Visualizing Ocular Morphogenesis by Lightsheet Microscopy Using rx3:GFP Transgenic Zebrafish.
Visualizing Ocular Morphogenesis by Lightsheet Microscopy Using rx3:GFP Transgenic Zebrafish.
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DOI：
10.3791/62296
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发表时间：
2021-04-05
期刊：
影响因子：
--
通讯作者：
Morris AC
中科院分区：
文献类型：
--
作者：
Petersen RA;Morris AC
脊椎动物的眼睛发育是一个复杂的过程，开始于胚胎原肠发育的末期，需要细胞迁移、增殖和分化的精确协调。延时成像提供了对眼睛发育过程中细胞行为的独特洞察，因为它允许我们可视化体内的眼球发生。斑马鱼是可视化这一过程的一个很好的模型，因为它们的脊椎动物眼睛高度保守，并且能够在保持光学透明的情况下快速和外部发育。转基因斑马鱼Tg(rx3：GFP)的使用大大促进了斑马鱼眼睛发育的时间推移成像研究。在发育中的前脑中，rx3：GFP的表达标志着单眼区的细胞，GFP在眼区外翻形成视泡，视泡内陷形成视杯时继续表达。因此，rx3：GFP表达的高分辨率时间推移成像允许我们跟踪眼睛原基发育到视网膜的时间。光片显微镜能够穿透较厚的样品进行荧光成像，最大限度地减少光漂白和光毒性，并且成像速度快，是一种理想的眼部形态发生随时间变化的成像方法。这里，提供了一种使用商用光片显微镜和图像处理工作站来分析所得到的数据的眼形态发生的时移成像的协议。该方案详细介绍了胚胎麻醉、低温琼脂糖包埋、显像室悬浮、设置成像参数，最后利用图像分析软件对成像数据进行分析的过程。由此得到的数据集可以提供对眼睛形态发生过程的有价值的见解，以及由于基因突变、暴露于药物或其他实验操作而对这一过程的扰动。
Vertebrate eye development is a complex process that begins near the end of embryo gastrulation and requires the precise coordination of cell migration, proliferation, and differentiation. Time-lapse imagining offers unique insight to the behavior of cells during eye development because it allows us to visualize oculogenesis in vivo. Zebrafish are an excellent model to visualize this process due to their highly conserved vertebrate eye and their ability to develop rapidly and externally while remaining optically transparent. Time-lapse imaging studies of zebrafish eye development are greatly facilitated by use of the transgenic zebrafish line Tg(rx3:GFP). In the developing forebrain, rx3:GFP expression marks the cells of the single eye field, and GFP continues to be expressed as the eye field evaginates to form an optic vesicle, which then invaginates to form an optic cup. High resolution time lapse imaging of rx3:GFP expression, therefore, allows us to track the eye primordium through time as it develops into the retina. Lightsheet microscopy is an ideal method to image ocular morphogenesis over time due to its ability to penetrate thicker samples for fluorescent imaging, minimize photobleaching and phototoxicity, and image at a high speed. Here, a protocol is provided for time-lapse imaging of ocular morphogenesis using a commercially available lightsheet microscope and an image processing workstation to analyze the resulting data. This protocol details the procedures for embryo anesthesia, embedding in low melting temperature agarose, suspension in the imaging chamber, setting up the imaging parameters, and finally analyzing the imaging data using image analysis software. The resulting dataset can provide valuable insights into the process of ocular morphogenesis, as well as perturbations to this process as a result of genetic mutation, exposure to pharmacological agents, or other experimental manipulations.
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DOI：
10.1242/dev.138305
发表时间：
2016-07-15
期刊：
Development (Cambridge, England)
影响因子：
--
作者：
Muthu V;Eachus H;Ellis P;Brown S;Placzek M
通讯作者：
Placzek M
DOI：
10.3791/4196
发表时间：
2012-11-18
期刊：
Journal of visualized experiments : JoVE
影响因子：
--
作者：
Avdesh A;Chen M;Martin-Iverson MT;Mondal A;Ong D;Rainey-Smith S;Taddei K;Lardelli M;Groth DM;Verdile G;Martins RN
通讯作者：
Martins RN
DOI：
10.3791/53966
发表时间：
2016-04-10
期刊：
Journal of visualized experiments : JoVE
影响因子：
--
作者：
Icha J;Schmied C;Sidhaye J;Tomancak P;Preibisch S;Norden C
通讯作者：
Norden C
影响因子：
9.8
作者：
Picker A;Cavodeassi F;Machate A;Bernauer S;Hans S;Abe G;Kawakami K;Wilson SW;Brand M
通讯作者：
Brand M
影响因子：
16.2
作者：
Cavodeassi F;Carreira-Barbosa F;Young RM;Concha ML;Allende ML;Houart C;Tada M;Wilson SW
通讯作者：
Wilson SW