细胞形态改变与细胞运动的影像<em>果蝇</em>原肠胚形成使用DE-cadherin的记者转基因果蝇
Özet
Herein we describe a procedure to capture live images of Drosophila gastrulation. This has enabled us to better understand the apical constriction involved in early development and further analyze mechanisms governing cellular movements during tissue structure modification.
Abstract
原肠是国内第一套多细胞动物，例如果蝇胚胎发育过程中出现形态的动态事件。这种形态学改变也被认为是上皮间质转化（EMT）。 EMT的失调与纤维化和癌症转移有关。有新出现的证据表明，EMT是由多个的分子机制来控制。作为控制根尖狭窄这样，许多关键基因也已知可用于在癌转移中观察到的EMT的重要因素。像果蝇原肠胚形成过程中的EMT，上皮细胞可被诱导以改变它们的形状和被重新编程的细胞命运重定向朝向各种其它类型的细胞。在这里，我们提供果蝇原肠胚形成的鲁棒成像方法以测定在此阶段的胚胎发育的形态发生细胞运动和细胞命运识别的开始。使用这种方法，我们确定Ç在原肠胚形成的时间ELL重排并用GFP在原肠胚形成示范根尖狭窄的重要性标记DE-cadherin的。
Introduction
原肠胚是所述第一组的多细胞动物如果蝇 1,2-胚胎发育过程中出现的形态动态事件。有趣的是，新的证据表明，这一过程是通过机械和分子机制3之间的相互作用调控。此外，上皮至间质转变（EMT），这是在原肠胚形成的关键过程，也牵连在人类疾病过程，如癌症转移4-8。作为控制根尖狭窄这样，许多基因也被称为是在癌症转移9中观察到的EMT的关键因素。因此，在原肠胚形成的时间根尖狭窄是调查前述监管机制，以提高我们的癌症转移的理解的优秀典范。该技术的优点是，我们可以在实时的，因此，原肠胚形成的时间观察细胞的运动，我们将能够筛选参与原肠胚以及肿瘤转移的基因。
虽然相对未知的，细胞-细胞粘附被认为在根尖狭窄1中发挥中心作用。 果蝇遗传学非常适合于单细胞水平调查探索调控的分子机制。这种模式将使我们在原肠揭开根尖狭窄的重要性。此外，这种方法可以用于筛选参与癌转移的基因。 果蝇原肠胚形成的捕获实时图像，进一步使我们更详细地了解管理组织重排的分子机制。在此，我们提供了一个简单的方法的全面说明实现这一目标。
Protocol
Representative Results
Discussion
Although we have previously reported a similar procedure to capture live images of the gastrulation process in Drosophilla1, the method we describe here is detailed and easy to trace endogenous cadherin expression and thus is quite useful for genetic screening of key factors involved in gastrulation. To maximize success with this imaging procedure, it is essential to use an indented slide. Mechanical pressure sometimes causes embryonic death. Therefore, it is also important to handle the embryos as ge…
Açıklamalar
The authors have nothing to disclose.
Acknowledgements
This study was supported by the Astellas Foundation for Research on Metabolic Disorders (HT), Takeda Science Foundation (HT), and MEXT-Supported Program for the Strategic Research Foundation at Private Universities (HT).
Materials
| Halocarbon oil 700 | Sigma | MKBH 5726 | |
| Vacuum grease Silicone | Beckman | 335148 | |
| Glass coverslip | Matsunami glass | Thickness No1 | 24-36mm |
| Embryo stariner | Corning | Corning3477 | |
| Plastic Drosophilla Stock Bottles | Hitec | MKC-100 | |
| DE-Cadherin knock-in flies | REF (10) |
Referanslar
- Haruta, T., Warrior, R., Yonemura, S., Oda, H. The proximal half of the Drosophila E-cadherin extracellular region is dispensable for many cadherin-dependent events but required for ventral furrow formation. Genes Cells. 15 (3), 193-208 (2010).
- Oda, H., Takeichi, M. Evolution: structural and functional diversity of cadherin at the adherens junction. J. Cell Biol. 193 (7), 1137-1146 (2011).
- Fernandez-Sanchez, M. E., Serman, F., Ahmadi, P., Farge, E. Mechanical induction in embryonic development and tumor growth integrative cues through molecular to multicellular interplay and evolutionary perspectives. Methods Cell Biol. 98, 295-321 (2010).
- Alderton, G. K. Metastasis: Epithelial to mesenchymal and back again. Nat. Rev. Cancer. 13 (1), 3 (2013).
- Fabregat, I., Malfettone, A., Soukupova, J. New Insights into the Crossroads between EMT and Stemness in the Context of Cancer. J. Clin. Med. 5 (3), (2016).
- Serrano-Gomez, S. J., Maziveyi, M., Alahari, S. K. Regulation of epithelial-mesenchymal transition through epigenetic and post-translational modifications. Mol. Cancer. 15, (2016).
- Yu, A. Q., Ding, Y., Li, C. L., Yang, Y., Yan, S. R., Li, D. S. TALEN-induced disruption of Nanog expression results in reduced proliferation, invasiveness and migration, increased chemosensitivity and reversal of EMT in HepG2 cells. Oncol. Rep. 35 (3), 1657-1663 (2016).
- Zheng, X., et al. Epithelial-to-mesenchymal transition is dispensable for metastasis but induces chemoresistance in pancreatic cancer. Nature. 527 (7579), 525-530 (2015).
- Kalluri, R., Weinberg, R. A. The basics of epithelial-mesenchymal transition. J. Clin. Invest. 119 (6), 1420-1428 (2009).
- Huang, J., Zhou, W., Dong, W., Watson, A. M., Hong, Y. Directed, efficient, and versatile modifications of the Drosophila genome by genomic engineering. Proc. Natl. Acad. Sci. U.S.A. 106 (20), 8284-8289 (2009).
