干细胞样<em>爪</em>胚胎外植体，研究早期神经发育特点<em>体外</em>和<em>在体内</em
Summary
In Xenopus embryos, cells from the roof of the blastocoel are pluripotent and can be programmed to generate various tissues. Here, we describe protocols to use amphibian blastocoel roof explants as an assay system to investigate key in vivo and in vitro features of early neural development.
Abstract
Understanding the genetic programs underlying neural development is an important goal of developmental and stem cell biology. In the amphibian blastula, cells from the roof of the blastocoel are pluripotent. These cells can be isolated, and programmed to generate various tissues through manipulation of genes expression or induction by morphogens. In this manuscript protocols are described for the use of Xenopus laevis blastocoel roof explants as an assay system to investigate key in vivo and in vitro features of early neural development. These protocols allow the investigation of fate acquisition, cell migration behaviors, and cell autonomous and non-autonomous properties. The blastocoel roof explants can be cultured in a serum-free defined medium and grafted into host embryos. This transplantation into an embryo allows the investigation of the long-term lineage commitment, the inductive properties, and the behavior of transplanted cells in vivo. These assays can be exploited to investigate molecular mechanisms, cellular processes and gene regulatory networks underlying neural development. In the context of regenerative medicine, these assays provide a means to generate neural-derived cell types in vitro that could be used in drug screening.
Introduction
脊椎动物神经系统出现从神经板作为神经上皮细胞的均质层。了解如何发展方案被诱导，编码，并建立了神经板的区域化过程中，目前，在发育生物学的一个主要目标。与其他系统相比，实验上适合爪蟾胚胎是选择用于分析早期步骤的神经发育1,2-一个模型。这是很容易获得的胚胎的大量涌现，以及外部发展允许访问神经胚3的第一个步骤。许多工具都可以通过实验操作非洲爪蟾 （ 非洲爪蟾）胚胎发育。微注射的mRNA或吗啉（MO），包括诱导型MOS管，以及生化和药理学工具，允许控制增益功能（GOF）和损失函数（LOF）和信号通路4,5的具体变化。该BLAstocoel屋顶外胚层，周围囊胚，或非常早期原肠胚的动物极，和被称为“动物帽'（AC）是多能干细胞的来源，可以由操纵基因在表达前的被编程外植体的准备。在这份手稿是用X的详细协议蟾交流植测试在体外和体内的分子机制和细胞过程底层神经发育。
一种技术被提出，允许精细观察的基因表达模式的非洲爪蟾蝌蚪的神经管，在命运决定的线索识别的预备步骤。而观察扁平式组织通常在鸡胚6的研究中使用的，它并没有被正确地在爪蟾说明。基因表达由注射合成mRNA或MO成的2个或4细胞期胚胎的卵裂球操纵允许交流的编程外植体4。为骨形态发生蛋白（BMP）通路被抗BMP因子头蛋白的表达的例子抑制，给出了一个神经同一性交流细胞3。该协议是详细说明用于通过用阴离子交换树脂珠粒直接接触进行交流外植体和本地时间控制暴露于外在线索。最后一个技术描述了由不同的编程细胞的分离和重新编写相关的混植体移植试验在体内的神经祖细胞发育功能。
青蛙胚胎是一个功能强大的模型来研究脊椎动物的早期神经发育。结合操纵基因表达的外植在体外培养物提供了在神经上皮区域化，增殖和形态7-12的研究的重要信息。交流外植体的编程允许的官能心脏体外 13,14发展。使用外植体移植的15导致的最小转录开关诱导神经嵴分化方案 16的识别。 透明带limitans intrathalamica（ZLI）是信令中心分泌音猬（SHH），以控制尾侧前脑的成长和区域化。当持续暴露于嘘，神经上皮细胞共表达三个转录因子基因- BARH样同源盒-2（barhl2），orthodenticle-2（OTX2）及易洛魁-3（irx3） –获取ZLI车厢的两个特点：有权表达SHH，和由前神经板细胞分离的能力。作为一个模型系统，感应一个ZLI命运的成神经上皮细胞将提交8。
这些协议旨在提供简单，廉价，有效的发育生物学家和其他研究人员的工具来探索的基本MEC关键的神经细胞行为的hanisms。这些协议是非常通用的，并允许大范围的外在和内在的神经判定线索调查。它允许长期在神经系承诺，诱导的相互作用和细胞行为的体内分析 。
Protocol
Representative Results
Discussion
神经发育是通过从周围组织（来自在3,31,32）蜂窝发展方案和信号之间的复杂的相互作用编排。在这里，我们描述了一组协议，可以在十中使用蟾胚胎探索外在和参与神经命运决定和在体外和体内神经形态发生内在因素。这些协议可以用作例如在 X上热带胚胎，但是X.热带胚胎小四倍然后X.蟾胚胎。这两种需要使用的镊子和钨针更细。如?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
The author thanks Hugo Juraver-Geslin, Marion Wassef and Anne Hélène Monsoro-Burq for their help and advice, and the Animal Facility of the Institut Curie. The author thanks Paul Johnson for his editing work on the manuscript. This work was supported by the Centre National de la Recherche Scientifique (CNRS UMR8197, INSERM U1024) and by grants from the “Association pour la Recherche sur le Cancer” (ARC 4972 and ARC 5115; FRC DOC20120605233 and LABEX Memolife) and the Fondation Pierre Gilles de Gennes (FPGG0039).
Materials
| Paraformaldehyde | VWR | 20909.290 | Toxic |
| anion exchange resin beads | Biorad | 140- 1231 | |
| Bovine Serum Albumin | SIGMA | A-7888 | For culture of animal cappH 7.6 |
| Gentamycine | GIBCO | 15751-045 | antibiotic |
| Bovine Serum Albumin | SIGMA | A7906 | for bead preparation |
References
- Nieuwkoop, P. D. IIB, Pattern formation in the developing central nervous system (CNS) of the amphibians and birds (English). Proceedings of the Koninklijke Nederlandse Akademie van Wetenschappen. 94, 121-127 (1991).
- Nieuwkoop, P. D. The neural induction process; its morphogenetic aspects. Int J Dev Biol. 43, 615-623 (1999).
- Harland, R. Neural induction. Curr Opin Genet Dev. 10, 357-362 (2000).
- Sive, H. L., Grainger, R. M., Harland, R. M. . Early development of Xenopus laevis : a laboratory manual. , (2000).
- Hoppler, S., Vize, P. D., Hoppler, S., Vize, P. D. . Xenopus protocols : post-genomic approaches. , (2012).
- Franklin Hughes, W., La Velle, A. The effects of early tectal lesions on development in the retinal gonglion cell layer of chick embryos. J Comp Neurol. 163, 265-283 (1975).
- Theveneau, E., Mayor, R. Beads on the run: beads as alternative tools for chemotaxis assays. Methods Mol Biol. 769, 449-460 (2011).
- Juraver-Geslin, H. A., Gomez-Skarmeta, J. L., Durand, B. C. The conserved barH-like homeobox-2 gene barhl2 acts downstream of orthodentricle-2 and together with iroquois-3 in establishment of the caudal forebrain signaling center induced by Sonic Hedgehog. Dev Biol. 396, 107-120 (2014).
- Green, J. B., New, H. V., Smith, J. C. Responses of embryonic Xenopus cells to activin and FGF are separated by multiple dose thresholds and correspond to distinct axes of the mesoderm. Cell. 71, 731-739 (1992).
- Wallingford, J. B., Ewald, A. J., Harland, R. M., Fraser, S. E. Calcium signaling during convergent extension in Xenopus. Curr Biol. 11, 652-661 (2001).
- Kiecker, C., Niehrs, C. A morphogen gradient of Wnt/beta-catenin signalling regulates anteroposterior neural patterning in Xenopus. DEVELOPMENT. 128, 4189-4201 (2001).
- Wilson, P. A., Hemmati-Brivanlou, A. Induction of epidermis and inhibition of neural fate by Bmp-4. Nature. 376, 331-333 (1995).
- Afouda, B. A., Hoppler, S. Xenopus explants as an experimental model system for studying heart development. Trends in cardiovascular medicine. 19, 220-226 (2009).
- Afouda, B. A. Stem-cell-like embryonic explants to study cardiac development. Methods Mol Biol. 917, 515-523 (2012).
- Milet, C., Monsoro-Burq, A. H. Dissection of Xenopus laevis neural crest for in vitro explant culture or in vivo transplantation. Journal of visualized experiments: JoVE. , (2014).
- Milet, C., Maczkowiak, F., Roche, D. D., Monsoro-Burq, A. H. Pax3 and Zic1 drive induction and differentiation of multipotent, migratory, and functional neural crest in Xenopus embryos. Proc Natl Acad Sci U S A. 110, 5528-5533 (2013).
- Nieuwkoop, P. D., Faber, J., Nieuwkoop, P. D., Faber, J. . Normal table of Xenopus laevis (Daudin): a systematical and chronological survey of the development from the fertilized egg till the end of metamorphosis. , (1994).
- Harland, R. M. In situ hybridization: an improved whole-mount method for Xenopus embryos. Methods Cell Biol. 36, 685-695 (1991).
- Turner, D. L., Weintraub, H. Expression of achaete-scute homolog 3 in Xenopus embryos converts ectodermal cells to a neural fate. Genes Dev. 8, 1434-1447 (1994).
- Sive, H. L., Grainger, R. M., Harland, R. M. Removing the Vitelline Membrane from Xenopus laevis Embryos. CSH protocols. , (2007).
- Sive, H. L., Grainger, R. M., Harland, R. M. Animal Cap Isolation from Xenopus laevis. CSH protocols. , (2007).
- Sive, H. L., Grainger, R. M., Harland, R. M. Embryo dissection and micromanipulation tools. CSH protocols. , (2007).
- Wilson, S. W., Houart, C. Review: Early Steps in the Development of the Forebrain. Developmental Cell. 6, 167-181 (2004).
- Juraver-Geslin, H. A., Durand, B. C. Early development of the neural plate: new roles for apoptosis and for one of its main effectors caspase-3. Genesis. 53, 203-224 (2015).
- Heasman, J. Patterning the early Xenopus embryo. Development. 133, 1205-1217 (2006).
- Rubenstein, J. L., Martinez, S., Shimamura, K., Puelles, L. The embryonic vertebrate forebrain: the prosomeric model. Science. 266, 578-580 (1994).
- Puelles, L., Rubenstein, J. L. R. Forebrain gene expression domains and the evolving prosomeric model. Trends in Neurosciences. 26, 469-476 (2003).
- Martinez-Ferre, A., Martinez, S. Molecular regionalization of the diencephalon. Frontiers In Neuroscience. 6, 73-73 (2012).
- Scholpp, S., Lumsden, A. Review: Building a bridal chamber: development of the thalamus. Trends in Neurosciences. 33, 373-380 (2010).
- Coffman, C., Harris, W., Kintner, C. Xotch, the Xenopus homolog of Drosophila notch. Science. 249, 1438-1441 (1990).
- Pera, E. M., Acosta, H., Gouignard, N., Climent, M., Arregi, I. Active signals, gradient formation and regional specificity in neural induction. Exp Cell Res. 321, 25-31 (2014).
- Stern, C. D. Neural induction: old problem, new findings, yet more questions. Development. 132, 2007-2021 (2005).
- Juraver-Geslin, H. A., Ausseil, J. J., Wassef, M., Durand, B. C. Barhl2 limits growth of the diencephalic primordium through Caspase3 inhibition of beta-catenin activation. Proc Natl Acad Sci U S A. 108, 2288-2293 (2011).
- Sive, H. L., Grainger, R. M., Harland, R. M. Dissociation and Reaggregation of Xenopus laevis Animal Caps. CSH protocols. , (2007).
- Harland, R. M., Grainger, R. M. Xenopus research: metamorphosed by genetics and genomics. Trends Genet. 27, 507-515 (2011).
- Beccari, L., Marco-Ferreres, R., Bovolenta, P. The logic of gene regulatory networks in early vertebrate forebrain patterning. Mech Dev. 130, 95-111 (2013).
- Pani, A. M., et al. Ancient deuterostome origins of vertebrate brain signalling centres. Nature. 483, 289-294 (2012).
- Holland, L. Z., et al. Evolution of bilaterian central nervous systems: a single origin?. Evodevo. 4, 27 (2013).
- Pratt, K. G., Khakhalin, A. S. Modeling human neurodevelopmental disorders in the Xenopus tadpole: from mechanisms to therapeutic targets. Disease models & mechanisms. 6, 1057-1065 (2013).
- Sasai, Y., Ogushi, M., Nagase, T., Ando, S. Bridging the gap from frog research to human therapy: a tale of neural differentiation in Xenopus animal caps and human pluripotent cells. Development, growth & differentiation. 50, s47-s55 (2008).
