Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 26 (3), 720-732.e4

TETs Regulate Proepicardial Cell Migration Through Extracellular Matrix Organization During Zebrafish Cardiogenesis

Affiliations

TETs Regulate Proepicardial Cell Migration Through Extracellular Matrix Organization During Zebrafish Cardiogenesis

Yahui Lan et al. Cell Rep.

Abstract

Ten-eleven translocation (Tet) enzymes (Tet1/2/3) mediate 5-methylcytosine (5mC) hydroxylation, which can facilitate DNA demethylation and thereby impact gene expression. Studied mostly for how mutant isoforms impact cancer, the normal roles for Tet enzymes during organogenesis are largely unknown. By analyzing compound mutant zebrafish, we discovered a requirement for Tet2/3 activity in the embryonic heart for recruitment of epicardial progenitors, associated with development of the atrial-ventricular canal (AVC). Through a combination of methylation, hydroxymethylation, and transcript profiling, the genes encoding the activin A subunit Inhbaa (in endocardium) and Sox9b (in myocardium) were implicated as demethylation targets of Tet2/3 and critical for organization of AVC-localized extracellular matrix (ECM), facilitating migration of epicardial progenitors onto the developing heart tube. This study elucidates essential DNA demethylation modifications that govern gene expression changes during cardiac development with striking temporal and lineage specificities, highlighting complex interactions in multiple cell populations during development of the vertebrate heart.

Keywords: DNA demethylation; endocardium; genetics; heart development.

Figures

Figure 1.
Figure 1.. tet2 and tet3 Have Overlapping Functions in PE Recruitment to the Heart
(A) WISH for PE marker wt1 at 40 hpf and 54 hpf. Arrows indicate PE cells with wt1 transcripts. (B) Lateral view of hearts showing GFP-labeled PE and epicardium in 46-hpf, 52-hpf, and 72-hpf larvae carrying the Tg(tcf21:NLS-EGFP) transgene. White arrow indicates the extracellular matrix bridge between AVC and the pericardial wall. (C) Ventral view of hearts showing GFP-labeled PE and epicardium in 48-hpf and 72-hpf sibling or tet2/3DM larvae carrying the Tg(tcf21:NLS-EGFP) transgene. White arrows indicate tcf21+ PE and epicardial cells located on the heart. Yellow arrows indicate tcf21+ PE and epicardial cells located on the yolk sac. Graph indicates the total number of PE and epicardial cells in 48-hpf or 72-hpf sibling and tet2/3DM larvae and the number of PE and epicardial cells located on heart or yolk sac at 72-hpf sibling and tet2/3DM larvae. (D) The PE migration defect is partially rescued by TET2 mRNA injection or 5-aza treatment. GFP-labeled PE and epicardium in 4-dpf sibling, tet2/3DM, and tet2/3DM injected with wild-type hTET2 mRNA, mutant hTET2 mRNA, or tet2/3DM exposed to 75 μM 5-aza larvae carrying the Tg(tcf21:NLS-EGFP) transgene. Graph indicating the number of epicardial cells located on the heart at 4 dpf is shown. Scale bars: (A) 50 μm; (B–D) 100 μm. **p < 0.01; ***p < 0.001; ns indicates not significant. Data are presented as mean ± SD derived from at least three independent biological replicates.
Figure 2.
Figure 2.. Epicardial Cells from Donor Hearts Do Not Migrate onto tet2/3DM Recipient Hearts
(A) Schematic of epicardial cell migration assay. Wild-type tcf21:DsRed donor hearts (isolated at 72 hpf) were co-cultured with either wild-type or tet2/3DM myl7:GFP recipient hearts (isolated at 48 hpf) for one week and then confocal images taken. (B)Confocal images showed epicardial cells from wild-type donor heart can migrate onto wild-type recipient hearts. In 30 pairs of co-cultured hearts, 6 of recipient hearts were observed having epicardial cells migrated from donor hearts. (C) Confocal images showed epicardial cells from wild-type donor heart failed to migrate onto tet2/3DM recipient hearts. In 60 pairs of co-cultured hearts, none of the recipient hearts were observed having epicardial cells from donor hearts. Scale bar: 50 μm.
Figure 3.
Figure 3.. Hypermethylation and Deregulation of Cardiac Developmental Genes in tet2/3DM Larvae
(A) Enrichment of various regulatory regions in 5hmC peak and hyper-DMR by 5hmC chromatin immunoprecipitation (ChiP)-sequencing and ERRBS. (B) Total number of hyper-and hypo-DMR (tet2/3DM heart versus wild-type heart) by ERRBS. (C) Overlapping locations between hyper-DMRs and 5hmC peaks. (D) Pathway enrichment analysis for genes associated with hyper-DMR. (E) Scatterplot of RNA sequencing data illustrating transcriptional changes in 48-hpf tet2/3DM heart as compared to wild-type heart. (F) Pathway enrichment analysis for downregulated genes in 48-hpf tet2/3DM heart as compared to wild-type heart by RNA sequencing.
Figure 4.
Figure 4.. AVC Development Shows Disruption in tet2/3DM Larvae
(A) GFP-labeled AVC endocardium represents Wnt activity in sibling heart, but not tet2/3DM heart. Hearts were dissected from 48-hpf larvae carrying the Tg(7xTCF-Xla.Siam:GFP) transgene. White arrows indicate AVC endocardial cells with Wnt activity. (B) WISH forAVC markers bmp4 and has2 at 48 hpf. (C) GFP-labeled endocardium represents AV valve formation in 4-dpf sibling, but not tet2/3DM larvae carrying the Tg(kdrl:EGFP) transgene. White arrows indicate the AV valve. Bottom left images show higher magnification views of AV valve regions. (D) WISH for AVC marker bmp4 in 48 hpf and confocal imaging for GFP-labeled PE and epicardium in 72-hpf control and IWR-1-treated larvae. Scale bars: 50 μm.
Figure 5.
Figure 5.. Tet2/3-Dependent Aberrant Promoter Hypermethylation and Deregulation of inhbaa and sox9b Leads to AVC and PE Migration Defects
(A) RT-PCR analysis of inhbaa and sox9b transcripts in 48-hpf embryonic heart. (B) DNA methylation status of inhbaa in 48-hpf isolated heart or isolated AVC. Diagram indicates inhbaa locus and the associated regulatory regions. Gray box represents 5hmC peak. Black box represents the coding sequence. White box represents hyper-DMR identified by ERRBS. Profiles of 5mC + 5hmC in hyper-DMR region were validated by bisulfite sequencing. n = 4 per condition. (C) DNA methylation status of sox9b in 48 hpf isolated heart. Diagram indicates sox9b locus and the associated regulatory regions. Gray box represents 5hmC peak. Black box represents the coding sequence. White box represents hyper-DMR. Profiles of 5mC + 5hmC in hyper-DMR region were validated by bisulfite sequencing. n = 4 per condition. (D) WISH for AVC markers bmp4 and has2 at 48-hpf sibling, tet2/3DM, and tet2/3DM injected with inhbaa mRNA or sox9b mRNA larvae. Scale bar: 50 μm. (E) Number of epicardial cells on the heart of 4-dpf sibling, tet2/3DM, and tet2/3DM injected with inhbaa mRNA, sox9b mRNA, or sox9b combined with inhbaa mRNA larvae carrying the Tg(tcf21:NLS-EGFP) transgene. Data are presented as the mean ± SD. The significance is indicated as *p < 0.05; **p < 0.01; ***p < 0.001; ns indicates not significant.
Figure 6.
Figure 6.. Tet2/3 Regulate PE Migration through Extracellular Matrix Organization
(A) WISH for ECM constituent gene vcana at 48 hpf. Black arrows indicate AVC-specific expression of vcana in sibling, but not tet2/3DM heart. (B) GFP-labeled PE and epicardium in 4-dpf larvae carrying the Tg(tcf21:NLS-EGFP) transgene. Sibling, tet2/3DM, and tet2/3DM injected with has2 mRNA and tet2/3DM injected with low concentration (0.5 mg/mL) collagen larvae were shown in lateral views. Sibling and tet2/3DM injected with high concentration (8 mg/mL) collagen larvae were shown in ventral views to represent collagen aggregate and heart clearly. The heart is outlined with white dashed line. The collagen aggregate is outlined with red dashed line. (C) Number of epicardial cells on the heart of 4-dpf sibling, tet2/3DM, and tet2/3DM injected with sox9b mRNA or tet2/3DM injected with low concentration (0.5 mg/mL) collagen larvae carrying the Tg(tcf21:NLS-EGFP) transgene. Numbers data are presented as the mean ± SD derived from 3 independent biological replicates. (D) Working model shows Tet2/3-dependent demethylation regulates the expression of inhbaa and sox9b, which subsequently regulate AVC ECM organization and PE migration. Scale bars: 50 μm. The significance is indicated as *p < 0.05; **p < 0.01; ***p < 0.001.

Similar articles

See all similar articles

Cited by 3 articles

References

    1. Ahuja S, Dogra D, Stainier DYR, and Reischauer S (2016). Id4 functions downstream of Bmp signaling to restrict TCF function in endocardial cells during atrioventricular valve development. Dev. Biol. 472, 71–82. - PubMed
    1. Akalin A, Garrett-Bakelman FE, Kormaksson M, Busuttil J, Zhang L, Khrebtukova I, Milne TA, Huang Y, Biswas D, Hess JL, et al. (2012). Base-pair resolution DNA methylation sequencing reveals profoundly divergent epigenetic landscapes in acute myeloid leukemia. PLoS Genet. 8, e1002781. - PMC - PubMed
    1. Anelli V, Villefranc JA, Chhangawala S, Martinez-McFaline R, Riva E, Nguyen A, Verma A, Bareja R, Chen Z, Scognamiglio T, et al. (2017). Oncogenic BRAF disrupts thyroid morphogenesis and function via twist expression. eLife 6, e20728. - PMC - PubMed
    1. Azhar M, Schultz, Jel J, Grupp I, Dorn GW 2nd, Meneton P, Molin DG, Gittenberger-de Groot AC, and Doetschman T (2003). Transforming growth factor beta in cardiovascular development and function. Cytokine Growth Factor Rev. 14, 391–407. - PMC - PubMed
    1. Backs J, and Olson EN (2006). Control of cardiac growth by histone acetylation/deacetylation. Circ. Res. 98, 15–24. - PubMed

Publication types

LinkOut - more resources

Feedback