FIGURE SUMMARY
Title

The DEAD box RNA helicase Ddx39ab is essential for myocyte and lens development in zebrafish

Authors
Zhang, L., Yang, Y., Li, B., Scott, I.C., Lou, X.
Source
Full text @ Development

Loss of ddx39ab leads to an embryonic lethal phenotype in zebrafish. (A) GFP expression pattern in zebrafish trapping line RP-011. Lateral views. S, somite. Arrowhead in inset indicates GFP expression in heart. (B) Morphological defects in RP-011 homozygous mutant embryos. RP-011 homozygous embryos did not show any morphological defects until 24 hpf; at 48 hpf, they showed a curved body axis, and at 72 hpf heart edema, dysmorphic jaw and widespread degeneration. At least 80 embryos of each genotype were observed and representative samples are shown. (C) The zebrafish ddx39ab genomic locus. Exons (blue boxes) are numbered. The transposon is inserted after the second intron. (D) Zebrafish Ddx39ab protein contains DEXDc and HELICc domains. Insertion of the gene-trapping element results in a fusion transcript that will be translated into a fusion protein containing the first 69 amino acids from Ddx39ab at the N-terminus. (E) RT-PCR results showing the presence of fusion transcript in RP-011 embryos. (F) qPCR results showing the absence of ddx39ab mRNA in RP-011 homozygous embryos. Data are mean±s.e.m. **P<0.01. n.s, not significant. (G) Injection of ddx39ab mRNA rescues the developmental defects of RP-011 homozygous embryos. (H) Quantification of the rescue of morphological defects of homozygous ddx39ab mutant embryos (Control) by injection of ddx39ab mRNA.

Loss of ddx39ab leads to cardiomyocyte differentiation defects in zebrafish embryos. (A) Ventral view of heart from whole-mount wild-type or ddx39ab mutant zebrafish embryos at 36 hpf, with cranial to the top. Myocardium was labeled with MF20 antibody. Scale bars: 200 μm. (B) RNA in situ hybridization and qPCR results for cardiogenic regulatory gene expression in wild-type and ddx39ab mutant zebrafish embryos at 26 hpf. (C) RNA in situ hybridization and qPCR results for cardiomyocyte structural gene expression in wild-type and ddx39ab mutant zebrafish embryos at 26 hpf. (B,C) Frontal views with dorsal side to the top. At least 15 (A) or 20 (B,C) embryos for each genotype were analyzed and representative samples are shown. For qPCR results, data are mean±s.e.m. n.s, not significant. *P<0.05, **P<0.01, ***P<0.001.

Loss of ddx39ab results in defective skeletal muscle differentiation in zebrafish. (A) Immunostaining demonstrates the organization of myofilaments in wild-type and ddx39ab mutant embryos at 24 hpf. Anti-MHC antibody (MF20) labels thick (myosin) filaments and F-actin was visualized by phalloidin staining. Scale bars: 20 μm. (B) RNA in situ hybridization for myogenic regulatory gene expression in wild-type and ddx39ab mutant zebrafish embryos at 32 hpf. (C,D) RNA in situ hybridization and qPCR analysis for myocyte structural gene expression in wild-type and ddx39ab mutant zebrafish embryos at 32 hpf. (B,C) Lateral views with anterior to the left. (D) Transverse section with dorsal side to top. At least 15 (A) or 20 (B-D) embryos of each genotype were analyzed and representative samples are shown. For qPCR results, data are mean±s.e.m. n.s, not significant. **P<0.01, ***P<0.001.

Lens fiber cell differentiation is defective in ddx39ab mutant embryos. (A) ddx39ab mutants exhibit defects in lens fiber morphogenesis. (Top) Bright-field images of the eyes from wild-type and ddx39ab mutant zebrafish embryos at 28 hpf. The scatter plot shows the diameter of individual lens fibers. (Middle) Equatorial sections through the lens center showing the organization of lens fibers; cell membrane is labeled with mRFP. Cell number was quantified on equatorial sections. (Bottom) Schematics showing the shape and arrangement of lens fiber cells. The convexity of lens fiber cells is plotted. Scale bars: 50 μm. (B,C) RNA in situ hybridization and qPCR results for lens gene expression in wild-type and ddx39ab mutant zebrafish embryos at 32 hpf. Frontal views with dorsal side to the top. (A-C) At least 20 embryos of each genotype were analyzed and representative samples are shown. All data are mean±s.e.m. n.s, not significant. **P<0.01, ***P<0.001.

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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.

Cell death in wild type or ddx39ab mutant embryo. Right panels, magnified view of the boxed region. Scale bars, 20um. For each stage, at least 10 embryos for each genotype were analyzed and representative samples are shown.

Zebrafish ddx39ab embryonic expression pattern. (A): In situ hybridization experiment revealed there is maternal deposit ddx39ab mRNA in early stage embryo. (B): Strong expression of ddx39ab could be observed in myotome at 18 somite stage. (C-D): At 24hpf enriched ddx39ab mRNA could be observed in lens, heart tube and trunk muscle. Triangle indicated expression in heart tube. (E-H): Later on, the expression was restrained to specific regions in brain, retina (i), pharyngeal arches (i) and endoderm derived organs (ii). A, lateral view. B, dorsal view. C, frontal view. D to H, head to left. i and ii are sections of H as indicated. hpf, hour past fertilization.

Retina development in ddx39ab mutants. (A): Frontal sections of retina at 32 hpf. (B): RNA in situ hybridization and qPCR results are shown for retinal ganglion marker gene expression in control and ddx39a mutant zebrafish embryos. Dorsal views with anterior to the top. For qPCR results, data are mean ± SEM. ns: not significant. (C): Confocal images of retinal ganglion cell (labeled with zn8 antibody) from wild type and ddx39ab mutant embryo at 48 hpf. Scale bars, 20um. A, B and C, at least 15 embryos for each genotype were analyzed and representative samples are shown.

Disorganization of primary lens fiber cell in ddx39a embryo. Cell membrane was labeled with mRFP; living images were taken at 28 hpf. Scale bars, 25 μM.

Ddx39ab mutant embryos exhibit normal mitoses. Mitotic cells stained with DAPI (blue) and α-tubulin (red). Scale bars, 5um.

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