Observing Mitotic Division and Dynamics in a Live Zebrafish Embryo.
Observing Mitotic Division and Dynamics in a Live Zebrafish Embryo.
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DOI：
10.3791/54218
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发表时间：
2016-07-15
期刊：
影响因子：
--
通讯作者：
Parant JM
中科院分区：
文献类型：
--
作者：
Percival SM;Parant JM
有丝分裂是生物体生长和分化的关键。这个过程是高度动态的，需要有序的事件来完成适当的染色质凝聚、微管-着丝点附着、染色体分离和细胞质分裂在很短的时间内。这一微妙过程中的错误可能导致人类疾病，包括出生缺陷和癌症。研究人类有丝分裂疾病状态的传统方法通常依赖于细胞培养系统，这在研究人类疾病时缺乏自然生理学和发育/组织特异性背景。该方案克服了许多障碍，提供了一种可视化的方法，具有高分辨率，染色体动力学的脊椎动物系统，斑马鱼。本协议将详细介绍一种可用于获得分裂细胞动态图像的方法，其中包括：体外转录，斑马鱼繁殖/收集，胚胎植入和延时成像。并对该协议的优化和修改进行了探讨。使用H2A。F/Z-EGFP（标记染色质）和mCherry-CAAX（标记细胞膜）mrna注射胚胎，观察AB野生型、auroraBhi1045和esco2hi2865突变斑马鱼的有丝分裂。在斑马鱼的高分辨率实时成像允许人们观察多个有丝分裂，统计量化有丝分裂缺陷和有丝分裂进展的时间。此外，还观察了定义不适当的有丝分裂过程（即聚集缺陷，染色体错误分离等）和不适当的染色体结果（即非整倍体，多倍体，微核等）的定性方面。该试验可用于观察组织分化/发育，并适用于突变斑马鱼和药理学试剂的使用。有丝分裂缺陷如何导致癌症和发育障碍的可视化将大大提高对疾病发病机制的理解。
Mitosis is critical for organismal growth and differentiation. The process is highly dynamic and requires ordered events to accomplish proper chromatin condensation, microtubule-kinetochore attachment, chromosome segregation, and cytokinesis in a small time frame. Errors in the delicate process can result in human disease, including birth defects and cancer. Traditional approaches investigating human mitotic disease states often rely on cell culture systems, which lack the natural physiology and developmental/tissue-specific context advantageous when studying human disease. This protocol overcomes many obstacles by providing a way to visualize, with high resolution, chromosome dynamics in a vertebrate system, the zebrafish. This protocol will detail an approach that can be used to obtain dynamic images of dividing cells, which include: in vitro transcription, zebrafish breeding/collecting, embryo embedding, and time-lapse imaging. Optimization and modifications of this protocol are also explored. Using H2A.F/Z-EGFP (labels chromatin) and mCherry-CAAX (labels cell membrane) mRNA-injected embryos, mitosis in AB wild-type, auroraBhi1045, and esco2hi2865 mutant zebrafish is visualized. High resolution live imaging in zebrafish allows one to observe multiple mitoses to statistically quantify mitotic defects and timing of mitotic progression. In addition, observation of qualitative aspects that define improper mitotic processes (i.e., congression defects, missegregation of chromosomes, etc.) and improper chromosomal outcomes (i.e., aneuploidy, polyploidy, micronuclei, etc.) are observed. This assay can be applied to the observation of tissue differentiation/ development and is amenable to the use of mutant zebrafish and pharmacological agents. Visualization of how defects in mitosis lead to cancer and developmental disorders will greatly enhance understanding of the pathogenesis of disease.
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DOI：
10.1083/jcb.201301006
发表时间：
2013-04-01
期刊：
The Journal of cell biology
影响因子：
--
作者：
Gascoigne KE;Cheeseman IM
通讯作者：
Cheeseman IM
影响因子：
2.7
作者：
Jeong, KilHun;Jeong, Jae-Yeon;Lee, Hyunsook
通讯作者：
Lee, Hyunsook
DOI：
10.1242/dev.115584
发表时间：
2014-12
期刊：
Development (Cambridge, England)
影响因子：
--
作者：
Irion U;Krauss J;Nüsslein-Volhard C
通讯作者：
Nüsslein-Volhard C
影响因子：
16
作者：
Beckouët F;Hu B;Roig MB;Sutani T;Komata M;Uluocak P;Katis VL;Shirahige K;Nasmyth K
通讯作者：
Nasmyth K
DOI：
10.1073/pnas.1308335110
发表时间：
2013-08-20
影响因子：
11.1
作者：
Jao, Li-En;Wente, Susan R.;Chen, Wenbiao
通讯作者：
Chen, Wenbiao