Trapping cardiac recessive mutants via expression-based insertional mutagenesis screening

Abstract

Rationale: Mutagenesis screening is a powerful genetic tool for probing biological mechanisms underlying vertebrate development and human diseases. However, the increased colony management efforts in vertebrates impose a significant challenge for identifying genes affecting a particular organ, such as the heart, especially those exhibiting adult phenotypes on depletion.

Objective: We aim to develop a facile approach that streamlines colony management efforts via enriching cardiac mutants, which enables us to screen for adult phenotypes.

Methods and results: The transparency of the zebrafish embryos enabled us to score 67 stable transgenic lines generated from an insertional mutagenesis screen using a transposon-based protein trapping vector. Fifteen lines with cardiac monomeric red fluorescent protein reporter expression were identified. We defined the molecular nature for 10 lines and bred them to homozygosity, which led to the identification of 1 embryonic lethal, 1 larval lethal, and 1 adult recessive mutant exhibiting cardiac hypertrophy at 1 year of age. Further characterization of these mutants uncovered an essential function of methionine adenosyltransferase II, α a (mat2aa) in cardiogenesis, an essential function of mitochondrial ribosomal protein S18B (mrps18b) in cardiac mitochondrial homeostasis, as well as a function of DnaJ (Hsp40) homolog, subfamily B, member 6b (dnajb6b) in adult cardiac hypertrophy.

Conclusions: We demonstrate that transposon-based gene trapping is an efficient approach for identifying both embryonic and adult recessive mutants with cardiac expression. The generation of a zebrafish insertional cardiac mutant collection shall facilitate the annotation of a vertebrate cardiac genome, as well as enable heart-based adult screens.

Figure 1. A collection of 15 GBT lines with cardiac expression of tagged genes

(A) Lateral views of GBT embryos at 2–4 days post fertilization (dpf) are shown. mRFP expression was observed in the heart (arrowhead) of 15 GBT lines but not in other GBT lines, such as GBT0365 (arrow), which exhibited neuronal expression. Scale bar=200 μm. (B-C) Cardiac expression analysis of tagged genes in either embryonic (B) or adult zebrafish hearts (C) by RT-PCR. (D) Cardiac expression analysis of human orthologs of tagged genes using RT-PCR. * indicates candidate genes and orthologs that have not been included in the present ZIC collection. (E) Whole-mount in situ hybridization was used to determine the expression patterns of the tagged genes. Lateral views are shown in left panels, and ventral views are shown in right panels. Arrowheads indicate heart expression in 3 cardiac lines, while the arrow indicates the heart area in the sncgb control embryo, which showed no detectable signal. Scale bar=200 μm.

Figure 2. mat2aa is disrupted in the embryonic lethal GBT0364 line

(A) A RP2 element was inserted into the first intron of the mat2aa gene in the GBT0364 line. (B) mat2aa transcripts were dramatically abolished in the GBT0364 homozygous mutant embryos, as indicated using RT-PCR. (C) GBT0364 homozygous mutants presented a cardiac edema (arrow) phenotype at 3 dpf. (D) Mat2aa ATG morpholino injection resulted in cardiac edema phenotype (arrow) in WT Wik embryos similar to that observed in GBT0364. Scale bars=0.5 mm. (E-F) Western blotting demonstrating that GBT0364 homozygous mutant embryos exhibited significantly reduced methylation levels on histone proteins at 4 dpf. n=3. (G) GBT0364 homozygous mutant embryos also exhibited a reduced global DNA methylation level at 4 dpf. The values are presented as the mean±standard deviation obtained from three independent biological repeats performed in triplicate. *P<0.05.

Figure 3. Myocardium-specific rescue of cardiac phenotypes in GBT0364 via cre-loxP system

(A-B) Myocardium-specific expression of Mat2aa is demonstrated by non-overlapping expression of mRFP with EGFP from Tg(fli1a:EGFP) (A) and overlapping expression with Tg(titin:actn2-EGFP) (B). Insets are images of higher magnification. Scale bar=20 μm. (C) Ventricular chamber size is reduced in GBT0364 homozygous mutant embryos, which can be rescued by the Tg(cmlc2:YFP-Cre) transgene. Lateral views of DIC images are shown. (D) cmlc2 expression is reduced in the hearts of GBT0364 homozygous mutant embryos, which can be rescued using the Tg(cmlc2:YFP-Cre) transgene. Ventral views of embryos following whole-mount in situ hybridization are shown. Scale bars=20 μm. (E-G) Quantification of the ventricular volume (E), percent fraction shortening (F), and heart rate (G). n=6, *P<0.05.

Figure 4. Mat2aa regulates cardiomyocyte cell size and cell number

(A) Representative images of embryonic hearts at 3 dpf after co-staining with anti-β-catenin (green) and anti-Mef2 (red) antibodies from a GBT0364 WT sibling and homozygous mutant with or without injection of 1 nL of p53_MO. Scale bar=20 μm. (B) Quantification of cardiomyocyte (CM) cell size. Injection of p53_MO did not alter the reduced CM size in GBT0364 homozygous mutants. (C) Quantification of CM cell circularity. No significant changes of circularity were observed. (D) Representative images of embryonic hearts at 3 dpf after co-staining with anti-TUNEL (green) and anti-Mef2 (red) antibodies from a GBT0364 WT sibling and homozygous mutant with or without injection of 1 nL p53_MO. (E) Quantification of CM cell number in the ventricle. Injection of p53_MO partially rescued the reduced CM number in GBT0364 homozygous mutant embryos. (F) Quantification of the CM cell apoptosis index in the ventricle. Injection of p53_MO partially rescued the increased TUNEL index of CMs in GBT0364 homozygous mutant embryos. (G) Representative images of embryonic hearts at 3 dpf after co-staining with anti-PCNA (green) and anti-Mef2 (red) antibodies from a GBT0364 WT sibling and homozygous mutants with or without injection of 1 nL p53_MO. Arrows indicate PCNA+/Mef2+ cells. (H) Quantification of the CM cell proliferation index in the ventricle. Injection of p53_MO did not alter the reduced PCNA index of CMs in GBT0364 homozygous mutant embryos. n=6, *P<0.05.

Figure 5. GBT0425 line uncovers essential functions of Mrps18b as a mitochondrial protein

(A) A RP2 element was inserted into the second intron of the mrps18b gene in the GBT0425 line. (B) The mrps18b transcript was dramatically reduced in GBT0425 homozygous embryos by RT-PCR using mrps18b-c-F/R primers for the entire transcripts amplification. (C) GBT0425 homozygotes appeared smaller at 10 dpf. Scale bar=200 μm. (D) Quantification of fraction shortening (FS%). FS is significantly reduced in GBT0425 homozygous mutant embryos at 10 dpf, but not at 6 dpf. n=6. (E-L) Fluorescent images of individual cells dissociated from either heterozygous (E-H) or homozygous (I-L) GBT0425 embryonic hearts are shown. Mitochondria are stained with MitoTracker Green FM, which appears to co-localize with the mRFP signal. E-L, scale bars=20 μm. (M-P) Transmission electronic micrographs indicating an apparently reduced mitochondrial number in the GBT0425 homozygous embryonic heart (M,N) and disrupted mitochondrial morphology in the remaining mitochondria (O,P) are shown. Arrowheads indicate representative mitochondria; N, nuclei. M-N, scale bars=2 μm; O-P, scale bars=200 nm. (Q) Quantification of mitochondrial number indicated a significantly reduction in the GBT0425 homozygous embryos at 8 dpf. n=3, *P<0.05.

Figure 6. Sarcomere localized Dnajb6b is disrupted in the GBT0411 line

(A) A RP2 element was inserted into the 6th intron of the dnajb6b gene in the GBT0411 line. (B) The dnajb6b entire transcript was dramatically ablated in GBT0411 homozygous embryos. Shown are results of RT-PCR. (C) Northern blotting hybridization results using either mRFP or the short isoform dnajb6b as a probe. The predominant dnajb6b long isoform mRNA in an adult wild type heart is disrupted, which results in truncated dnajb6b mRNA encoded by exon 1–6 that is similar to the dnajb6b short isoform. (D) Shown are lateral views of a live embryo to indicate heart and eye-specific expression of mRFP at 3 dpf. Scale bar=200 μm. (E-F) Fluorescent images of adult heart sections from GBT0411 line after crossing with either Tg(titin:actn2-EGFP) (E) or Tg(fli1a:EGFP) (F). Scale bars=20 μm.

Figure 7. GBT0411 is a recessive adult mutant that exhibits cardiac hypertrophy phenotype

(A) Representative images of dissected hearts from GBT0411 WT sibling, heterozygous and homozygous fish at 12 months old. Scale bar=1 mm. (B) Quantification of the ventricular surface area to body weight index (VSA/BW) showed significant heart enlargement in the GBT0411 homozygous fish compared to that in WT sibling control at 12 months but not at 4 months old. n=6. (C) Representative images of adult heart sections stained with anti-β-catenin antibodies to indicate cardiomyocyte cell size at 12 months old stage. Scale bar=10 μm. (D) Quantification of cardiomyocyte cell size from (C), which is significantly increased in GBT0411 homozygous compared with that in WT fish at 12 months old. 20–30 cardiomyocytes were measured. (E) Representative images of adult heart sections at 12 months old stained with anti-αactinin antibodies. Muscular disarray was detected in the GBT0411 homozygous fish. Insets are images of higher magnification. Scale bar=20 μm. (F) The quantitative RT-PCR results indicating the re-activated expression of atrial natriuretic factor (anf) in GBT0411 homozygous fish heart at 12 months, but not 4 months old, are shown. V, ventricle; A, atrium; OFT, outflow tract. n=3, *P<0.05.