RESUMO
Zebrafish is a powerful model for forward genetics. Reverse genetic approaches are limited by the time required to generate stable mutant lines. We describe a system for gene knockout that consistently produces null phenotypes in G0 zebrafish. Yolk injection of sets of four CRISPR/Cas9 ribonucleoprotein complexes redundantly targeting a single gene recapitulated germline-transmitted knockout phenotypes in >90% of G0 embryos for each of 8 test genes. Early embryonic (6 hpf) and stable adult phenotypes were produced. Simultaneous multi-gene knockout was feasible but associated with toxicity in some cases. To facilitate use, we generated a lookup table of four-guide sets for 21,386 zebrafish genes and validated several. Using this resource, we targeted 50 cardiomyocyte transcriptional regulators and uncovered a role of zbtb16a in cardiac development. This system provides a platform for rapid screening of genes of interest in development, physiology, and disease models in zebrafish.
Assuntos
Técnicas de Inativação de Genes/métodos , Coração/embriologia , Proteína com Dedos de Zinco da Leucemia Promielocítica/genética , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/genética , Animais , Sequência de Bases , Sistemas CRISPR-Cas/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Subunidades alfa G12-G13 de Proteínas de Ligação ao GTP/genética , Engenharia Genética/métodos , Morfolinos/genética , Miócitos Cardíacos/citologia , Transcrição Gênica/genética , Peixe-Zebra/embriologiaRESUMO
We report an unexpected role for protease signaling in neural tube closure and the formation of the central nervous system. Mouse embryos lacking protease-activated receptors 1 and 2 showed defective hindbrain and posterior neuropore closure and developed exencephaly and spina bifida, important human congenital anomalies. Par1 and Par2 were expressed in surface ectoderm, and Par2 was expressed selectively along the line of closure. Ablation of G(i/z) and Rac1 function in these Par2-expressing cells disrupted neural tube closure, further implicating G protein-coupled receptors and identifying a likely effector pathway. Cluster analysis of protease and Par2 expression patterns revealed a group of membrane-tethered proteases often coexpressed with Par2. Among these, matriptase activated Par2 with picomolar potency, and hepsin and prostasin activated matriptase. Together, our results suggest a role for protease-activated receptor signaling in neural tube closure and identify a local protease network that may trigger Par2 signaling and monitor and regulate epithelial integrity in this context.
Assuntos
Sistema Nervoso Central/embriologia , Sistema Nervoso Central/metabolismo , Desenvolvimento Embrionário/genética , Tubo Neural/embriologia , Tubo Neural/metabolismo , Receptor PAR-2/metabolismo , Animais , Diferenciação Celular/fisiologia , Células Cultivadas , Sistema Nervoso Central/citologia , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Proteínas de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Humanos , Camundongos , Camundongos Mutantes , Tubo Neural/citologia , Defeitos do Tubo Neural/genética , Defeitos do Tubo Neural/metabolismo , Defeitos do Tubo Neural/fisiopatologia , Peptídeo Hidrolases/genética , Peptídeo Hidrolases/metabolismo , Receptor PAR-1/genética , Receptor PAR-1/metabolismo , Receptor PAR-2/genética , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais/fisiologia , Células-Tronco/citologia , Células-Tronco/metabolismo , Proteínas rac1 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/metabolismoRESUMO
Procoagulant activity on tumor cells can enhance their ability to spread via the circulation to colonize distant organs. Toward defining the relative importance of the main host responses to coagulation for hematogenous metastasis, we examined lung metastases after intravenous injection of melanoma cells in Nf-E2(-/-) mice, which have virtually no circulating platelets; Par4(-/-) mice, which have platelets that fail to respond to thrombin; Par1 and Par2(-/-) mice, which have markedly attenuated endothelial responses to coagulation proteases; and Fib(-/-) mice, which lack fibrinogen. In a severe combined immunodeficiency (SCID) background, median lung tumor count in Nf-E2(-/-), Par4(-/-), and Fib(-/-) mice was 6%, 14%, and 24% of wild type, respectively; total tumor burden was only 4%, 9%, and 3% of wild type, respectively. Similar results were seen in a syngeneic C57BL6 background. By contrast, deficiencies of protease-activated receptor 1 (PAR1) or PAR2 did not provide protection. These results provide strong genetic evidence that platelets play a key role in hematogenous metastasis and contribute to this process by both thrombin-dependent and -independent mechanisms. Importantly, PAR4 heterozygosity conferred some protection against metastasis in this model. Thus even partial attenuation of platelet function may be sufficient to provide benefit.