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Acquisition of Leftward Flow in Xenopus laevis
非洲爪蟾GRP左像移动的观测   

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参见作者原研究论文

本实验方案简略版
Current Biology
Jan 2007

Abstract

In Xenopus, the left-right axis is established following an extracellular vectorial leftward flow driven by monocilia at the gastrocoel roof plate (GRP) during late gastrulation / neurulation (Schweickert et al., 2007). As the GRP lies inside the developing archenteron, imaging of flow is challenging. Here we present the detailed procedure to visualize leftward flow in Xenopus laevis embryos.

Keywords: Cilia (纤毛), Left-right asymmetry (左右不对称), Imaging (成像), Leftward flow (向左流), Gastrocoel roof plate (原肠腔顶板)

Materials and Reagents

  1. Fluorescent micro beads (e.g. FluoSpheres 0.5 μm) (Life Technologies, catalog number: F-8813 )
  2. Agarose
  3. Penicillin/Streptomycin (10.000 Units/ml; Gibco®, catalog number: 15140 )
  4. HEPES Pufferan (Carl Roth, catalog number: HN78.3 )
  5. Bead solution (see Recipes)
  6. 5x MBSH (Modified Barth‘s Saline) (see Recipes)

Equipment

  1. Sharp forceps (Fine Science Tools, catalog number: Dumont #5 )
  2. Micro blade (Fine Science Tools, Dissecting Knife - Fine Tip)
  3. Sectioning dish (Petri Dish with bottom covered in 1% Agarose in 1x MBSH)
  4. Transfer pipet (Carl Roth, model: EA65.1 )
  5. Glass staining block (Karl-Hecht Assistent (Lymphbecken) 2020)
  6. Microscope slides
  7. Coverslips
  8. 5 ml syringe
  9. Vaseline
  10. Fluorescence microscope equipped with a digital camera (wide field)
  11. Large Petri dish filled with PBS or MBSH for explant retrieval

Procedure

  1. Raise Xenopus embryos to stage 17 (Nieuwkoop et al., 1994).
  2. Build a flexible imaging chamber (Figure 1) by putting the opening of a vaseline filled syringe directly onto a microscope slide. Gently press the piston to release vaseline as you draw a rectangle (approximately 1.5 x 1 cm) onto the slide. Make sure that the rectangle is closed. Fill the rectangle with the bead solution until it has a convex meniscus.


    Figure 1. A flexible imaging chamber

  3. Cut dorsal explant (cf. Figure 2 in Blum et al., 2009):
    1. Transfer a stage 17 embryo into a sectioning Petri dish filled with 1x MBSH.
    2. With a micro blade, remove the head by transversally cutting the anterior part of the embryo.
    3. Erect the cup-shaped embryo such that you can see inside. Then place small alternating cuts at the left and right ‘lateral lines’ until you reach the area of the circumblastoporal collar.
    4. With forceps and the micro blade, pull apart the dorsal and ventral halves of the embryo until you see the opening of the blastopore from the inside.
    5. Place a final cut ventral to the blastopore to separate the dorsal explant from the rest of the embryo. Make sure not to touch the dorsal explant with the forceps or blade from the inside.
    6. You should now be left with a bathtub shaped dorsal explant with the GRP placed at its deepest point.
  4. With a transfer pipet, carefully bring the dorsal explant into the glass staining block that contains the FluoSphere bead solution and gently pipet the explant up and down in this medium to make sure that FluoSpheres reach the GRP.
  5. Transfer the dorsal explant into the FluoSphere solution filled chamber by carefully pipetting. Make sure that the explant never touches the surface of any liquid as surface tension disintegrates the tissue. Orient the explant with the inside facing up, i.e. towards you.
  6. Seal the flexible chamber with a coverslip by gently pushing it down with opened forceps. Carefully push until the coverslip touches the rim of the dorsal explant, i.e. the left and right ‘lateral line’ and the ventral side of the circumblastoporal collar.
  7. While pushing down, sometimes the ventral circumblastoporal collar folds over and blocks the GRP from view. If that happens, a slight drag of the coverslip towards the posterior pole can shear the explant to allow imaging of the GRP.
  8. Let the setup rest for 5-10 min to allow the explant to deform, which otherwise would suggest false particle movements during early acquisition.
  9. Put the slide in your (wide-field) fluorescence microscope and focus on the GRP.
  10. Excite with the required wavelength of the FluoSpheres - you should see a ‘starry sky’. Now adjust focus to the focal plane slightly above the GRP.
    Note: as the GRP cilia are just 5 μm in length, leftward flow occurs only in the plane right above the epithelium (Schweickert et al., 2007). To facilitate finding the right plane, focus into the tissue until you do not see fluorescence of any bead. Now carefully change the focal plane until the first beads appear in focus.
  11. In wild type embryos, you should see particles moving to the left of the GRP. In Xenopus laevis, leftward flow is relatively slow (~2.5 μm/s) and therefore best visualized using time-lapse videography (Schweickert et al., 2007). Depending on further analysis and hence temporal resolution, acquire a movie with at least 2 fps.
  12. For retrieving the dorsal explant after investigation, submerge the complete slide in a large Petri dish filled with 1x MBSH or 1x PBS and carefully remove the coverslip with forceps. The explant should float into the buffer from which it can be pipetted into fixative for further analysis (in situ hybridization, immunohistochemical staining, etc.).

Recipes

  1. Bead solution
    Dilute FluoSpheres 1:2,500 in 1x MBSH (prepare ~5 ml)
  2. 5 x MBSH (1 L)
    Note: Before usage, dilute to 1x with H2O
    25.7 g NaCl
    0.375 g KCl
    1 g NaHCO3
    1 g MgSO4.7H2O
    0.39 g (CaNO3)2.4H2O
    0.3 g CaCl2.2H2O
    11.9 g Hepes
    5 ml penicillin/streptomycin

Acknowledgments

This work was supported by Deutsche Forschungsgemeinschaft grants to M.B.

References

  1. Blum, M., Beyer, T., Weber, T., Vick, P., Andre, P., Bitzer, E. and Schweickert, A. (2009). Xenopus, an ideal model system to study vertebrate left-right asymmetry. Dev Dyn 238(6): 1215-1225.
  2. Nieuwkoop, P. and Faber, J. Normal Table of Xenopus laevis (Daudin), 1994, GARLAND PUBLISHING, New York&London, USA.
  3. Schweickert, A., Weber, T., Beyer, T., Vick, P., Bogusch, S., Feistel, K. and Blum, M. (2007). Cilia-driven leftward flow determines laterality in Xenopus. Curr Biol 17(1): 60-66. 

简介

在非洲蟾蜍属中,左后轴是在晚期胃泌乳/神经发生期间在胃粘膜顶板(GRP)处由单纤维细胞驱动的细胞外向量向左流动之后建立的(Schweickert等人 >,2007)。 由于GRP位于发育中的室内,流动的成像是具有挑战性的。 在这里我们提出详细的过程可视化向左流动在非洲爪蟾胚胎。

关键字:纤毛, 左右不对称, 成像, 向左流, 原肠腔顶板

材料和试剂

  1. 荧光微珠(例如 FluoSpheres0.5μm)(Life Technologies,目录号:F-8813)
  2. 琼脂糖
  3. 青霉素/链霉素(10.000单位/ml; Gibco ,目录号:15140)
  4. HEPES Pufferan(Carl Roth,目录号:HN78.3)
  5. 珠子解决方案(参见配方)
  6. 5x MBSH(Modified Barth's Saline)(参见配方)

设备

  1. 尖锐钳(Fine Science Tools,目录号:Dumont#5)
  2. 微刀片(Fine Science Tools,Dissecting Knife - Fine Tip)
  3. 切片(培养皿底部覆盖1%MBSH的1%琼脂糖)
  4. 移液管(Carl Roth,型号:EA65.1)
  5. 玻璃染色块(Karl-Hecht Assistent(Lymphbecken)2020)
  6. 显微镜载玻片
  7. 盖舌
  8. 5毫升注射器
  9. 凡士林
  10. 配备数码相机(广角镜头)的荧光显微镜
  11. 大培养皿充满PBS或MBSH用于外植体检索

程序

  1. 将非洲爪蟾胚胎提升到阶段17(Nieuwkoop等人,1994)。
  2. 通过将凡士林填充的注射器的开口直接放置在显微镜载玻片上,构建柔性成像室(图1)。 轻轻按下活塞释放凡士林,在滑块上绘制一个矩形(约1.5 x 1厘米)。 确保矩形已关闭。 用小珠溶液填充矩形,直到它有一个凸弯月面

    图1.柔性成像室

  3. 切割背外植体(参见Blum等人,2009年的图2):
    1. 将阶段17胚胎转移到填充1x MBSH的切片培养皿中
    2. 用微型刀片,通过横向切割胚胎的前部来移除头。
    3. 直立杯形胚胎,让你可以看到里面。 然后在左侧和右侧的"侧线"放置小交替切口,直到您到达外环凸缘区域。
    4. 用钳子和微型刀片,拉开胚胎的背侧和腹侧两半,直到你看到从里面打开blastopore。
    5. 将最后切口腹部到胚胎孔分离背部外植体与胚胎的其余部分。 确保不要用镊子或刀片从里面触摸背外植体。
    6. 你现在应该留下一个浴缸形的背部外植体,GRP放在最深的点
  4. 使用移液管,小心地将背外植体带入含有FluoSphere珠溶液的玻璃染色块,轻轻地吸取外植体上下在此培养基中,以确保FluoSpheres达到GRP。
  5. 通过小心吸取转移背外植体到FluoSphere溶液填充室。确保外植体从不接触任何液体的表面,因为表面张力分解组织。定向外植体,内部朝上,即。向你。
  6. 用盖子轻轻地向下推动弹性腔室,用打开的钳子密封。小心推动,直到盖玻片接触背侧外植体的边缘,即左右侧线和腹外侧环。
  7. 在向下推时,有时腹侧环绕颈环折叠并阻挡GRP。如果发生这种情况,盖玻片向后极的轻微阻力可以剪切外植体以允许GRP的成像。
  8. 让设置休息5-10分钟,以允许外植体变形,否则会在早期采集期间暗示假颗粒运动
  9. 将幻灯片放在您的(广角)荧光显微镜,并专注于GRP
  10. 激发与所需的FluoSpheres波长 - 你应该看到一个"星空"。现在将焦点调整到略高于GRP的焦平面。
    注意:由于GRP纤毛的长度仅为5μm,向左流动只发生在上皮正上方的平面上(Schweickert et al。,2007)。为了方便找到正确的平面,聚焦到组织中,直到你看不到任何珠的荧光。现在小心改变焦平面,直到第一个珠子出现在焦点。
  11. 在野生型胚胎中,您应该看到移动到GRP左侧的粒子。在非洲爪蟾(Xenopus laevis)中,向左流动相对较慢(〜2.5μm/s),因此使用延时摄影术最好地可视化(Schweickert等人,2007)。根据进一步的分析,因此时间分辨率,获取至少2 fps的电影
  12. 为了在检查后检索背外植体,将完整的载玻片浸没在装有1x MBSH或1x PBS的大培养皿中,并用镊子小心地除去盖玻片。外植体应漂浮到缓冲液中,可以将其从移液管移至固定液中以进行进一步分析(原位杂交,免疫组织化学染色,等)。

食谱

  1. 珠溶液
    稀释FluoSpheres 1:2,500在1x MBSH中(制备〜5ml)
  2. 5×MBSH(1L)
    注意:使用前,用H 2 O 稀释为1x/
    25.7g NaCl
    0.375克KCl
    1g NaHCO 3 3/h 1g MgSO 4。 7H 2 0.39g(CaNO 3)sub 2+。 4H 2 O 0.3g CaCl 2 。 2H 2 11.9g Hepes
    5ml青霉素/链霉素

致谢

这项工作得到了德意志交响乐团对M.B.

参考文献

  1. Blum,M.,Beyer,T.,Weber,T.,Vick,P.,Andre,P.,Bitzer,E。和Schweickert,A。(2009)。 非洲蟾蜍,一个理想的模型系统,研究脊椎动物的左右不对称性。 Dev Dyn 238(6):1215-1225。
  2. Nieuwkoop,P。和Faber,J。非洲爪蟾正常表(Daudin),1994,GARLAND PUBLISHING,New York& London,美国。
  3. Schweickert,A.,Weber,T.,Beyer,T.,Vick,P.,Bogusch,S.,Feistel,K。和Blum,M。(2007)。 纤毛驱动的向左流动决定了非洲爪蟾的偏侧性 。 Curr Biol 17(1):60-66。 
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Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用:Thumberger, T. and Blum, M. (2013). Acquisition of Leftward Flow in Xenopus laevis. Bio-protocol 3(23): e996. DOI: 10.21769/BioProtoc.996.
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