The careg element reveals a common regulation of regeneration in the zebrafish myocardium and fin

Abstract

The existence of common mechanisms regulating organ regeneration is an intriguing concept. Here we report on a regulatory element that is transiently activated during heart and fin regeneration in zebrafish. This element contains a ctgfa upstream sequence, called careg, which is induced by TGFβ/Activin-β signalling in the peri-injury zone of the myocardium and the fin mesenchyme. In addition, this reporter demarcates a primordial cardiac layer and intraray osteoblasts. Using genetic fate mapping, we show the regenerative competence of careg-expressing cells. The analysis of the heart reveals that the primordial cardiac layer is incompletely restored after cryoinjury, whereas trabecular and cortical cardiomyocytes contribute to myocardial regrowth. In regenerating fins, the activated mesenchyme of the stump gives rise to the blastema. Our findings provide evidence of a common regenerative programme in cardiomyocytes and mesenchyme that opens the possibility to further explore conserved mechanisms of the cellular plasticity in diverse vertebrate organs.

Figure 1. A transgenic reporter of the peri-injured myocardium and the fin stump.

(a–i) Transversal ventricle sections of transgenic fish ctgfa^careg:EGFP immunostained for embCMHC (red) at different days post-cryoinjury (d.p.ci.). The cardiac muscle is detected by F-actin staining (Phalloidin, blue). (b,e,h) The injury-remote part of the ventricular wall displays a subcortical layer of ctgfa^careg:EGFP cells that is located between the thin cortical myocardium (CoM) and the inner trabecular myocardium (TrM). This layer does not express embCMHC and remains unaltered during regeneration. (c,f,i) The injury-abutting zone of the ventricular wall shows transient expression of ctgfa^careg:EGFP and embCMHC within a distance of 100 μm from the wound border during regeneration. N≥4. (j–o) Live-imaging of fins at different days post amputation (d.p.a.). Bright-field (BF) was combined with fluorescence. (k,m,o) Higher magnifications of the region at the amputation plane (yellow dashed line) show transient expression of ctgfa^careg:EGFP in the stump and the regenerate. N=4. (p–s) Immunofluorescence staining of longitudinal fin sections. (p,r) At 0 d.p.a., ctgfa^careg:EGFP⁺ cells are associated with Tenascin C fibres along the border between the bones (B) and mesenchymal cells (MES), but not the epidermis (EP). Some of these cells express an osteoblast marker visualized with Zns5 antibody (red). (q,s) At 3 d.p.a., Tenascin C demarcates the zone of tissue remodelling in the stump and the outgrowth. ctgfa^careg:EGFP is upregulated in the activated MES at the peri-injury zone, but not in Zns5-labelled osteoblasts of the outgrowth. N≥4. Post-infarcted ventricle is encircled with a dotted line. Fin amputation plane is shown with a dashed line. The same rules apply to all subsequent figures.

Figure 2. ctgfa^careg:EGFP does not reproduce endogenous ctgfa expression in peri-injury tissues.

(a–h) In situ hybridization using ctgfa and enhanced green fluorescent protein (egfp) probes on sections of hearts at 14 d.p.ci. and fins at 3 d.p.a. of ctgfa^careg:EGFP transgenic fish shows distinct expression patterns. (a,b) ctgfa mRNA is not detected in the myocardium, but in a subset of non-myocytes in the post-infarcted tissue. (c,d) egfp mRNA is expressed in the outer wall of the myocardium and at the peri-injury zone (yellow arrowheads). (e,f) ctgfa mRNA is present in a few bone-associated cells of the fin, while (g,h) egfp mRNA is detected in the blastema. N=6. (i–m) careg:EGFP and careg:dmKO2 double transgenic fish display an overlapping expression pattern in the regenerating heart at 7 d.p.ci. and the fin at 1 d.p.a. N=4.

Figure 3. Sequence analysis of the careg element.

(a) Comparison of the careg element and the ctgfa genomic sequence of wild-type AB strain zebrafish, which was used to generate transgenic fish. The careg element includes a 3,179 bp sequence immediately upstream of the transcriptional start site (TSS) and 10 bp of the 5′-untranslated region of ctgfa. (b) LASTz net pairwise alignment of the ctgfa genomic region between Zebrafish (Danio rerio, Ensembl version 87.10, Chromosome 20; 25′438′500 - 25′444′700) and 9 other fish species (highlighted in blue), Xenopus (yellow), chicken (green) and 2 mammals (orange). The pink boxes indicate the conserved regions in the majority of the species. Black and white bars within the pink boxes represent gaps in the alignments. The careg sequence is indicated with a green line above the alignment. The gap in the careg sequence corresponds to additional 465 bp in the sequence from the Ensembl database. This sequence is absent in the genome of AB zebrafish strain. The scale bar indicates the genomic position in the chromosome 20 of zebrafish. Two conserved regions of ∼400 and 150 bp are indicated with pink boxes at the bottom of the alignment. (c) Prediction of transcription factor (TF) binding sites in the careg sequence (green) with MatInspector (Genomatix). The pink boxes indicate the conserved regions identified in b. A SMAD3 binding site is highlighted in red. The complete list of TF binding sites is given in Supplementary Data 1.

Figure 4. The careg reporter is activated in other injury models of the zebrafish heart and fin.

(a,b) Ventricle section of careg:EGFP heart at 7 days post amputation (d.p.a.) labelled with F-actin (blue) and antibodies against GFP (green) and embCMHC (red). careg:EGFP expression is activated in the ventricular trabeculae close to the amputation plane of the resected apex (dashed line). N=3. (c–l) Live-imaging of the same fin at different time points post-cryoinjury. Cryoinjury of the caudal fin results in spontaneous sloughing of destroyed tissue within two days after the damage induction, followed by resumed regeneration. Bright-field was combined with fluorescence. (c,d) At 3 h.p.ci. (hours post-cryoinjury), careg:EGFP is not detected in the fin. Ischaemic tissue, which is distal to the cryoinjury plane (blue line), remains integrated with the rest of the body at this early stage. (e,f) At 1 d.p.ci., the majority of the damaged tissue detached from the stump. careg:EGFP is detected proximally to the damaged zone. (g,h) At 2 d.p.ci., the reparation of the distorted margin is accompanied by careg:EGFP expression. (i,j) At 4 d.p.ci., the protruding blastema displays enhanced expression of the careg reporter. (k,l) The advanced regeneration is associated with downregulation of careg:EGFP expression. N=3.

Figure 5. The regeneration biosensor careg is dependent on TGFβ/Activin-β signalling.

(a–c) Immunofluorescence staining of careg:EGFP ventricle at 7 d.p.ci. with antibodies against GFP (green), pSmad3 (red) and Tropomyosin (blue) revealed the presence of TGFβ/Activin-β-activated cells in the careg:EGFP-expressing tissue. N=6. (d–g) careg:EGFP heart sections at 7 d.p.ci. treated with 0.1% DMSO or 20 μM SB431542, an inhibitor of TGFβ type I receptors, and immunostained with antibodies against GFP (green) and embCMHC (red). The intact myocardium was detected with F-actin staining (blue). In the magnified images, the upper dotted line demarcates a 100 μm-thick margin of the remaining myocardium from the injury border (lower dotted line). (h) Percentage of careg:EGFP⁺ and embCMHC⁺ area within a distance of 100 μm from the post-infarcted tissue in hearts at 7 d.p.ci. treated with DMSO or SB431542. The inhibitor treatment resulted in a significant reduction of careg:EGFP and embCMHC expression in the peri-injured zone, compared to control hearts treated with DMSO. N=8. ***P<0.001; unpaired t-test. Error bars correspond to s.e. of the mean (s.e.m.). (i–n) Live-imaging of careg:EGFP fins at 3 d.p.a. in a wild-type (WT) background treated with DMSO or SB431542, and in the fgf20a (dob) mutant background. careg:EGFP expression is suppressed in the stump by TGFβ inhibition, but it remains normal in fgf20a mutant fins. (o–t) Longitudinal sections of the fins shown in the left panels display a reduction of pSmad3-positive nuclei (red) and careg:EGFP in the stump of SB431542-treated fins, whereas both markers are unaltered in fgf20a mutants. BV, blood vessel. N=4.

Figure 6. careg is expressed in embryonic CMs and the outer wall of developing ventricle.

(a–l) Longitudinal sections of careg:EGFP;cmlc2:DsRed2-Nuc double transgenic hearts at different time points during development. Cardiac nuclei are marked by DsRed expression. The endogenous fluorescence was quenched with HCl treatment before immunostaining. GFP and DsRed were detected by antibody staining. (a–f) At 1 and 3 d.p.f. (days post-fertilization), careg:EGFP and endogenous embCMHC are co-expressed in embryonic CMs. (g–i) At 12 d.p.f., a few careg:EGFP⁺ CMs delaminate from the outer heart surface and invade into the ventricle (V) chamber, as seen by the residual EGFP. (j–l) At 30 d.p.f., careg:EGFP is restricted to the outer layer of the ventricular wall, and is downregulated in the trabecular myocardium. careg:EGFP is also expressed in non-myocytes of the bulbus arteriosus (BA) and a few cells of the atrium (A). (m–o) Longitudinal sections of careg:dmKO2;cmlc2:EGFP transgenic heart at 30 d.p.f. immunostained for GFP (green) and dmKO2 (red). The careg:dmKO2 transgenic line has a cardiac developmental expression pattern similar to that of careg:EGFP (Supplementary Fig. 10). N≥5.

Figure 7. Embryonic careg-positive CMs contribute to the trabecular myocardium.

(a) Schematic representation of the transgenic strains used for lineage tracing. (b) Experimental design. (c,d) Longitudinal sections of hearts at 21 d.p.f. immunostained against mCherry (red), GFP (green) and Tropomyosin (blue). (c) Control embryos treated with the vehicle do not display mCherry fluorescence. (d) mCherry⁺ CMs are present in the trabecular and outer myocardium at 21 d.p.f. in embryos treated with 4-OHT, suggesting that the trabecular myocardium derives from embryonic careg⁺ CMs. N=9.

Figure 8. Monitoring the primordial CM layer between the compact and trabecular myocardium.

(a) Longitudinal sections of careg:dmKO2;cmlc2:EGFP transgenic adult hearts labelled with antibodies against KO2 (red) and Tropomyosin (blue, white). V, ventricle; A, atrium; BA, bulbus arteriosus. (b) careg:dmKO2 expression is maintained during adulthood in a single layer of thin subcortical CMs (arrowheads) in the junctional zone (JZ) between the compact/cortical (CoM) and the trabecular myocardium (TrM). N=4. (c) A higher magnification of the adult ventricle wall of double transgenic fish careg:dmKO2; careg:EGFP displays an overlapping expression of both markers in subcortical myocytes (arrowheads). N=4. (d) A magnified adult ventricle wall of careg:dmKO2 transgenic fish displays an abundant expression of N-cadherin (green, white) on the surface of the junctional CMs on the side facing the trabecular myocardium. N=4. (e) A fragment of the adult ventricle wall of careg:dmKO2;wt1a-6.8kb:GFP transgenic fish displays the separation of compact myocardium and subcortical careg:dmKO2⁺ layer by wt1a-6.8kb:GFP-labelled fibroblasts of the junctional zone. N=4. (f–i) At 90 d.p.ci., transversal heart sections of careg:EGFP hearts display a gap in the primordial layer within the regenerated myocardium. The extent of the gap is indicated with arrowheads. N=4.

Figure 9. careg-expressing cells contribute to the regenerating myocardium and fin mesenchyme.

(a,b) Schematic representation of the transgenic strains and the experimental design for lineage tracing of careg-expressing primordial CMs during ventricle regeneration. (c–f) Representative sections of hearts treated with 4-OHT before cryoinjury to label the primordial layer, stained with antibody against Tropomyosin (blue). (c,d) At 10 d.p.ci., no zsYellow is detected in the trabecular myocardium at the injury site, indicating that primordial CMs do not contribute to the regeneration zone along the post-cryolesioned tissue. (e,f) At 30 d.p.ci., the zsYellow-positive primordial layer regenerates incompletely. (g) Experimental design for lineage tracing of careg-expressing dedifferentiated CMs during ventricle regeneration. (h–k) Representative sections of hearts treated with 4-OHT after cryoinjury hearts immunostained against Tropomyosin (blue) (h,i) At 15 d.p.ci., zsYellow is expressed in the trabecular myocardium at the injury site. (j,k). At 30 d.p.ci., zsYellow-positive CMs are detected in the trabecular (TrM) and cortical myocardium (CoM) of the regenerated muscle. N≥8. (l) Transgenic strains and the design of lineage tracing experiments during fin regeneration. (m) Longitudinal section of lineage-labelled fins at 3 d.p.a., stained for osteoblasts using the Zns5 antibody (green) and for nuclei with DAPI (blue). mCherry is detected in the entire mesenchyme of the regenerative outgrowth and below the amputation plane. N=6. (n–u) Live-imaging of the same lineage-labelled fin at different time points of regeneration. The regenerative outgrowth and the stump tissue are labelled with mCherry. N=6.

Figure 10. CMs and stump MES activate a common regulatory element during regeneration.

(a,b) A schematic representation of ventricle sections and whole fins at different time points during regeneration. The peri-injury zone of the ventricle and the stump of the fin activate a transient expression of the careg reporter in a TGFβ/Activin-β-dependent manner. In the heart, the careg reporter also demarcates the primordial layer, which fails to be completely restored in the new myocardium.