Method Article
We present a protocol for capturing the dynamics of zebrafish larval tail fin regeneration on a whole-tissue scale using brightfield-based stereomicroscopy. This technique enables capturing the regeneration dynamics with single cell resolution. This methodology can be adapted to any stereomicroscope equipped with a CCD camera and time-lapse software.
The zebrafish larval tail fin is ideal for studying tissue regeneration due to the simple architecture of the larval fin-fold, which comprises of two layers of skin that enclose undifferentiated mesenchyme, and because the larval tail fin regenerates rapidly within 2-3 days. Using this system, we demonstrate a method for capturing the repair dynamics of the amputated tail fin with time-lapse video brightfield stereomicroscopy. We demonstrate that fin amputation triggers a contraction of the amputation wound and extrusion of cells around the wound margin, leading to their subsequent clearance. Fin regeneration proceeds from proximal to distal direction after a short delay. In addition, developmental growth of the larva can be observed during all stages. The presented method provides an opportunity for observing and analyzing whole tissue-scale behaviors such as fin development and growth in a simple microscope setting, which is easily adaptable to any stereomicroscope with time-lapse capabilities.
The ability of an organism to orchestrate tissue repair processes after injury is crucial for its survival 1. While all animals have the capacity to heal their wounds, the extent to which tissues regenerate differs greatly among species. Vertebrate species such as zebrafish, salamanders and frog tadpoles have the remarkable ability to regenerate lost tissues, including their appendages, portions of their eyes, heart, and central nervous system 2-4. Mammalian species, such as the African spiny mouse and rabbits, are capable of regenerating holes in their pinnae 5-7, and humans and mice regenerate portions of their liver as well as their digit tips during fetal and juvenile stages 8-12. Although it is not well understood yet why and how certain species regenerate tissues more effectively than others, the presence of similar genetic pathways suggests that these mechanisms may lie dormant in species without great regeneration potential 13,14. Thus elucidating tissue repair and regeneration mechanisms in species with satisfactory regeneration outcomes will benefit regeneration in humans.
We have chosen the larval zebrafish tail fin as a paradigm to demonstrate its regeneration with time-lapse brightfield stereomicroscopy. The zebrafish larval tail fin is anatomically simple as compared to the more complex adult structures, consisting of a two-layered, infolded epithelium with somatosensory axons innervating the skin that surrounds medially located mesenchymal cells 15. Despite the anatomical differences, larval tail fin regeneration is somewhat comparable to adult fin regeneration in terms of the molecular signatures and the outgrowth responses 16,17. As compared to the adult fin, imaging larval tail fin regeneration has however several advantages: 1) larval fin regeneration is completed within just 2-3 days 16, 2) larvae can be mounted in low-melt agarose, and 3) larvae do not require feeding until ~ 5 days post fertilization (dpf) due to the presence of the yolk sac. This makes zebrafish larvae ideal for observing tissue repair dynamics in vivo.
The presented method enables the capture of detailed dynamics underlying the early processes of fin regeneration. Many studies have utilized fluorescence-based confocal microscopy to study cellular and subcellular biological processes in embryonic and larval zebrafish. Sophisticated confocal imaging setups are however often not accessible to everyone and highly expensive as compared to other imaging techniques. In contrast, the presented methodology utilizes a Discovery V12 stereomicroscope equipped with Axiovision software and a time-lapse module, thus providing a more affordable alternative to expensive imaging equipment to examine tissue behaviors. We demonstrate that this method can be utilized for imaging tissue regeneration with high temporal resolution at a minimal cost. The implications for this method could extend beyond basic biology to advance mammalian regeneration studies using organ cultures, for therapeutic development through pharmacological and genetic screens, and it can serve as a teaching tool in a classroom setting.
斑马鱼( 珍珠层株)根据既定协议孕育和提高。所作的所有努力,尽量减少痛苦,使用0.4毫米的三卡因麻醉和1 mM的三卡因安乐死。斑马鱼胚胎和幼虫严格按照良好的动物的做法被处理为经适当委员会(MDI生物实验室动物的核心IACUC数13-20)。这项研究是根据协议,#14-09经国家人类基因组研究所动物护理和使用委员会,MDIBL制度保障#A-3562-01。
注意:成像程序,捕捉在斑马鱼幼体总结在下面的步骤鳍再生:
1.提高斑马鱼幼虫阶段
2.准备成像室的
3.安装和预注射的成像置的幼虫(这一步是可选)
注意:此步骤是适合的截肢和再生翅片长度之间的比较,如鳍再生后的截肢平面不识别在斑马鱼的幼虫。
4.截肢分析
5.安装幼虫的时间推移成像
6.时间推移成像
7.数据分析
所提出的技术是适用于阐明组织修复的动态响应截肢。电影表明,翅片的截肢最初触发荷包的效果,通过肌动蛋白-肌球蛋白电缆中存在的特征在于收缩翅片折28( 图5的A,B)。伴随地,将细胞从伤口(见电影)挤出。因而收缩可以是驱逐细胞很可能注定要经历细胞死亡的方法。我们的研究结果进一步表明,幼虫的生长发育出现独立再生(电影),而鳍再生不会启动,直到大约14小时后截肢由鳍片长度和面积超过36小时的时间过程以下截肢( 图5C测,D)。 1.5天后的总再生鳍生长为约60%的原始翼片长度( 图5E)的。两者合计,这些结果表明,截肢TR iggers翅片收缩,从伤口,并且在时间上延迟的再生反应的细胞挤出。而挤出的细胞可能注定要经历细胞死亡,需要这些细胞的性质,以进一步澄清。
图1.成像室环组件
(A)中显示的是一个连接到硅润滑脂盖玻片一个塑料环。的塑料网格连接于所述腔室的与硅润滑脂四个小点的内部。 (B)中含有的安装幼虫的腔室填充有三卡因溶液和载玻片被附连到顶部。 (C)在安装2天岁的幼虫(箭头)显示在更高的放大倍率来描绘它的尺寸相对于网格。"_blank">点击此处查看该图的放大版本。
图2.成像室组件从培养皿中进行 。 (A)中显示的是一个商用玻璃顶玻璃底培养皿与附连到玻璃盖玻片一塑料网。 (B)中显示的是一个自建培养皿室钻入盖和连接的从硅润滑脂外侧的盖玻片的孔。网格和幼虫安装包含三卡因溶液室内。密封室,硅脂适用于底部室和连接顶盖的上方,外缘。 请点击此处查看该图的放大版本。
图截肢和安装幼虫的成像3.计划 。 (A)中对于截肢,放置一个麻醉幼虫于琼脂糖被覆培养皿和截肢的尾鳍用注射器针头。 (B)的安装时,用移液管转移幼虫到1.5毫升管填充有42℃的液体琼脂糖和吸取含有幼虫到成像室中的下降,定位鱼和覆盖胚胎培养基中的固化琼脂糖。 (C)的刮去从尾鳍琼脂糖使用封微加载枪头或类似的工具,并用新鲜培养基替换培养基的胚胎。 (D)图像尾鳍在立体显微镜下。 请点击此处查看该图的放大版本。
图4.自建加热孵化室 。 (AC)显示的是一个温水孵化室用纸板,泡沫包装和魔术贴。有线圆顶加热器(最初设计用于鸡卵孵化)连接到使用铝带室。 请点击此处查看该图的放大版本。
图5.鳍再生动力 。所利用的尾鳍截肢测定和定量方法(A) 的计划,以确定该翼片长度(红色箭头)和面积(翅片的红色轮廓)。 (B)的尾鳍截肢最初触发收缩翅片,接着雷杰nerative组织生长。散热片也经历了发展和成长,就证明了横向尺寸增加。 (c)中所示的翼片长度为时间的函数,露出一个线性再生开始生长在〜14 HPA。翅片面积(D)的定量揭示一个最初减小尺寸,这可以归因于该鳍的收缩。后〜14小时,以线性速率翅片尺寸增大。截肢前的片长,36小时后(E)的比较显示〜60%的再生。比例尺:100微米简称:前置放大器,前置截肢;小时后截肢,HPA;再生,再生;放大器,截肢 请点击这里查看这个数字的放大版本。
电影 。鳍再生超过36的时间过程小时。显示的是再生的过程中,一个尾鳍的2.5天龄幼虫。开始30分钟后截肢再生的增长是使用3.5倍物镜成像在立体显微镜30分钟一班。
所提出的方法允许观察伤口愈合和组织再生中在明视显微镜活斑马鱼幼虫体内延时成像,使用相对简单的设置。这个过程需要我们已经测试过的某些重要方面,这将优化结果:1)低浓度琼脂糖(〜0.5%)将减少不断增长的幼虫斑马鱼的生长障碍,2)清除周围的散热片的琼脂糖重要的是不要模糊愈合过程,3)捕获在一个塑料网状琼脂糖保留在整个手术过程中稳定的位置琼脂糖和动物,以及4)一个适当的温度控制的环境中,这是幼虫存活必不可少的。我们已经适应加热的孵育室23,24,其利用正在录音到纸板泡沫包装,以及有线圆顶加热器来控制温度和适当的空气流通用在最小的波动成像过程。这个简单的和有成本效益室可以制备以适应任何显微镜。类似的温水孵化室也已用于成像老鼠和小鸡发展24,29。
我们建议预截去的幼虫被安装为一个预截肢图像,拆下截肢,和重新安装用于延时成像。虽然它是在最终的成像室中的单个步骤执行这些步骤是可行的,在我们的经验,我们发现,截肢尾鳍在玻璃盖玻片不是最佳的,因为它泪组织并且不会导致在一个干净的切口。用注射器针头琼脂糖为基础的截肢方法最初是由川和他的同事(2004)16中描述,也是在我们的经验,非常适合进行截肢。因此,在相当复杂的一系列步骤,我们介绍的是有充分理由的,并确保最佳的再生效果。
我们发现,larv人斑马鱼在2 DPF可以被成像到在琼脂糖和三卡因溶液1.5天。我们使用制备即时海洋盐,其不与试样的健康干扰对所呈现的成像期间的pH优化的三卡因(pH7的)解决方案。我们先前然而还证明了在Danieau介质使用三卡因允许在共聚焦显微镜对至少2天30 2.5延时成像DPF幼虫斑马鱼。因此,最佳的缓冲液条件可以延长幼虫健康和成像的长度。或者,可用于麻醉,或2-苯氧基乙醇,我们发现在幼虫和成虫阶段的耐受性良好,在28℃下进行至少60小时下三卡因的浓度。
为了避免在散热片再生的缺陷,我们删除从之前的成像尾鳍琼脂糖。我们的数据表明,在1.5天鳍已再生至约60%。这种再生率与以前的研究定义3天一致s的平均时间尾鳍再生的斑马鱼幼虫多达6 DPF 16。替代方法琼脂糖却可能被用来装鱼的成像。例如,等离子体细凝块31或氟化乙烯丙烯(FEP)管涂覆有甲基纤维素和填充有非常低的琼脂糖浓度(0.1%),已被建议用于光片镜32和可适合于我们提出的方法。然而,我们不推荐甲基纤维素和0.1%琼脂糖,因为它们需要该样品被安装在所述腔室的底部,由于缺乏这些介质的凝固。非常高浓度的甲基纤维素将另外产生根据我们的经验气穴,并且这些可以与成像过程产生干扰。如果这些媒体是优选使用的底腔,重要的是,在物镜和试样之间的适当的工作距离是否存在。应当注意的是,米乙基纤维素作为安装介质,建议只为1天,因为它可能与幼虫健康干预32。
安装在盖的检体可能会导致缓慢的引力向下漂移。因此,在每一个时间点,这可以被投影成一个单一的平面或仅是在焦平面可以提取用于组装最终电影图像推荐图像的多个部分。成像试样在底部室可以是一种替代方法,以避免潜在的向下漂移。血浆凝块可能是有用的,以避免漂移,作为等离子体会粘到外层包封层(EVL,周皮)31,因此,可以稳定的样品。然而,这需要进行测试,以及多久幼虫斑马鱼可以保持在血浆凝块而不与健康幼虫或鳍再生的干扰。
我们的电影是利用组装各个部分(26微米)的一个记录,Z堆叠,其覆盖所述鳍(〜10微米),并且在成像过程期间占鳍的潜在的z漂移的整个厚度。为了保留的3-D信息,但也可以突出的z栈成单个图像。因为这可能会导致在图像的模糊程度,明去卷积可以期望。软件,如反卷积或Autoquant X3可以被用于此目的。或者,数学算法(在Tadrous 33描述的)可被用于获得高的信号-噪声比(SNR)的点扩散函数。获得高信噪比表示在明解卷积的主要障碍之一。虽然这种方法需要高对比度和细样品的厚度,这将是适当的尾鳍的成像,由于其宽度减小。
所提出的成像方法的一个明显的优点是,它是快速适应装备有CCD照相机的任何立体显微镜ð时间推移软件,并提供一种低成本的替代更昂贵的共焦成像系统。虽然这种方法不使用荧光进行小区检测,它可以扩展为这样的应用,利用一种自动系统,用于快门控制和后摄像卷积软件34。这将使用户能够进一步的观察与单细胞或亚细胞分辨率创面的修复和再生过程在更长的时间段。
光学清晰度和缓解与胚胎和幼虫的斑马鱼可以处理了,这种方法对任何立体的适应能力使得它适合于教学的基本脊椎动物生物学课堂上。这种方法可以为学生提供一个更好地了解组织修复和再生背后的基本生物过程。已经捕获用类似的方法等生物学过程是斑马鱼胚胎发育23,34和心函数(未公布的)。这种方法还提供了可能性监测伤口修复和再生中的幼虫已进行遗传和药理学上操纵。
作者什么都没有透露。
We thank the MDI Biological Laboratory animal core service facility for zebrafish maintenance. Research reported in this publication was supported by Institutional Development Awards (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant numbers P20GM104318 (for COBRE) and P20GM103423 (INBRE) and Department of Defense – USAMRAA (W81XWH-BAA-1) grant.
Name | Company | Catalog Number | Comments |
Reagents | |||
Bullseye Agarose (MidSci, Cat. No. BE-GCA500) | |||
Low-melt agarose (Fisher BioReagents, Cat No. BP1360-100) | |||
1-phenyl-2-thiourea [Alfa Aesar, Cat No. L06690] | |||
Instant Ocean Aquarium Salt (Pet store) | |||
Methylene Blue (0.1% solution) (Sigma, Cat. No. M9140) | |||
Tricaine (Ethyl 3-aminobenzoate methanesulfonate, Sigma-Aldrich, Cat. No. E10505) | |||
2-Phenoxyethanol (Sigma-Aldrich, Cat. No. 77699) | |||
Petri Dish 35 x 15 mm (BD Falcon, Cat. No 351008) | |||
Petri Dish 60 x 15 mm (BD Falcon, Cat. No 351007) | |||
Petri Dish 100 x 25 mm (BD Falcon, Cat. No 351013) | |||
5.75 inch boroschillate glass pipets (Fisher) | |||
35 mm Glass Top Glass Bottom Dish (MatTek Corporation, Cat No. D35-20-0-TOP) Glass: 0.085-0.115mm | |||
Superfrost/Plus microscope slides (Fisherbrand, Cat No. 12-550-15) | |||
Glass coverslips (Electron Microscopy Services, Cat No. 72191-75) | |||
Glass coverslips (Warner Instruments, Cat. No. CS-18R15) | |||
Phifer Phiferglass Insect Screen Charcoal - 48" (Home Depot) | |||
DOW CORNING® HIGH VACUUM GREASE | |||
Microloader pipette tips 20 µl (Eppendorf, Cat. No. 930001007) | |||
Fine Scissors - Sharply Angled Up (Fine Science Tools, Cat. No. 14037-10) | |||
3 mL Luer-Lok™ disposable syringe (BD, Cat. No. 309657) | |||
60 mL Luer-Lok™ disposable syringe (BD, Cat. No. 309653) | |||
23-gauge syringe needles (BD, Cat. No. 305145) | |||
Dumont #5 Forceps (Fine Science Tools, Cat. No. 11295-00) | |||
Equipment | |||
LabDoctor Mini Dry Bath (MidSci) | |||
Zeiss Discovery.V12 compound microscope | |||
Zeiss Plan Apo S 3.5X objective | |||
Zeiss AxioCam MRm | |||
Zeiss Axiovision software, Release 4.8.2SP1 (12-2011) |
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