来自胚胎GFP-表达小鼠的Ex Vivo Oculo运动切片培养,用于对Oculo运动神经外生长进行时移成像
1Department of Ophthalmology,Boston Children’s Hospital, 2Department of Ophthalmology,Harvard Medical School, 3F.M. Kirby Neurobiology Center,Boston Children’s Hospital, 4Department of Neurology,Boston Children’s Hospital, 5Department of Neurology,Harvard Medical School, 6Howard Hughes Medical Institute
Summary
前体切片测定允许实时成像奥库运动神经外生。切片通过嵌入E10.5 Isl MN:GFP胚胎在生长中,在振动体上切片,并在一个顶的培养箱中生长。斧子引导通路的作用是通过向培养培养物添加抑制剂来评估的。
Abstract
精确的眼动对视力至关重要,但眼动系统的发展,特别是控制斧突引导的分子通路,尚未得到充分阐明。这部分是由于传统斧子制导测定的技术限制。为了识别影响奥库运动神经的其他斧子引导线索,开发了一种前体切片测定,以实时图像向眼睛生长的奥孔运动神经。E10.5 IslMN-GFP胚胎用于生成体外切片,将其嵌入到胶质中,在振动体上切片,然后在显微镜级顶培养箱中生长,用延时显微显微镜进行 24-72 h。从原子核到轨道的斧子生长的体内时间。小分子抑制剂或重组蛋白可以添加到培养培养物中,以评估不同斧头引导途径的作用。该方法的优点是维护轴子遍历的更多局部微环境,而不是对生长的轴子进行轴向,并沿其轨迹的多个点评估轴子。它还可以识别对斧子特定子集的影响。例如,对 CXCR4 的抑制会导致仍在中脑内的斧头生长,而不是口向生长,但已经退出的斧头不受影响。
Introduction
目视电机系统为研究斧子导向机构提供了一个优雅的系统。它相对简单,包括三个颅神经内侧六个移动眼睛的眼外肌肉(EOMs),以及抬起眼睑的悬浮肌(LPS)。oculo运动神经内枢LPS和四个EOMs – 下等斜和中,下等,和优越的直肠肌肉。其他两种神经, 三角和腹肌, 每个只有内瓦特一个肌肉, 优越的斜和侧直肠肌肉, 分别.眼动提供了一个简单的读出,显示内侧是否适当、缺失或异常。此外,还有由于神经元发育或斧突指导的不足而导致的人眼运动障碍,统称为先天性颅内疾病(CCDDs)1。
尽管有这些优点,目状电机系统很少用于斧子制导研究2,3,4,5,6,7,8, 9,10,由于技术缺陷。体外斧子制导测定有许多缺点11。共培养性测定,其中神经元外植与目标组织12或转染细胞13的外植一起培养,取决于外植的对称性和外植与目标组织之间的精确定位。条纹测定14,15,其中两个线索被放置在交替条纹和斧子被评估为优先增长一个条纹,只表明一个基板优于另一个,而不是说两个是有吸引力的或排斥,或生理相关。微流体室可以形成精确的化学梯度,但受试者生长的斧子会剪切应力16,17,18,这会影响它们的生长。此外,在每种方法中,收集外植或分离的细胞需要对生长的轴虫进行轴状化,因此这些测定实际上检查轴子再生,而不是初始轴子生长。最后,这些体外方法消除了影响斧子的微环境及其对不同点的线索的反应,传统上只单独测试一个线索。使这些缺点更加复杂的是,眼部运动系统中每个原子核的体积小,使得解剖在技术上对植物外或分离的培养物都具有挑战性。此外,眼动神经元的主要培养物通常是异构的,有显著的细胞死亡,并且密度依赖,需要从多个胚胎中汇集细胞(藤井裕久,个人交流)。然而,由于需要时间和费用,体内方法(包括敲除小鼠模型)不适合用于筛选。
开发培养整个胚胎的方法19允许标记迁移细胞20或阻断特定分子21,但整个胚胎培养需要在滚筒瓶中孵育,从而排除了标记的实时成像结构。允许操纵胚胎,然后在子宫或母亲腹部(维持胎盘连接)22的外科技术,允许操纵胚胎,然后进一步发育22,但这些也不允许延时成像。
为了克服体外检测的障碍,允许快速筛选信号通路,23日开发了一种体外胚胎切片培养技术,该技术改编自先前公布的外周神经外生长方案24。使用该协议,在沿其轨迹的许多周围结构(包括EOM目标)存在的情况下,可以随时间而对发育中的oculo运动神经进行成像。通过在培养介质中添加小分子抑制剂、生长因子或引导提示,我们可以评估沿斧子轨迹的多个点的制导扰动,从而能够更快速地评估潜在的生长和指导因素。
Protocol
此处描述的所有动物工作均符合波士顿儿童医院机构动物护理和使用委员会 (IACUC) 协议的批准和执行。 1. 分时配接 放置ISLMN:GFP (岛运动神经元绿色荧光蛋白;MGI: J:132726;Jax Tg (Isl-EGFP®)1Slp/J 库存号: 017952) 雄性小鼠和雌性小鼠一起过夜。在交配前称量雌性并记录重量。注:ISL MN:GFP专门标记运动神经元与无细胞毒性?…
Representative Results
正常结果:图1提供了实验的原理图。早在E9.5在小鼠,第一个斧头开始从oculmotor核26出现。通过E10.5,一种包含早期先驱神经元的迷法性神经,可以在中枢可见。E10.5的胚胎(即使在同一垃圾内)在神经向轨道前进的步数上存在显著差异,这可能是由于几个小时的发育差异。在正常子宫发育期间,第一个GFP阳性oculmotoraxons在接下来的18-24小时(E11.5)到达轨道/眼睛,然?…
Discussion
这种外生切片培养方案与传统的斧子导样测定23相比具有显著优势。每个颅骨运动核的大小不是一个限制因素,不需要任何困难的解剖。维持斧子传播的内源微环境,允许修改一个信令通路,同时保持其他信令通路。此外,可以在沿斧子轨迹的不同点评估效果。由于斧子制导需要多个线索和线索的组合,沿着路径29,这提供了一个显著的优势。与分离或外植培养不同,这?…
Divulgaciones
The authors have nothing to disclose.
Acknowledgements
资金由国家眼科研究所 [5K08EY027850], 国家儿童健康与发展研究所 [U54HD090255], 哈佛-视觉临床科学家发展计划 [5K12EY016335], 圣殿骑士眼科基金会 [职业入门者]格兰特*和儿童医院眼科基金会[教师发现奖]。欧洲经委会是霍华德·休斯医学研究所的调查员。
Materials
| 24-Well Tissue Culture Plate | Genesee Scientific | 25-107 | |
| 6-Well Tissue Culture Plate | Genesee Scientific | 25-105 | |
| Disposable Pasteur Pipet (Flint Glass) | VWR | 14672-200 | |
| Fine Forceps | Fine Science Tools | 11412-11 | |
| Fluorobrite DMEM | Thermo Fisher Scientific | A1896701 | |
| Glucose (200 g/L) | Thermo Fisher Scientific | A2494001 | |
| Hank's Balanced Salt Solution (1X) | Thermo Fisher Scientific | 14175-095 | |
| Heat Inactivated Fetal Bovine Serum | Atlanta Biologicals | S11550H | |
| HEPES Buffer Solution (1M) | Thermo Fisher Scientific | 15630106 | |
| L-Glutamine (250 nM) | Thermo Fisher Scientific | 25030081 | |
| Loctite Superglue | Loctite | ||
| Low Melting Point Agarose | Thermo Fisher Scientific | 16520050 | |
| Millicell Cell Culture Insert (30mm, hydrophilic PTFE, 0.4 um) | Millipore Sigma | PICM03050 | |
| Moria Mini Perforated Spoon | Fine Science Tools | 10370-19 | |
| Penicillin/Streptomycin (10,000 U/mL) | Thermo Fisher Scientific | 15140122 | |
| Petri Dish (100 x 15mm) | Genesee Scientific | 32-107G | |
| Phosphate Buffered Saline (1X, pH 7.4) | Thermo Fisher Scientific | 10010049 | |
| Razor Blades | VWR | 55411-050 | |
| Surgical Scissors – Blunt | Fine Science Tools | 14000-12 | |
| Ti Eclipse Perfect Focus with TIRF | Nikon | ||
| Vibratome (VT 1200S) | Leica | 1491200S001 | |
| Vibratome Blades (Double Edge, Stainless Steel) | Ted Pella, Inc. | 121-6 |
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Citar este artículo
Whitman, M. C., Bell, J. L., Nguyen, E. H., Engle, E. C. Ex Vivo Oculomotor Slice Culture from Embryonic GFP-Expressing Mice for Time-Lapse Imaging of Oculomotor Nerve Outgrowth. J. Vis. Exp. (149), e59911, doi:10.3791/59911 (2019).