Song et al., 2019 - HDAC1-mediated repression of the retinoic acid-responsive gene ripply3 promotes second heart field development. PLoS Genetics   15:e1008165 Full text @ PLoS Genet.

Fig 1 <italic>Crg</italic> 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 SHF markers of the arterial pole are reduced in <italic>crg</italic> 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 SHF-derived VC and smooth muscle development is impaired in the <italic>crg</italic> 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 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 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 <italic>Ripply3</italic> is expanded anteriorly into <italic>nkx2</italic>.<italic>5+</italic> cells in <italic>crg</italic> 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 Loss of <italic>ripply3</italic> in <italic>crg</italic> 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. 4 ZFIN is incorporating published figure images and captions as part of an ongoing project. Figures from some publications have not yet been curated, or are not available for display because of copyright restrictions.

EXPRESSION / LABELING:
Antibody:
Fish:
Stage Range: Prim-15 to Day 4
PHENOTYPE:
Fish:
Observed In:
Stage Range: Prim-15 to Day 4

Fig. 9 ZFIN is incorporating published figure images and captions as part of an ongoing project. Figures from some publications have not yet been curated, or are not available for display because of copyright restrictions.

Fig. S1

CM specification and early differentiation are overtly unaffected in crg mutants.

(A-D) ISH for CM specification markers gata4 and hand2 at the 8s stage. n = 5 WT and n = 5 crg mutants embryos examined. (E,F) ISH for the CM differentiation marker myl7 at the 20s stage. n = 10 WT and n = 5 crg mutants embryos examined. Views are dorsal with anterior up.

Fig. S2

The SHF marker mef2cb has increased expression adjacent to differentiated CMs at the arterial pole of the heart in crg mutants.

(A-B”) Two-color FISH for myl7 and mef2cb in WT sibling and crg mutant embryos at 30 hpf. Brackets in A and B indicate mef2cb at the arterial pole of in WT and crg mutant hearts, respectively. n = 5 WT and n = 5 crg mutants embryos examined. (C) RT-qPCR for the SHF marker mef2cb from embryos at 36 hpf.

Fig. S3

Later-differentiating VCs are reduced in crg mutants.

(A-B”) Images of hearts from photoconverted WT sibling and crg mutant myl7:Kaede embryos at 48 hpf following photoconversion at 36 hpf. The arterial poles (brackets) are to the right. (C) Quantification of the area of later-differentiating VCs (Yellow+/Purple-). (n = 11 for WT and crgmutants).

Fig. S5

Hdac1 depletion and treatment with TSA produce similar phenotypes as crgmutants.

(A,B) Control and Hdac1-depleted embryos at 48 hpf. Lateral views with anterior to the left. (C,D) Hearts from control and Hdac1-depleted myl7:NLS-DsRed2 embryos at 48 hpf. Frontal views. Purple alone indicates ventricle. Yellow indicates atrium. Arrows indicate arterial pole of the ventricle. (E) Quantification of CMs in the atria and ventricles of control and Hdac1-depleted myl7:NLS-DsRed2 embryos at 48 hpf (n = 19 for control and Hdac1-depleted embryos). (F,G) Control and TSA-treated embryos at 48 hpf. Lateral views with anterior to the left. (H,I) Hearts from control and TSA-treated myl7:NLS-DsRed2 embryos at 48 hpf. Frontal views. Purple alone indicates ventricle. Yellow indicates atrium. Arrows indicate arterial pole of the ventricle. (J) Quantification of CMs in the atria and ventricles of control and TSA-treated myl7:NLS-DsRed2 embryos at 48 hpf (n = 14 for both control and TSA-treated embryos).

Fig. S7

Expression of nkx2.5 and ripply3 in WT and crg mutants.

(A-D”) Confocal images of two-color FISH for nkx2.5 and ripply3 in WT and crg mutant embryos at 30 and 36 hpf. Images are dorsal views with anterior up. Insets in A-D indicate lateral views of the confocal images. n = 10 WT and n = 9 crg mutants embryos for 30 hpf and n = 4 WT and n = 4 crg mutants embryos for 36 hpf examined. Scale bar is 100 μm.

Fig. S9

Generation and validation of the heat-shock inducible GFP-ripply3 transgenic line.

(A) Schematic of the heat-shock inducible hsp70l:GFP-ripply3 transgene. (B-E) Heat-shock induction of GFP-ripply3 at 24 hpf. Control embryos are heat-shocked non-transgenic siblings. (F,H) Control and hsp70l:GFP-ripply3 embryos at 48 hpf following heat-shock at 10 hpf. Lateral views with anterior to the left. (G,I) Hearts from control and hsp70l:GFP-ripply3myl7:NLS-DsRed2 embryos at 48 hpf following heat-shock at 10 hpf. Frontal views. Purple alone indicates ventricle. Yellow indicates atrium. Arrows indicate arterial pole of the ventricle. (J) Quantification of CMs from control and hsp70l:GFP-ripply3myl7:NLS-DsRed2 embryos at 48 hpf following heat-shock at 10 hpf (n = 25 for control and GFP-ripply3+). (K,M) Control and ripply3 mRNA-injected embryos at 48 hpf. Lateral views with anterior to the left. (L,N) Hearts from control and ripply3 mRNA-injected myl7:NLS-DsRed2 embryos at 48 hpf. Frontal views. Purple alone indicates ventricle. Yellow indicates atrium. Arrows indicate arterial pole of the ventricle. (O) Quantification of CMs from control and ripply3 mRNA-injected myl7:NLS-DsRed2 embryos at 48 hpf (n = 21 for control and ripply3 mRNA-injected embryos).

Fig. S10

Generation of ripply3 mutants.

(A) Schematic of the ripply3 exons and the gRNA targeting exon of the ripply3 gene. (B) The ripply3 mutant allele used deletes 61bp including the start codon. (C,D) WT sibling and ripply3mutants at 48 hpf. (E,F) Ripply3 transcripts are not detectable in ripply3 mutants at 30 hpf. Arrows indicate posterior pharyngeal region where ripply3 is expressed in WT embryos. Views are dorsal with anterior up. (G) RT-qPCR for ripply3 in ripply3 mutants at 36 hpf indicates the transcripts are essentially undetectable. Primers used do not bind within the deleted region. (H) Quantification of atrial CMs in crgwt+hetripply3wt+hetcrgwt+hetripply3-/-crg-/-ripply3wt+het, and crg-/-ripply3-/- embryos at 48 hpf (n = 37 for crgwt+hetripply3wt+het, n = 10 for crgwt+hetripply3-/-, n = 48 for crg-/-ripply3wt+het, n = 18 for crg-/-ripply3-/-).

Acknowledgments:
ZFIN wishes to thank the journal PLoS Genetics for permission to reproduce figures from this article. Please note that this material may be protected by copyright. Full text @ PLoS Genet.