摘要
AIM： To investigate the role of tumor necrosis factoralpha （TNF-α） in zebrafish retinal development and myelination. METHODS： Morpholino oligonucleotides （MO）, which are complementary to the translation start site of the wild-type embryonic zebrafish TNF-α mRNA sequence, were synthesized and injected into one to four-cell embryos. The translation blocking specificity was verified by Western blotting using an anti-TNF-α antibody, whole-mount in sltuhybridization using a hepatocytespecific mRNA probe ceruloplasmin （cp）, and coinjection of TNF-α MO and TNF-α mRNA. An atonel homolog 7 （atoh7） mRNA probe was used to detect neurogenesis onset. The retinal neurodifferentiation was analyzed by immunohistochemistry using antibodies Zn12, Zprl, and Zpr3 to label ganglion cells, cones, and rods, respectively. Myelin basic protein （mbp）was used as a marker to track and observe the myelination using whole-mount in situ hybridization. RESULTS： Targeted knockdown of TNF-α resulted in specific suppression of TNF-α expression and a severely underdeveloped liver. The co-injection of TNF-α MO and mRNA rescued the liver development. Retinal neurogenesis in TNF-cc morphants was initiated on time. The retina was fully laminated, while ganglion cells, cones, and rods were well differentiated at 72 hours post-fertilization （hpf）. mbp was expressed in Schwann cells in the lateral line nerves and cranial nerves from 3 days post -fertilization （dpf） as well as in oligodendrocytes linearly along the hindbrain bundles and the spinal cord from 4 dpf, which closely resembled its endogenous profile. CONCLUSION： TNF-α is not an essential regulator for retinal neurogenesis and optic myelination.
AIM： To investigate the role of tumor necrosis factoralpha （TNF-α） in zebrafish retinal development and myelination. METHODS： Morpholino oligonucleotides （MO）, which are complementary to the translation start site of the wild-type embryonic zebrafish TNF-α mRNA sequence, were synthesized and injected into one to four-cell embryos. The translation blocking specificity was verified by Western blotting using an anti-TNF-α antibody, whole-mount in sltuhybridization using a hepatocytespecific mRNA probe ceruloplasmin （cp）, and coinjection of TNF-α MO and TNF-α mRNA. An atonel homolog 7 （atoh7） mRNA probe was used to detect neurogenesis onset. The retinal neurodifferentiation was analyzed by immunohistochemistry using antibodies Zn12, Zprl, and Zpr3 to label ganglion cells, cones, and rods, respectively. Myelin basic protein （mbp）was used as a marker to track and observe the myelination using whole-mount in situ hybridization. RESULTS： Targeted knockdown of TNF-α resulted in specific suppression of TNF-α expression and a severely underdeveloped liver. The co-injection of TNF-α MO and mRNA rescued the liver development. Retinal neurogenesis in TNF-cc morphants was initiated on time. The retina was fully laminated, while ganglion cells, cones, and rods were well differentiated at 72 hours post-fertilization （hpf）. mbp was expressed in Schwann cells in the lateral line nerves and cranial nerves from 3 days post -fertilization （dpf） as well as in oligodendrocytes linearly along the hindbrain bundles and the spinal cord from 4 dpf, which closely resembled its endogenous profile. CONCLUSION： TNF-α is not an essential regulator for retinal neurogenesis and optic myelination.
基金
Supported by the National Natural Science Foundation of China (No.81301080)
the Tianjin Natural Science Foundation (No.15JCYBJC24400, No.15JCQNJC10900)
the Scientific Research Foundation for the Returned Overseas Chinese Scholars (No.2012-1707)
