FIGURE SUMMARY
Title

Sin3a associated protein 130 kDa, sap130, plays an evolutionary conserved role in zebrafish heart development

Authors
DeMoya, R.A., Forman-Rubinsky, R.E., Fontaine, D., Shin, J., Watkins, S.C., Lo, C.W., Tsang, M.
Source
Full text @ Front Cell Dev Biol

sap130a and sap130b have non-overlapping functions in the zebrafish heart (A, B) A simple distance matrix phylogeny tree of Sap130a and Sap130b in broad or teleost specific contexts (C) Schematic of Sap130a and Sap130b protein sequences from the UniProt database highlighting the conserved regions and predicted mutant proteins. Unorganized sequence in pink, C-terminal conserved domain in blue, which contains the binding domain for SIN3A and HDAC1 (D, E) Representative images of Tg(myl7:EGFP), MZsap130a;Tg(myl7:EGFP) and MZsap130b;Tg(myl7:EGFP) mutant hearts at 48hpf. V and A are ventricle and atria, respectively. Scale bar 100 μm.

MZsap130a;sap130bpt35b/pt35b mutants are not healthy (A) sap130a;sap130b double heterozygous adults, male (top) and female (bottom). (B) sap130a;sap130b double homozygous adults, male (top) and female (bottom). (C) Graph quantifying weight to length ratio for adults from a sap130a;sap130b double heterozygous in-cross, pvals are for one-way ANOVA, error bars are standard error mean (SEM). Red points represent females and males in black. (D) Graph quantifying the heart phenotype proportions for Tg(myl7:EGFP), MZsap130a;Tg(myl7:EGFP), MZsap130b;Tg(myl7:EGFP), and MZsap130a;sap130bpt35b/+;Tg(myl7:EGFP), pvals are for fisher’s exact test. Scale bar 5 mm.

Cardiac gene expression in MZsap130a and MZsap130b(A) WISH of nkx2.5 at 10 somite stage for AB*, MZsap130a and MZsap130b. (B) WISH of myh6 and myh7 at 24hpf for AB*, MZsap130a and MZsap130b. (C) WISH of myh7 at 36hpf in AB* and MZsap130a. Scale bar 100 μm.

RNAseq reveals cardiac contraction and conduction is altered in MZsap130a(A) Representative images of Tg(myl7:EGFP) and MZsap130a;Tg(myl7:EGFP) embryos collected for whole embryo RNAseq at 36hpf. (B) Volcano plot of 36hpf whole embryo RNAseq data. (C) Heatmap of sarcomere and conduction genes (Supplementary Figure S6) at 36hpf from whole embryos. (D) DAVID functional annotation cluster 8 from downregulated genes in MZsap130a, showing DAVID calculated p-values. (E) Heatmap of 48hpf heart tissue RNAseq data for the same genes found in panel C. V and A are ventricle and atria, respectively. Scale bar 100 μm.

MZsap130a show cardiac functional deficits (A–C) Shows systole and diastole frames from recordings of live ventricular contractions in Tg(myl7:EGFP), MZsap130a;Tg(myl7:EGFP) and MZsap130b;Tg(myl7:EGFP) at 48hpf. (D, E) Quantified cardiac parameters total stroke volume (TSV) and cardiac output (CO), pvals from one-way ANOVA, error bars are SEM. Each point represents individual ventricle and color coded for 3+ experiments. For Tg(myl7:EGFP), n = 115; MZsap130a NV, n = 36; MZsap130a SV, n = 30; MZsap130b NV, n = 57; MZsap130b SV, n = 13. Scale bar 20 μm.

Lineage tracing reveal changes to SHF in MZsap130a at 48hpf (A) Diagram showing how the FHF heart tube at 24hpf was photoconverted to red, leaving SHF progenitors unlabeled in green and imaging at 48hpf. (B, C) Confocal imagines of Tg(nkx2.5:kedge) and MZsap130a;Tg(nkx2.5:kedge) at 48hpf with the heart tube being photoconverted at 24hpf, the red outlined region represents area measurements collected. (D) Quantified SHF (green area) accreted by 48hpf, pval is from a one ANOVA, error bars are SEM. Each point represents a single embryo, Tg(nkx2.5:kedge), n = 15; MZsap130a;Tg(nkx2.5:kedge) NV, n = 14; MZsap130a;Tg(nkx2.5:kedge) SV, n = 10. V and A are ventricle and atria, respectively. Scale bar 100 μm.

Tg(myl7:memGFP) reveals longer OFT by 72hpf in MZsap130a(A) Quantified OFT lengths at 72hpf, pval is from a one ANOVA, SEM error bars. Each point represents a single embryo, Tg(myl7:memGFP), n = 18; MZsap130a;Tg(myl7:memGFP) NV, n = 20; MZsap130a;Tg(myl7:memGFP) SV, n = 9 (B) Representative images of Tg(myl7:memGFP) and MZsap130a;Tg(myl7:memGFP), white lines demarcate OFT length, pvals from one way ANOVA, SEM error bars. (C)Tg(myl7:memGFP)+;H2b:mCherry + ventricular CM counts for three WT or MZsap130a SV heart, pval is from a t-test, SEM error bars (D)Tg(myl7:memGFP)+;H2b:mCherry + out-flow tract cell counts for three WT or MZsap130a SV heart, pval is from a t-test, SEM error bars. V and A are ventricle and atria, respectively. Scale bar 100 μm.

Adult MZsap130a hearts have large bulbus arteriosus (A, B)AB* and MZsap130a adult SV hearts extracted at approximately 4–6 months post fertilization. (C) Quantification of ventricle area of unfixed hearts, pval is from a one ANOVA solid black bars are mean, dotted lines represent up and lower 25th percentiles. Each point represents a single heart, AB*, n = 14; MZsap130a NV, n = 7; MZsap130a SV, n = 12. (D) Quantification of BA area. of unfixed hearts, pval is from a one ANOVA solid black bars are mean, dotted lines represent up and lower 25th percentiles. Each point represents a single heart, n = 10 for all groups. V, A, and BA are ventricle, atria, and bulbus arteriosus respectively, Scale bar 200 μm.

sap130a shows an association with hdac1 and MZsin3ab mutants have SVs (A) Representative image of Tg(myl7:EGFP) and MZsap130a;hdac1b382/+;Tg(myl7:EGFP) NV and SVs at 48hpf. (B) Representative image of Tg(myl7:EGFP) and MZsin3ab;Tg(myl7:EGFP) NV and SVs at 48hpf. (C) Quantification of heart phenotype proportions in Tg(myl7:EGFP), MZsap130a;Tg(myl7:EGFP), hdac1b382/+;Tg(myl7:EGFP) and MZsap130a;hdac1b382/+;Tg(myl7:EGFP). The p-values are fisher’s exact test. (D) Quantification of heart phenotype proportions in Tg(myl7:EGFP) and MZsin3ab;Tg(myl7:EGFP). The p-values are fisher’s exact test. V and A are ventricle and atria, respectively. Scale bar 100 μm.

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PHENOTYPE:
Fish:
Observed In:
Stage: Long-pec

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.

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Acknowledgments
This image is the copyrighted work of the attributed author or publisher, and ZFIN has permission only to display this image to its users. Additional permissions should be obtained from the applicable author or publisher of the image. Full text @ Front Cell Dev Biol