Lei Xu-Dan, Sun Yan, Cai Shi-Jiao, Fang Yang-Wu, Cui Jian-Lin, Li Yu-Hao
Key Laboratory of Tumor Microenvironment and Neurovascular Regulation, Nankai University School of Medicine, Tianjin 300071, China.
Int J Ophthalmol. 2016 Jun 18;9(6):831-7. doi: 10.18240/ijo.2016.06.07. eCollection 2016.
To investigate the role of tumor necrosis factor-alpha (TNF-α) in zebrafish retinal development and myelination.
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 situ hybridization using a hepatocyte-specific mRNA probe ceruloplasmin (cp), and co-injection of TNF-α MO and TNF-α mRNA. An atonal homolog 7 (atoh7) mRNA probe was used to detect neurogenesis onset. The retinal neurodifferentiation was analyzed by immunohistochemistry using antibodies Zn12, Zpr1, 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.
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-α 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.
TNF-α is not an essential regulator for retinal neurogenesis and optic myelination.
研究肿瘤坏死因子-α（TNF-α）在斑马鱼视网膜发育和髓鞘形成中的作用。
合成与野生型斑马鱼胚胎TNF-α mRNA序列翻译起始位点互补的吗啉代寡核苷酸（MO），并将其注射到一至四细胞期胚胎中。通过使用抗TNF-α抗体的蛋白质免疫印迹法、使用肝细胞特异性mRNA探针铜蓝蛋白（cp）的全胚胎原位杂交法以及共注射TNF-α MO和TNF-α mRNA来验证翻译阻断特异性。使用无调性同源物7（atoh7）mRNA探针检测神经发生的起始。通过免疫组织化学分别使用抗体Zn12、Zpr1和Zpr3标记神经节细胞、视锥细胞和视杆细胞，分析视网膜神经分化。使用髓鞘碱性蛋白（mbp）作为标记物，通过全胚胎原位杂交追踪和观察髓鞘形成。
靶向敲低TNF-α导致TNF-α表达的特异性抑制以及肝脏严重发育不全。共注射TNF-α MO和mRNA挽救了肝脏发育。TNF-α morphants中的视网膜神经发生按时启动。在受精后72小时（hpf），视网膜完全分层，而神经节细胞、视锥细胞和视杆细胞分化良好。从受精后3天（dpf）开始，mbp在侧线神经和颅神经的施万细胞中表达，从4 dpf开始，沿着后脑束和脊髓呈线性表达于少突胶质细胞中，这与内源性表达模式非常相似。
TNF-α不是视网膜神经发生和视神经髓鞘形成的必需调节因子。
