Characterization of Müller glia and neuronal progenitors during adult zebrafish retinal regeneration.
Characterization of Müller glia and neuronal progenitors during adult zebrafish retinal regeneration.
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DOI：
10.1016/j.exer.2008.07.009
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发表时间：
2008-11
影响因子：
3.4
通讯作者：
Hyde, David R.
中科院分区：
文献类型：
--
作者：
Thummel, Ryan;Kassen, Sean C.;Enright, Jennifer M.;Nelson, Craig M.;Montgomery, Jacob E.;Hyde, David R.
关键词：
成年斑马鱼视网膜在光诱导光感受器细胞死亡后表现出强大的再生反应。这种反应是由内核层(INL)中的Müler胶质细胞增殖启动的，它产生了神经元前体细胞，这些细胞继续分裂并迁移到外核层(ONL)，在那里它们分化为视杆和视锥感受器。我们之前对持续强光处理16、31、51、68和96小时的视网膜基因表达进行了微阵列分析，以确定可能与神经前体细胞的生成和增殖有关的基因及其相应的蛋白质。我们检测了两个候选转录因子Pax6和Ngn1，以及一个候选转基因OLIG2：EGFP在光损伤再生视网膜中的表达。我们比较了这些标记相对于增殖细胞核抗原(增殖细胞核抗原)的时空表达模式，增殖细胞核抗原是斑马鱼视网膜中已建立的细胞增殖标记，而TG(GFAP：EGFP)nt11转基因系专门标记Müler胶质细胞。我们发现，Müller胶质细胞在再生过程中会去分化，这是因为在它们重新进入细胞周期以产生神经前体细胞后，细胞特异性标记物如GFAP(胶质纤维酸性蛋白)和谷氨酰胺合成酶的丢失。在51小时的持续光照处理中，增殖的神经前体细胞首次检测到Pax6的表达，这是在31小时的光照后Müler胶质细胞首次进入细胞周期之后的显著表现。这表明Pax6在神经元前体细胞中的表达增加，而不是在增殖的Müler神经胶质细胞中。在去分化的Müller胶质细胞中，通过68小时的持续光照处理，首次检测到OLIG2启动子的EGFP表达，而Pax6在密切相关的增殖神经前体细胞迁移到ONL中表达。Pax6和OLIG2的表达持续到光疗后三天，神经前体细胞开始分化为新的视杆和视锥感受器。在光照处理68小时后，在增殖的神经前体细胞中首次检测到Ngn1蛋白的表达。然而，Ngn1的表达持续存在于INL核的一个子集，直到光处理后17天。利用TG(GFAP：EGFP)nt11转基因株系，将Ngn1定位于再生反应后重建的Müler神经胶质核。因此，这些标记物可以用来识别视网膜再生特定阶段的不同细胞类型：神经前体细胞的形成、增殖和Müler胶质细胞的重建。这些标记对于进一步表征其他视网膜损伤模型中的再生反应以及阐明与破坏再生反应的突变体和变异体相关的缺陷将是重要的。
The adult zebrafish retina exhibits a robust regenerative response following light-induced photoreceptor cell death. This response is initiated by the Müller glia proliferating in the inner nuclear layer (INL), which gives rise to neuronal progenitor cells that continue to divide and migrate to the outer nuclear layer (ONL), where they differentiate into rod and cone photoreceptors. We previously conducted a microarray analysis of retinal gene expression at 16, 31, 51, 68, and 96 hours of constant intense-light treatment to identify genes and their corresponding proteins that may be involved in the generation and proliferation of the neuronal progenitor cells. We examined the expression of two candidate transcription factors, Pax6 and Ngn1, and one candidate transgene, olig2:EGFP, in the regenerating light-damaged retina. We compared the temporal and spatial expression patterns of these markers relative to PCNA (proliferating cell nuclear antigen), an established marker for proliferating cells in the zebrafish retina, and the Tg(gfap:EGFP)nt11 transgenic line that specifically labels Müller glial cells. We found that Müller glial cells dedifferentiate during regeneration, based on the loss of cell-specific markers such as GFAP (glial fibrillary acidic protein) and glutamine synthetase following their reentry into the cell cycle to produce neuronal progenitors. Pax6 expression was first detected in the proliferating neuronal progenitors by 51 hours of constant light treatment, which is significantly after the Müller glia first reenter the cell cycle after 31 hours of light. This suggests that Pax6 expression increases in neuronal progenitors, rather than in the proliferating Müller glia. EGFP expression from the olig2 promoter was first detected by 68 hours of constant light treatment in the dedifferentiated Müller glia, with Pax6 expressed in the closely-associated proliferating neuronal progenitors migrating to the ONL. Both Pax6 and olig2 expression persisted until three days post light-treatment, when the neuronal progenitors begin differentiating into new rod and cone photoreceptors. Ngn1 protein expression was initially detected in proliferating neuronal progenitors at 68 hours of light treatment. However, Ngn1 expression persisted in a subset of the INL nuclei until 17 days post-light treatment. Using the Tg(gfap:EGFP)nt11 transgenic line, Ngn1 was localized to the Müller glial nuclei that were reestablished following the regenerative response. These markers, therefore, can be used to identify different cell types at particular stages of retinal regeneration: neuronal progenitor formation, proliferation, and the reestablishment of the Müller glia cells. These markers will be important to further characterize the regeneration response in other retinal damage models and to elucidate the defects associated with mutants and morphants that disrupt the regeneration response.
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