Dusp6 Attenuates Ras/MAPK Signaling to Limit Zebrafish Heart Regeneration Maria A

STEM CELLS AND REGENERATION RESEARCH ARTICLE Dusp6 attenuates Ras/MAPK signaling to limit zebrafish heart regeneration Maria A. Missinato1, Manush Saydmohammed1, Daniel A. Zuppo1, Krithika S. Rao2, Graham W. Opie1, Bernhard Kühn2,3 and Michael Tsang1,*

ABSTRACT Unlike mammals, zebrafish (Danio rerio) can regenerate the heart Zebrafish regenerate cardiac tissue through proliferation of pre- throughout the lifespan (Itou et al., 2012a; Poss et al., 2002). After existing cardiomyocytes and neovascularization. Secreted growth resection of the ventricular apex, a blood clot is formed to seal the factors such as FGFs, IGF, PDGFs and Neuregulin play essential wound that is subsequently replaced by fibrin and collagen (Poss roles in stimulating cardiomyocyte proliferation. These factors et al., 2002). Within hours of the injury, the epicardium, a layer of activate the Ras/MAPK pathway, which is tightly controlled by cells surrounding the heart, is activated and these cells proliferate the feedback attenuator Dual specificity phosphatase 6 (Dusp6), an and migrate to the injury area after undergoing epithelial-to- ERK phosphatase. Here, we show that suppressing Dusp6 mesenchymal transition (EMT). The function of activated epicardial function enhances cardiac regeneration. Inactivation of Dusp6 by cells is to promote the formation of new blood vessels that support small molecules or by gene inactivation increased cardiomyocyte the regenerating myocardium (González-Rosa et al., 2012; Lepilina proliferation, coronary angiogenesis, and reduced fibrosis after et al., 2006). Between 4 and 14 days post amputation (dpa), spared ventricular resection. Inhibition of Erbb or PDGF receptor signaling cardiomyocytes dedifferentiate and proliferate (Jopling et al., 2010; suppressed cardiac regeneration in wild-type zebrafish, but had a Kikuchi et al., 2010). By 30-60 dpa, the heart is fully regenerated, milder effect on regeneration in dusp6 mutants. Moreover, in rat with the fibrotic tissue resolved and replaced by new cardiac muscle primary cardiomyocytes, NRG1-stimulated proliferation can be and vasculature. enhanced upon chemical inhibition of Dusp6 with BCI. Our results The molecular mechanisms activated after injury that drive suggest that Dusp6 attenuates Ras/MAPK signaling during cardiomyocyte proliferation are complex and involve ligands such regeneration and that suppressing Dusp6 can enhance cardiac repair. as Fibroblast growth factors (FGFs) (Lepilina et al., 2006; Lien et al., 2006), Neuregulins (Nrg) (Gemberling et al., 2015), KEY WORDS: Zebrafish, Heart regeneration, Cardiac repair, Dual Transforming growth factor β (TGFβ) (Chablais and Jazwinska, specificity phosphatase 6, Ras/MAPK signaling, Cardiomyocyte 2012b), Platelet derived growth factor β (PDGFβ) (Lien et al., proliferation 2006), Insulin-like growth factor (IGF) (Huang et al., 2013) and Bone morphogenetic proteins (BMPs) (Wu et al., 2016). A number INTRODUCTION of these secreted factors bind to receptor tyrosine kinases (RTKs), Myocardial infarction (MI) is a leading cause of morbidity and resulting in activation of the mitogen-activated protein kinase mortality in industrialized countries (Mozaffarian et al., 2016). The (MAPK) pathway. For example, the epidermal growth factor Nrg1 effects of MI include massive cardiomyocyte death, which leads to has been shown to promote cardiomyocyte dedifferentiation and the formation of a non-contractile scar (Mill et al., 1990). Humans proliferation in zebrafish (Gemberling et al., 2015), mice (D’Uva have a poor ability to replace damaged cardiac tissue after an MI et al., 2015) and infant human hearts (Polizzotti et al., 2015). event, mostly because adult cardiomyocytes are postmitotic in Likewise, FGFs activate MAPK signaling to promote cellular mammals (Ahuja et al., 2007). Therefore, a major goal of cardiac proliferation and differentiation (Tsang and Dawid, 2004). During regenerative medicine is to induce cardiomyocyte proliferation or zebrafish heart regeneration, FGFs stimulate epicardial cell to transdifferentiate cardiac fibroblasts into beating cardiomyocytes activation and EMT together with neovascularization (Choi et al., (Bersell et al., 2009; Ebelt et al., 2008; Zhou et al., 2015). Although 2013; Lepilina et al., 2006). there have been intensive studies towards the direct reprogramming To control proper signaling levels, a number of feedback of fibroblasts, including the use of chemical agents, there is still inhibitors, including Dual specificity phosphatase 6 (Dusp6; also much progress to be made before an effective treatment is achieved known as Mkp3) function to attenuate FGF-stimulated MAPK (Cao et al., 2016b; Qian et al., 2012; Zhang et al., 2016). signaling during embryonic development (Kawakami et al., 2003; Li et al., 2007; Tsang and Dawid, 2004). Dusp6 is a cytoplasmic ERK1/2 phosphatase and its expression is regulated by active FGF 1Department of Developmental Biology, University of Pittsburgh, School of signaling during development (Li et al., 2007). Two groups have Medicine, Pittsburgh, PA 15213, USA. 2Pediatric Institute for Heart Regeneration and Therapeutics (I-HRT), Richard King Mellon Foundation Institute for Pediatric independently generated Dusp6 mutant mice. Li et al. (2007) Research and Division of Cardiology, Children’s Hospital of Pittsburgh of UPMC reported that Dusp6 mutant mice have higher pERK levels and and Department of Pediatrics, University of Pittsburgh, Pittsburgh, PA 15224, USA. phenotypes reminiscent of hyperactive FGFR signaling, such as 3McGowan Institute of Regenerative Medicine, Pittsburgh, PA 15219, USA. corona craniosynotosis and dwarfism. By contrast, Maillet et al. *Author for correspondence ([email protected]) (2008) noted that Dusp6 mutant mice were phenotypically normal in appearance, but had an enlarged heart owing to increased M.S., 0000-0001-7850-0732; D.A.Z., 0000-0002-4229-0849; K.S.R., 0000-0003- 2307-8583; G.W.O., 0000-0003-4406-3161; M.T., 0000-0001-7123-0063 cardiomyocyte proliferation during development. Importantly, after permanent ligation of the left coronary artery, Dusp6 mutant mice

Received 11 July 2017; Accepted 29 January 2018 have a smaller area of scar tissue and improved cardiac output, DEVELOPMENT

1 STEM CELLS AND REGENERATION Development (2018) 145, dev157206. doi:10.1242/dev.157206 showing that deletion of Dusp6 can promote cardiac repair in Western blot analysis confirmed the absence of Dusp6 protein in mammals. Consistent with these findings, mice overexpressing dusp6pt30a/pt30a embryos, implicating pt30a as a null allele Dusp6 in the heart have myocardial failure, increased fibrosis and (Fig. 2D). apoptosis (Purcell et al., 2007). In agreement, regenerative capacity In all cases, homozygous dusp6 mutants developed normally, is suppressed in zebrafish overexpressing dusp6 (Han et al., 2014). survived to adulthood at the expected Mendelian frequency and Here, we report that Dusp6 function limits the regenerative were fertile. Although we did not observe defects during development, response in the heart. dusp6 mutant zebrafish were generated and we did detect a mild increase in heart size by 5 months of age in showed increased cardiomyocyte proliferation and angiogenesis dusp6 mutants (Fig. 2E-G). This was accompanied by a thicker of the coronary vasculature, accompanied by faster resolution of compact myocardium (Fig. 2H-J, Fig. S2A) compared with WT fibrotic tissue after ventricular resection. Importantly, dusp6 mutant zebrafish matched by age and length. This was only noted in adults zebrafish can efficiently regenerate the heart even in the presence of older than 5 months, as younger and length-matched controls had Nrg or PDGF receptor (PDGFR) inhibitor, which block regeneration hearts of normal size. To understand the mechanism underlying the in wild-type (WT) zebrafish. These findings suggest that Dusp6 increase in heart size in dusp6 mutants, we measured cardiomyocyte functions to attenuate Nrg1 and PDGFR signaling after cardiac injury. proliferation at 3 and 5 months of age. Cardiomyocyte proliferation Moreover, chemical inhibition of Dusp6 using small molecules index was quantified by immunostaining for both Proliferating cell enhanced cardiac regeneration, offering potential new tools for nuclear antigen (Pcna) and Mef2c (cardiomyocyte marker). dusp6 therapeutic treatments. mutant hearts have significantly more proliferating cardiomyocytes than WT hearts at 3 months (Fig. S2B,C). However, by 5 months RESULTS there was no difference, suggesting that the dusp6 mutant hearts do dusp6 is expressed in endothelial cells and cardiomyocytes not proliferate indefinitely. In addition, measuring cardiomyocyte in the adult heart cell size with wheat germ agglutinin (WGA) staining showed that To determine if dusp6 is expressed in the zebrafish heart and there was no difference between WT and dusp6 mutant hearts, during regeneration, we performed Q-PCR before and after ventricle suggesting that the increase in heart mass was a result of increased apex amputation. Within 1 dpa, dusp6 expression was induced total cardiomyocyte number (Fig. S2D,E). after injury and continued to 7 dpa (Fig. 1A). To determine the cell- Next, we crossed the dusp6 mutants into Tg(fli1a:EGFP)y1,a specific expression of dusp6 we used RNAscope in cryosectioned transgenic line that marks endothelial cells, and noted that mutant Tg(fli1a:EGFP)y1 to delineate endothelial cells from cardiomyocytes hearts had a thicker compact myocardium containing more vessels in adult hearts. In uninjured hearts, dusp6 expression was detected than WT hearts (Fig. 2K,L, Fig. S2F). Moreover, the vessels in the diffusely within the myocardium and compact myocardium compact myocardium of dusp6pt30a/pt30a hearts show increased (Fig. 1B). dusp6 transcripts were also localized to endothelial activated (phosphorylated) ERK (pERK) when compared with WT cells, as evidenced by colocalization with EGFP+ cells (Fig. 1B). At hearts (Fig. 2M,N, Fig. S3A,B). These findings reveal that dusp6 3 dpa, dusp6 expression was detected in the myocardium and within mutants are phenotypically normal during development but as endothelial cells (Fig. 1B, Fig. S1). Studies have shown that Dusp6 adults they develop mild cardiomegaly with thickened compact is expressed in the myocardium and epicardium, thus expression in myocardium. This phenotype has features similar to those described vessel-like structures was not previously noted (Han et al., 2014). for ectopic expression of Nrg1 in the cardiomyocytes of adult We next generated transgenic lines using the 10 kb dusp6 zebrafish (Gemberling et al., 2015). nrg1 has been shown to be promoter driving membrane-targeted green fluorescent protein induced in perivascular cells that surround blood vessels in the (memGFP) [Tg(dusp6:memGFP)pt21] in order to reveal the activity compact myocardium after ventricular resection. Given the of dusp6 regulatory sequences by activation of exogenous gene induction of dusp6 expression in endothelial cells after injury, we expression in the adult heart. In uninjured adult hearts, weak hypothesized that Dusp6 could attenuate Nrg1 signaling during expression of memGFP in cardiomyocytes and stronger expression heart regeneration. in the blood vessels were noted in the compact myocardium (Fig. 1C). To confirm memGFP expression in the vasculature, dusp6 mutant zebrafish exhibit increased cardiomyocyte Tg(dusp6:memGFP)pt21; Tg(kdrl:NLS:mCherry)is5 hearts were proliferation and enhanced cardiac regeneration analyzed, in which endothelial cell nuclei are labeled in red and Since Dusp6 suppresses ERK activity and this pathway is activated dusp6 regulatory sequences drive memGFP expression in green. by a number of growth factors during regeneration, we reasoned that After injury, memGFP/mCherry double-positive cells were observed loss of dusp6 could augment cardiac regeneration. Ventricular apex in the nascent vessels in the injured area (Fig. 1D). Moreover, resection in dusp6pt30a/pt30a zebrafish was performed, and at 7 dpa memGFP+ cells were detected as early as 3 dpa, suggesting the a significant increase in proliferating cardiomyocytes was noted presence of endothelial cells within the regenerate (Fig. 1D). These (Fig. 3A,B). This observation was confirmed in dusp6pt30d/pt30d observations suggest that dusp6 might serve a crucial early function mutants (Fig. S4A-D). We next determined whether the increased during cardiac regeneration and is expressed in cardiomyocytes and cardiomyocyte proliferation in dusp6 mutants was prevalent in the endothelial cells after injury. throughout heart regeneration. At 4 and 7 dpa, a significant increase in cardiomyocyte proliferation index was noted, but not at later stages dusp6 mutant hearts develop cardiomegaly and have a (12 and 20 dpa) (Fig. S5A,B), indicating that increased proliferation thicker compact myocardium in dusp6 mutants was limited to the first 10 days after injury. To examine the role of Dusp6 in development and heart regeneration, In order for proper heart regeneration to occur, new blood vessels we generated mutant alleles of dusp6 using transcriptional activator- are required to form to support the regenerating myocardium like effector nucleases (TALENs) (Fig. 2A-C) (Bedell et al., 2012; (Lepilina et al., 2006). We therefore crossed the dusp6pt30a mutation Cade et al., 2012). We generated TALEN constructs targeting 30 bp into the Tg(fli1a:EGFP)y1 line. We observed an increased downstream of the dusp6 initiation codon (Fig. 2A). Four dusp6 presence of GFP+ cells within the injury by 8 dpa in mutant + mutant alleles (named pt30a-d) were identified (Fig. 2B,C). hearts (Fig. 3C,D). The increased presence of fli1a:EGFP vessels DEVELOPMENT

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Fig. 1. dusp6 is induced upon cardiac injury and expressed in cardiomyocytes and endothelial cells. (A) Q-PCR analysis of dusp6 in adult zebrafish hearts at 1 and 7 days post amputation (dpa), compared with uninjured hearts. dusp6 is induced after ventricle apex amputation. Values are normalized to β-actin and rnap expression. Data represent three independent replicates. *P<0.05, one-way ANOVA. (B) dusp6 expression in uninjured hearts and at 3 dpa using RNAscope. In uninjured hearts, dusp6 (red) is weakly detected throughout the heart including endothelial cells [Tg(fli1a:EGFP)y1], myocardium (Myo) and compact myocardium (CM). At 3 dpa, dusp6 expression was detected in the myocardium and in endothelial cells. Dotted line demarcates the amputation plane. Sections were counterstained with DAPI (blue) to visualize nuclei. Boxed areas are magnified beneath. (C) Confocal image from Tg(dusp6:memGFP)pt21 heart immunostained with anti-GFP and anti-Mef2C, showing that cardiomyocytes express memGFP in uninjured hearts. (D) AFOG images (left) and confocal images (center and right) of Tg(dusp6:memGFP)pt21; Tg(kdrl:NLS:mCherry)is5 hearts at multiple time points after ventricle apex amputation (n=3 for each time point). memGFP is expressed in the vessels of the compact myocardium in uninjured hearts and in the nascent vessels inside the clot after injury. Sections were counterstained with DAPI (blue) to visualize nuclei. Scale bars: 100 µm.

in the regenerate at an earlier time point suggests that heart appeared normal in dusp6 mutant hearts based on decreased regeneration was accelerated in the dusp6 mutant. arrhythmic (irregular heart beat rhythm) events as measured by At 20 dpa, dusp6 mutants consistently showed reduced scar electrocardiography (ECG) at 20 dpa (Fig. 4D,E, Fig. S6). This was tissue in the resected hearts (Fig. 4A-C). Moreover, cardiac function detected as variation in the R-R interval, which represents the time DEVELOPMENT

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Fig. 2. dusp6 mutant hearts exhibit mild cardiomegaly. (A) The TALENs targeting dusp6 exon 1. (B) Recovery of dusp6 mutant alleles pt30a-d. Mismatches created by the 1 bp insertion in pt30d are indicated in red. (C) Predicted amino acid sequence of dusp6pt30 alleles. (D) Western blot showing absence of Dusp6 protein in dusp6pt30a/pt30a embryos. β-actin was used as a loading control. (E,F) Whole-mount images of uninjured WT (E) and dusp6pt30a/pt30a (F) hearts at 5 months of age. dusp6 mutant hearts show cardiomegaly. A, atrium; V, ventricle; BA, bulbus arteriosus. (G) Quantification of the ratio of ventricle area/body weight (VA/BW) in WT (n=5) and dusp6 mutant (n=5) fish. dusp6pt30a/pt30a fish have a larger VA/BW ratio than WT fish. **P<0.01, Student’s t-test. (H,I) AFOG staining of uninjured heart sections at 5 months of age. dusp6 mutant hearts (I) have a thicker compact myocardium (brackets) than WT hearts (H). (J) Quantification of compact myocardium thickness in uninjured hearts at 5 months of age for WT (n=4) and dusp6 mutant (n=5). ****P<0.0001, Student’s t-test. (K,L) Sections of uninjured WT (n=4) and dusp6 mutant (n=4) fish with the Tg(fli1a: EGFP)y1 background to visualize the endothelium. dusp6 mutant hearts (L) have a thicker compact myocardium (red arrows) containing more vessels than WT hearts (K). (M,N) pERK is detected in fli1a:EGFP+ vessels of the compact myocardium. dusp6 mutant hearts (n=4) (N) have a thicker compact myocardium with more vessels showing pERK staining than in WT uninjured hearts (n=4) (M). Scale bars: 100 µm.

between heart beats. Uninjured zebrafish had normal heart rhythm lipoprotein lipase (lpl) and periostin ( postna and postnb) (Ito et al., with a regular R-R interval, and thus the standard deviation of the 2014) (Fig. 4F, Table S1). By contrast, expression of these fibrosis delta R-R values was low. After ventricular apex amputation, heart genes in the dusp6 mutant hearts had already returned to the rhythm variation in the R-R interval was high in both WT and baseline levels of uninjured hearts, suggesting that regeneration dusp6pt30a/pt30a zebrafish. By 20 dpa, WT hearts showed irregular was almost complete at 20 dpa. We performed Q-PCR experiments R-R intervals, whereas dusp6 mutant heart presented regular R-R on several of these genes and confirmed the RNA-seq findings intervals, suggesting a restoration of cardiac function (Fig. 4D,E, (Fig. 4G). Taken together, these data show that genetic disruption of Fig. S5). dusp6 results in enhanced cardiac regeneration. To confirm that dusp6pt30a/pt30a hearts regenerate faster than WT we performed transcriptome analysis of uninjured hearts at 20 dpa Chemical inhibition of Dusp6 increases cardiomyocyte (Fig. 4F). In WT hearts, genes known to be associated with cardiac proliferation and improves cardiac regeneration fibrosis were significantly upregulated at 20 dpa. This includes Previously, we employed a transgenic FGF reporter chemical fibronectin ( fn1a and fn1b) (Wang et al., 2013), collagen (col1a1b, screen for small molecules that modulate FGF/Ras/MAPK activity col1a2, col5a1, col12a1a) (Chablais and Jazwinska, 2012a), TGFβ (Molina et al., 2007; Saydmohammed et al., 2011). We identified 2- signaling (smad3b) (Chablais and Jazwinska, 2012b), lysyl oxidase benzylidene-3-(cyclohexylamino)-3H-inden-1-one chloride (BCI),

(loxl2a), tenascin C (tnc), caveolin 1 (cav1) (Cao et al., 2016a), which enhanced Ras/MAPK signaling (Molina et al., 2009). BCI, DEVELOPMENT

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2014; Molina et al., 2009). To confirm the specificity of BCI in suppressing Dusp6 function we tested whether lower BCI215 doses could enhance cardiac regeneration in heterozygous dusp6pt30a/+ zebrafish. As predicted, a suboptimal dose of BCI215 did not increase cardiomyocyte proliferation in WT hearts at 7 dpa (Fig. S9A,B). However, this suboptimal dose of BCI215 significantly increased cardiomyocyte proliferation in dusp6pt30a/+ hearts (Fig. S9A,B). To confirm BCI215 specificity, we treated dusp6pt30a/pt30a mutants with BCI215 and did not observe increased cardiomyocyte proliferation (Fig. S9C,D). Our data show that chemical inhibition of Dusp6 can enhance cardiac regeneration, offering a potential therapeutic target for enhancing cardiac repair.

PDGFR inhibition reduces the effects of dusp6 mutations on injury-induced cardiomyocyte proliferation Studies have shown that during zebrafish heart regeneration, several growth factors activating the Ras/MAPK pathway are crucial for proper regeneration to occur. To determine the ligands that Dusp6 attenuates after ventricular resection, we tested the effects of suppressing specific RTKs with small molecules. PDGFβ is induced after cardiac injury and has been demonstrated to stimulate cardiomyocyte proliferation (Lien et al., 2006). WT and dusp6pt30a/pt30a mutant zebrafish were treated with a specific inhibitor of PDGFRs called PDGFR tyrosine kinase inhibitor III (PTKI III) (Matsuno et al., 2002). In WT hearts, we observed a significant decrease in the cardiomyocyte proliferation index after pt30a/pt30a Fig. 3. Increased cardiomyocyte proliferation and angiogenesis in dusp6 treatment with PTKI III (Fig. 6A,C,E). In the dusp6 mutant mutant hearts after cardiac injury. (A) Hearts at 7 dpa, immunostained for hearts, by contrast, cardiomyocyte proliferation was not affected by Mef2c (green; cardiomyocyte nuclei) and Pcna (red; proliferation marker). PTKI III treatment (Fig. 6B,D,E). Moreover, in WT hearts PTKI III Cardiomyocyte proliferation is increased in dusp6 mutant hearts (n=23) treatment caused a failure to resolve fibrotic tissue at 20 dpa, as compared with WT hearts (n=35). Arrowheads indicate proliferating noted from fibrin deposition in the injury area (Fig. 6F,G,J). cardiomyocytes. (B) Quantification of cardiomyocyte proliferation index in WT pt30a/pt30a and dusp6 mutant hearts. ****P<0.0001, Student’s t-test. (C) EGFP+ vessels However, in dusp6 hearts, the injured area was much were visualized in Tg(fli1a:EGFP)y1; dusp6pt30a/pt30a (n=16) and Tg(fli1a: reduced and lacked fibrin, suggesting that even in the presence EGFP)y1 (n=10) hearts at 8 dpa. Dashed line demarcates the resection plane. of a PDGFR inhibitor, normal cardiac regeneration can occur (D) Quantification of new vessels formed inside the clot area of hearts at 8 dpa. (Fig. 6H-J). These observations suggest that Dusp6 attenuates **P<0.01, Student’s t-test. Scale bars: 100 µm. PDGFR signaling during cardiac regeneration. and its analog BCI215, are allosteric inhibitors of Dusp6 Erbb2 inhibition reduces the effects of dusp6 mutation on (Korotchenko et al., 2014). Since genetic disruption of dusp6 injury-induced cardiomyocyte proliferation resulted in accelerated heart regeneration, we examined whether The mild cardiomegaly and thickened compact myocardium chemical suppression of Dusp6 activity would have similar effects. phenotypes in the dusp6 mutants resemble the effects of Because we observed toxicity in embryos with long-term treatments transgenic Nrg1 overexpression in the zebrafish heart of BCI, our first objective was to determine the maximum tolerated (Gemberling et al., 2015). We hypothesized that Dusp6 attenuates dose (MTD) in adult zebrafish of BCI and BCI215. Retro-orbital Nrg/Erbb activity. To test this, we monitored expression of errfi1b injections were performed daily for 4 days in adult zebrafish with (also known as mig6), a reported target and feedback attenuator of 0.5, 2.5, 12.5 and 25 mg/kg BCI and BCI215. At 0.5 and 2.5 mg/kg, EGF signaling (Chung et al., 2004; Ferby et al., 2006). Q-PCR BCI and BCI215 were well tolerated (Fig. S7A,B), but higher BCI analysis showed no difference in errfi1b expression in WT hearts doses (12.5 and 25 mg/kg) resulted in more deaths than BCI215, upon injury (Fig. 7A). However, in dusp6 mutant hearts, errfi1b confirming our previous observations in embryos (Korotchenko expression was significantly increased, suggesting that Nrg1 et al., 2014). signaling was elevated (Fig. 7A). One day after ventricle apex amputation, BCI (0.5 mg/kg), BCI215 We next injected AG1478, a known inhibitor of EGF receptors (0.5 mg/kg) or vehicle DMSO was injected for 6 consecutive including Erbb2 (Gemberling et al., 2015), into dusp6 mutant days and hearts were extracted at 7 dpa to assess cardiomyocyte zebrafish after ventricular amputation to determine if cardiac proliferation, at 8 dpa to assess angiogenesis, and at 25 dpa to regeneration can be suppressed. Retro-orbital injection of AG1478 measure fibrotic tissue area. Both molecules improved cardiac efficiently decreased cardiomyocyte proliferation in WT hearts regeneration in adult zebrafish by increasing the cardiomyocyte (Fig. 7B-F, Fig. S10A,B). Although injecting the same dose of proliferation index (Fig. 5A-D) and blood vessel formation (Fig. 5E- AG1478 reduced cardiomyocyte proliferation in dusp6 mutant H), and reducing scar size (Fig. 5I-L). Importantly, BCI and BCI215 hearts (Fig. 7D-F), the proliferation index was similar to that of did not increase cell proliferation in uninjured hearts (Fig. S8A,B) and control DMSO-treated WT hearts. We injected AG1478 into did not cause cardiomegaly (Fig. S8C). dusp6pt30a/pt30a; Tg(fli1a:EGFP)y1 to monitor endothelial cells in Several studies have reported that BCI and BCI215 can inhibit the regenerate. EGF receptor inhibition blocked new vessel other Dusp family members, including Dusp1 (Korotchenko et al., formation in WT hearts (Fig. 7G,H,K), but did not affect vessel DEVELOPMENT

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Fig. 4. dusp6 mutant hearts show accelerated regeneration. (A,B) Sections of hearts at 20 dpa stained with AFOG to visualize the deposition of fibrotic tissue (arrows). dusp6 mutant hearts (n=13) (B) have reduced fibrotic tissue area compared with WT hearts (n=17) (A). (C) Quantitation of fibrotic tissue area at 20 dpa. ***P<0.001, Student’s t-test. (D) Representative ECG from WT (n=25) and dusp6 mutants (n=24) at 20 dpa. dusp6 mutant fish exhibit fewer arrhythmic events than WT. ECG are shown from four fish (#1, #10, #11, #13). (E) Quantitation of the arrhythmic events, measured as R-R intervals, in WT and dusp6pt30a/pt30a zebrafish at 20 dpa. ****P<0.0001, Student’s t-test. (F) RNA-seq analysis of uninjured hearts and hearts at 20 dpa. At 20 dpa, WT hearts express fibrosis genes, but in dusp6pt30a hearts fibrosis genes are downregulated, suggesting faster regeneration. (G) Q-PCR analysis of selected fibrosis genes in hearts at 20 dpa. dusp6pt30a hearts show downregulation of fibrosis genes. Values are normalized to β-actin and rnap expression. Data are from one representative experiment from three independent biological replicates.

formation in dusp6pt30a/pt30a hearts (Fig. 7I-K). Fibrotic tissue was is increased in dusp6 mutant hearts and that suppressing Nrg still present in WT hearts after AG1478 treatment, suggesting receptor activity is not sufficient to block regeneration. that heart regeneration was inhibited by blocking Nrg signaling We next tested whether cardiomyocyte proliferation can be (Fig. S11). However, dusp6pt30a/pt30a hearts exhibited much reduced rescued in zebrafish harboring heterozygous mutations in erbb2 fibrotic tissue area (Fig. S11). These data suggest that Nrg signaling (erbb2st61/+). Homozygous erbb2 mutants do not survive to adults

Fig. 5. Chemical inhibition of Dusp6 increases cardiomyocyte proliferation and angiogenesis and reduces fibrosis after cardiac injury. (A-C) Adult zebrafish hearts at 7 dpa, injected for 6 days with DMSO (n=16) (A), 0.5 mg/kg BCI (n=13) (B) or 0.5 mg/kg BCI215 (n=12) (C) and stained for Mef2c and Pcna. (D) Quantification of proliferating cardiomyocytes at 7 dpa. BCI and BCI215 increased cardiomyocyte proliferation compared with DMSO vehicle. (E-G) Tg(fli1a:EGFP)y1 hearts at 8 dpa injected for 6 days with BCI (n=16) (F), BCI215 (n=19) (G) or DMSO (n=14) (E) and stained for Mef2c. (H) Quantification of new vessels formed inside the clot area of hearts at 8 dpa. (I-K) Sections of hearts at 25 dpa stained with AFOG to visualize the scar. Intact cardiac muscle stains orange-brown, fibrin stains red and collagen blue. Fish injected with BCI (n=12) (J) and BCI215 (n=18) (K) resolved the injury faster than DMSO-injected fish (n=14) (I). (L) Quantification of clot areas at 25 dpa. (D,H,L) ****P<0.0001, ***P<0.001, **P<0.01, *P<0.05, one-way ANOVA. Scale bars: 100 µm. DEVELOPMENT

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Fig. 6. dusp6 mutant hearts are mildly affected by PDGFR inhibition. (A-D) Hearts at 7 dpa were injected for 6 days with PTKI III (C,D) or vehicle DMSO (A,B) and stained for Mef2c and Pcna to determine cardiomyocyte proliferation. WT hearts (DMSO, n=22; PTKI III, n=19) (A,C) and dusp6pt30a/pt30a hearts (DMSO, n=21; PTKI III, n=15) (B,D). Arrowheads indicate proliferating cardiomyocytes. (E) Quantification of cardiomyocyte proliferation in WT and dusp6 mutant hearts after injection of PTKI III or DMSO. Each point represents the average proliferation index from one heart. (F-I) AFOG staining of WT or dusp6pt30a/pt30a hearts at 20 dpa injected for 6 days with PTKI III (WT, n=8; dusp6pt30a/pt30a, n=12) (H,I) or DMSO (WT, n=10; dusp6pt30a/pt30a, n=7) (F,G). Dashed line demarcates injury zone. (J) Quantification of fibrotic tissue area in hearts at 20 dpa. (E,J) ****P<0.0001, ***P<0.001, **P<0.01; ns, not significant; one-way ANOVA. as they fail to trabeculate their hearts (Lyons et al., 2005). cardiac injury, is induced in endothelial cells at sites juxtaposed Because Nrg1 is induced during heart regeneration in zebrafish to cells expressing nrg1. Marin-Juez et al. (2016) demonstrated a (Gemberling et al., 2015), we reasoned that heterozygous erbb2st61/+ requirement for rapid revascularization in zebrafish heart regeneration carriers might show decreased cardiomyocyte proliferation. This was and that suppressing this process affected cardiomyocyte confirmed as there was a significant decrease in the presence of Pcna+ proliferation. These findings suggest that a positive interaction cardiomyocytes at 7 dpa (Fig. 7L). Treatment of heterozygous exists between endothelial cells and cardiomyocytes that supports erbb2st61/+ zebrafish with BCI215 resulted in a mild rescue of heart regeneration. cardiomyocyte proliferation (Fig. 7L). To delineate Dusp6 function in zebrafish heart regeneration, we To directly test the effects of BCI on Nrg1 activity, we employed disrupted Dusp6 using TALENs. dusp6 mutants are overtly normal the rat primary cardiomyocyte proliferation assay. In these and do not show embryonic or adult lethality. Previous studies have experiments, primary neonatal rat ventricular cardiomyocytes shown that Dusp6 attenuates FGF signaling during early (NRVM) are cultured in vitro in the presence or absence of development (Li et al., 2007; Tsang et al., 2004). We anticipated NRG1, a known cardiomyocyte mitogen (Bersell et al., 2009). NRG1 developmental defects including dorsal/ventral patterning, heart, treatment increased cardiomyocyte proliferation as marked by central nervous system, craniofacial and fin formation as FGFs are increased phosphorylated Histone H3 (H3P) (Fig. 8). The addition known to be crucial for the proper development of these tissues. of BCI together with NRG1 resulted in a significant increase in However, analysis of dusp6 mutant embryos did not reveal any cardiomyocyte proliferation (Fig. 8). Taken together, our data show developmental defects normally associated with increased FGF that the effects of blocking Nrg and PDGF signaling during heart activity. It might not be surprising that zebrafish dusp6 mutant regeneration are significantly reduced in the absence of Dusp6. embryos are normal, as a similar lack of developmental defects was described for mouse Dusp6 mutants (Li et al., 2007; Maillet et al., DISCUSSION 2008). These findings suggest that either dusp6 is dispensable for Dusp6 is an important regulator of Ras/MAPK signaling, and embryonic development or that redundancy with other Dusp family this pathway is utilized to control epicardial cell activation, members can compensate for the absence of dusp6. For example, cardiomyocyte proliferation and angiogenesis. Previous reports Dusp2, Dusp4, Dusp5 and Dusp7 can dephosphorylate ERK1/2 and based on in situ hybridization staining have revealed dusp6 compensate for the loss of dusp6 (Kidger and Keyse, 2016; Kondoh expression within cardiomyocytes and in the epicardial layer (Han and Nishida, 2007). Alternatively, since several FGF negative- et al., 2014; Itou et al., 2012b). Here, using double-transgenic feedback regulators such as sef (also known as il17rd) and sprouty zebrafish, we found that the dusp6 promoter can drive memGFP family genes are expressed early in development, the loss of expression in endothelial cells of the compact myocardium in dusp6 may be compensated by their activity (Fürthauer et al., 2001, uninjured hearts and, after amputation, that memGFP is induced in 2002; Tsang et al., 2002). Although early embryonic and larval the nascent vessels in the injury area. This expression in endothelial development appeared normal, we did observe differences between cells is compelling given a recent study that showed nrg1 induction dusp6 mutant and WT adult zebrafish. After ventricular resection, in perivascular cells and the rapid appearance of vessels soon after dusp6pt30a/pt30a mutants showed accelerated regeneration, in line injury (Gemberling et al., 2015; Marin-Juez et al., 2016). Thus, with the observations from Dusp6 null mice subjected to MI dusp6 is expressed in the epicardium and cardiomyocytes and, after (Li et al., 2007; Maillet et al., 2008). In addition, dusp6 mutant DEVELOPMENT

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Fig. 7. EGF receptor inhibition in dusp6 mutants dampens cardiac regeneration. (A) Q-PCR analysis of errfi1b at 0 and 3 dpa. After amputation, dusp6 mutant hearts show higher errfi1b expression compared with WT. Data represent six independent replicates. (B-E) Hearts at 7 dpa injected for 6 days with AG1478 (C,E), or DMSO vehicle (B,D) and stained for Mef2c and Pcna to determine cardiomyocyte proliferation. Arrowheads indicate proliferating cardiomyocytes. For WT: DMSO, n=36; 10 µM and 25 µM AG1478, n=17 and n=14, respectively. For dusp6pt30a/pt30a: DMSO, n=31; 10 µM and 25 µM AG1478, n=19 and n=13, respectively. (F) Quantification of cardiomyocyte proliferation in WT and dusp6 mutant hearts after injection of AG1478 or DMSO. (G-J) Tg(fli1a:EGFP)y1 hearts at 8 dpa injected with AG1478 (H,J) or DMSO (G,I). MHC (red) marks the resection plane. AG1478 treatment blocked angiogenesis in WT hearts, but not in dusp6 mutant hearts. Dashed line demarcates resection plane. n=17 for DMSO in WT and dusp6pt30a/pt30a; n=7 for 25 µM AG1478 in WT and dusp6pt30a/pt30a. (K) Quantification of new vessels formed at 8 dpa. (L) Quantification of cardiomyocyte proliferation in erbb2st61/+ hearts at 7 dpa, injected with BCI215 (n=13) or DMSO (n=14) as compared with sibling WT hearts (DMSO, n=17). erbb2st61/+ hearts have a diminished cardiomyocyte proliferation index compared with WT hearts, and this is rescued by BCI215. (A,F,K,L) ****P<0.0001, ***P<0.001, *P<0.05; ns, not significant; one-way ANOVA. Scale bars: 100 µm.

zebrafish show elevated cardiomyocyte proliferation and physiological growth of the heart. Both dusp6 mutant and Nrg1 angiogenesis. The induction of GFP expression by the dusp6 transgenic adult hearts show cardiomegaly and an expanded compact enhancer in endothelial cells (Fig. 1) coupled with increased pERK myocardium, albeit much milder in the dusp6 mutants (Gemberling levels (Fig. 2N) suggests that the main function of Dusp6 is in the et al., 2015). For this reason, we hypothesized that dusp6 mutant coronary vasculature after injury. zebrafish have higher levels of Nrg1/Erbb2 signaling. This was To determine which growth factor signaling pathway Dusp6 confirmed by monitoring the expression of a specific target of EGF attenuates during heart growth and regeneration, we blocked RTK signaling, errfi1b, which was increased in dusp6 mutant hearts. A signaling with specific small molecule inhibitors of PDGFR and consequence of Dusp6 deficiency is that the threshold concentration Erbb2. In the absence of Dusp6, PTKI III, an inhibitor of PDGFR, of ligand required to stimulate proliferation is reduced. Previous work was unable to suppress heart regeneration. A similar effect was noted has shown a role for FGFR, IGF and PDGFR in angiogenesis and with the Erbb2 inhibitor AG1478. Our findings point to a general role cardiomyocyte proliferation after injury (Kim et al., 2010; Lepilina for Dusp6 as an attenuator of Ras signaling via multiple ligands et al., 2006). It will be of interest to determine in the future whether during heart regeneration. The phenotypic similarity between dusp6 Dusp6 can also impact these pathways for heart regeneration. mutant hearts and Nrg1 overexpression in zebrafish is intriguing and Furthermore, the exact cell type in which Dusp6 functions is not suggests that Dusp6 might attenuate Nrg1 signaling during clearly defined in this study. One model is that Dusp6 has multiple DEVELOPMENT

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signaling could prove to be important in the treatment of heart disease. One complication with Nrg1 therapies is that they have been implicated to induce tumor formation: in NRG1 trials it was found that breast cancer can arise from activated mutation in the NRG1 receptor ERBB2 (Rimawi et al., 2015). The utility of recombinant human NRG1 in cardiac regenerative therapy could be limited owing to this potential side effect of increasing cancer risk. However, a recent study in mice points to an absence of neoplastic growth with Nrg1 administration (Ganapathy et al., 2016). Nevertheless, using lower concentrations of Nrg1 together with chemical inhibition of Dusp6 could stimulate cardiomyocyte proliferation for cardiac repair.

MATERIALS AND METHODS Generation of dusp6 mutant zebrafish Two pairs of TALENs were designed against the translational initiation site in exon 1 of the dusp6 gene. TALEN repeat variable diresidues (RVDs) were assembled in two steps by the Golden Gate method using pT3TS (Cermak et al., 2015) and subcloned into pCS2+ containing the Fok1 homodimer Fig. 8. BCI augments NRG1 activity in primary rat cardiomyocytes. domain. mRNA was synthesized using the mMESSAGE mMACHINE Kit Postnatal day 1 rat neonatal ventricular myocytes were isolated and cultured in (Ambion). mRNAs encoding each TALEN were co-injected into one-cell 10% FBS. Cells were exposed to DMSO vehicle (A), NRG1 (50 ng/ml) (B) or stage zebrafish embryos. Heritable TALEN-induced mutations in the F1 and NRG1 plus BCI at 5 µM (C) for 3 days. All treatments were performed in the F2 generation were evaluated by genomic DNA PCR (primers 5′-GTGG- presence of 10% FBS. Cardiomyocyte proliferation was quantified by H3P CTCGCGCACTCACAGGCTA-3′ and 5′-GGAGCCGCCGTCGATGTT- staining (n=2). (D) BCI treatment augments the effect of NRG1 on neonatal TTCA-3′) followed by restriction fragment length polymorphisms that cardiomyocyte proliferation. ****P<0.0001, ***P<0.001, ANOVA followed by detect disruption of the ClaI site in exon 1 of dusp6. Bonferroni post-hoc test.

Zebrafish maintenance, ventricular amputation, and retro- roles in attenuating growth factors and this is achieved by its activity orbital injections in different cells. Another interpretation from this work is based on The zebrafish experiments were performed according to protocols approved the observation of strong dusp6 enhancer activity in endothelial cells by the Institutional Animal Care and Use Committee (IACUC) at the after injury. This is crucial in regulating cardiomyocyte proliferation, University of Pittsburgh, which conforms to NIH guidelines. Adult (6- to 18-month-old) wild-type AB* and Tü, transgenic Tg(fli1a:EGFP)y1 suggesting that any role of Dusp6 is indirect. Tissue-specific is5 inactivation of Dusp6 is likely to uncover the exact role of this (Lawson and Weinstein, 2002), Tg(kdrl:NLS:mCherry) (Wang et al., 2010), and mutant dusp6pt30a-d and erbb2st61 (Lyons et al., 2005) zebrafish phosphatase during heart regeneration. were maintained at 28°C. Tg(dusp6:memGFP)pt21 lines were generated We confirmed that inhibiting Dusp6 with small molecules could using the 10 kb dusp6 promoter as previously described (Molina et al., enhance heart regeneration in zebrafish (Han et al., 2014). Increased 2007). Both homozygous and heterozygous dusp6pt30a and dusp6pt30d cardiomyocyte proliferation, the earlier formation of coronary alleles were used in the experiments. vessels within the regenerate, and reduced scar tissue deposition To measure compact myocardium thickness and cardiomyocyte within the wound apex with BCI or BCI215 treatment was proliferation rate in uninjured hearts, zebrafish of 3 and 5 months of age observed. The increased proliferation of cardiomyocytes in were used. Ventricle area normalized to body weight (VA/BW) was amputated hearts was observed within 4-7 dpa and not detected measured for each fish at the indicated time and expressed in mm2/g. beyond 12 dpa. This is likely to reflect growth factor activity being Ventricle apex amputation was performed as described previously (Poss at its highest within this time frame to support cardiomyocyte et al., 2002). Approximately 20% of the ventricle apex was resected and zebrafish returned to aquaria with standard feeding and husbandry. Retro- proliferation, and this is the period when Dusp6 functions to orbital injections were performed as previously described (Pugach et al., attenuate the Ras pathway. Han et al. (2014) reported that cardiac 2009). After surgery, fish were allowed to recover for 24 h, and compounds injury resulted in induction of Dusp6 expression through H2O2 were retro-orbitally injected daily, for a maximum of 6 days. Control fish generation, and that this induction is a limiting factor in heart were injected with 3 µl 50% DMSO (Sigma, D8418) in filtered PBS. Fish regeneration. In their studies, the focus was on Dusp6 activity after were injected with 3 µl 0.5 mg/kg BCI (Sigma, 317496) (Molina et al., 2009) 7 dpa and they noted that BCI could increase the cardiomyocyte or its analog BCI215 (chemically synthesized by the University of Pittsburgh proliferation index at 14 dpa in the absence of H2O2. In our study, Chemistry Department) (Korotchenko et al., 2014) dissolved in DMSO. For we focused on the earlier events (from 1 to 7 dpa) and noted that the suboptimal dose experiment, fish were injected with 0.25 mg/kg blocking Dusp6 early was also sufficient to enhance cardiac BCI215. To inhibit PDGFR, 10 µM 4-(6,7-dimethoxyquinazolin-4-yl)-N- regeneration. Importantly, this enhancement in proliferation was (4-phenoxyphenyl) piperazine-1-carboxamide (PTKI III; Santa Cruz, sc- 204173) was injected daily for 6 days. To inhibit Erbb2, 10 µM or 25 µM observed only after cardiac amputation and not in uninjured hearts, AG1478 (Sigma, T4182) was injected daily for 6 days. implying that the effects of these compounds are dependent upon injury. It will be interesting to determine whether BCI and BCI215 RNAscope can augment heart regeneration in other injury models, such as RNAscope [Advanced Cell Diagnostics (ACD)] was performed on uninjured cryoinjury (Chablais and Jazwinska, 2012a), cardiomyocyte and injured (3 or 7 dpa) hearts isolated from WT and Tg(fli1a:EGFP)y1 adult ablation (He et al., 2016; Wang et al., 2011) or in the neonatal zebrafish. Hearts were fixed overnight at 4°C in 4% paraformaldehyde (PFA), mouse (Polizzotti et al., 2016). Recent findings show that Nrg1 can transferred into a sucrose gradient (10-20-30% sucrose in PBS) the following stimulate heart repair in mammals (D’Uva et al., 2015; Polizzotti day at 4°C before cryopreservation overnight. Tissue was embedded in et al., 2015), so the possibility of using BCI to augment Nrg1 Surgipath Cryo-Gel (Leica, 39475237) and sectioned at 14 µm. RNAscope DEVELOPMENT

9 STEM CELLS AND REGENERATION Development (2018) 145, dev157206. doi:10.1242/dev.157206 probe hybridization, amplification and immunostaining were performed RNA extraction, cDNA synthesis and quantitative PCR (Q-PCR) following the protocol provided in the RNAscope Multiplex Fluorescent Total RNA was extracted from uninjured hearts and hearts at 6 h post Reagent Kit v2 user manual (ACD). ACD designed the dusp6 probe used in amputation or 1, 3 and 7 dpa using TRIzol (Invitrogen) and the RNeasy this study. Following the final wash step of the RNAscope probe hybridization Micro Kit (Qiagen). Eight hearts were pooled together for each condition. protocol, immunofluorescent staining was performed to better visualize RNA (1 µg) was reverse transcribed to cDNA with SuperScript reverse endogenous GFP or cardiomyocyte nuclei. Primary antibodies for transcriptase (Invitrogen) using random hexamers. Q-PCR was performed immunostaining were chicken anti-GFP (1:1000; Aves Labs, GFP-1020) as described previously (Missinato et al., 2015). β-actin (actb2) and RNA and rabbit polyclonal anti-Mef2c (1:500; Santa Cruz, sc-313). Secondary polymerase (rnap; also known as polr2d) were used to normalize gene antibodies for immunostaining were fluorescein goat anti-chicken 488 expression in the Q-PCR experiments. Primers for Q-PCR are listed in (1:1000; Aves Labs, F-1005) and Alexa Fluor 488 goat anti-rabbit IgG Table S2. At least three independent biological replicates were performed. peroxidase conjugate (1:1000; Invitrogen, A11008). Slides were sealed using ProLong Diamond Antifade Mountant with DAPI (Invitrogen, P36962). RNA-seq sample preparation and data analysis Images were taken on a Zeiss 700 confocal microscope at 20× and 40×. Image For the RNA-seq experiment, only ventricles were collected, dissecting out analysis was performed using ImageJ Fiji (NIH). atria and outflow tract with the use of a micro-scalpel. Total RNA was extracted from uninjured and 20 dpa hearts using TRIzol (Invitrogen) and Immunostaining, fibrotic tissue area, cell counting and size the RNeasy Micro Kit (Qiagen). Two ventricles were pooled for each measurement condition. Tufts Genomic Core (http://tucf-genomics.tufts.edu) prepared the For histological examination, hearts were collected in cold PBS and fixed in libraries and ran RNA-seq using an Illumina HiSeq 2500 with 150 bp 4% PFA in PBS for 2 h at room temperature. After two washes in PBS, a single-end reads. RNA-seq reads were analyzed using the CLC Genomics 30% sucrose solution in PBS was used to cryopreserve the tissues before Workbench software package (Qiagen). immersion in embedding medium (Leica). 14 µm cryosections were collected and consecutive sections used for immunostaining, and for Acid Western blot Fuchsin Orange G (AFOG) staining as previously described (Poss et al., WT and dusp6 mutant zebrafish embryos (24 h post fertilization) were 2002). Images were taken with a Leica MZ16 microscope and Q Imaging dechorionated and deyolked by gently pipetting in ice-cold PBS with Retiga 1300 camera. Fibrotic areas were measured using ImageJ. For each proteinase/phosphatase inhibitor. Deyolked embryos were centrifuged at heart, the average of the sum of the scar area was calculated from the four 5000 rpm (2300 g) for 5 min. After removing PBS, embryos were lysed in largest central sections. Laemmli buffer. Equal amounts of protein (50 µg) were heat denatured and Primary antibodies used for immunostaining were chicken anti-GFP separated by 10% SDS-PAGE and transferred to nitrocellulose membrane (Aves Labs, GFP-1020; 1:100), rabbit polyclonal anti-Mef2c (Santa Cruz, using a semi-dry blot system (Bio-Rad). Blots were scanned using the Li- sc-313; 1:500), mouse monoclonal anti-PCNA (Sigma, P8825; 1:3000), COR Odyssey CLx infrared imaging system, and band intensities were mouse monoclonal anti-MAPK, activated (diphosphorylated ERK1/2) quantified and normalized using Image Studio software (Li-COR). (Sigma, M8159; 1:100) and mouse anti-MHC (DSHB, F59; 1:50). Antibodies were diluted in Odyssey Blocking Buffer containing 0.2% Secondary antibodies were Alexa Fluor 488 goat anti-rabbit IgG Tween 20 (National Diagnostics). Primary antibodies were mouse peroxidase conjugate (Invitrogen, A11008; 1:1000), Alexa Fluor 594 monoclonal Dusp6 (Sigma, WH0001848M1; 1:50) and goat polyclonal goat anti-mouse IgG (H+L) (Invitrogen, A11005; 1:1000), Alexa Fluor 488 β-actin (Santa Cruz, sc-1615; 1:100). Secondary antibodies (1:15,000) were goat anti-mouse IgG (H+L) (Invitrogen, A11001; 1:1000) and Alexa Fluor IRDye 680 donkey anti-goat (Li-COR, 926-68024) and IRDye 800 donkey 488 goat anti-chicken (Thermo Fisher, A11039; 1:300). Slides were anti-mouse (Rockland, 610-731-002). mounted with Vectashield mounting medium with DAPI (Vector Laboratories, H-1200). Images were taken with a Zeiss LSM 700 Electrocardiogram (ECG) confocal microscope. For each experiment, at least four sections were ECG was performed in adult zebrafish 1 day prior to ventricle amputation, analyzed for each heart. Slides stained with only secondary antibody were and zebrafish were kept in single tanks to allow tracking. At day 0, surgery used as negative control. was performed and ECG was recorded at 20 dpa using the iWorx ECG Cardiomyocyte proliferation index (%) was calculated from the number system, and selecting the zebrafish ECG settings. Each adult zebrafish was of Mef2c+ Pcna+ cells among Mef2c+ cells. Nascent vasculature was anesthetized for 2 min in Tricaine (MS-222, Sigma) and positioned on its quantified by measuring (ImageJ) the areas of vessels formed in the back on the pedestal located on top of the grounded aluminium base. Gently, regenerate in Tg(fli1a:EGFP)y1 hearts and dividing by the injury area. the Ag/AgCl surface electrodes were placed on the fish axially along the Vessel density in uninjured hearts was quantified by measuring (ImageJ) the center line of the ventral surface. One electrode was placed near the gills and areas of vessels within the compact myocardium in Tg(fli1a:EGFP)y1 hearts the other next to the heart. ECG was recorded for 3 min. Data were analyzed and dividing by the compact myocardium area, to give the percentage of with Labscribe3 software (iWorx) and the R-R interval was measured as vessels per compact myocardium. Four images were taken for each heart previously described (Zhang et al., 2014). Data were expressed as standard section and the average was calculated for each heart. deviation of delta R-R values. pERK1/2 staining (%) was measured with ImageJ as pERK1/2+ immunostained area divided by compact myocardium area. Two to ten Neonatal ventricular cardiomyocyte (NRVM) isolation and sections were used for each heart and the average was calculated for each treatments heart. The thickness of the compact myocardium was measured from the NRVM from 1-day-old Sprague Dawley rats (Charles River) were isolated average of 16 measurements for each heart section. using a commercially available kit (Cellutron, nc-6031). Cells were digested To measure cardiomyocyte size, sections were stained with mouse anti- to yield single-cell suspensions and pre-plated for 1 h to purify MHC (DSHB, F59; 1:50), Rhodamine wheat germ agglutinin (WGA) cardiomyocytes from other cell populations. Cells were cultured for (Vector Laboratories, RL-1022; 1:500) for the detection of cell borders, and 3 days in NS medium containing 5% FBS (Cellutron, m-8031) on glass DAPI (1:1000) to label nuclei. We followed the methods described by coverslips coated with 10 µg/ml fibronectin (BD Biosciences). Isolated Nguyen et al. (2014) to measure cardiomyocyte size. Only round cells triple cardiomyocytes were treated with vehicle control (DMSO), recombinant positive for MHC, WGA and DAPI staining were measured using ImageJ. Neuregulin 1 (NRG1, 50 ng/ml), or a combination of NRG1 and BCI For each ventricle, four confocal images were taken using a 60× objective (5 µM) for 72 h. and the cardiomyocyte size was averaged. For each heart, at least four sections were analyzed and 120-150 cells were measured. The ventricular Immunofluorescence of cultured cardiomyocytes wall thickness was measured in transverse section after AFOG staining The cultured cells were fixed with 4% PFA for 12 min at room temperature. using ImageJ. Four measurements were made for each heart section, and for Samples were blocked with 5% goat serum containing Triton X-100 each heart at least four sections were analyzed. (0.5 μl/ml) for 30 min at room temperature. Following blocking they were DEVELOPMENT

10 STEM CELLS AND REGENERATION Development (2018) 145, dev157206. doi:10.1242/dev.157206 incubated in primary antibody solutions overnight at 4°C: anti-phospho- generation of heritable zebrafish gene mutations using homo- and heterodimeric Histone H3 (rabbit IgG, Millipore, 06-570; 1:200), anti-α-actinin (mouse TALENs. Nucleic Acids Res. 40, 8001-8010. IgG1, Sigma, A7811; 1:200). Cells were then washed with PBS and Cao, J., Navis, A., Cox, B. D., Dickson, A. L., Gemberling, M., Karra, R., Bagnat, M. and Poss, K. D. (2016a). Single epicardial cell transcriptome sequencing incubated with secondary antibodies in PBS for 1 h at room temperature: identifies Caveolin 1 as an essential factor in zebrafish heart regeneration. Alexa Fluor 488 goat anti-rabbit IgG (Thermo Fisher, A11008; 1:500), Development 143, 232-243. Alexa Fluor 594 goat anti-mouse IgG (Thermo Fisher, A11032; 1:500); Cao, N., Huang, Y., Zheng, J., Spencer, C. I., Zhang, Y., Fu, J.-D., Nie, B., Xie, M., followed by nuclear counterstaining in Hoechst solution (Invitrogen; Zhang, M., Wang, H. et al. (2016b). Conversion of human fibroblasts into 1:1000) for 5 min at room temperature. The cells were mounted in 10 μl functional cardiomyocytes by small molecules. Science 352, 1216-1220. mounting medium containing 1% n-propyl gallate (NPG) dissolved in Cermak, T., Starker, C. G. and Voytas, D. F. (2015). Efficient design and assembly of custom TALENs using the Golden Gate platform. Methods Mol. Biol. 1239, glycerol and sealed with nail polish. All imaging was performed on a Nikon 133-159. A1-R confocal using a 40× oil immersion objective followed by analysis Chablais, F. and Jazwinska, A. (2012a). Induction of myocardial infarction in adult + using Nikon Elements software. Scoring of H3P cells was performed in a zebrafish using cryoinjury. J. Vis. Exp. 62, 3666. blinded fashion for quantitative analysis. Rat NRVM were isolated from two Chablais, F. and Jazwinska, A. (2012b). The regenerative capacity of the zebrafish separate litters for the experiment. In each litter, the animals were divided heart is dependent on TGFbeta signaling. Development 139, 1921-1930. into two groups and NRVM were isolated independently from each group. Choi, W.-Y., Gemberling, M., Wang, J., Holdway, J. E., Shen, M.-C., Karlstrom, Isolated cells were plated and treated on duplicate coverslip wells for R. O. and Poss, K. D. (2013). In vivo monitoring of cardiomyocyte proliferation to identify chemical modifiers of heart regeneration. Development 140, 660-666. staining and quantification. Imaging of 20× fields was performed on eight Chung, H. A., Hyodo-Miura, J., Kitayama, A., Terasaka, C., Nagamune, T. and random fields per coverslip (∼1000 cardiomyocytes per field) and then Ueno, N. (2004). Screening of FGF target genes in Xenopus by microarray: averaged between the duplicate coverslips. Final data from isolations from temporal dissection of the signalling pathway using a chemical inhibitor. Genes the two different donor litters were statistically analyzed. Cells 9, 749-761. D’Uva, G., Aharonov, A., Lauriola, M., Kain, D., Yahalom-Ronen, Y., Carvalho, S., Weisinger, K., Bassat, E., Rajchman, D., Yifa, O. et al. (2015). ERBB2 Statistical analysis triggers mammalian heart regeneration by promoting cardiomyocyte Statistical analyses were performed with GraphPad Prism version 7.0. dedifferentiation and proliferation. Nat. Cell Biol. 17, 627-638. Statistical significance was analyzed by unpaired Student’s t-test and one- Ebelt, H., Zhang, Y., Kampke, A., Xu, J., Schlitt, A., Buerke, M., Muller-Werdan, way ANOVA. Data are shown as mean±s.e.m. P<0.05 was considered U., Werdan, K. and Braun, T. (2008). E2F2 expression induces proliferation of significant. Statistical significance between the multiple treatments on rat terminally differentiated cardiomyocytes in vivo. Cardiovasc. Res. 80, 219-226. ̀ cardiomyocytes was assessed by one-way ANOVA followed by post-hoc Ferby, I., Reschke, M., Kudlacek, O., Knyazev, P., Pante, G., Amann, K., Sommergruber, W., Kraut, N., Ullrich, A., Fassler, R. et al. (2006). Mig6 is a Bonferroni correction. negative regulator of EGF receptor-mediated skin morphogenesis and tumor formation. Nat. Med. 12, 568-573. Acknowledgements Fürthauer, M., Reifers, F., Brand, M., Thisse, B. and Thisse, C. (2001). sprouty4 We thank Takis Benos for discussion on RNA-seq data analysis; Donghun Shin and acts in vivo as a feedback-induced antagonist of FGF signaling in zebrafish. Neil Hukriede for critical reading of the manuscript. Development 128, 2175-2186. Fürthauer, M., Lin, W., Ang, S. L., Thisse, B. and Thisse, C. (2002). Sef is a Competing interests feedback-induced antagonist of Ras/MAPK-mediated FGF signalling. Nat. Cell The authors declare no competing or financial interests. Biol. 4, 170-174. Ganapathy, B., Nandhagopal, N., Polizzotti, B. D., Bennett, D., Asan, A., Wu, Y. and Kühn, B. (2016). Neuregulin-1 administration protocols sufficient for Author contributions stimulating cardiac regeneration in young mice do not induce somatic, organ, or Conceptualization: M.A.M., M.S., D.A.Z., K.S.R., B.K., M.T.; Methodology: M.A.M., neoplastic growth. PLoS ONE 11, e0155456. M.S., D.A.Z., K.S.R., G.W.O., B.K., M.T.; Validation: M.A.M., M.T.; Formal analysis: Gemberling, M., Karra, R., Dickson, A. L. and Poss, K. D. (2015). Nrg1 is an M.A.M., M.S., D.A.Z., K.S.R., G.W.O., B.K., M.T.; Investigation: M.A.M., M.S., injury-induced cardiomyocyte mitogen for the endogenous heart regeneration D.A.Z., K.S.R., M.T.; Data curation: M.T.; Writing - original draft: M.A.M.; Writing - program in zebrafish. Elife 4, e05871. review & editing: M.A.M., M.S., D.A.Z., K.S.R., G.W.O., B.K., M.T.; Supervision: González-Rosa, J. M., Peralta, M. and Mercader, N. (2012). Pan-epicardial M.T.; Project administration: M.T.; Funding acquisition: B.K., M.T. lineage tracing reveals that epicardium derived cells give rise to myofibroblasts and perivascular cells during zebrafish heart regeneration. Dev. Biol. 370, Funding 173-186. This work was supported by funding from the American Heart Association Han, P., Zhou, X.-H., Chang, N., Xiao, C.-L., Yan, S., Ren, H., Yang, X.-Z., Zhang, (14GRNT20480183) and the National Institutes of Health (NIH) (R01HD053287). M.-L., Wu, Q., Tang, B. et al. (2014). Hydrogen peroxide primes heart D.A.Z. is supported by a T32 training grant from the NIH (T32 EB001026). This regeneration with a derepression mechanism. Cell Res. 24, 1091-1107. research was supported by the Richard King Mellon Foundation Institute for He, J., Wang, Y., Missinato, M. A., Onuoha, E., Perkins, L. A., Watkins, S. C., St Pediatric Research (Children’s Hospital of Pittsburgh of UPMC, to B.K.) and by a Croix, C. M., Tsang, M. and Bruchez, M. P. (2016). 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