实时和重复测量电活动改变的个体活斑马鱼的骨骼肌生长
Summary
光学透明度是斑马鱼细胞生物学和生理学工作的主要优势。描述了用于测量个体动物细胞生长的稳健方法，该方法允许对骨骼肌和邻近组织的生长如何与全身生长相结合的新见解。
Abstract
许多方法可用于可视化活体胚胎、幼虫或幼年斑马鱼全身的单个细胞。我们表明，可以在共聚焦激光扫描显微镜中扫描具有荧光标记质膜的活鱼，以确定肌肉组织的体积和存在的肌肉纤维的数量。描述并验证了随时间推移测量活体动物细胞数量和大小的有效方法，并针对更艰巨的分割方法进行了验证。描述了允许控制肌肉电活动的方法，从而控制收缩活动。骨骼肌收缩活动的丧失大大降低了肌肉生长。在幼虫中，描述了一种允许重新引入模式化的电诱发收缩活动的协议。所描述的方法最大限度地减少了个体间变异性的影响，并允许分析电，遗传，药物或环境刺激对生物体背景下的各种细胞和生理生长参数的影响。随后可以对定义的早期生活干预对个体的测量效果进行长期随访。
Introduction
调节的组织生长，包括细胞数量（增生）和/或细胞大小（肥大）的增加，是发育、再生以及生态和进化适应的关键因素。尽管近几十年来在细胞和发育生物学的分子遗传学理解方面取得了巨大进步，但对组织和器官大小调节的机制理解仍处于起步阶段。这种知识空白的一个原因是难以以必要的空间和时间精度量化生物体中的组织生长。
随着时间的推移，可以反复测量整个生物体生长的各个方面，揭示每个个体的生长曲线1，2，3，4，5。日益复杂的扫描方法，如双X射线吸收测定法（DXA），计算机断层扫描（CT）和磁共振成像（MRI），允许跟踪单个个体（包括人类和模式生物）的整个器官和其他身体区域（例如，个体识别的骨骼肌）的生长6，7，8，9，10.然而，这些方法尚不具有揭示单个细胞的分辨率，因此很难辨别细胞行为与组织水平生长之间的联系。为了建立这种联系，传统研究通常依赖于类似个体动物的队列，其中一些在连续的时间点被处死，然后进行细胞学细节分析。这种方法需要平均跨组（最好是相似但可变的）个体观察到的变化，因此缺乏时间和空间分辨率，因此很难在细胞水平上找到暗示因果关系的相关事件。
对无脊椎动物模式生物的研究，最初在秀丽隐杆线虫和D. melanogaster中，通过开发光学显微镜来实现细胞分辨率并准确测量单个个体随时间推移的生长，从而规避了这些问题。这些研究揭示了这些小型模式生物生长过程中惊人的不变细胞谱系行为11，12，13，14，15，16，17。然而，许多动物，包括所有脊椎动物，具有不确定的细胞谱系，并通过神秘的反馈过程控制组织生长，这些反馈过程有助于将基因编码的生长程序转变为功能性三维生物体，其所有组成组织和器官的大小都适当匹配。为了理解这些复杂的生长过程，需要对单个个体的整个组织或器官进行随时间推移的成像，这些组织或器官可以在选择的时间通过遗传，药理学或其他干预措施进行实验操作，并随后分析效果。
每块脊椎动物骨骼肌都有确定的大小、形状和功能，以及与相邻组织（如骨骼、肌腱和神经）的相互作用。有些肌肉很小，就在皮肤下，因此是高分辨率成像研究的良好候选者。与大多数器官类似，每块肌肉在达到稳定的成年大小之前，都会在整个胚胎、出生后和青少年生命中生长。然而，肌肉在成年后也具有改变大小的独特能力，这取决于使用和营养18，这种特性对机体健康，运动表现和独立生活有重大影响。老年肌肉质量和功能的丧失，肌肉减少症，是面临人口老龄化的社会日益关注的问题19，20，21。
我们和其他人专注于斑马鱼幼虫节段重复体内骨骼肌组织块的生长，作为一个包含数百个细胞的明显封闭系统，其中可以观察和操纵组织生长，维持和修复22，23，24，25，26。虽然之前已经报道了一些定量工作25，26，27，28，29，30，31，32，33，34，35，但没有详细和经过验证的方法来测量单个脊椎动物生物的细胞细节肌肉生长。这里描述了如何执行这种重复测量的有效协议以及验证，并提供了一个用于分析肥大和增生生长变化以响应改变的电活动的示例。
Protocol
所描述的所有研究均按照机构指南进行，并根据1986年《动物（科学程序）法》和随后的修改获得英国内政部的适当许可。胚胎/幼虫应在28.5°C下饲养，直到原肠胚形成完成，但随后可以保持在22-31°C以控制发育速度。可以在室温下扫描或刺激鱼。 1.麻醉斑马鱼幼虫 将合适的荧光报告成鱼如 Tg（Ola.Actb：Hsa.HRAS-EGFP）vu119Tg 参考文?…
Representative Results
快速精确地测量体细胞体积描述了一种样品制备、数据采集和体积分析的方法，该方法可以快速测量斑马鱼幼虫的肌肉生长。可以在活体动物中使用用膜靶向GFP（β-actin：HRAS-EGFP） 或mCherry（α-actin：mCherry-CAAX ）标记在其质膜上的鱼来测量肌肉大小。使用三卡因暂时麻醉幼虫，安装在低熔点琼脂糖中，并使用共聚焦荧光显微镜成像。选择Somite 17来分析肌肉大小，因为它…
Discussion
在这里，我们报告了一种准确有效地估计活斑马鱼幼虫肌肉体积的方法，在阶段或遗传变异中，色素沉着不是成像的一大障碍，并且当短暂麻醉和/或固定耐受性良好时。虽然我们采用了激光扫描共聚焦显微镜，但所描述的方法适用于转盘共聚焦或光片显微镜以及在不同焦平面上创建图像堆栈的任何其他方法。描述了一系列越来越复杂的组织大小和细胞含量估计方法。每种方法都有优点和局限性，…
Disclosures
The authors have nothing to disclose.
Acknowledgements
作者非常感谢Hughes实验室成员Seetharamaiah Attili，Jana Koth，Fernanda Bajanca，Victoria C. Williams，Yaniv Hinits，Giorgia Bergamin和Vladimir Snetkov博士为开发所述协议所做的努力，以及Henry Roehl，Christina Hammond，David Langenau和Peter Currie共享质粒或斑马鱼系。SMH是医学研究委员会（MRC）科学家，计划资助G1001029，MR / N021231 / 1和MR / W001381 / 1支持。MA拥有伦敦国王学院的MRC博士培训计划博士生奖学金。这项工作得益于学者、导师和朋友大卫·M·罗宾逊（David M. Robinson）的三角输入。
Materials
| Adhesive, Blu Tack | Bostik | – | – |
| Aerosol vacuum | – | – | – |
| Agarose | Sigma-Aldrich | A9539 | – |
| Agarose, low gelling temperature | Sigma-Aldrich | A9414 | Once melted, keep at 37oC in a block heater to remain in liquid form for repeated use. |
| Block heater | Cole-Parmer | SBH130 | – |
| BODIPY FL C5-ceramide | Thermo Scientific | D3521 | To be diluted in fish water and used at 5 µM for overnight incubation. |
| Crocodile clips and wires | – | – | – |
| Fiji/imageJ | National Institutes of Health, NIH | – | – |
| Fish medium, Fish water | – | – | Circulating system water collected from the fish facility. |
| Fish medium, E3 medium | – | – | E3 is described in The Zebrafish Book. http://zfin.org (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, and 0.33 mM MgSO4 in distilled water). |
| Fluorescence microscope | Leica | Leica MZ16F | Fluorescence microscope of other kind are also expected to be suitable. |
| Glass needle | World Precision Instruments, Inc. | 1B100-6 | To be fire-polished to prevent damage of the embryos during manipulation. |
| Grass stimulator | Grass Instruments | S88 | Stimulators of other kind are also expected to be suitable. |
| Kimwipes, Delicate Task Wipers | Kimberly-Clark Professional | 13258179 | – |
| Laser scanning microscope (LSM) | Zeiss | Zeiss LSM 5 Exciter Zeiss LSM 880 |
LSM of other kind are also expected to be suitable. |
| Nunc Cell-Culture Treated, 6-well plate | Thermo Scientific | 140675 | – |
| Objective, 20×/1.0W water immersion | Zeiss | – | – |
| Pasteur Pipette, Graduated 1 mL | Starlab Group | E1414-0100 | – |
| Pasteur Pipette, Micro Fine Tip 1 mL | Starlab Group | E1414-1100 | – |
| Petri dish, 60 mm | Sigma-Aldrich | P5481 | – |
| Plasmid, CMV-Cerulean | Christina L. Hammond (University of Bristol) | pCS2+_cerulean_kanR plasmid injected at 25-75 pg at one-cell stage. Citation: Bussman J, and Schulte-Merker S. (2011) Development 138:4327-4332. doi: 10.1242/dev.068080. | |
| Plasmid, pCS-mCherry-CAAX | Henry Roehl (University of Sheffield) | – | For in vitro transcription using the SP6 promoter (plasmids containing other membrane labelling markers can be used); synthesised capped mRNA to be injected at 100-200 pg at one-cell stage. |
| Pulse Controller | Hoefer Scientific Instruments | PC750 | – |
| Soldering iron | – | – | – |
| Tricaine | Sigma-Aldrich | E10521 | Ethyl 3-aminobenzoate methanesulfonate/ MS-222; to be dissolved in fish water and used at 0.6 mM. |
| Volocity | Perkin Elmer/Quorum Technologies Inc | – | – |
| Watchmaker forceps, No. 5 | – | – | – |
| Wire, Platinum | Goodfellow | PT005142/12 | 0.40 mm in diameter; an expensive alternative of silver. |
| Wire, Silver | Acros Organics | 317730010 | 0.25 mm in diameter (a range of diameter i.e. 0.25-0.5 mm had been tested, which produced similar results). |
| Zebrafish, myog:H2B-mRFP | David M. Langenau (Massachusetts General Hospital; Harvard Stem Cell Institute) | – | ZFIN official name: Tg(myog:Hsa.HIST1H2BJ-mRFP), fb121Tg. http://zfin.org/ZDB-ALT-160803-2 Citation: Tang Q, Moore JC, Ignatius MS, Tenente IM, Hayes MN, Garcia EG, Torres Yordán N, Bourque C, He S, Blackburn JS, Look AT, Houvras Y, Langenau DM. Imaging tumour cell heterogeneity following cell transplantation into optically clear immune-deficient zebrafish. Nat Commun. 2016 Jan 21;7:10358. doi: 10.1038/ncomms10358. |
| Zebrafish, α-actin:mCherry-CAAX | Peter D. Currrie (ARMI, Monash University) | – | ZFIN official name: Tg(actc1b:mCherry-CAAX), pc22Tg. http://zfin.org/ZDB-ALT-150224-2 Citation: Berger J, Tarakci H, Berger S, Li M, Hall TE, Arner A, and Currie PD. Loss of Tropomodulin4 in the zebrafish mutant träge causes cytoplasmic rod formation and muscle weakness reminiscent of nemaline myopathy. Dis Model Mech. 2014 Dec;7(12):1407-15. doi: 10.1242/dmm.017376. |
| Zebrafish, β-actin:HRAS-EGFP | – | – | ZFIN official name: Tg(Ola.Actb:Hsa.HRAS-EGFP), vu119Tg. http://zfin.org/ZDB-ALT-061107-2 Citation: Cooper MS, Szeto DP, Sommers-Herivel G, Topczewski J, Solnica-Krezel L, Kang HC, Johnson I, and Kimelman D. Visualizing morphogenesis in transgenic zebrafish embryos using BODIPY TR methyl ester dye as a vital counterstain for GFP. Dev Dyn. 2005 Feb;232(2):359-68. doi: 10.1002/dvdy.20252. |
| ZEN software | Zeiss | – | – |
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Cite This Article
Attwaters, M., Kelu, J. J., Pipalia, T. G., Hughes, S. M. Real Time and Repeated Measurement of Skeletal Muscle Growth in Individual Live Zebrafish Subjected to Altered Electrical Activity. J. Vis. Exp. (184), e64063, doi:10.3791/64063 (2022).