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, 15 (5), e1008165
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HDAC1-mediated Repression of the Retinoic Acid-Responsive Gene ripply3 Promotes Second Heart Field Development

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HDAC1-mediated Repression of the Retinoic Acid-Responsive Gene ripply3 Promotes Second Heart Field Development

Yuntao Charlie Song et al. PLoS Genet.

Abstract

Coordinated transcriptional and epigenetic mechanisms that direct development of the later differentiating second heart field (SHF) progenitors remain largely unknown. Here, we show that a novel zebrafish histone deacetylase 1 (hdac1) mutant allele cardiac really gone (crg) has a deficit of ventricular cardiomyocytes (VCs) and smooth muscle within the outflow tract (OFT) due to both cell and non-cell autonomous loss in SHF progenitor proliferation. Cyp26-deficient embryos, which have increased retinoic acid (RA) levels, have similar defects in SHF-derived OFT development. We found that nkx2.5+ progenitors from Hdac1 and Cyp26-deficient embryos have ectopic expression of ripply3, a transcriptional co-repressor of T-box transcription factors that is normally restricted to the posterior pharyngeal endoderm. Furthermore, the ripply3 expression domain is expanded anteriorly into the posterior nkx2.5+ progenitor domain in crg mutants. Importantly, excess ripply3 is sufficient to repress VC development, while genetic depletion of Ripply3 and Tbx1 in crg mutants can partially restore VC number. We find that the epigenetic signature at RA response elements (RAREs) that can associate with Hdac1 and RA receptors (RARs) becomes indicative of transcriptional activation in crg mutants. Our study highlights that transcriptional repression via the epigenetic regulator Hdac1 facilitates OFT development through directly preventing expression of the RA-responsive gene ripply3 within SHF progenitors.

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Crg mutants have a specific deficit in VCs.
(A-B) WT sibling and crg mutants at 48 hpf. Lateral views with anterior to the left. Arrow in B indicates pericardial edema. (C-D) Hearts from WT sibling and crg mutant myl7:NLS-DsRed2 embryos at 48 hpf. Frontal views. Purple alone indicates ventricle. Yellow indicates atrium. Arrows indicate arterial pole of the ventricle. (E-G) Quantification of CMs in the atria and ventricles of WT sibling and crg mutant myl7:DsRed2-NLS embryos at 36, 48, and 72 hpf. For 36 hpf, n = 14 for WT and crg mutants. For 48 hpf, n = 20 for WT and crg mutants. For 72 hpf, n = 17 for WT and crg mutants. Asterisk in all graphs indicates p<0.05 as determined by Student’s t-test. Error bars for all graphs indicate s.e.m.
Fig 2
Fig 2. SHF markers of the arterial pole are reduced in crg mutants.
(A,B) RT-qPCR for the pan-cardiac differentiation marker myl7 and SHF marker ltbp3 from embryos at 36 and 48 hpf. (C-D”) Two-color FISH for myl7 and ltbp3 in WT sibling and crg mutant embryos at 30 hpf. Brackets in C and D indicate presence and absence of ltbp3 at the arterial pole of WT and crg mutant hearts, respectively. n = 5 WT and n = 5 crg mutants embryos were examined.
Fig 3
Fig 3. SHF-derived VC and smooth muscle development is impaired in the crg mutants.
(A-B”) Representative images of hearts from photoconverted WT sibling and crg mutant myl7:NLS-KikGR embryos at 48 hpf. The arterial poles (brackets) are to the right. (C) Quantification of later-differentiating VCs (Yellow+/Purple- cells) (n = 10 for WT and crg mutants). (D,E) Confocal images of IHC for MHC and Elnb in WT and crg mutant embryos at 72 hpf. n = 10 WT and n = 10 crg mutants embryos examined. (F,G) Confocal images of DAF-2DA staining coupled with IHC for Vmhc in WT sibling and crg mutant embryos at 96 hpf. n = 10 WT and n = 10 crg mutants embryos examined. Images in D-G are frontal views with anterior up. Arrows indicate Elnb and DAF-2DA staining of the bulbous arteriosus.
Fig 4
Fig 4. Crg mutants contain a loss of function mutation in hdac1.
(A) Summary of the region containing the crg mutation from positional cloning. Fractions indicate recombinants for SSLPs. (B) Reads from RNA-seq data indicating the mutation and effect on the hdac1 exon 7. Boxes indicate mutation with T to A (green bar) change affecting splice donor site. (C) Schematic indicating the skipping of exon 7 or the retention of intron 6 and 7 found in hdac1 transcripts from crg mutants. (D) Predicted consequences on the proteins generated from the improper splice forms of the hdac1 transcripts, if they were translated. The red bar indicates the 32 amino acid deletion predicted to occur from the transcript that skips exon 7 in crg mutants. The gray bar indicates a 9 amino acid extension after going out of frame at amino acid 213 for the hdac1 transcript that retains introns 6 and 7 in crg mutants. (E) Western blot for Hdac1 protein in WT sibling and crg mutants.
Fig 5
Fig 5. Hdac1 is required for the proliferation of SHF progenitors.
(A-B”‘) Confocal images of IHC for hearts and Nkx2.5+ SHF progenitors in WT sibling and crg mutant embryos at 33 hpf. Nkx2.5+ (green), PHH3 (blue) and MHC (purple). Outline in A and B indicates Nkx2.5+/MHC- SHF. Arrows indicate Nkx2.5+/MHC-/PHH3+ cells. Yellow arrow indicates Nkx2.5+/MHC-/pHH3+ cell of the higher magnification inset. (A”“and B”“) Schematic indicating IHC from A and B. Green indicates Nkx2.5+/MHC- cells. Blue indicates Nkx2.5+/MHC-/PHH3+ cells. Purple indicates MHC+ cells. Anterior is up in A-B”“. (C) Quantification of SHF progenitors (Nkx2.5+/MHC-). (D) Percentage of PHH3+ SHF progenitors. For C and D, n = 14 for WT and n = 15 crg mutants. (E) RT-qPCR for cdkn1a from sorted nkx2.5:ZsYellow+ cells.
Fig 6
Fig 6. Hdac1 is required both cell and non-cell autonomously to promote VCs.
(A) Schematic of the blastula cell transplantation strategy. Vmhc (purple) indicates ventricle. Arrows indicate myl7:GFP+ cells (yellow). (B) Frequency of host WT embryos with WT and Hdac1-depleted donor GFP+ CMs at 48 hpf. n = 245 WT donor into WT host transplants; n = 244 Hdac1 deficient into WT host transplants. (C) Quantification of donor CMs (GFP+) found in host embryos hearts from transplants (n = 19 for WT in WT and n = 18 Hdac1-depleted in WT). (D) Frequency of host WT and Hdac1-depleted embryos with WT donor GFP+ CMs at 48 hpf. n = 169 WT donor into WT host transplants; n = 177 WT donor into Hdac1-depleted host transplants. (E) Quantification of donor CMs (GFP+) found in host embryos hearts from transplants (n = 18 for WT in WT and n = 12 WT in Hdac1-depleted). Fisher exact test was used to compare significance of frequencies in B and D.
Fig 7
Fig 7. Ripply3 is expanded anteriorly into nkx2.5+ cells in crg mutants.
(A-D) RT-qPCR for ripply3 expression in whole embryos at 48 hpf and sorted nkx2.5:ZsYellow+ cells at 28 hpf. (E-H”) Confocal images of two-color FISH for nkx2.5 and ripply3 in WT and crg mutant embryos at 18 and 24 hpf. Images are dorsal views with anterior up. Insets in F-H indicate lateral views of the confocal images. Bracket in E indicates space between posterior nkx2.5 and anterior ripply3 domains. Arrow in F indicates border between nkx2.5 in pharyngeal mesoderm and ripply3 in pharyngeal endoderm. Brackets with arrows in G and H indicate overlap in nkx2.5 and ripply3 domains in crg mutant embryos. n = 22 WT and n = 5 crg mutants embryos for 18 hpf and n = 19 WT and n = 11 crg mutants embryos for 24 hpf examined. Scale bars in E and G are 50 μm. Scale bars in F and H are 100 μm.
Fig 8
Fig 8. Loss of ripply3 in crg mutants partially restores VC number.
(A,B) Control and transgenic hsp70l:GFP-ripply3 embryos at 48 hpf following heat-shock at 20 hpf. (C,D) Hearts from control and hsp70l:GFP-ripply3 transgenic embryos with the myl7:DsRed2-NLS transgene at 48 hpf following heat-shock at 20 hpf. Purple alone indicates ventricle. Yellow indicates atrium. Arrows indicate arterial pole of the ventricle. (E) Quantification of CMs in control and hsp70l:GFP-ripply3 transgenic embryos at 48 hpf following GFP-ripply3 induction at 20 hpf (n = 18 for control, n = 29 for GFP-ripply3+). (F) Quantification of VCs in crgwt+het; ripply3wt+het, crgwt+het; ripply3-/-, crg-/-; ripply3wt+het, and crg-/-; ripply3-/- embryos at 48 hpf (n = 37 for crgwt+het; ripply3wt+het, n = 10 for crgwt+het; ripply3-/-, n = 48 for crg-/-; ripply3wt+het, n = 18 for crg-/-; ripply3-/-). (G) Quantification of VCs in WT (crgwt+het—uninjected) sibling, crgwt+het–Tbx1 depleted, crg-/-uninjected, and crg-/-Tbx1-depleted embryos at 48 hpf (n = 11 for crgwt+hetcontrol uninjected, n = 10 for crgwt+het–Tbx1-depleted, n = 10 for crg-/-control uninjected, n = 10 for crg-/-Tbx1-depleted). For all CM quantification, embryos contained the myl7:DsRed2-NLS transgene.
Fig 9
Fig 9. Hdac1 promotes transcriptional repression at RAREs within the ripply3 promoter.
(A) Schematic of DR1 and DR4 RAREs within the ripply3 promoter. (B) EMSAs with RARs for the ripply3 DR1 and DR4 sites. The positive control is a DR5 site found in promoters of other direct targets [133]. (C) ChIP-qPCR for induced GFP-VP16-RARab at the DR1 and DR4 sites. (D-F) ChIP-qPCR for HDAC1, H3K27ac, H3K27me3 at the ripply3 DR1 and DR4 sites in WT sibling and crg mutants. Fold enrichment for C-F was normalized versus IgG pull-down. (G) Deletions of the ripply3 promoter DR1 and DR4 sites using multiplexed gRNAs and Cas9. Asterisks indicate deletions, which were confirmed with Sanger sequencing. (H) RT-qPCR for ripply3 expression in WT sibling and crg mutant embryos with ripply3 DR1 and DR4 promoter deletions at 36 hpf. The control gRNAs target a region ~100kb away from the ripply3 promoter. (I) Model depicting Hdac1 and RAR function regulating ripply3 in SHF development.

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