RESUMO
In the past two decades, purinergic signaling has emerged as a key regulator of hematopoiesis in physiological and pathological conditions. ADP receptor P2y12 is a crucial component of this signaling, but whether it is involved in primitive hematopoiesis remains unknown. To elucidate the function of P2y12 and provide new insights for drug development, we established a zebrafish P2y12 mutant by CRISPR/Cas 9-based genetic modification system, and investigated whether P2y12 acted as an important regulator for primitive hematopoiesis. By using mass spectrometry (MS) combined with RNA sequencing, we showed that absence of P2y12 induced excessive erythropoiesis, evidenced by significantly increased expression of mature erythrocytes marker α-globin (Hbae1 and Hbae3), ß-globin (Hbbe1 and Hbbe3). Expression pattern analysis showed that P2y12 was mainly expressed in red blood cells and endothelial cells of early zebrafish embryos. Further studies revealed that primitive erythroid progenitor marker Gata1 was markedly up-regulated. Remarkably, inhibition of Gata1 by injection of Gata1 morpholino could rescue the erythroid abnormality in P2y12 mutants. The present study demonstrates the essential role of purinergic signaling in differentiation of proerythrocytes during primitive hematopoiesis, and provides potential targets for treatment of blood-related disease and drug development.
Assuntos
Fator de Transcrição GATA1/antagonistas & inibidores , Hematopoese/fisiologia , Receptores Purinérgicos P2Y12/fisiologia , Proteínas de Peixe-Zebra/antagonistas & inibidores , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados , Sistemas CRISPR-Cas , Diferenciação Celular/fisiologia , Embrião não Mamífero/fisiologia , Endotélio Vascular/fisiologia , Eritrócitos/fisiologia , Feminino , Fator de Transcrição GATA1/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Técnicas de Inativação de Genes , Hematopoese/genética , Masculino , Mutação , Receptores Purinérgicos P2Y12/genética , Regulação para Cima/fisiologia , Peixe-Zebra , Proteínas de Peixe-Zebra/metabolismoRESUMO
OBJECTIVE: MicroRNA-126 (miR-126) is an endothelium-enriched miRNA and functions in vascular integrity and angiogenesis. The application of miRNA as potential biomarker and therapy target has been widely investigated in various pathological processes. However, its role in lymphatic diseases had not been widely explored. We aimed to reveal the role of miR-126 in lymphangiogenesis and the regulatory signaling pathways for potential targets of therapy. APPROACH AND RESULTS: Loss-of-function studies using morpholino oligonucleotides and CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) system showed that silencing of miR-126a severely affected the formation of parachordal lymphangioblasts and thoracic duct in zebrafish embryos, although their development in miR-126b knockdown embryos was normal. Expression analyses by in situ hybridization and immunofluorescence indicated that miR-126a was expressed in lymphatic vessels, as well as in blood vessels. Time-lapse confocal imaging assay further revealed that knockdown of miR-126a blocked both lymphangiogenic sprouts budding from the posterior cardinal vein and lymphangioblasts extension along horizontal myoseptum. Bioinformatics analysis and in vivo report assay identified that miR-126a upregulated Cxcl12a by targeting its 5' untranslated region. Moreover, loss- and gain-of-function studies revealed that Cxcl12a signaling acted downstream of miR-126a during parachordal lymphangioblast extension, whereby Flt4 signaling acts as a cooperator of miR-126a, allowing it to modulate lymphangiogenic sprout formation. CONCLUSIONS: These findings demonstrate that miR-126a directs lymphatic endothelial cell sprouting and extension by interacting with Cxcl12a-mediated chemokine signaling and Vegfc-Flt4 signal axis. Our results suggest that these key regulators of lymphangiogenesis may be involved in lymphatic pathogenesis of cardiovascular diseases.
Assuntos
Quimiocina CXCL12/metabolismo , Linfangiogênese , MicroRNAs/metabolismo , Transdução de Sinais , Ducto Torácico/metabolismo , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/metabolismo , Animais , Animais Geneticamente Modificados , Sistemas CRISPR-Cas , Movimento Celular , Proliferação de Células , Quimiocina CXCL12/genética , Biologia Computacional , Regulação da Expressão Gênica no Desenvolvimento , Inativação Gênica , Genótipo , Linfografia , MicroRNAs/genética , Microscopia Confocal , Morfolinos/genética , Morfolinos/metabolismo , Fenótipo , Ducto Torácico/embriologia , Fatores de Tempo , Imagem com Lapso de Tempo , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/genética , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genéticaRESUMO
BACKGROUND AND AIMS: ApoEb is a zebrafish homologous to mammalian ApoE, whose deficiency would lead to lipid metabolism disorders (LMDs) like atherosclerosis. We attempted to knock out the zebrafish ApoEb, then establish a zebrafish model with LMD. METHODS: ApoEb was knocked out using the CRISPR/Cas9 system, and the accumulation of lipids was confirmed by Oil Red O staining, confocal imaging, and lipid measurements. The lipid-lowering effects of simvastatin (SIM), ezetimibe (EZE) and Xuezhikang (XZK), an extract derived from red yeast rice, were evaluated through in vivo imaging in zebrafish larvae. RESULTS: In the ApoEb mutant, significant vascular lipid deposition occurred, and lipid measurement performed in the whole-body homogenate of larvae and adult plasma showed significantly increased lipid levels. SIM, EZE and XZK apparently relieved hyperlipidemia in ApoEb mutants, and XZK had a significant inhibitory effect on the recruitment of neutrophils and macrophages. CONCLUSIONS: In this study, an LMD model has been established in ApoEb mutant zebrafish. We suggest that this versatile model could be applied in studying hypercholesterolemia and related vascular pathology in the context of early atherosclerosis, as well as the physiological function of ApoE.