基因功能分析和可视化的纤毛产生的流体流动的在枯否囊泡中
Summary
纤毛产生的流体流动在枯否囊泡（KV）控制的斑马鱼胚胎的左右模式。在这里，我们描述了一种技术，调节基因的功能，特别是在KV细胞。此外，我们展示了如何提供KV可视化流体流动的荧光珠。
Abstract
内部器官，如心脏，大脑和肠道左右（LR）为他们的正常功能是至关重要的不对称性。运动纤毛参与建立LR的不对称性在脊椎动物胚胎，包括老鼠，青蛙和斑马鱼2-6。这些'LR的纤毛'产生不对称的流体流动是必要的，以触发一个保守的不对称交点（TGF-β超家族）的信号级联在左侧侧板中胚层，这被认为是提供开发机关7 LR构图信息。因此，理解机制LR构图，它是必不可少的，以确定基因调节的LR纤毛细胞的组织，运动和长度LR纤毛和他们的能力，以产生强大的非对称流。
在斑马鱼胚胎中，LR纤毛位于巨噬泡（KV）2,4,5。 KV是由一个单一的层monociliated上皮细胞，附上一个充满液体的腔。 KV是来自一组被称为的背先行者细胞（DFCS）迁移外包阶段在背胚率在8,9〜20-30细胞的命运映射。在早期的体节阶段，DFC的集群，并分化成纤毛上皮细胞，形成KV中的胚胎10,11 tailbud。结合的光学透明度和快速发展的斑马鱼胚胎斑马鱼KV一个很好的模型系统来研究，LR纤毛细胞能够识别和跟踪八间指定流感诊所求诊。
有趣的是，DFC / KV细胞谱系的祖细胞保留蛋黄细胞的细胞质之间的桥梁，受精后4小时（HPF）的，而蛋黄细胞和其他接近8高倍视野2后的胚胎干细胞的细胞质之间的桥梁。利用这些细胞的桥梁，我们开发了一个特定阶段的喷油策略提供吗啉代寡otides（密苏里州）专门DFC的和击倒的目标基因的功能，在这些细胞中12。这技术创造嵌合体胚胎基因的功能被撞倒在发展的背景下，野生型胚胎的DFC / KV血统的。在KV，我们要分析非对称流体流动注射荧光微球到KV珠流明，并记录运动时，通过电视显微镜2。流体流动是易于可视化和可量化的跟踪珠位移随着时间的推移。
在这里，使用特定的阶段，的DFC-针对性的基因敲除技术和注射荧光微球KV可视化流程，我们提出了一个协议，提供了一个有效的方法来描述一个特定基因的作用在KV的发育和功能。
Protocol
概述特定阶段的斑马鱼胚胎注射液反义吗啉代寡核苷酸（MO），结合到靶mRNA和蛋白表达，转录干扰，被广泛用于基因敲除（损失函数）的研究斑马鱼13,14。基因的工具，LLC提供的MO，无论是羧基发出绿色荧光的或丽丝胺（发出红色荧光）的检测MO注射的胚胎中，使用荧光显微镜的标签。通过注入MO到蛋黄细胞在斑马鱼发育的不同阶段中，它能够提供的MO胚胎的具体的室中（…
Representative Results
图1给出了一个流程图，用于测试基因功能DFC / KV细胞和如何引入荧光珠的可视化流体流的喷射策略阶段特异性的MO注射提供了一种有用的方法来分析基因功能的特定的室中的胚胎。在KV。在成功阶段特异性注射胚胎的是，荧光的MO分布示意性地示出在图2中，在图1D-F和在活胚胎。一个不成功的MO注射，在其中的MO仍然在蛋黄细胞聚集， 如图2D</…
Discussion
使用特定阶段的目标MO注射到DFC / KV细胞系是一个非常有用的方法来研究基因功能的细胞自主性，避免多效性所造成的全球基因敲除的表型。然而，这些注射可以在技术上具有挑战性。注射液的MO之间的256细胞和1000细胞的阶段，可能会导致在三种可能的结果：1）在MO仍然聚集在注射部位，2）的MO整个蛋黄扩散并进入DFC / KV细胞或3）的MO整个蛋黄，进入DFC / KV细胞和胚胎干细胞的扩散。因此，在这个?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们感谢菲奥娜优良的实验室支持和斑马鱼的保健福利。这项工作是一个AHA博士前奖学金GW（11PRE5730027）的和NHLBI补助金的HJY（R01HL66292）和JDA（R01HL095690）。
Materials
| Name of Reagent/Material | Company | Catalogue Number |
| Standard Control oligo-Lissamine tagged | Gene Tools, LLC | |
| Custom Rock2b morpholino oligo | Gene Tools, LLC | |
| Fluoresbrite Multifluorescent 0.5 micron Microspheres | Polysciences, Inc. | 24054 |
References
- Sutherland, M. J., Ware, S. M. Disorders of left-right asymmetry: heterotaxy and situsinversus. Am. J. Med. Genet. C Semin. Med. Genet. 151C (4), 307-317 (2009).
- Essner, J. J., Amack, J. D., Nyholm, M. K., Harris, E. B., Yost, H. J. Kupffer’s vesicle is a ciliated organ of asymmetry in the zebrafish embryo that initiates left-right development of the brain, heart and gut. Development. 132 (6), 1247-1260 (2005).
- Nonaka, S., et al. Randomization of left-right asymmetry due to loss of nodal cilia generating leftward flow of extraembryonic fluid in mice lacking KIF3B motor protein. Cell. 95 (6), 829-837 (1998).
- Essner, J. J., et al. Conserved function for embryonic nodal cilia. Nature. 418 (6893), 37-38 (2002).
- Kramer-Zucker, A. G., et al. Cilia-driven fluid flow in the zebrafish pronephros, brain and Kupffer’s vesicle is required for normal organogenesis. Development. 132 (8), 1907-1921 (2005).
- Schweickert, A., et al. Cilia-driven leftward flow determines laterality in Xenopus. Curr. Biol. 17 (1), 60-66 (2007).
- Tabin, C. J. The key to left-right asymmetry. Cell. 127 (1), 27-32 (2006).
- Cooper, M. S., D’Amico, L. A. A cluster of noninvolutingendocytic cells at the margin of the zebrafish blastoderm marks the site of embryonic shield formation. Dev. Biol. 180 (1), 184-198 (1996).
- Melby, A. E., Warga, R. M., Kimmel, C. B. Specification of cell fates at the dorsal margin of the zebrafish gastrula. Development. 122 (7), 2225-2237 (1996).
- Amack, J. D., Wang, X., Yost, H. J. Two T-box genes play independent and cooperative roles to regulate morphogenesis of ciliated Kupffer’s vesicle in zebrafish. Dev. Biol. 310 (2), 196-210 (2007).
- Oteiza, P., Koppen, M., Concha, M. L., Heisenberg, C. P. Origin and shaping of the laterality organ in zebrafish. Development. 135 (16), 2807-2813 (2008).
- Amack, J. D., Yost, H. J. The T box transcription factor no tail in ciliated cells controls zebrafish left-right asymmetry. Curr. Biol. 14 (8), 685-690 (2004).
- Nasevicius, A., Ekker, S. C. Effective targeted gene ‘knockdown’ in zebrafish. Nat. Genet. 26 (2), 216-220 (2000).
- Bill, B. R., Petzold, A. M., Clark, K. J., Schimmenti, L. A., Ekker, S. C. A primer for morpholino use in zebrafish. Zebrafish. 6 (1), 69-77 (2009).
- Kimmel, C. B., Law, R. D. Cell lineage of zebrafish blastomeres. I. Cleavage pattern and cytoplasmic bridges between cells. Dev. Biol. 108 (1), 78-85 (1985).
- Arrington, C. B., Yost, H. J. Extra-embryonic syndecan 2 regulates organ primordia migration and fibrillogenesis throughout the zebrafish embryo. Development. 136 (18), 3143-3152 (2009).
- Caron, A., Xu, X., Lin, X. Wnt/beta-catenin signaling directly regulates Foxj1 expression and ciliogenesis in zebrafish Kupffer’s vesicle. Development. 139 (3), 514-524 (2012).
- Wang, G., et al. The Rho kinase Rock2b establishes anteroposterior asymmetry of the ciliated Kupffer’s vesicle in zebrafish. Development. 138 (1), 45-54 (2011).
- Aamar, E., Dawid, I. B. Sox17 and chordin are required for formation of Kupffer’s vesicle and left-right asymmetry determination in zebrafish. Dev. Dyn. 239 (11), 2980-2988 (2010).
- Neugebauer, J. M., Amack, J. D., Peterson, A. G., Bisgrove, B. W., Yost, H. J. FGF signalling during embryo development regulates cilia length in diverse epithelia. Nature. 458 (7238), 651-654 (2009).
- Schneider, I., et al. Zebrafish Nkd1 promotes Dvl degradation and is required for left-right patterning. Dev. Biol. 348 (1), 22-33 (2010).
- Matsui, T., et al. Canopy1, a positive feedback regulator of FGF signaling, controls progenitor cell clustering during Kupffer’s vesicle organogenesis. Proc. Natl. Acad. Sci. U.S.A. 108 (24), 9881-9886 (2011).
- Shu, X., et al. Na,K-ATPase alpha2 and Ncx4a regulate zebrafish left-right patterning. Development. 134 (10), 1921-1930 (2007).
- Esguerra, C. V. Ttrap is an essential modulator of Smad3-dependent Nodal signaling during zebrafish gastrulation and left-right axis determination. Development. 134 (24), 4381-4393 (2007).
- Rosen, J. N., Sweeney, M. F., Mably, J. D. Microinjection of Zebrafish Embryos to Analyze Gene Function. J. Vis. Exp. (25), e1115 (2009).
- Yuan, S., Sun, Z. Microinjection of mRNA and Morpholino Antisense Oligonucleotides in Zebrafish Embryos. J. Vis. Exp. (27), e1113 (2009).
- Molina, G., et al. Zebrafish chemical screening reveals an inhibitor of Dusp6 that expands cardiac cell lineages. Nat. Chem. Biol. 5 (9), 680-687 (2009).
- Kimmel, C. B., Ballard, W. W., Kimmel, S. R., Ullmann, B., Schilling, T. F. Stages of embryonic development of the zebrafish. Dev. Dyn. 203 (3), 253-310 (1995).
- Schneider, I., Houston, D. W., Rebagliati, M. R., Slusarski, D. C. Calcium fluxes in dorsal forerunner cells antagonize beta-catenin and alter left-right patterning. Development. 135 (1), 75-84 (2008).
- Clement, A., Solnica-Krezel, L., Gould, K. L. The Cdc14B phosphatase contributes to ciliogenesis in zebrafish. Development. 138 (2), 291-302 (2011).
- Matsui, T., Bessho, Y. Left-right asymmetry in zebrafish. Cell Mol. Life Sci. , (2012).
Cite This Article
Wang, G., Yost, H. J., Amack, J. D. Analysis of Gene Function and Visualization of Cilia-Generated Fluid Flow in Kupffer’s Vesicle. J. Vis. Exp. (73), e50038, doi:10.3791/50038 (2013).