue. Bottom, quantification of EGFP+ area per 100 µm2 cardiac tissue in uninjured hearts (C), border zone (D) and remote zone (E) of 3 dpa hearts. Data are presented as the mean±s.d. Numbers of animals are shown in Table S3. *P<0.05, one-way ANOVA with Tukey's post-hoc test. Scale bar: 100 µm in B.

Evidence of the presence of a repressive element within a 22 bp cLEN sequence. (A) Transgene constructs examined for reporter gene expression. Endocardial expression results are summarized on the right. EC, endocardial cell. Uninj. and Reg. correspond to uninjured and 3 dpa regenerating hearts, respectively. (B) Images of sections of transgenic fish carrying cLEN deletion transgenes. Top, uninjured hearts. Middle, the remote zone of 3 dpa hearts. Bottom, the border zone of 3 dpa hearts. The arrows indicate endocardial EGFP expression. (C) Quantification of EGFP+ area per 100 µm2 cardiac tissue in uninjured hearts. (D) Quantification of EGFP+ area per 100 µm2 cardiac tissue in the border zone of 3 dpa hearts. (E) Quantification of EGFP+ area per 100 µm2 cardiac tissue in the remote zone of 3 dpa hearts. The data are presented as the mean±s.d. Numbers of animals are shown in Table S3. *P<0.05; **P<0.01; ***P<0.001; N.S., not significant. One-way ANOVA with Tukey's post-hoc test. Scale bar: 100 µm in B.

The cLEN repressive element, but not a neighboring sequence, is present in other Danio species. (A) Alignment of orthologous sequences of cLEN11-1 to cLEN11-5 in Danio species, including Danio rerio, Danio aesculapii, Danio kyathit and Danio albolineatus. cLEN-22 contains 6 or 9 bp insertions in non-zebrafish sequences. Asterisks indicate conserved base pairs. The conserved GTCA sequence overlapping cLEN-11-1 and cLEN-11-2 is marked in red. Dendrogram indicates phylogenetic relationship. (B) Images of cardiac sections of cLENins6bp:EGFP transgenic fish harboring a 6 bp insertion (TGTGTA). (C) Images of cardiac sections of cLENmgtca:EGFP transgenic fish harboring the mutation of GTCA to CATT. The arrows indicate endocardial EGFP expression. Numbers of animals are shown in Table S3. (D) RT-qPCR analysis of lepb in uninjured and 3 dpa hearts of D. aesculapii. (E) RT-PCR of samples from the uninjured and 3 dpa hearts of D. kyathit. actb2 was used as the loading control. Uninj., uninjured hearts. (F) RT-qPCR analysis of lepb in the uninjured hearts of zebrafish, D. aesculapii, D. kyathit and D. albolineatus. The data are presented as mean±s.d., n=3; lepb transcript levels were normalized to actb2 levels in D,F. *P<0.05; N.S., not significant. Student's unpaired, two-tailed t-test. Scale bars: 100 µm in B,C.

Developmental emergence of the repression mechanism. Images of sections of the developing hearts of cLEN (A-D) and cLENΔ47 (E-H) reporter fish. Numbers of animals are shown in Table S3. The arrows indicate endocardial EGFP expression. Scale bars: 20 µm in A,E; 50 µm in D,H.

Proposed model of regulation of the activity of cardiac TREEs in uninjured and regenerating hearts. In uninjured embryonic hearts, cardiac TREEs are inactive; they are activated upon injury. Repression of cardiac TREEs is not functional in the heart in early development. During maturation, cardiac TREEs are actively repressed to prevent aberrant activation in uninjured tissues. Upon injury, the dual function of distinct cis-regulatory elements restricts cardiac TREE activation to the wounded area.

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 @ Development