体外实时成像在小鼠神经上皮单细胞分裂
Summary
在这里，我们开发所需的工具<em>体外</em>实时成像跟踪单E8.5的小鼠神经上皮细胞分裂
Abstract
我们开发了一个系统，集成了实时成像的荧光标记物和小鼠胚胎神经上皮的培养切片。我们利用现有的鼠标线的遗传细胞谱系追踪：他莫昔芬诱导的Cre线和红色荧光蛋白的酶Cre记者表达Cre重组酶介导的重组后。通过使用他莫昔芬的相对较低的水平，我们能够在一个小数量的细胞以诱导重组，允许我们按照个别的细胞分裂。此外，我们观察到的转录反应刺猬（SHH）信号使用OLIG2-EGFP转基因株系1-3，我们监测纤毛的形成与病毒感染培养的片表达的纤毛标记，SSTR3-GFP 4。为了图像的神经上皮，我们收获胚胎E8.5，分离出神经管，安装在适当的培养条件下成神经切片成像室和执行时间推移共聚焦成像。我们的前活体内成像方法，使我们能够跟踪单细胞分裂，初级纤毛的形成和Shh响应有关生理的方式评估的相对时序。这种方法可以很容易地适应用不同的荧光标记物，并提供该字段，用以监测细胞行为的原产地和实时的工具。
Protocol
成年小鼠安乐死机械颈椎脱位。所有动物的程序批准由IACUC和埃默里大学生物安全委员会。 1。胚胎生成过他莫昔芬诱导表达Cre重组酶行CAGGCreER，dsRedCre记者线（玻璃转化温度（CAG-BGEO的，红色荧光蛋白* MST）1Nagy）（ 图1）5,6。要监视的Shh的响应中的子细胞，跨CAGGCreER和DsRed的配合的OLIG2-eGFP的BAC转基因小鼠（Tg的（OLIG2-EGFP）EK23Gsat）（ 图1?…
Representative Results
在这里，我们进行体外实时成像单细胞分裂E8.5小鼠神经上皮内。要标记单个细胞，诱导Cre重组酶在细胞内含有一个的酶Cre记者行表达红色荧光蛋白重组后，5,6（ 图3A）的一个子集。因此，48小时后，我们能够观察到在体外成像（ 图4A-D）的单细胞分裂。随之而来的是，我们监测标记细胞成为嘘响应的包括修改BAC转基因OLIG2-EGFP线（ 图3C-D;图4G-…
Discussion
我们的体外系统，使我们在发展中的神经上皮实时直接观察单个细胞分裂。作为一个例子，我们研究小鼠胚胎神经管内的细胞分裂和监控无论是纤毛形成或嘘响应。我们确认我们的成像效果组（n = 24）与固定部分（N = 178），表明我们的技术提供有关生理数据的结果是一致的。
我们的技术依赖于被限制酶Cre诱导的细胞的一个子集，因此，他莫昔芬的剂量必?…
Declarações
The authors have nothing to disclose.
Acknowledgements
该研究项目得到了一个的ARRA补充，5 R01 NS056380。通过病毒载体核心和埃默里大学神经科学NINDS核心设施补助，P30NS055077显微镜核心提供额外的支持。我们感谢埃默里大学的转基因小鼠和基因打靶核心的鼠标线从GENSAT的派生格雷格帕佐尔SSTR3-GFP的稳定IMCD3细胞株; SSTR3-GFP慢病毒布拉德利尤德构建。单克隆抗体从发展研究杂交瘤细胞银行，开发的的NICHD的主持下，获得和维护由美国爱荷华大学生物科学系，爱荷华州爱荷华市52242。所有动物的程序批准由IACUC和埃默里大学生物安全委员会。
Materials
| Name of the reagent | Company | Catalogue number | Comments (optional) |
| Z/RED line (STOCK Tg(CAG-Bgeo,-DsRed*MST)1Nagy/J | Jackson Laboratory | 005438 | |
| Olig2-eGFP line (STOCK Tg(Olig2-EGFP)EK23Gsat/Mmcd | MMRRC, | 010555-UCD | |
| CAGGCreER | Jackson Laboratory | 003724 | |
| Tamoxifen | Sigma | T5648 | |
| DMEM/F12 (1:1) | GIBCO | 21041-025 | |
| Newborn calf serum | Lonza | 14-416F | |
| Penicillin/Streptomycin | Invitrogen | 15140-122 | |
| Rat Serum SD male | Harlan Bioproducts | 4520 | |
| 1M Hepes | BioWhittaker | 17-737E | |
| L-Glutamine | GIBCO | 21041-025 | |
| Light mineral oil | Sigma | M8410 | |
| Sstr3-GFP lentivirus | Emory Viral Core | ||
| Micro-knife, size 0.025 mm | Electron Microscopy Sciences | 62091 | |
| 35 mm poly-L-lysine coated glass bottom dish | MatTek | P35GC-0-10-C | |
| 100% petroleum jelly | Kroger | FL9958c | |
| A1R Laser Scanning Confocal Inverted Microscope | Nikon | ||
| NIS Elements software | Nikon | ||
| Imaris 3D software | Bitplane AG | Imaris 7.2.3 | |
| OCT | Tissue-Tek | 4583 | |
| Cryostat | Leica | CM1850 | |
| Heat-inactivated sheep serum | Invitrogen | 16210-072 | |
| Triton X-100 | Fisher Scientific | BP151 | |
| Parafolmaldehyde | Sigma | P6148 | |
| Phosphate Buffer | Lab made | ||
| Rat monoclonal anti-RFP (5F8) | Chromotek | 110411 | |
| Rabbit anti-Arl13b serum | NeuroMab | ||
| mouse monoclonal anti-Arl13b 1:5 | NeuroMab | ||
| Rabbit anti-Olig2 | Chemicon | AB9610 | |
| Mouse monoclonals Pax6 | Developmental Hybridoma Bank | Pax6 | |
| Mouse monoclonalsShh | Developmental Hybridoma Bank | 5E1 | |
| Mouse monoclonals Nkx2.2 | Developmental Hybridoma Bank | 74.5A5 | |
| Rabbit polyclonal Ki67 | Abcam | AB15580 | |
| Alexa Fluor 488 | Molecular Probes | A11029 | |
| Alexa Fluor 568 | Molecular Probes | A11031 | |
| Alexa Fluor 350 | Molecular Probes | A11046 | |
| Hoechst | Fisher | AC22989 | |
| TO-PRO-3 | Invitrogen | T3605 | |
| ProLong Gold anti-fade reagent | Invitrogen | P36934 |
Referências
- Yamada, T., Pfaff, S. L., Edlund, T., Jessell, T. M. Control of cell pattern in the neural tube: motor neuron induction by diffusible factors from notochord and floor plate. Cell. 73, 673-686 (1993).
- Ericson, J., Muhr, J., Placzek, M., Lints, T., Jessell, T. M., Edlund, T. Sonic hedgehog induces the differentiation of ventral forebrain neurons: A common signal for ventral patterning within the neural tube. Cell. 81, 747-756 (1995).
- Briscoe, J. A. Homeodomain Protein Code Specifies Progenitor Cell Identity and Neuronal Fate in the Ventral Neural Tube. Cell. 101, 435-445 (2000).
- Berbari, N. F., Johnson, A. D., Lewis, J. S., Askwith, C. C., Mykytyn, K. Identification of ciliary localization sequences within the third intracellular loop of G protein-coupled receptors. Mol. Biol. Cell. 19 (4), 1540-1547 (2008).
- Vintersten, K. Mouse in red: red fluorescent protein expression in mouse ES cells, embryos, and adult animals. Genesis. 40 (4), 241-246 (2004).
- Hayashi, S., McMahon, A. P. Efficient recombination in diverse tissue by a tamoxifen-inducible form of Cre: a tool for temporally regulated gene activation/inactivation in the mouse. Dev. Biol. 244 (2), 305-318 (2002).
- Gong, S., Zheng, C., Doughty, M. L., Losos, K., Didkovsky, N., Schambra, U. B., Nowak, N. J., Joyner, A., Leblanc, G., Hatten, M. E., Heintz, N. A gene expression atlas of the central nervous system based on bacterial artificial chromosomes. Nature. 425 (6961), 917-925 (2003).
- Mukouyama, Y. S., Deneen, B., Lukaszewicz, A., Novitch, B. G., Wichterle, H., Jessell, T. M., Anderson, D. J. Olig2+ neuroepithelial motoneuron progenitors are not multipotent stem cells in vivo. Proc. Natl. Acad. Sci. U.S.A. 103 (5), 1551-1556 (2006).
- Jones, E. A. V., Crotty, D., Kulesa, P. M., Waters, C. W., Baron, M. H., Fraser, S. E., Dickinson, M. E. Dynamic in vivo imaging of postimplantation mammalian embryos using whole embryo culture. Genesis. 34, 228-235 (2002).
- Piotrowska-Nitsche, K., Caspary, T. Live imaging of individual cell divisions in mouse neuroepithelium shows asymmetry in cilium formation and Sonic hedgehog response. Cilia. 1 (6), (2012).
- Nowotschin, S., Ferrer-Vaquer, A., Hadjantonakis, A. -. K. Imaging mouse development with confocal time-lapse microscopy. Methods in Enzymology. 476, 351-377 (2010).
Tags
Ex Vivo Live ImagingSingle Cell DivisionsMouse NeuroepitheliumFluorescent MarkersCulturing SlicesEmbryonic Mouse NeuroepitheliumGenetic Cell Lineage TracingTamoxifen-inducible Cre LineCre Reporter LineDsRed ReporterCre-mediated RecombinationTamoxifen InductionIndividual Cell DivisionsTranscriptional ResponseSonic Hedgehog SignalingOlig2-eGFP Transgenic LineCilia MarkerSstr3-GFP ReporterNeuroepithelium ImagingTime-lapse Confocal ImagingPrimary Cilia FormationShh Response
Citar este artigo
Piotrowska-Nitsche, K., Caspary, T. Ex vivo Live Imaging of Single Cell Divisions in Mouse Neuroepithelium. J. Vis. Exp. (74), e4439, doi:10.3791/4439 (2013).