Role of tumor necrosis factor-alpha in zebrafish retinal neurogenesis and myelination.
Role of tumor necrosis factor-alpha in zebrafish retinal neurogenesis and myelination.
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DOI：
10.18240/ijo.2016.06.07
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发表时间：
2016-06
影响因子：
1.4
通讯作者：
Xu Lei;Yan Sun;Shi-Jiao Cai;Yangwu Fang;Jian-Lin Cui;Yu-Hao Li
中科院分区：
文献类型：
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作者：
Xu Lei;Yan Sun;Shi-Jiao Cai;Yangwu Fang;Jian-Lin Cui;Yu-Hao Li
目的探讨肿瘤坏死因子-α (TNF-α)在斑马鱼视网膜发育和髓鞘形成中的作用。方法合成与野生型胚胎斑马鱼TNF-α mRNA序列翻译起始位点互补的Morpholino oligonucotides (MO)，并将其注射到一至四细胞胚胎中。通过抗TNF-α抗体的Western blotting、肝细胞特异性mRNA探针铜蓝蛋白（ceruloplasmin, cp）的全载原位杂交以及TNF-α MO和TNF-α mRNA的联合注射验证了翻译阻断的特异性。无调性同源7 (atoh7) mRNA探针用于检测神经发生的发生。采用免疫组化方法分析视网膜神经分化，分别用抗体Zn12、Zpr1和Zpr3标记神经节细胞、视锥细胞和视杆细胞。髓鞘碱性蛋白（Myelin basic protein, mbp）作为标记物，采用原位全载杂交技术对髓鞘形成过程进行跟踪和观察。结果靶向敲低TNF-α可特异性抑制TNF-α表达，导致肝脏严重发育不全。联合注射TNF-α MO和mRNA可挽救肝脏发育。TNF-α变异体视网膜神经发生准时开始。在受精后72小时，视网膜完全层合，神经节细胞、视锥细胞和视杆细胞分化良好。mbp从受精后3天开始在侧线神经和脑神经的雪旺细胞中表达，从受精后4天开始在沿后脑束和脊髓呈线性分布的少突胶质细胞中表达，与其内源性谱非常相似。结论TNF-α不是视网膜神经发生和视神经髓鞘形成的必要调节因子。
AIM To investigate the role of tumor necrosis factor-alpha (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 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. 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-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. CONCLUSION TNF-α is not an essential regulator for retinal neurogenesis and optic myelination.