Trapping Cardiac Recessive Mutants via Expression-Based Insertional Mutagenesis ScreeningNovelty and Significance
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.
On completion of the human genome project, the identification of 20 000+ genes facilitated a systematic approach for characterizing genes that participate in a particular biological process and disease.1 The Cardiovascular Gene Ontology Annotation Initiative represents such an effort that summarizes the functional knowledge of gene products across all species using structured biological vocabularies2 (http://www.geneontology.org/GO.cardio.shtml). More than 4000 genes implicated in heart development, cardiovascular processes, and cardiac diseases have been compiled through the extraction of human genes associated with cardiovascular-related gene ontology processes, genomewide cardiovascular gene association analyses, and knockout studies of developmentally important genes. However, the current cardiac gene list is incomplete, and the expression and function of each individual gene have not been annotated.
Mutagenesis screening is a powerful genomewide gene discovery tool used in lower model organisms, such as yeast, Caenorhabditis elegans and Drosophila, which has successfully been extended to vertebrates.3,4 An ethylnitrosourea-based mutagenesis screen has been conducted to seek mouse mutants that mimic congenital heart diseases5 and identify adult modifiers of cardiomyopathy.6 The scale of a mutagenesis screen in vertebrates is rather limited because of the significant increase of resources required for colony management compared with lower model organisms. Moreover, most recessive mutants exhibit adult phenotypes that are only detectable either with the use of sophisticated phenotyping techniques or when animals are under certain stresses.7 Therefore, most screens in vertebrates have focused on embryonic lethal mutants, which only account for 5% to 10% of the genome.8,9
Because of its low-cost maintenance and highly prolific nature, the zebrafish (Danio rerio) has emerged as an increasingly popular vertebrate model for mutagenesis screening. The first 2 large-scale mutagenesis screens using ethylnitrosourea as a mutagen generated hundreds of mutants, which laid the foundation for the zebrafish to become a premier animal model.8,10 To address the limitations of molecular cloning, mutagens with a sequence tag have been exploited, including either retrovirus-9,11 or transposon-based vectors.12,13 In mice, the transposon-based gene-trapping approach has been successfully exploited in embryonic stem cells, and this approach has been recently extended to an entire animal.14–16 Efficient transposon-based gene trapping has been recently established in zebrafish using pGBT-RP2.1 (RP2), which contains both gene reporting and gene-breaking cassettes.17 When inserted into an intron in an endogenous gene, RP2 hijacks the targeted splicing donor sequence, truncates the encoded protein, and disrupts gene function with high knockdown efficiency.
Here, we leveraged the simplified colony management and high mutagenicity of the RP2 cassette to establish zebrafish as a useful model vertebrate animal for introducing biases in the genes that might be screened for adult cardiac phenotypes. To enrich the cardiac mutants, we integrated an expression-based enriching strategy with mutagenesis screening. Ten insertional lines with embryonic heart expression were bred into homozygosity and then raised to adulthood. Three mutants exhibiting embryonic lethal, larval lethal, and adult recessive phenotypes were identified. Our results demonstrate that transposon-based gene trapping in zebrafish is an efficient approach to identify genes with cardiac expression. The generation of a zebrafish insertional cardiac (ZIC) mutant collection shall facilitate the annotation of the genes in the cardiac genome, and open the door for systematic identifying genetic modifiers of major cardiac diseases.
For a detailed description, see the Online Data Supplement Methods.
Zebrafish (D. rerio) were maintained and handled according to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (Publication No. 85-23, revised 1996) and the Mayo Clinic Institutional Animal Care and Use Committee. The approved Institutional Animal Care and Use Committee protocol number is A17610. The Institutional Review Board protocol that describes the method for extracting RNA from the human heart is 10-000216.
Generating and Cloning GBT Lines
The gene-break transposon (GBT) mutagenesis vector GBT-RP2.1 (RP2) plasmid solution (1 nL of a 50 ng/µL) was combined with 100 ng/µL of Tol2 transposase mRNA and injected into zebrafish embryos at the single-cell stage to generate insertional lines as described previously.17
Three methods, including inverse polymerase chain reaction (PCR), rapid amplification of 5′-complementary DNA, and rapid amplification of 3′-complementary DNA, were combined to clone and validate the GBT integration sites. Inverse PCR was conducted to determine the exact integration positions of RP2. Genotyping primers flanking the predicted integration site were designed to confirm the integration position using a RP2 vector-specific primer combined with a gene-specific primer. Occasionally, the exact integration site of the transposon within the targeted intron was determined using a series of PCR primers spanning the intron.17
Fraction shortening, ventricle chamber size, and the heart rate of an embryonic zebrafish heart were measured by documenting videos of the beating heart.18 To quantify the cardiomyocyte (CM) cell size, number, proliferation, and apoptosis, dissected embryonic hearts were subjected to immunostaining as described.19 To determine the adult heart enlargement phenotype in the GBT0411 line, the index of ventricular surface area to body weight was used according to the previously described method.20,21 Frozen sections (10–12 µm) of adult heart ventricles were subjected to immunostaining using previously described methods.21 The stained images were captured using a Zeiss Axioplan II microscope equipped with ApoTome and AxioVision software (Carl Zeiss).
All values are presented as the mean±SD. Student t test was used for comparisons between special 2 groups, and ANOVA was used for assessment differences among ≥3 groups. Significance was accepted at P<0.05.
Fifteen GBT Lines Exhibit Cardiac mRFP Expression
The RP2 plasmid and transposase mRNA was injected into 4000 embryos to initiate the mutagenesis screen. Embryos showing a mosaic monomeric red fluorescent protein (mRFP) signal in the heart at 2 to 4 days postfertilization (dpf) were preselected to enrich for lines with cardiac expression. Among the 545 preselected embryos, 441 embryos survived to adulthood and were defined as F0 founders. Subsequently, outcrosses of these F0 founders were performed, and 67 stable F1 insertional lines were generated (Online Figure I). The mRFP expression in the heart during the first 4 days of embryogenesis was detected in 15 F1 lines using fluorescence microscopy (Figure 1A). Other lines, such as GBT0365, which exhibited mRFP-tagged gene expression in the central nervous system, did not show detectable expression in the embryonic heart.
To facilitate molecular cloning of the tagged genes, we outcrossed 15 potential cardiac insertional lines to obtain fish with a single copy insertion. Southern blotting was used to identify fish with a lower copy number of insertions (Online Figure II). After 2 to 4 generations of outcrossing, 13 of the 15 lines contained a single insertion. The remaining insertional lines, GBT0363 and GBT0421, contained multiple insertions that could not be separated using simple crossing (Table). We cloned the candidate RP2 integration loci using a combination of rapid amplification of 5′-complementary DNA, rapid amplification of 3′-complementary DNA, and inverse PCR technologies (Table). In 10 of the 13 lines, the loci were integrated within well-annotated genes in the zebrafish genome (Zv9). As expected, 9 of the 10 insertions were located in introns downstream of the ATG start codon, with one exception in which a RP2 element was inserted into the first intron located upstream of the ATG translational start site of the cysteine-serine–rich nuclear protein 1b (csrnp1b) gene in the GBT0416 line. An upstream AUG was located within the reading frame of the mRFP coding sequence, which likely accounts for the mRFP expression in GBT0416. The RP2 insertions in 3 lines, GBT0413, GBT0418, and GBT0423, were within repetitive genomic regions based on the Zv9 genomic database. Further annotation of these genomic regions is required to determine the molecular nature of these 3 lines (Table). In this study, we primarily focus on the 10 cardiac GBT lines for which the molecular nature was clear.
It was expected that the splice acceptor in the RP2 vector would hijack a splice donor of each tagged gene, which would truncate the encoded protein and lead to functional disruption.17 We assessed the efficiency of gene disruption at the level of RNA splicing. Homozygous embryos were identified using genotyping, and the expression level of tagged transcripts was subsequently measured via quantitative reverse transcriptase (RT)-PCR using a primer pair targeted to the flanking exons. Compared with wild-type siblings, native transcriptional splicing events in all 10 cardiac mutant lines were reduced by ≥99% (Table). Similarly, the targeted splicing event in synuclein, γ b (sncgb) was completely disrupted in the homozygous GBT0365 noncardiac control line. These data demonstrate efficient knockdown of the tagged genes in the individual GBT lines.
Assessment of the Expression of 10 Tagged Genes
Among the 10 RP2-tagged genes, only 5 have been listed in the current Cardiovascular Gene Ontology Annotation Initiative (Table). To verify that the 10-tagged genes were indeed expressed in the heart, we examined their expression via RT-PCR using RNA extracted from zebrafish embryonic hearts. All of the 10 tagged genes showed positive signals in RT-PCR assays, whereas the sncgb control did not (Figure 1B). Positive signals were also detected for the 3 candidate genes in GBT0418, GBT0363, and GBT0421, including zgc:113030, ATP synthase, H+ transporting, mitochondrial F0 complex, subunit b, isoform 1(atp5f1), and the family with sequence similarity 78, member B (fam78b). We also validated their expression in adult zebrafish hearts using both RT-PCR (Figure 1C) and fluorescence microscopy in dissected hearts (Online Figure III). Then, we assessed the corresponding human orthologs for these 13 genes and sncgb as a control. Based on the RT-PCR analysis using RNA extracted from normal human heart tissue, we detected cardiac expression for all 13 orthologous genes (Figure 1D). Taken together, these findings suggest that all 10 tagged genes exhibited cardiac expression in both embryonic and adult zebrafish, as well as in adult humans.
According to the published whole-mount in situ hybridization (ISH) data, 3 genes, insulin receptor b (insrb/GBT0422), vomeronasal 2 receptor l1 (v2rl1/GBT0424), and mitochondrial ribosomal protein S18B (mrps18b/GBT0425), exhibited ubiquitous expression and 4 genes, DnaJ (Hsp40) homolog, subfamily B, member 6b (dnajb6b/GBT0411), csrnp1b/GBT0416, retinoid X receptor, α a (rxraa/GBT0419), and methionine adenosyltransferase II, α a (mat2aa/GBT0364) showed restricted expression but with no reported heart signal at the embryonic stage. No ISH expression data have been reported for the remaining 3 genes, vesicle-associated membrane protein-associated protein A like (vapal/GBT0410), exportin 7 (xpo7/GBT0412), and arrestin domain-containing 1b (arrdc1b/GBT0415). We hypothesized that the lack of documented cardiac expression could reflect the relatively low sensitivity of ISH assays, low abundance of the transcripts, or dynamic expression patterns. To test these possibilities, we performed ISH assays for the 3 tagged genes, dnajb6b, csrnp1b, and mrps18b, and sncgb as a control. A short color developing time revealed RNA expression patterns similar to those that have been previously reported22 (and www.zfin.org) (Figure 1E), whereas a prolonged color reaction showed cardiac signals for all 3 genes but not for the sncgb noncardiac control (Figure 1A and 1E).
We documented the dynamic expression of mRFP in the heart from these insertional lines. As exemplified by GBT419, mRFP signal was observed in the posterior hindbrain and somite at 20 hours postfertilization, which gradually expanded to the tectum and pharyngeal arch at 36 hours postfertilization (Online Figure IV). As the embryo developed, clear cardiac signals started to emerge at 72 hours postfertilization, whereas the pharyngeal arch signal faded at 96 hours postfertilization. The dynamic mRFP pattern reflected the endogenous expression profile of the tagged gene rxraa, as revealed by ISH using an rxraa riboprobe (Online Figure IV).22,23 In addition, Tg(fli1a: enhanced green fluorescent protein [EGFP]), a transgenic reporter strain that labels the entire endocardium layer was crossed with GBT lines to determine the cell type-specific expression in the hearts of zebrafish from these cardiac lines. Based on the comparison with the EGFP signal, we identified tagged fusion proteins that were expressed in both layers (GBT0416), only in the endocardium (GBT0422), or only in the myocardium (GBT0411) (Online Figure IV). The myocardium expression of the tagged Dnajb6b-mRFP fusion in the GBT0411 line was further confirmed by crossing with Tg(titin:actn2-EGFP), a myocardium marker transgenic fish (Online Figure IV).24 Thus, these analyses demonstrated that GBT lines with affected cardiac genes can be reliably identified, and their cardiac expression can be faithfully revealed using the mRFP reporter.
The Embryonic Lethal GBT0364 Line Uncovers an Essential Function of Mat2aa in Cardiogenesis
We conducted incrosses for all 10 characterized cardiac lines and identified GBT0364 as an embryonic lethal line with ≈25% offspring exhibiting a pericardiac edema phenotype at 3 dpf (Figure 2C). The RP2 insertion was present in the first intron of mat2aa, which encodes a highly conserved protein showing 89% amino acid identity with the human ortholog (Figure 2A and Online Figure V). Although a strong maternal mRFP signal was detected at the 1-cell stage in the offspring of a female (Online Figure VI), the mRFP signals emerged in the offspring of a male parent at 50% epiboly, indicating the onset of zygotic mat2aa expression (Online Figure VI). The mRFP expression was restricted to particular tissues including the eye, somites, pharyngeal arch, brain, pineal gland, and heart at later stages (Online Figure VI). A similar tissue-restricted and dynamic RNA expression pattern was detected using ISH with either an mRFP or a mat2aa riboprobe (Online Figure VI), confirming that the mRFP tag in the RP2 vector faithfully reports the expression of the endogenous gene.
The RP2 insertion dramatically abolished the expression of mat2aa transcripts in GBT0364 homozygous mutants, which resulted in pericardiac edema at 3 dpf and eventual death at 8 dpf (Figure 2B and 2C, Online Figure VII and data not shown). The results from the genotyping PCR analysis revealed that this insertion was tightly associated with embryonic lethal phenotypes (Online Figure VII). The linkage between the aberrant RNA splicing event and the phenotypes was also validated using a rescue experiment using morpholinos (MOs) that blocked the RP2-disrupting splicing event17 (Online Figure VII). Additionally, the cardiac phenotypes in the GBT0364 were faithfully recapitulated by knocking down Mat2aa expression via the injection of an ATG translational MO (Figure 2D and Online Figure VII). We assessed the functional disruption of the Mat2aa, which is an enzyme that catalyzes the synthesis of S-adenosylmethionine, a principal methyl donor for both DNA and protein methylation.25 We detected a significant reduction of the methylation levels of the histone proteins H3k9 me3 and H3k27 me3 (Figure 2E and 2F) and a compromised global DNA methylation level in GBT0364 homozygous embryos (Figure 2G).
The expression of Mat2aa in the heart is restricted to the myocardium, as demonstrated by crossing GBT0364 line with either Tg(fli1a:EGFP) or Tg(titin:actn2-EGFP) lines (Figure 3A and 3B). We sought to perform a cardiac-specific rescue assay by exploiting the loxP sites integrated into the RP2 vector, and generated the Tg(cmlc2:yellow fluorescent protein (YFP)-Cre) transgenic fish line in which Cre recombinase expression was driven specifically in CMs (Online Figure VIII). We further characterized the cardiac phenotypes in GBT0364 and revealed a reduced ventricular chamber size, decreased cardiac myosin light chain 2 (cmlc2) expression, and compromised cardiac functions, as indicated by reduced fraction shortening and heart rates (Figure 3C–3G). Consistent with the multiple-tissue expression of mat2aa, noncardiac defects were also observed in GBT0364 homozygous mutants, including reduced body length and pineal gland mRFP signal (Online Figure VII). In Tg(cmlc2:YFP-Cre); GBT0364/GBT0364 fish, the cardiac-specific Cre recombinase could effectively excise the mutagenic core of the RP2 vector in GBT0364/GBT0364 fish, as indicated by a switch in fluorescence from mRFP to YFP in CMs (Online Figure VIII). Consequently, the cardiac phenotypes, including pericardial edema, reduced fraction shortening, and reduced heart rates, were rescued (Figure 3C–3G). The decreases in cmlc2 expression levels and ventricular chamber size were also restored, whereas noncardiac phenotypes, such as the decreased mRFP expression level in the pineal gland, were not rescued (Online Figure VII). Taken together, our data established a direct causality between the myocardial expression of Mat2aa and its role in regulating cardiogenesis and cardiac functions.
To gain further mechanistic insight into the function of mat2aa in the control of ventricular chamber size, we assessed the CM cell size and cell number by immunostaining dissected embryonic hearts with anti-β-catenin (to quantify individual CM cell size) and anti-Mef2 (to define CM identity) antibodies (Figure 4A). We detected significant reductions of the CM cell size and cell number in homozygous embryos at 3 dpf (Figure 4A–4E). The results of proliferation cell nuclear antigen costaining and terminal deoxynucleotidyl transferase dUTP nick end labeling assays revealed that reduced CM proliferation and increased CM apoptosis, respectively, could potentially explain the decreased CM cell number (Figure 4D–4H). Previous studies have suggested that S-adenosylmethionine affects apoptosis in hepatocytes25,26; therefore, we injected a p53 (MO into GBT0364 homozygous mutant embryos to inhibit apoptosis. The injection of the p53 MO delayed the occurrence of pericardial edema at 3 to 4 dpf. The cellular analysis indicated that the injection of the p53 MO effectively rescued the CM cell number but not the CM size, and affected CM apoptosis but not CM proliferation (Figure 4). Taken together, the results suggested a role for Mat2aa in regulating CM cell size and cell number during cardiogenesis. Consistent with its function in the liver, the function of Mat2aa in regulating CM number is partially ascribed to p53-regulated apoptosis.
The Larval Lethal GBT0425 Line Uncovers an Essential Function of Mrps18b in Cardiac Mitochondrial Homeostasis
During our inbreeding efforts for the remaining 9 cardiac lines, we found that the GBT0425 line failed to yield any viable homozygous adults. A detailed analysis of the homozygous GBT0425 larvae revealed an apparent developmental delay concomitant with a significantly compromised cardiac contractility at 10 dpf, and ultimately resulted in death at 12 dpf (Figure 5C and 5D). This mutant line showed no visible phenotypes during early embryogenesis and was, therefore, not detected in our initial phenotype-based screen. A single RP2 insertion was identified in the 2nd intron of the mrps18b gene (Figure 5A). The mrps18b gene encodes 1 of the 29 components of the conserved small subunit of the mitochondrial ribosome27 (Online Figure IX). The occurrence of the normal splicing event for the full-length transcript was dramatically reduced in GBT0425 homozygous mutant embryos (Figure 5B and Table). The genetic linkage between the mrps18b gene and the observed phenotype was validated by genotyping the 16 larvae exhibiting these phenotypes, which were all confirmed at the molecular level to be homozygous mutants (Online Figure IX).
Consistent with the identity of MRPS18B as a nuclear-encoded mitochondrial protein,27 we detected strong and long-lasting maternal mRFP expression in the GBT0425 embryos (Online Figure IX). Because the RP2 insertion generates a truncated Mrps18b-mRFP fusion protein containing 75 N-terminal amino acid residues (Online Figure IX and Table), with the first 35 residues encoding the predicted mitochondrial targeting sequence (http://ihg.gsf.de/ihg/mitoprot.html), it was expected that mRFP would report mitochondria-specific expression. Indeed, we observed punctate perinuclear expression of mRFP in a dissected GBT0425 heart, and the mRFP was colocalized with the mitochondrial stain MitoTracker Green FM (Figure 5E–5L and Online Figure IX). The truncation of the C-terminal 167 residues in Mrps18b will most likely disrupt the function of the mitochondria ribosome.
In GBT0425 homozygous mutant hearts, reduced number of mitochondria in the dissociated cells was revealed by either MitoTracker Green FM signal or mRFP fluorescence (Figure 5E–5L). As expected, the mitochondrial number appeared to be also reduced in the somite cells (Online Figure IX). The mitochondrial defects in the heart were confirmed by transmission electron microscopy analysis (Figure 5M–5Q). In addition, the morphologies of the individual mitochondria were disturbed, appearing degenerated with undefined cristae borders (Figure 5O and 5P). These observed phenotypes in GBT0425 homozygous fish are unlikely caused by nonspecific effects of fluorescence proteins, because no detectable phenotypes were found in other ZIC lines, such as GBT0410 with comparable level of fluorescence protein expression (Online Figure IX). Taken together, our data revealed that Mrps18b is an essential component of the nuclear-encoded small subunit of the mitochondrial ribosome. The prolonged maternal expression of Mrps18b might protect homozygous GBT0425 mutants from early embryonic lethality, resulting in larval phenotypes when maternal Mrps18b expression is diminished at ~8 dpf.
The Adult Recessive GBT 0411 Line Links Dnajb6b to Cardiac Hypertrophy
We continued to examine the offspring of the remaining 8 cardiac lines, but we did not detect any fish with visually detectable phenotypes. We reasoned that invasive phenotyping methods were required to identify the adult mutants. We were particularly interested in GBT0411, which displayed a rather specific mRFP expression in the heart at the embryonic stage (Figure 1A and 1E). An RP2 insertion was identified in the intron between exon 7 and 8 of dnajb6b, which encodes a protein with 53% amino acid identity to human DNAJB6 (Figure 6A and Online Figure V). In humans, exons 1 to 8 encode the short isoform of the cytosolic DNAJB6(S), whereas exons 1 to 10 encode the long isoform, DNAJB6(L), which harbors a nuclear localization signal encoded by the last 2 exons.28 This RP2 insertion in the GBT0411 line dramatically reduced the normal splicing event between exon 6 and 7 (Figure 6B and Table), and switched most Dnajb6b(L) to Dnajb6b(S)-like isoform, as revealed by Northern blotting (Figure 6C). In the heart, Dnajb6b was expressed only in the myocardium in both embryonic and adult stages (Figure 6D–6F and Online Figure IV). At the subcellular level, the mRFP fluorescence exhibited a striated expression pattern in the sarcomere that colocalized with the Actn2-EGFP, which was consistent with the Z-disc localization reported for DNAJB6(S) in human muscles.29 Interestingly, mutations in human DNAJB6 caused limb-girdle muscular dystrophy type 1D.29,30
We euthanized some of adult cardiac GBT lines and assessed the morphology of their hearts. We observed significantly enlarged ventricles in homozygous GBT0411 fish at 1 year old (Figure 7A and 7B). Immunostaining using anti–β-catenin and anti-proliferation cell nuclear antigen antibodies revealed an increase of CM cell size, but not cell proliferation in the homozygous GBT0411 mutants (Figure 7C and 7D and Online Figure X). Hallmarks of cardiac hypertrophy, including muscular disarray, were also detected by α-actinin antibody staining (Figure 7E), and reactivation of fetal gene atrial natriuretic factor (anf) was detected at 1 year but not at 4 months old (Figure 7F). The cardiac hypertrophy phenotypes in GBT0411 is less likely caused by side effects of fluorescence proteins, as other 7 ZIC lines do not exhibit any noticeable cardiac phenotypes at ≥1 year old, despite stronger mRFP expression in some ZIC lines (Online Figure X). We also did not notice any significant heart size change in either the GBT0031 heterozygous fish17 or Tg(titin:actn2-EGFP) transgenic fish24 at >1 year old, despite their stronger mRFP or EGFP expression level than GBT0411. It is noteworthy to point out that promoter-specific toxicity of fluorescent proteins in CMs might still exist in zebrafish and need to be evaluated by generating transgenic lines containing fluorescent proteins only. Nevertheless, our data strongly suggest that GBT0411 is a recessive mutant for dnajb6b, with an adult phenotype that implicates a role for dnajb6b in cardiac hypertrophy.
Expression-Based Insertional Mutagenesis Screen Strategy Can be Used to Annotate the Cardiac Genome
The current study presents an efficient system to identify cardiac genes and to annotate their expression and functions via a transposon-based insertional mutagenesis screening strategy. Our data demonstrated that RP2-based gene trapping is a sensitive and reliable method for identifying genes with cardiac expression. Five of the 10 tagged genes identified in our fluorescence-based approach are novel cardiac genes that had not previously been detected by ISH and other methods, and have not been included in the Cardiac Gene Ontology database. The cardiac expression of all 10 genes has been validated using RT-PCR in both zebrafish and humans. Our data suggested that the compromised sensitivity of the ISH technology can be partially ascribed to difficulties in distinguishing specific signals from background. Moreover, the dynamic expression profile and subcellular localization of the tagged genes can be easily revealed by following the mRFP reporter.
Each GBT line with both cardiac expression and cardiac phenotypes served as an opportunity for elucidating the molecular mechanisms of heart development and cardiac diseases. The characterization of the GBT0364 line revealed the myocardium-specific expression of Mat2aa in the heart and suggested essential roles for this protein in cardiogenesis and cardiac functions. Both cardiac expression and functions for mat2a have not previously been reported, despite a number of in vitro studies concerning the role of Mat2a in the apoptosis of leukemic T-cells31 and as a transcriptional corepressor of the Maf oncoprotein.32 Our observations prompted further investigations to elucidate functions involving methylation in CM differentiation. The characterization of the GBT0425 line demonstrated the mitochondrial expression of Mrps18b in the heart and revealed an essential function for Mrps18b protein in mitochondrial homeostasis and larval survival. The characterization of the GBT0411 line revealed the CM-specific expression of dnajb6b and suggested its function in adult cardiac hypertrophy. Based on the interaction of DNAJB6 with the hsp70 complex and its implication in regulating protein recycling,33 defective toxic protein aggregation warrants further investigation as a candidate molecular mechanism.
The expression of many cardiac genes is not restricted only to the heart. Therefore, although the cardiac phenotypes can still be specific, it is desirable to separate the primary phenotypes attributable to gene disruption within the heart from secondary consequence of gene disruption in the other tissues. Because each GBT line represented a potential revertible mutant that enables tissue-specific rescue experiments, the causality between the observed cardiac phenotypes and their cardiac expression can be conveniently established, as our experimentation with GBT0364 demonstrated. Moreover, our data prompted the generation of additional tissue-specific Cre transgenic lines that can be used to determine the primary functions of genes expressed in other cardiac cell types, including those of the epicardium, endocardium, cardiac cushion, and cardiac conduction system.
Expression-Based Insertional Mutagenesis Screen Strategy Facilitates the Identification of Recessive Cardiac Mutants in Vertebrates
The present study also demonstrates that the in vivo protein trapping methodology effectively reduces colony management efforts, the major bottleneck for recessive screens performed in vertebrates, thereby offering an appealing alternative method complementary to other mutagenesis approaches. Because fish with GBT insertions can be easily identified using an mRFP tag, the tagged genes can be cloned using PCR-based approaches. The majority of the generated GBT lines represent a near-null mutant allele for the tagged gene, as indicated by the ≥99% knockdown efficiency observed in the GBT lines generated here and the ≥97% knockdown efficiency reported in a previous study.17 Integration of an expression-based enriching strategy further simplifies colony management. Because of our expertise in the heart, we selectively worked on 15 candidate lines with cardiac expression. Deprioritizing the other 52 lines at an early mutagenesis screen step reduced our colony management efforts by >75%. The current preselection strategy was conducted during embryogenesis, based on the general belief that many fetal genes play important roles during the pathogenesis of adult diseases. At the expense of experimental convenience, this approach can be limited by its intrinsic biases toward genes with embryonic cardiac expression. Interestingly, all 10 ZIC genes identified here based on their embryonic expression also express in both adult zebrafish and adult human hearts. If needed, this preselection strategy can be complemented by the selection of GBT lines with mRFP expression in dissected adult hearts.
A plausible solution for conducting recessive adult screens in vertebrates is to establish an international consortium with an accessible central location for generating mutants. Different categories of the mutants shall be distributed to individual labs with related expertise for downstream phenotyping. The European Mouse Disease Clinic program represents such an ongoing collaborative effort,7 with the aim of breeding 500 mutant mice lines into homozygosity. Our expression-based preselection offers an efficient decision-making strategy to disseminate the proper mutant lines to downstream laboratories. Currently, thousands of stable GBT lines are being generated at the Mayo Clinic. Images of the tagged genes are being documented and deposited in the website (www.zfishbook.org).34 Therefore, insertional lines with particular expression patterns can be digitally screened, and each category of insertional lines can be disseminated to laboratories with matching expertise for downstream breeding and phenotyping. Indeed, our initial digital screen of 322 GBT lines identified 18 candidate cardiac lines (Online Figure XI). Because each downstream laboratory only needs to handle a limited number of fish lines, it is anticipated that the recessive adult screen can be conducted on a much larger scale.
Besides increased colony management efforts, the focus of the present study, the other major bottleneck for an adult screen is phenotyping. It is likely that cardiac phenotypes will be detected in many or all of the remaining 7 homozygous GBT lines with the use of adequate phenotyping assays or stressing tools. To test this hypothesis, we plan to maintain and expand a ZIC mutant collection, starting from the 10 cardiac GBT lines reported here (Table). Meanwhile, we are developing cardiac phenotyping assays and stressing tools, as represented by the generation of the first 2 adult zebrafish models of cardiomyopathies.20,21 An exciting future direction is to test whether the ZIC lines can be screened to identify genetic modifiers for adult cardiomyopathy, as well as other major cardiovascular diseases.
We thank the Mayo Clinic’s Electron Microscopy Core Facility for the transmission electronic microscopy analysis and the Mayo Clinic Zebrafish Core Facility for managing the fish.
Sources of Funding
This work was supported in part by funds from the National Institutes of Health (HL107304/GM63904 to X. Xu, GM63904/DA14546/HG006431 to S.C. Ekker), the Mayo Foundation (to X. Xu and X. Lin), and CMST2013CB945300/NSFC31172173 to T.P. Zhong.
In December 2012, the average time from submission to first decision for all original research papers submitted to Circulation Research was 15 days.
The online-only Data Supplement is available with this article at http://circres.ahajournals.org/lookup/suppl/doi:10.1161/CIRCRESAHA.112.300603/-/DC1.
- days postfertilization
- gene-break transposon
- in situ hybridization
- monomeric red fluorescent protein
- reverse transcriptase polymerase chain reaction
- zebrafish insertional cardiac
- Received August 5, 2012.
- Revision received December 20, 2012.
- Accepted January 2, 2013.
- © 2013 American Heart Association, Inc.
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- Barve S
- Clark KJ,
- Argue DP,
- Petzold AM,
- Ekker SC
Novelty and Significance
What Is Known?
Parts of the human genome are implicated in the development of the heart and cardiovascular diseases; however, the functions of these genes are yet to be annotated.
Mutagenesis screening in model organisms is a powerful tool for genomewide gene discovery and functional annotation.
A transposon-based insertional mutagenesis system has been established in zebrafish to generate mutants and to annotate gene functions.
What New Information Does This Article Contribute?
Integration of an expression-based preselection step into insertional mutagenesis screening provides an effective strategy to enrich mutants affecting a particular organ such as the heart.
The reduced colony management efforts enable the identification of adult recessive mutants with cardiac expression.
Methionine adenosyltransferase II, α a (mat2aa) is essential for cardiogenesis; ribosomal protein S18B (mrps18b) is needed for cardiac mitochondrial homeostasis at the larva stage; and DnaJ (Hsp40) homolog, subfamily B, member 6b (dnajb6b) is implicated in adult cardiac hypertrophy.
Mutagenesis screening in model organisms is a powerful tool for genomewide gene discovery and functional annotation; however, it is inefficient in identifying genes that affect a particular organ, such as the heart, and is limited in identifying vertebrate recessive adult mutants because of challenges in colony management. Here, we address these 2 bottlenecks through the integration of an expression-based selection strategy with insertional mutagenesis screening. We isolated 10 zebrafish insertional cardiac mutant lines with clear molecular nature and identified both known and novel genes presenting cardiac expression and functions. In addition to an embryonic lethal mutant, we isolated a mutant exhibiting larval phenotypes, and a mutant with adult recessive phenotypes at 1 year of age. These cardiac phenotypes can be potentially reverted by the deletion of the transposon insert using a cardiomyocyte-specific Cre transgene, enabling the determination of the causality between myocardial expression of the tagged gene and the observed cardiac phenotype. In summary, our data demonstrate an expression-based mutagenesis screening strategy for efficiently generating both embryonic and adult recessive mutants in a particular organ; suggesting an approach that will facilitate the annotation of vertebrate genomes.