(a) Schematic of cryoinjury procedure on adult TgBAC(nppa:mCitrine) fish and immunohistochemistry on section of injured TgBAC(nppa:mCitrine) heart 7 dpi. Overview image on left and zoom-in of boxed …
Single-cell mRNA sequencing data from cryoinjured TgBAC(nppa:mCitrine) zebrafish hearts at 7 dpi.
Raw reads are represented per gene (rows) and single cell (columns).
List of the clusters in the adult injured heart (raw data see SD1), as identified by StemID and the associated cells per cluster.
List of differentially expressed genes between cardiomyocytes clusters 2 and 7 of the adult heart dataset.
Only genes with a p-value<0.05 are listed. Per gene, the log2 fold change, adjusted p-value (padj) and associated gene name are given. GO-terms for genes upregulated between clusters 2 and 7 (p<0.01) are listed in separate excel sheets.
(a) Design of the bacterial artificial chromosome (BAC) used for generation of the transgenic line TgBAC(nppa:mCitrine). (b) Transgenic mCitrine expression in the heart in relation to RFP expression …
tSNE maps visualizing log2-transformed read-counts of genes with high expression in cardiomyocytes (mt-co1, tnnc1a, tnnt2a), in fibroblasts (fn1b), in endothelial cells (cdh5) and immune cells (ctsd)…
(a) tSNE map of the cardiomyocyte clusters derived from the single-cell mRNA-sequencing. (b) mCitrine fluorescence levels of the nppa:mCitrine FACS sorted cells from injured hearts that were used …
(a) In situ hybridizations for border zone genes nppb, mustn1, nppa and vmhc in zebrafish hearts at seven dpi compared to uninjured hearts. (b) In situ hybridizations for glycolysis genes hk1 and ldh…
(a) Cartoon to illustrate the experimental procedure for single cell analysis of embryonic and adult cardiac cells. (b) tSNE map of combined adult (red) and embryonic datasets (light blue). (c) tSNE …
Single-cell mRNA sequencing data from Tg(cmlc2:GFP) zebrafish hearts at 2 days post fertilization.
Raw reads are represented per gene (rows) and single cell (columns).
List of the clusters from the embryonic heart data (SD5) identified by StemID and the associated cells for each cluster.
Combined single-cell mRNA sequencing data from embryonic and adult hearts.
Raw reads represented per gene (rows) and single cell (columns).
List of pairwise differentially expressed genes between all cardiomyocyte clusters from the combined embryonic and adult datasets.
Only genes with a p-value<0.01 are listed. Per gene, the log2 fold change, adjusted p-value (padj) and associated gene name are given. GO-terms for genes upregulated between clusters 5 and 6 (p<0.01) are listed in separate excel sheets.
(a) Bar plot of StemID scores for the cardiomyocyte clusters (clusters #2, 1, 4 and 7) calculated by the formula: number of significant links for each cluster multiplied by the median transcriptome …
List of genes that are differentially upregulated in the respective modules identified in the pseudo timeline analysis (related to Figure 3c).
In situ hybridization for the glycolysis gene hexokinase1 (a,b) and the embryonic cardiac gene myomesin1b (c,d) on sections of injured zebrafish hearts at 3 dpi (a,c) and 7 dpi (b,d). While hk1 …
(a) Succinate dehydrogenase (SDH) enzyme staining on a seven dpi heart section with injury area separated by dashed line. Quantification of SDH activity in remote zone (RZ), border zone (BZ) and …
(a) Plot showing differentially expressed genes between cluster 7 versus cluster 2 cells. Differentially expressed genes (p-value<0.05) are highlighted in red (upregulated in cluster 7 cells) and …
(a) Experimental design for the 2-DG injections to inhibit glycolysis in injured zebrafish hearts. (b) Confocal image of injured zebrafish hearts at seven dpi either injected with PBS or 2-DG …
(a) Cartoon showing experimental procedure to induce cardiomyocyte specific Nrg1 expression in zebrafish. Panels show in situ hybridization for hexokinase 1 (hk1) and lactate dehydrogenase a (ldha) …
Oligonucleotide sequences for real-time PCR analysis.
(a) Overview picture of phd3:GFP heart 7dpi co-stained for expression of ldha by fluorescent ISH. (b) Zoom-ins of trabecular border zone cardiomyocytes. Note high ldha expression and low expression …
(a) Cartoon showing the experimental procedure to analyse the effects of glycolysis inhibitors (2-DG and lonidamine) on cardiomyocyte proliferation. (b) Immunofluorescence analysis on P7 cardiac …
(a) Cartoon showing experimental procedure. Myocardial infarction (MI) was induced by left anterior descending coronary artery ligation at the same day when Dox was removed to induce cardiomyocyte …
(A) Distribution of reads per cell. Green line represents 3,500 reads, red line indicates the mean number of reads per cell (10,443). (B) tSNE maps generated with different read cutoffs. Read …
Representative GO-terms for cardiomyocyte clusters. GO-terms in upper table are obtained from differentially expressed genes between adult clusters 2 and 7 (p<0.01). Lower table GO-terms are …
Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
---|---|---|---|---|
Strain, strain background (Danio rerio) | Tupfel Long Fin (TL) | ZIRC | ZDB-GENO-990623–2 | |
Genetic reagent (Danio rerio) | Tg(myl7:dsRED)s879Tg | Chi et al., 2008 | ZDB-FISH-150901–3078 | |
Genetic reagent (Danio rerio) | Tg(myl7:GFP)twu34Tg | Huang et al., 2003 | ZDB-FISH-150901–212 | |
Genetic reagent (Danio rerio) | Tg(phd3:GFP)sh144 | Santhakumar et al., 2012 | ZDB-FISH-150901–26851 | |
Genetic reagent (Danio rerio) | Tg(gata4:EGFP)ae1 | Heicklen-Klein and Evans, 2004 | ZDB-FISH-150901–14762 | |
Genetic reagent (Danio rerio) | TgBAC(nppa:mCitrine) | This paper | More info on generation of this line can be obtained from the Materials and methods section ‘Transgenic zebrafish lines and cryoinjury’. | |
Cell line (Mus musculus) | TetRE-caErbb2 X MYH6-tTA X MYH6-cre ROSA26-tdTomato | D'Uva et al., 2015 | More info on how to obtain primary cell line can be obtained from Materials and methods sections ‘Transgenic mouse lines and animal procedures’ and ‘Pharmacological inhibition of glycolysis’. | |
Biological sample (Mus musculus) | TetRE-caErbb2 X MYH6-tTA hearts | D'Uva et al., 2015 from: Xie et al., 1999 and Yu et al., 1996. | The Jackson Laboratory, stock no. 010577; | |
Biological sample (Danio rerio) | Tg(cmlc2:CreER)pd10 x Tg(β-act2:BSNrg1)pd107hearts | Gemberling et al., 2015 | ZDB-FISH-150901–25249 x ZDB-FISH-150901–25354 | |
Biological sample (Danio rerio) | Tg(gata4:EGFP)ae1hearts | Lin et al. 2009 | ZDB-FISH-150901–14762 | |
Biological sample (Danio rerio) | Tg(phd3:GFP)sh144hearts | Santhakumar et al., 2012 | ZDB-FISH-150901–26851 | |
Antibody | Anti-AuroraB kinase (mouse monoclonal) | BD Transduction Laboratories | #611082, RRID:AB_2227708 | 1:200 |
Antibody | Anti-Ki67 (Rabbit monoclonal) | Cell Marque | #275R | 1:200 |
Antibody | Anti-MCT4 (rabbit polyclonal) | Santa Cruz | #SC50329, RRID:AB_2189333 | 1:200 |
Antibody | Anti-PCNA (mouse monoclonal) | DAKO | #M0879, RRID:AB_2160651 | 1:800 |
Antibody | Anti-GFP (chicken polyclonal) | Aves | #GFP-1010, RRID:AB_2307313 | 1:1000 |
Antibody | Anti-Mef2C (rabbit polyclonal) | Santa Cruz/Biorbyt | #SC313, RRID:AB_631920 / #orb256682 | Both 1:1000 |
Chemical compound, drug | 2-Deoxyglucose | Sigma-Aldrich | #D6134 | 1 mg/g or 3 mM |
Chemical compound, drug | Lonidamine | Sigma-Aldrich | L4900 | 80 uM |
Chemical compound, drug | 2NBDG | Caymanchem | #11046 | 400 uM |
Chemical compound, drug | AG1478 | Selleck Chemical | S2728 | 5M, from 10 mM stock in DMSO |
Chemical compound, drug | Doxycycline | Harlan Laboratories | TD02503 | |
Chemical compound, drug | Trizol | Life technologies | #15596026 | |
Chemical compound, drug | Fast SYBR Green Master Mix | Applied Biosystems | #4385612 | |
Chemical compound, drug | Collagenase type II | Gibco | 17101015 | 0.1% |
Chemical compound, drug | TrypLE Express Enzyme (1x), phenol red | Gibco | 12605036 | |
Software, algorithm | FIJI | Schindelin et al., 2012 | Version 2.0.0, RRID:SCR_002285 | |
Software, algorithm | RaceID2/StemID | Grün, D. et al. De Novo Prediction of Stem Cell Identity using Single-Cell Transcriptome Data. Cell Stem Cell 19, 266–277 (2016). | RRID:SCR_017242 | |
Software, algorithm | Rstudio | Rstudio | RRID:SCR_000432 | Version 1.2.1335 |
Software, algorithm | Imaris | Bitplane | RRID:SCR_0007370 | V9.3.1 |
Software, algorithm | Gene Set Enrichment Analysis | Genepattern, Broad Institute | RRID:SCR_003199 | # of permutations = 1000 |
Commercial assay, kit | Superscript III First strand synthesis system | Thermo Fisher Scientific | #18080051 | Input 200 ng RNA |
Commercial assay, kit | MiRNeasy | Qiagen | 217004 | |
Commercial assay, kit | High capacity cDNA Reverse Transcription kit | Applied Biosystems | 4374966 | Input 1 ug RNA |
Commercial assay, kit | Neonatal Dissociation kit | Miltenyi Biotec | 130-098-373 | |
Commercial assay, kit | TruSeq small RNA primers | Illumina | 20005613 |