Citations

The zebrafish has emerged as an excellent model for cardiovascular research thanks to its ex-utero and rapid embryonic development, its embryonic transparent nature, and its capacity to survive in the absence of a functional cardiovascular system during the first week of development, which enables functional characterization of mutations that would otherwise induce lethality in traditional murine models. The aim of this project is to identify and characterize novel genes involved in zebrafish cardiovascular development using mutagenic gene trapping, a technique that generates random insertional mutations across the genome. We use the well-defined RP2 gene-breaking transposon system, which not only mutates, but also fluorescently tags the trapped gene product(s) (Clark, Nat Methods, 2011). RP2 also introduces loxP sites into the mutant locus, which can be used for Cre-mediated phenotype rescue by microinjection of Cre recombinase, or by crossing to tissue-specific Cre lines. From over 3000 RP2-injected embryos, 141 fish showed germline transmission. Among them, 51 expressed strong fluorescence in different tissues, including the heart, vessels, notochord, central nervous system (CNS), and eyes. Three cardiovascular lines were selected for phenotypic characterization and functional studies. RP2#C2 strain expressed fluorescence in the heart valves, CNS, eyes and pectoral fin buds. Inverse PCR in RP2#C2 demonstrated a trapped gene at meis4.1a, which encodes a homeobox transcription factor that has not been previously studied in zebrafish. RP2#121 strain expressed strong fluorescence in cardiac and skeletal muscles. RP2#91 strain showed expression in the vasculature and demonstrated a trapped gene at pdgfra. Homozygous RP2#91 mutants showed severe defects in the heart, blood flow, and other body parts including the head and musculature. We are currently creating a panel of tissue-specific Cre lines targeting tissues such as cardiomyocytes, endothelial cells and smooth muscle cells for spatiotemporal rescuing of the mutant phenotype. The generated zebrafish protein-trap lines are invaluable tools to annotate gene function, dissect the molecular mechanisms of cardiovascular development, and potentially serve as disease models.

Abstract Colorectal cancer (CRC) is the second leading cause of cancer death worldwide. Growing evidence supports gene fusions as good candidates for molecularly targeted therapy in CRC. Here we describe a case of a 63-year-old man who had a radical right hemicolectomy procedure 24 months ago. Pathological diagnosis indicated colorectal adenocarcinoma with stage pT4N2bMx. During re-examination in December 2016, positron emission tomography/computed tomography scans indicated relapse with multiple lymph nodes metastasis. Then the patient received a nine-cycle combination treatment of XELOX and bevacizumab and showed progressive disease (PD). Subsequently, the patient was treated with bevacizumab plus FOLFIRI for 2 months before discontinuation because of adverse events. Paraffin sections of postoperative colorectal tissue were subjected to next-generation sequencing, and epidermal growth factor receptor (EGFR) amplification and rare EGFR–SEPT14 fusion were identified. The patient then received erlotinib, an EGFR tyrosine kinase inhibitor (TKI), and achieved a partial response. However, the patient subsequently showed PD, and a new variant, EGFRvIII, appeared in metastasis, which may be involved in erlotinib resistance. We suggest that there is value in treating patients harboring EGFR fusions with EGFR TKI therapy, and EGFR–SEPT14 fusion may be used as a therapeutic target for CRC. Key Points To the authors' knowledge, this is the first report of EGFR–SEPT14 fusion in colorectal cancer. The patient achieved a partial response after treatment with the epidermal growth factor receptor tyrosine kinase inhibitor erlotinib. This report expands the list of gene fusions in colorectal cancer and highlights new targets for the therapeutic intervention. EGFRvIII may be involved in erlotinib resistance, which is rare in colorectal cancer.

Cardiac development in vertebrates is a finely tuned process regulated by a set of conserved signaling pathways. Perturbations of these processes are often associated with congenital cardiac malformations. Platelet-derived growth factor receptor α (PDGFRα) is a highly conserved tyrosine kinase receptor, which is essential for development and organogenesis. Disruption of Pdgfrα function in murine models are embryonic lethal due to severe cardiovascular defects, suggesting a role in cardiac development, thus necessitating the use of alternative models to explore its precise function. In this study, we generated a zebrafish pdgfra mutant line by gene trapping, in which the Pdgfra protein is truncated and fused with mRFP (Pdgfra-mRFP). Our results demonstrate that pdgfra mutants have defects in cardiac morphology as a result of abnormal fusion of myocardial precursors. Expression analysis of the developing heart at later stages suggested that Pdgfra-mRFP is expressed in the endocardium. Further examination of the endocardium in pdgfra mutants revealed defective endocardial migration to the midline, where cardiac fusion eventually occurs. Together, our data suggests that pdgfra is required for proper medial migration of both endocardial and myocardial precursors, an essential step required for cardiac assembly and development.

Summary Cellular senescence, which is known to halt proliferation of aged and stressed cells, plays a key role against cancer development and is also closely associated with organismal aging. While increased insulin‐like growth factor ( IGF ) signaling induces cell proliferation, survival and cancer progression, disrupted IGF signaling is known to enhance longevity concomitantly with delay in aging processes. The molecular mechanisms involved in the regulation of aging by IGF signaling and whether IGF regulates cellular senescence are still poorly understood. In this study, we demonstrate that IGF ‐1 exerts a dual function in promoting cell proliferation as well as cellular senescence. While acute IGF ‐1 exposure promotes cell proliferation and is opposed by p53, prolonged IGF ‐1 treatment induces premature cellular senescence in a p53‐dependent manner. We show that prolonged IGF ‐1 treatment inhibits SIRT 1 deacetylase activity, resulting in increased p53 acetylation as well as p53 stabilization and activation, thus leading to premature cellular senescence. In addition, either expression of SIRT 1 or inhibition of p53 prevented IGF ‐1‐induced premature cellular senescence. Together, these findings suggest that p53 acts as a molecular switch in monitoring IGF ‐1‐induced proliferation and premature senescence, and suggest a possible molecular connection involving IGF ‐1‐ SIRT 1‐p53 signaling in cellular senescence and aging.

Hacs1, a SH3 and SAM domain‐containing adaptor protein, is up‐regulated by IL‐4 in activated B cells and strongly expressed in dendritic cells. To elucidate the function of Hacs1 in immune regulation, we generated Hacs1 − / − mice by deletion of the SH3 and SAM domains. Hacs1 − / − mice were viable and fertile and had normal bone marrow B‐cell development and normal splenic T‐ and B‐cell populations. However, adult Hacs1 − / − mice had increased peritoneal Bla cells (IgM + CD5 + ). On immunization with T‐cell‐independent antigen TNP‐Ficoll, Hacs1 − / − mice had increased production of anti‐TNP IgM and IgG3. Purified splenic B cells from Hacs1 − / − mice showed increased cell proliferation on BCR (B‐cell receptor) stimulation. We further demonstrate that the Hacs1 − / − B cells had increased global tyrosine phosphorylation, including tyrosine kinases Lyn and Akt. Both T‐helper type 1 (T h 1) and T‐helper type 2 (T h 2) humoral responses were enhanced in Hacs1 − / − mice. In vitro bone marrow‐derived Hacs1 − / − dendritic cells showed increased IL‐12 production on stimulation with ovalbumin (OVA). This study suggests that Hacs1 is an immunoinhibitory adaptor that might be a useful target for immune suppression therapy.—Wang, D., Stewart, A. K., Zhuang, L., Zhu, Y., Wang, Y., Shi, C., Keating, A., Slutsky, A., Zhang, H., Wen, X.‐Y. Enhanced adaptive immunity in mice lacking the immunoinhibitory adaptor Hacs1. FASEBJ. 24, 947–956 (20l0). www.fasebj.org