Yang, Jingchun

The present forward mutagenesis screen strategies in vertebrates are difficult to identify genes and mutants affecting a specific organ of interest. Here, we report a strategy to enrich cardiac mutants by leveraging the transparency of zebrafish embryos and the high efficiency of pGBT-RP2 (RP2), a gene-breaking transposon-based in vivo protein trap cassette. By selectively raising up founder fish with Red Fluorescence Protein (RFP) reporter expression in the heart, we successfully generated 15 cardiac mutant lines. All tagged loci exhibit cardiac expression and the overall knockdown efficiency at transcript level is greater than 95%. One of the cardiac lines, RP2_ #135, is a homozygous embryonic lethal line that is caused by a RP2 insertion in methionine adenosyltransferase 2a (mat2a) . Taking advantage of the LoxP sites inside the RP2 vector, we further demonstrated that the cardiac phenotypes in RP2_ #135 is due to a disrupted expression of mat2a in cardiomyocytes, as indicated by a conditional rescue assay using Tg(cmlc2:Cre-YFP) , a myocardium specific Cre transgenic line. Finally, we show that the reduced cardiomyocyte number, but not size, in the mat2a mutant is conveyed by the p53-mediated apoptosis pathway. Together, our data prove the feasibility of a facile approach to generate a vertebrate cardiac mutant collection with the following two unique features. First, the expression dynamics of each tagged gene is reported by RFP. Second, the causality between the tissue-specific gene expression and the resulted cardiac phenotypes can be attested genetically.

Background: While proper heart contraction requires an intact sarcomere structure, contraction reciprocally regulates sarcomere assembly and cardiomyocyte shape during heart morphogenesis and heart remodeling. However, how contraction modulates heart morphogenesis and the underlying molecular mechanism remains unclear. Here we address this question by manipulating cardiac contractility in a developing zebrafish embryo. Results: Cessation of heart contraction was achieved by treating zebrafish embryos with blebbistatin, a myosin II specific inhibitor, or Niferdipine, a Ca 2+ channel inhibitor. Loss of cardiac contractility reduces ventricular chamber size in 1 dpf embryo, but significantly increases ventricular chamber size in 2 dpf and 3 dpf embryos. In both circumstances, cessation of contraction consistently prevents or disrupts the lateral fusion of myofibril. Cessation of contraction-induced heart enlargement is reversible and also dependent on the time of drug treatment. This heart-remodeling event in 2dpf embryo, which manifests as increased cardiomyocyte surface area accompanied by reduced thickness, can be attenuated by reducing the transmural pressure towards the ventricle wall. This mechanotransductive process occurs in the myocardium without need of endocardium. At the molecular level, by screening several compounds, we found that PI3K signaling confers this contraction-regulated cardiomyocyte shape remodeling process without affecting the lateral fusion of sarcomere. Conclusion: Our data in an in vivo animal model distinguish two different and relatively independent functions of contraction in regulating lateral fusion of sarcomere and cardiomyocytes shape remodeling. The contraction-regulated heart shape remodeling is conveyed by PI3k signaling mediated mechanotransduction independent of endocardium.

Abstract Background and Aims: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a member of the tumor necrosis factor superfamily, is predominantly expressed on immune cells. Although TRAIL biology has garnered considerable interest as a potential anti-cancer strategy, TR agonists have had very limited anti-cancer activity in humans. TRAIL signaling in T cells may also potentially provide an immune checkpoint function as it can inhibit T cell activation and proliferation by interfering with T cell receptor signaling. However, this potential immune checkpoint function of TRAIL has not been examined in cancer biology. Cholangiocarcinoma (CCA), a malignancy of the bile ducts, provides a model to examine the potential immune checkpoint function of TRAIL. Methods: Using a syngeneic, orthotopic murine model of CCA (PMID: 29464042), murine CCA cells (SB cells) that express both TRAIL and TRAIL receptor (TR) were implanted into livers of WT C57BL/6J and Tr−/− mice. Hence, in this model the host immune cells express TRAIL but not the receptor; they would be capable of inducing TRAIL-mediated apoptosis in CCA cells but would be resistant to potential TRAIL-mediated immunosuppression. After 4 weeks of tumor growth, mice were sacrificed and tumor and immune characterization (via flow cytometry) was conducted. Results: Implantation of SB cells into Tr−/− mice result in a significant reduction in tumor volumes compared to WT mice. Tumor-bearing Tr−/− mice had a significant infiltration of cytotoxic T lymphocytes (CTLs) (CD45+CD3+CD8+CD11a+) and enhanced CTL effector function. Moreover, Tr−/− mice tumors had a significant decrease in granulocytic and monocytic myeloid-derived suppressor cell (MDSCs) as well as tumor-associated macrophages (TAMs) (CD45+F4/80+CD11b+CD206+) compared to WT mice tumors. Conclusion: Using a unique syngeneic orthotopic implantation model of murine CCA, we demonstrate that CCA cells expressing TRAIL in a mouse genetically deficient for TR exhibit reduced tumor volumes enhanced CTL infiltration and function, and reduced MDSC and TAM infiltration into the tumors. These data suggest that TRAIL has a potential immune checkpoint function, and targeting TRAIL signaling with consequent augmentation of CTL function maybe a promising therapeutic approach in human cancers. Citation Format: Emilien Loeuillard, Jingchun Yang, Haidong Dong, Gregory J. Gores, Sumera Ilyas. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) mediates tumor immune evasion in cholangiocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2738.

Background Sorbs2b (sorbin and SH3 domain‐containing 2b) was recently identified as a cardiomyopathy gene from a zebrafish mutagenesis screen. However, cardiac functions of its mammalian ortholog remain elusive. Methods and Results We conducted a detailed expression and subcellular localization analysis of Sorbs2 ortholog in mice and a phenotypic characterization in Sorbs2 knockout mice. Sorbs2 is highly expressed in the mouse heart and encodes an adhesion junction/desmosome protein that is mainly localized to the intercalated disc. A mutation with near complete depletion of the Sorbs2 protein in mice results in phenotypes characteristic of human arrhythmogenic cardiomyopathy (ACM), including right ventricular dilation, right ventricular dysfunction, spontaneous ventricular tachycardia, and premature death. Sorbs2 is required to maintain the structural integrity of intercalated disc. Its absence resulted in profound cardiac electrical remodeling with impaired impulse conduction and action potential derangements. Targeted sequencing of human patients with ACM identified 2 rare splicing variants classified as likely pathogenic were in 2 unrelated individuals with ACM from a cohort of 59 patients with ACM. Conclusions The Sorbs2 knockout mouse manifests several key features reminiscent of human ACM. Although the candidacy of SORBS2 as a new ACM‐susceptibility gene is supported by preliminary human genetics study, future validation in larger cohorts with ACM is needed.

Heart disease is the leading cause of mortality in the U.S. with approximately 610,000 people dying every year. Effective therapies for many cardiac diseases are lacking, largely due to an incomplete understanding of their genetic basis and underlying molecular mechanisms. Zebrafish (Danio rerio) are an excellent model system for studying heart disease as they enable a forward genetic approach to tackle this unmet medical need. In recent years, our team has been employing electrocardiogram (ECG) as an efficient tool to study the zebrafish heart along with conventional approaches, such as immunohistochemistry, DNA and protein analyses. We have overcome various challenges in the small size and aquatic environment of zebrafish in order to obtain ECG signals with favorable signal-to-noise ratio (SNR), and high spatial and temporal resolution. In this paper, we highlight our recent efforts in zebrafish ECG acquisition with a cost-effective simplified microelectrode array (MEA) membrane providing multi-channel recording, a novel multi-chamber apparatus for simultaneous screening, and a LabVIEW program to facilitate recording and processing. We also demonstrate the use of machine learning-based programs to recognize specific ECG patterns, yielding promising results with our current limited amount of zebrafish data. Our solutions hold promise to carry out numerous studies of heart diseases, drug screening, stem cell-based therapy validation, and regenerative medicine.

Animal models have played a critical role in validating human dilated cardiomyopathy (DCM) genes, particularly those that implicate novel mechanisms for heart failure. However, the disease phenotype may be delayed due to age-dependent penetrance. For this reason, we generated an adult zebrafish model, which is a simpler vertebrate model with higher throughput than rodents. Specifically, we studied the zebrafish homologue of GATAD1, a recently identified gene for adult-onset autosomal recessive DCM. We showed cardiac expression of gatad1 transcripts, by whole mount in situ hybridization in zebrafish embryos, and demonstrated nuclear and sarcomeric I-band subcellular localization of Gatad1 protein in cardiomyocytes, by injecting a Tol2 plasmid encoding fluorescently-tagged Gatad1. We next generated gatad1 knock-out fish lines by TALEN technology and a transgenic fish line that expresses the human DCM GATAD1-S102P mutation in cardiomyocytes. Under stress conditions, longitudinal studies uncovered heart failure (HF)-like phenotypes in stable KO mutants and a tendency toward HF phenotypes in transgenic lines. Based on these efforts of studying a gene-based inherited cardiomyopathy model, we discuss the strengths and bottlenecks of adult zebrafish as a new vertebrate model for assessing candidate cardiomyopathy genes.

ABSTRACTMutations in sarcomere genes have been found in many inheritable human diseases, including hypertrophic cardiomyopathy. Elucidating the molecular mechanisms of sarcomere assembly shall facilitate understanding of the pathogenesis of sarcomere‐based cardiac disease. Recently, biochemical and genomic studies have identified many new genes encoding proteins that localize to the sarcomere. However, their precise functions in sarcomere assembly and sarcomere‐based cardiac disease are unknown. Here, we review zebrafish as an emerging vertebrate model for these studies. We summarize the techniques offered by this animal model to manipulate genes of interest, annotate gene expression, and describe the resulting phenotypes. We survey the sarcomere genes that have been investigated in zebrafish and discuss the potential of applying this in vivo model for larger‐scale genetic studies. Anat Rec, 297:1681–1693, 2014. © 2014 Wiley Periodicals, Inc.

Contraction regulates heart development via a complex mechanotransduction process controlled by various mechanical forces. Here, we exploit zebrafish embryos as an in vivo animal model to discern the contribution from different mechanical forces and identify the underlying mechanotransductive signaling pathways of cardiogenesis. We treated 2 days postfertilization zebrafish embryos with Blebbistatin, a myosin II inhibitor, to stop cardiac contraction, which induces a response termed cessation of contraction-induced cardiomyocyte (CM) enlargement (CCE). Accompanying the CCE, lateral fusion of myofibrils was attenuated within CMs. The CCE can be blunted by loss of blood in tail-docked zebrafish but not in cloche mutant fish, suggesting that transmural pressure rather than shear stress is accountable for the chamber enlargement. By screening a panel of small molecule inhibitors, our data suggested essential functions of phosphoinositide 3-kinase signaling and protein synthesis in CCE, which are independent of the sarcomere integrity. In summary, we defined a unique CCE response in genetically tractable zebrafish embryos. A panel of assays was established to verify the contribution from extrinsic forces and interrogate underlying signaling pathways.

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.