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Frese et al., 2015 - RNA splicing regulated by RBFOX1 is essential for cardiac function in zebrafish. Journal of Cell Science   128(16):3030-40 Full text @ J. Cell Sci.

Fig. 1

Expression analysis of rbfox1 in zebrafish. (A) Amino acid sequence alignments of human, mouse and zebrafish rbfox1 demonstrating the high cross-species homology. Black background, amino acid identity; gray background, amino acids with similar chemical properties. (B,C) RNA antisense in situ hybridization against rbfox1 demonstrates a specific mRNA expression in neuronal, heart cells and skeletal muscle at 48hpf (V, ventricle; A, atrium). wt, wild-type. (D) Relative expression analysis of rbfox mRNAs shows that rbfox1 is highly expressed in the heart in comparison to the other rbfox family members. WF, whole fish. (E) qRT-PCR analysis of rbfox1 in different tissues of adult zebrafish reveals the highest expression in brain and eye tissue, and an equal expression in heart, skeletal muscle (SKM), intestine and liver tissue. Data are shown as mean±s.d. (pooled tissue of adult zebrafish n=8, two replicates each). ΔCt values compared to elfa1 as a reference gene.

EXPRESSION / LABELING:
Genes:
Fish:
Anatomical Terms:
Stage Range: Long-pec to Adult

Fig. 2

Knockdown of rbfox1 leads to cardiomyopathy and heart failure. (A–C) Lateral view of MO-control- and MO1- and MO2-rbfox1-injected embryos. After injection of 3ng MO1-rbfox1, 85% (D) and MO2-rbfox1, 72% (E) of morphants embryos develop cardiomyopathy and heart failure. Results are mean±s.d. (n=3). (F) cDNA analysis of rbfox1 morphants after injection of MO2-rbfox1 shows skipping of exon 6 (198-bp product) compared to a product including exon 6 (259bp product) in control-treated embryos. Sanger sequencing reveals that exon 6 is completely excluded, predictably leading to a frame shift of the coding sequence and premature stop in exon 7. (H,I) Fractional shortening (FS) of the ventricular chamber of MO-control- and MO1-rbfox1-injected embryos measured at the indicated developmental stages. Fractional shortening is significantly reduced in rbfox1 morphants after 48hpf and further declines at 96hpf. Results are mean±s.d. (n=10).

Fig. 3

rbfox1 deficency does not influence heart morphology. (A,B) H&E-stained sagittal histological sections of MO-control and MO1-rbfox1 morphant hearts at 72hpf. Morphants display normal heart morphology with distinct endocardial and myocardial cell layers in the atrium (A) and ventricle (V), and a clear differentiation and demarcation of the atrium and ventricle by the atrio-ventricular ring (AV). OFT, outflow tract. (C,D) Atrial- and ventricle-specific myosin heavy chains are expressed normally, also suggesting that there is normal molecular chamber specification [green, antibody against atrial-specific myosin (S46); red, antibody against ventricular and atrial myosin (MF20)]. (E–H) Ultrastructural analysis of heart and skeletal muscle cells of MO-control- and MO1-rbfox1-injected embryos at 48hpf showing organized sarcomere units (SU) with thin and thick myofilaments in well-aligned bundles and discernible AI-, M- and Z-bands.

EXPRESSION / LABELING:
Antibodies:
Fish:
Knockdown Reagent:
Anatomical Terms:
Stage: Long-pec
PHENOTYPE:
Fish:
Knockdown Reagent:
Observed In:
Stage Range: Long-pec to Protruding-mouth

Fig. 5 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. 6

Correctly balanced huG and actn3a isoform expression is essential for proper heart function. (A,B) Lateral view of a MO-huG-injected (MO-zhug) zebrafish embryo at 48hpf. Fractional shortening (FS) shows a progressive reduction of cardiac contractility. Inset, cDNA splice-site analysis of huG morphant. (C,D) Splice-site blockage in actn3a leads to mild cardiac dysfunction. actn3a-depleted zebrafish embryos develop heart failure with dilation of the atrium and reduced ventricular contractility beginning at the 72h developmental stage. Inset, cDNA splice-site analysis of actn3a morphant. Results in B and D are mean±s.d. (n=10).

Acknowledgments:
ZFIN wishes to thank the journal Journal of Cell Science for permission to reproduce figures from this article. Please note that this material may be protected by copyright. Full text @ J. Cell Sci.