Analysis of retinal cell development in chick embryo by immunohistochemistry and in ovo electroporation techniques.
Analysis of retinal cell development in chick embryo by immunohistochemistry and in ovo electroporation techniques.
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通过免疫组织化学和卵内电穿孔技术分析鸡胚视网膜细胞发育。
DOI：
10.1186/1471-213x-10-8
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发表时间：
2010-01-20
影响因子：
--
通讯作者：
Cai, Li
中科院分区：
文献类型：
--
作者：
Doh, Sung Tae;Hao, Hailing;Loh, Stephanie C.;Patel, Tapan;Tawil, Haim Y.;Chen, David K.;Pashkova, Anna;Shen, Andy;Wang, Huimin;Cai, Li
视网膜细胞的发育已经得到了广泛的研究；然而，目前关于动态形态和分子变化的知识还不完整。本研究旨在利用靶向视网膜注射、卵内电穿孔和免疫组织化学技术，揭示胚胎阶段视网膜细胞发育过程中的动态形态和分子变化。将表达绿色荧光蛋白(GFP)的质粒DNA送入视网膜下间隙，在胚胎第3天(E3)或第4天(E4)，借助脉冲电流将其导入鸡视网膜干/祖细胞。对转基因的视网膜组织在雏鸡发育的不同阶段进行了分析，从E4的神经发生开始到E18的神经发生接近结束。GFP的表达可以清楚地显示细胞形态和视网膜板层位置，以指示视网膜细胞的身份。使用细胞类型特异性标记(如Visinin、XAP-1、LIM1+2、PKCα、NeuN、Pax6、Brn3a、Vimentin等)的免疫组织化学。进一步确认了视网膜细胞的类型。然后通过计数观察到的具有各种视网膜细胞类型特有的形态特征的GFP表达细胞的数量，来确定随着时间的推移视网膜细胞类型的组成。在E3-E4的视网膜注射和电穿孔的新方法允许可视化所有类型的视网膜细胞，包括晚出生的神经元，例如在单细胞水平上的双极细胞，这在E1.5的注射和电穿孔的传统方法中是困难的。基于对细胞形态、视网膜板层位置、免疫组织化学和GFP表达细胞计数的分析，确定了视网膜细胞发育的时间线和动态的形态和分子变化。这些数据提供了更完整的视网膜细胞发育信息，可以为正常视网膜发育和疾病的研究提供参考。
Retinal cell development has been extensively investigated; however, the current knowledge of dynamic morphological and molecular changes is not yet complete. This study was aimed at revealing the dynamic morphological and molecular changes in retinal cell development during the embryonic stages using a new method of targeted retinal injection, in ovo electroporation, and immunohistochemistry techniques. A plasmid DNA that expresses the green fluorescent protein (GFP) as a marker was delivered into the sub-retinal space to transfect the chick retinal stem/progenitor cells at embryonic day 3 (E3) or E4 with the aid of pulses of electric current. The transfected retinal tissues were analyzed at various stages during chick development from near the start of neurogenesis at E4 to near the end of neurogenesis at E18. The expression of GFP allowed for clear visualization of cell morphologies and retinal laminar locations for the indication of retinal cell identity. Immunohistochemistry using cell type-specific markers (e.g., Visinin, Xap-1, Lim1+2, Pkcα, NeuN, Pax6, Brn3a, Vimentin, etc.) allowed further confirmation of retinal cell types. The composition of retinal cell types was then determined over time by counting the number of GFP-expressing cells observed with morphological characteristics specific to the various retinal cell types. The new method of retinal injection and electroporation at E3 - E4 allows the visualization of all retinal cell types, including the late-born neurons, e.g., bipolar cells at a level of single cells, which has been difficult with a conventional method with injection and electroporation at E1.5. Based on data collected from analyses of cell morphology, laminar locations in the retina, immunohistochemistry, and cell counts of GFP-expressing cells, the time-line and dynamic morphological and molecular changes of retinal cell development were determined. These data provide more complete information on retinal cell development, and they can serve as a reference for the investigations in normal retinal development and diseases.