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本文内容

  • 摘要
  • 摘要
  • 引言
  • 研究方案
  • 结果
  • 讨论
  • 披露声明
  • 致谢
  • 材料
  • 参考文献
  • 转载和许可

摘要

激光扫描与波长多光子荧光显微镜的先进光学技术组合的实施是为了捕获高分辨率的、 三维的、 实时成像中甘油三酯 (sox10:EGFP) 和甘油三酯 (foxd3:GFP) 斑马鱼胚胎神经嵴迁移。

摘要

先天性眼和颅颌面畸形反映神经嵴，流动人口的洄游的干细胞，导致整个身体的许多细胞类型的中断。了解神经嵴的生物学已经有限，反映出缺乏可以研究体内的转基因听话模型和实时。斑马鱼是特别重要的发展模式，为研究洄游的细胞群，如神经嵴。若要检查神经嵴迁移到眼睛发育，实施的结合激光扫描显微镜与长波长多光子荧光激发的先进光学技术是拍摄高分辨率的、 三维的、 实时的转基因斑马鱼胚胎，即甘油三酯 (sox10:EGFP) 和甘油三酯 (foxd3:GFP)，在眼睛发育如sox10和foxd3已经在众多的动物模型，以调节早期神经嵴分化，他们可能代表神经嵴细胞的标记所示。多光子延时成像用来辨别的行为和早期眼发展作出贡献的两个神经嵴细胞群的迁徙模式。本议定书提供关于作为一个例子，斑马鱼神经嵴在迁移期间，生成的视频信息，并可进一步用于可视化斑马鱼和其他模式生物的很多建筑的早期发展。

引言

先天性眼疾导致儿童失明，往往是由于异常的颅神经嵴。神经嵴细胞是瞬态的干细胞，起身从神经管形成许多机体全身。1,2,3,4,5前脑的两侧和中脑，神经嵴细胞，引起的骨和软骨的中部和额叶区和虹膜、 角膜、 小梁网和前段眼睛的巩膜。4,6,7,8神经嵴细胞从蛤蚧形式咽弓、 下巴和心脏流出道。1,3,4,9,10研究强调了贡献于眼部神经嵴和眼周发展，强调这些细胞在脊椎动物眼睛发育的重要性。事实上，中断的神经嵴细胞迁移和分化导致颅面部、 眼部异常如在阿克森费尔德丽格综合征和彼得斯加综合征。11,12,13,14,15,16,17因此，全面了解迁移、 增殖和分化的这些神经嵴细胞将提供洞察潜在先天性眼疾的复杂性。

斑马鱼是强大的模式生物研究眼发展，斑马鱼眼的结构类似于哺乳动物同行，以及许多基因进化保守斑马鱼和哺乳动物之间。18,19,20此外，斑马鱼胚胎是透明和卵生，促进眼发育的实时可视化。

扩大以前发表的作品，6，，720神经嵴细胞的迁移模式描述了使用多光子荧光延时成像上带有 SRY （性别决定区 Y） 的转录控制下的绿色荧光蛋白 (GFP) 的转基因斑马鱼线-框 10 (sox10) 或叉头框 D3 (foxd3) 基因调控区域。21,22,23,24.多光子荧光延时成像是一项强大的技术，结合先进的光学技术的激光扫描显微镜与长波长多光子荧光激发来捕获的标本，用荧光标记的高分辨率的三维图像。25,26,27多光子激光的使用已经超过标准的共焦显微镜，包括增加的组织的渗透和减少的荧光漂白明显的优势。

使用此方法，两个不同种群的神经嵴细胞迁移和洄游通道的时间变了可区分，即 foxd3 阳性神经嵴细胞在眼周间质和眼睛发育和颅面间质 sox10 阳性神经嵴细胞。使用此方法，介绍了可视化的斑马鱼的眼部和面部神经嵴迁移迁移方法，使其易于观察发展过程中实时调节的神经嵴迁移。

此协议提供信息以便在早期眼发育的甘油三酯 (sox10:EGFP) 和甘油三酯 (foxd3:GFP) 转基因斑马鱼作为示例生成定时视频。此协议可进一步用于任何来自神经嵴细胞在斑马鱼的眼部和面部结构的早期发展的高分辨率的、 三维的、 实时可视化。此外，这种方法进一步可以用于发展的其它组织和器官在斑马鱼和其他动物的模型可视化。

研究方案

The protocol described here was performed in accordance with the guidelines for the humane treatment of laboratory animals established by the University of Michigan Committee on the Use and Care of Animals (UCUCA).

1. Embryo Collection for Time-lapse Imaging

  1. Between 3 and 9 pm, set up male and female adult Tg(sox10:EGFP) or Tg(foxd3:GFP) transgenic zebrafish in a divided breeding tank for pairwise mating.
    NOTE: The Tg(sox10:EGFP) and Tg(foxd3:GFP) fish, kind gifts from Thomas Schilling and Mary Halloran, respectively, were crossed into the Casper (roy -/-, nacre -/-) background to decrease auto-fluorescence and interference with pigmentation.
    1. Assemble the breeding tank (slotted inner tank + solid outer tank) and fill it with reverse osmosis (RO) system water.
    2. Transfer up to 3 females and 3 males into opposing sides of the tank separated by a divider; up to 6 fish can be bred pairwise in a single breeding tank.
    3. The next morning, remove the divider shortly after the lights turn on in the vivarium.
      NOTE: In the present study, the lights are on a 14-hour light (on at 9 am) and 10-hour dark (off at 11 pm) cycle.
    4. Allow undisturbed mating for 20 min or until sufficient numbers of embryos are produced; the embryos will be at the bottom of the breeding tank. When eggs are observed, lift the slotted inner tank out along with the fish and quickly place them (fish and inner tank) into a clean solid outer tank filled with RO water.
  2. Prepare standard 1X embryo medium by adding the following per 1 L of RO water: 0.287 g (4.9 mM) NaCl, 0.127 g (0.17 mM) KCl, 0.048 g (0.33 mM) CaCl2.2H2O, 0.04 g (0.33 mM) MgSO4 (anhydrous). Stir solution until the salts are completely dissolved.
    1. Add 100 µL of 0.1% methylene blue as a fungicide. Add 0.25 g of sodium bicarbonate to adjust the pH to 7.75. Store medium at room temperature.
  3. Collect eggs from the outer tank using an egg-collecting screen, and transfer the eggs to Petri dishes (~50 embryos per dish) containing 30 mL of 1X embryo medium. Incubate the collected eggs at 28.5 °C.
  4. Embryo preparation
    1. Tg(sox10:EGFP) embryos.
      1. For Tg(sox10:EGFP) embryos, at 12 h post-fertilization (hpf), remove the dead eggs and assess the developmental stage of the embryos. Screen for GFP-positive embryos using a dissecting fluorescent stereomicroscope with a standard 460-490 nm band-pass excitation filter. Using forceps, remove the chorions from 3-4 embryos that express GFP, and transfer these embryos to a separate Petri dish containing 30 mL of fresh 1X embryo medium.
    2. Tg(foxd3:GFP) embryos.
      1. For Tg(foxd3:GFP) embryos, at 22-24 hpf, remove the dead eggs and assess the developmental stage of the embryos. Screen for positive embryos using a dissecting fluorescent stereomicroscope with a standard 460-490 nm band-pass excitation filter. Using forceps, remove the chorions from 3-4 embryos that express GFP.
      2. Prepare 1000X Phenylthiourea (PTU) stock solution by dissolving 0.75 g (3%) of PTU in 25 mL of Dimethylsulfoxide (DMSO). Aliquot and store the stock solution at 4 °C. Add 30 µL of 1000X PTU (3%) stock solution to 30 mL of 1X embryo medium to generate 0.003% PTU solution.
      3. Place the screened and dechorionated GFP-positive embryos in 0.003% PTU solution to inhibit pigmentation. Do not initiate treatment of the embryos with PTU prior to 20 hpf, as early treatment (at <20 hpf) can have adverse effects on neural crest and neuroepithelial development.28
    3. Prior to conducting the time-lapse experiment, monitor the embryos through live imaging using a standard stereomicroscope at 12-24 h intervals in age-matched comparisons as a control for temperature drift.

2. Mounting of Embryo for Time-lapse Imaging

  1. Preparation of time-lapse embryo media
    1. Prepare standard 0.4% tricaine stock solution by adding 0.004 g of tricaine to 100 mL of RO water. Aliquot (~1 mL) the stock solution into fresh 1.5-mL centrifuge tubes and store at -20 °C.
    2. Prepare time-lapse embryo medium (50 mL of 1X embryo medium, 0.016% tricaine) by adding 2 mL of 0.4% tricaine solution to 48 mL of 1X embryo medium. If using embryos >22 hpf, then also add 50 µL of 3% PTU stock solution (final concentration.003% PTU) to the embryo medium.
  2. Preparation of 2% low-melt agarose
    1. Add 0.4 g of low-melt agarose powder to 20 mL of 1X embryo medium. Heat for 1-2 min or until the solution is clear and all particles are dissolved.
      NOTE: Increasing the percentage of the agarose gel decreases the porosity of the matrix, which may physically impede the growth and development of the embryo. Therefore, the agarose solution can be lowered to 1.5% to minimize these effects. However, when the excitation beam is held stationary during laser-scanning microscopy, greater heating can occur, increasing rapidly in a logarithmic relationship with time. In this protocol, heating due to fluorophore absorption is highly localized to the focal region. Thus, in the region of interest, the temperature could increase to a value high enough (≥ 30 °C) to melt agarose solutions made at percentages lower than 1.5, thereby directly exposing the embryo to the laser or enabling conditions in which the embryo floats out of the focal plane. For this reason, the use of agarose solutions lower than 1.5% is not recommended.
    2. Aliquot (~1 mL) the agarose solution into fresh 1.5-mL centrifuge tubes for storage. Store excess agarose solution in liquid form on a heat block (60-70 °C) for 2-3 weeks.
  3. Mounting the Embryo
    1. To set up the open bath chamber, place a small amount of high vacuum grease on the base of the open bath chamber. Place a circular glass coverslip onto the base and screw the top of the open bath chamber onto the base until tight ( Figure 1A, B).
    2. Pipet (~500-700 µL) of 2% agarose solution (60-70 °C) into the open bath chamber until the base is ~3/4 filled ( Figure 1C). Note that once the agarose solution is in the open bath chamber on the lab bench, the temperature of the solution decreases approximately 1 °C/s.
    3. Wait 30 s to allow the agarose to cool slightly (~30-40 °C), without completely polymerizing, and subsequently transfer a single embryo to the center of the base ( Figure 1D, E).
    4. Under a fluorescent stereomicroscope, position the embryo using a 1-10 µL micropipette tip, making sure that the embryo is placed near the bottom of the agarose, as the embryo may float away when covered with embryo media if it is placed too near the surface of the agarose.
      NOTE: In the presented videos (Videos 1-3), the embryos are oriented laterally, but depending on the area of interest, the embryos can be oriented ventrally or dorsally.
    5. Monitor and reposition the embryo until the agarose has set. Once the agarose has set, use a transfer pipet to fill the assembled open chamber entirely with time-lapse embryo media ( Figure 1F).
    6. Place the open bath chamber in the quick exchange platform, which fits onto the stage adapter ( Figure 1G). Place the entire setup (embryo, open bath chamber, quick exchange platform and stage adaptor) onto the stage of the microscope immediately above the condenser ( Figure 1H).

3. Microscope Set-up for Time-lapse Imaging

  1. Determining laser settings
    1. Determining laser wavelength to use. Use a wavelength that is twice the excitation wavelength of the fluorophore of interest (e.g. wavelength between 880 and 940 nm for GFP).
      NOTE: In the present study, the wavelength setting for GFP was between 880 and 940 nm. The higher the wavelength, the lower the output power of the laser.
    2. Determine laser transmission. A high percent of laser transmission will kill the embryo, use the lowest level of transmission (recommended). For 24 to 48 h time-lapse studies, as presented herein, keep transmission below 5%.
    3. Determine microscope detection systems and ensure that the correct filters for the fluorophore are in place.
      NOTE: For multi-photon microscopes, there are multiple detection systems with various sensitivities for the emitted fluorescence. In general, an internal detection system has less sensitivity than an external detection system. For transgenic lines with high levels of GFP expression, the internal detection system is adequate. For transgenic lines with low levels of GFP expression or with other fluorophores (e.g., red fluorescent protein), an external detection system may be required to maintain the percent of laser transmission at a reasonable level. Regardless the detection system used, the correct filters for the fluorophore must be in place.
  2. Adjusting software settings on the multi-photon microscope
    1. Using the 5X objective, locate the embryo. Manually raise the stage to the highest position, and use the fine focus to position the embryo in the middle of the microscope range.
    2. Manually lower the stage, and change the 5X objective to the 25X water immersion objective (numerical aperture NA, 0.95). Carefully raise the stage to bring the embryo back into focus.
    3. In the software, click on the "xyzt" mode for obtaining multiple images at time intervals (t) in the x-y plane over a depth of "z". Use the epifluorescence or brightfield view to find the depth of focus in the area of interest, which will demarcate the Z-stack.
      1. In the software, click on "begin" button; for these experiments, the lateral edge of the eye was the beginning of the Z-stack. Click on "end" button; the midline of the embryo was the end of the Z-stack.
        NOTE: The step size was 0.3-0.6 µm and there was a total of ~200 steps for a z-stack size of 60 to 120 µm).
    4. Click on the menu for adjusting acquisition time and imaging frequency. For the present system, ~200 steps requires approximately 5 min for each z-stack acquisition. For adequate recovery of the fluorophore and survival of the embryo, allow for a ratio of at least 1:3 between z-stack acquisition (laser power on) and recovery time (laser power off).
      NOTE: For example, z-stacks are acquired every 20 min with 5 min of z-stack acquisition and 15 min of recovery. For this protocol, larger z-stacks can be obtained, but would appropriately increase the time between z-stack acquisitions, resulting in fewer images over the time-lapse course.
      1. With appropriate time for embryo recovery, set the time between z-stacks in the designated window. Set the total length of time for the experiment in the appropriate window.
    5. Final software, laser, and embryo adjustments.
      1. Turn on the live image setting to make final adjustments to the laser settings. Adjust laser transmission (see 3.1.2), gain and offset slider bars within the software to optimize the fluorescent image. Also, adjust the orientation of the embryo, as needed, depending on the length of the experiment, anticipated growth of the embryo, etc. Make sure that the area of interest remains within the frame through the duration of the experiment.
      2. Turn off the epifluorescent light source as it is no longer needed during time-lapse acquisition. Cover the stage with the laser safety box ( Figure 1I). When using the internal detection system, the laser safety box is adequate for protection against background light. Press "start".
        NOTE: However, with more sensitive external detection systems, the laser safety box does not block enough background light, and additional covers are required to prevent the disruption of image acquisition.

4. During Time-lapse Acquisition

  1. Refill the open bath chamber with time-lapse embryo media every 8-12 h (at least 2 times per day) during time-lapse acquisition through the sliding doors on the laser safety box ( Figure 1I).
  2. Before opening the doors of the laser safety box, ensure that the microscope is not actively acquiring an image.
    NOTE: The use of heaters and circulating media systems is not necessary for time-lapse imaging experiments lasting 24 to 48 h. Indeed, the temperatures of both stage and in-line heaters are difficult to control, and during image acquisition the embryo exhibits an adequate development rate at a temperature range from 25-28 °C. Moreover, circulating media systems tend to overflow and potentially damage the equipment. Thus, all embryos are routinely staged post-acquisition.

5. Post-acquisition processing

  1. In the software, click on the "file" menu and choose "save".
  2. Open the file in image processing software (see the Table of Materials). Highlight the correct image series. In the software, choose the "Process" menu. Click on 3D Deconvolution and "Apply" to deconvolve each z-stack.
    NOTE: The file is large; therefore, this step may take many hours.
  3. In the software, under the "Process" menu, click on "maximum projection." Click on "Apply" to initiate maximum projection to generate 1 image per z-stack. Export each maximum projected file (1 image per z-stack) as a tiff.
  4. Import individual tiff files into video processing software. Select all tiff files and drag them into the video editor. Adjust length of each image within the video to 0.1s. Export video as mov or mp4 file.

结果

多光子荧光延时成像产生一系列的视频显示斑马鱼线到眼前段中的甘油三酯 (sox10:EGFP) 和甘油三酯 (foxd3:GFP) 与颅面结构引起的颅神经嵴细胞的迁移模式。作为一个例子， sox10-12 和 30 hpf 之间的积极的神经嵴细胞迁移从神经管的边缘到颅面地区 (视频 1， 图 2)。来自前脑的两侧和中脑细胞迁移的背、 腹面的...

讨论

多光子延时成像使体内追踪的瞬态和洄游的细胞群体。这个强大的技术可用于研究胚胎过程在真正的时间，并在本研究中，这种方法的结果加强的神经嵴细胞迁移和发展的当前知识。以前的延时成像研究通常利用共聚焦激光扫描显微镜。29,30,31,32在这里，我们目前使用的多光子技术，相比传统?...

披露声明

这项工作提供了通过视觉研究的核心 (P30 EY007003) 与美国国立卫生研究院 (K08EY022912-01) 国家眼科研究所的赠款资助。

致谢

作者感谢托马斯 · 席林请送礼的甘油三酯 (sox10:eGFP) 鱼和玛丽哈洛伦的慈祥礼物甘油三酯(foxd3:GFP)鱼。

材料

NameCompanyCatalog NumberComments
Breeding Tanks with DividersAquaneeringZHCT100Crossing Tank Set (1.0-liter) Clear Polycarbonate with Lid and Insert
M205 FA Combi-ScopeLeica Microsystems CMS GmbHStereofluorescence Microscope - FusionOptics and TripleBeam
Sodium ChlorideMillipore (EMD)7760-5KGDouble PE sack. CAS No. 7647-14-5, EC Number 231-598-3
Potassium ChlorideMillipore (EMD)1049380500Potassium chloride 99.999 Suprapur. CAS No. 7447-40-7, EC Number 231-211-8.
Calcium Chloride DihydrateFisher ScientificC79-500Poly bottle; 500 g. CAS No. 10035-04-8
Magnesium Sulfate (Anhydrous)Millipore (EMD)MX0075-1Poly bottle; 500 g. CAS No. 7487-88-9, EC Number 231-298-2
Methylene BlueMillipore (EMD)284-12Glass bottle; 25 g. Powder, Certified Biological Stain
Sodium BicarbonateMillipore (EMD)SX0320-1Poly bottle; 500 g. Powder, GR ACS. CAS No. 144-55-8, EC Number 205-633-8
N-PhenylthioureaSigmaP7629-25G>98%. CAS Number 103-85-5, EC Number 203-151-2
DimethylsulfoxideSigmaD8418-500MLMolecular Biology grade. CAS Number 67-68-5, EC Number 200-664-3
Tricaine MethanesulfonateWestern Chemical Inc.MS222Tricaine-S
Low-Melt AgaroseISC BioexpressE-3112-25GeneMate Sieve GQA Low Melt Agarose, 25 g
Open Bath ChamberWarner InstrumentsRC-40HPHigh Profile
Glass CoverslipsFisher Scientific12-545-102Circle cover glass. 25 mm diameter
High Vacuum GreaseFisher Scientific14-635-5C2.0-lb. tube. DOW CORNING CORPORATION
1658832
Quick Exchange PlatformWarner InstrumentsQE-135 mm
Stage AdapterWarner InstrumentsSA-20LZ-AL16.5 x 10 cm
TC SP5 MP multi-photon microscopeLeica Microsystems CMS GmbH
Mai Tai DeepSee Ti-Sapphire LaserSpectraPhysics
Laser Safety BoxLeica Microsystems CMS GmbH
Leica Application Suite X (LAS X)  SoftwareLeica Microsystems CMS GmbH
Photoshop CS 6 Version 13.0 x64 SoftwareAdobe
iMovie Version 10.1.4 SoftwareApple

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