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In vitro Explant Cultures to Interrogate Signaling Pathways that Regulate Mouse Lung Development
探寻调控小鼠肺部发育的信号转导途径的体外外植体培养   

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Development
Jan 2017

Abstract

Early mouse lung development, including specification of primordia, patterning of early endoderm and determination of regional progenitor cell fates, is tightly regulated. The ability to culture explanted embryonic lung tissue provides a tractable model to study cellular interactions and paracrine factors that regulate these processes. We provide up-to-date protocols for the establishment of this culture model and its application to investigate hedgehog signaling in the developing lung.

Keywords: Mouse embryonic lung (小鼠胚胎肺), In vitro (体外), Explant culture (外植体培养)

Background

Mouse lung development initiates as an endodermal diverticulum of the anterior foregut endoderm at day 9.5 postcoitum (E9.5), with subsequent closure of a proximal tracheoesophageal septum for the formation of distinct tracheal and esophageal tubes (Minoo and King, 1994). Subsequent branching of primitive endodermal tubes yields a planar lung structure by E12.5, with subsequent orthogonal branches yielding three-dimensional structure characteristic of the mature lung (Metzger et al., 2008). The planar structure of lung rudiments isolated prior to E12.5 is suitable for in vitro culture at an air liquid interface (Carraro et al., 2010; Del Moral and Warburton, 2010). Embryonic lung is isolated by dissection using a stereo microscope either under bright field illumination or by fluorescence illumination when coupled with lineage tracing and fluorescent reporters. Herein we describe the use of a ShhCre/RosamTmG reporter mouse allowing Cre-mediated activation of membrane-localized GFP within anterior foregut endoderm from approximately E8.75 (Montgomery et al., 2007; Goss et al., 2009; Yao et al., 2017). Accordingly lung endoderm is visualized by green fluorescence and surrounding tissue by red fluorescence, allowing clear identification and microdissection of developing endodermal structures, including the lung, and imaging during in vitro culture.

Materials and Reagents

  1. Whole embryonic lung isolation
    1. BD 1 ml TB syringe 26 G (BD, catalog number: 309625 )
    2. 50 ml conical tube (Denville Scientific, catalog number: C1062-P (1000799))
    3. Petri dish (Greiner Bio One International, catalog number: 663161 )
    4. ShhCre mice (THE JACKSON LABORATORY, catalog number: 005622 )
    5. RosamTmG/+ mice (THE JACKSON LABORATORY, catalog number: 007576 )
    6. Mouse embryonic lungs from ShhCre/+, RosamTmG/+ mice
      Note: Mouse embryonic lungs from ShhCre/+; RosamTmG/+ mice were harvested between E10.5 and E12.5. The day of vaginal plug detection was considered to be E0.5
    7. General anesthesia: Ketamine (VET one, NDC 13985-702-10) and xylazine (AnaSed Injection, NDC 59339-110-20)
    8. 70% ethanol (Fisher Scientific, catalog number: BP8201500 )
    9. Phosphate buffered saline (PBS) (1x), liquid, without calcium and magnesium (Corning, catalog number: 21-040-CV )
    10. Penicillin-streptomycin (Thermo Fisher Scientific, GibcoTM, catalog number: 15070063 )
    11. PBS with P/S (see Recipes)

  2. Whole embryonic lung culture
    1. 12 wells plate (Denville Scientific, catalog number: T1012 )
    2. Disposable transfer pipettes, sterile (VWR, catalog number: 414004-036 )
    3. Razor blade (VWR, catalog number: 55411-050 )
    4. Nuclepore Polycarbonate Track-Etch membrane (13 mm, 8 μm) (GE Healthcare, catalog number: 150446 )
    5. Dulbecco’s modified Eagle medium: Nutrient Mix F-12 (DMEM/F12) (1x), liquid, 1:1 Contains GlutaMAX, but no HEPES buffer (Thermo Fisher Scientific, GibcoTM, catalog number: 10565042 )
    6. Penicillin-streptomycin (P/S) (Thermo Fisher Scientific, GibcoTM, catalog number: 15070063 )
    7. BenchMark fetal bovine serum (Gemini Bio-Products, catalog number: 100-106 )
    8. Embryonic lung culture medium (see Recipes)

Equipment

  1. Surgical instruments including:
    1. 2 Dumont #5 forceps (Fine Science Tools, catalog number: 11295-10 )
    2. Moria® spoon (perforated) (Fine Science Tools, catalog number: 10370-17 )
  2. Mouse necropsy instrument set including:
    1. Metzenbaum (Lahey) Scissors (Roboz Surgical Instrument, catalog number: RS-6950 )
    2. Micro Dissecting Scissors 4" Blunt (Roboz Surgical Instrument, catalog number: RS-5980 )
    3. Graefe Forceps Straight (Roboz Surgical Instrument, catalog number: RS-5130 )
    4. Graefe Forceps Curved (Roboz Surgical Instrument, catalog number: RS- 5135 )
  3. CO2 Incubator (Thermo Fisher Scientific, model: HeracellTM 150i )
  4. Fluorescent Stereo Microscope (Carl Zeiss, model: Zeiss SteREO Discovery.V8 , with 8x magnification) equipped with 5 MP, 36 bit, Peltier cooled Zeiss Axiocam MRc5 camera (Carl Zeiss, model: AxioCam MRc 5 )

Software

  1. Zen blue software (Carl Zeiss)
  2. PRISM software version 7

Procedure

  1. Embryonic lung isolation
    1. Timed-pregnant mice (11.5-12.5 days post-coitum) are placed under general anesthesia by intraperitoneal injection of ketamine/xylazine (ketamine 100 mg/kg, xylazine 10 mg/kg) with 30 G needle according to the Institutional Animal Care and Use Committee-approved protocol (see Note 1).
    2. Once in a surgical plane (determined by lack of toe pinch and eye blink reflexes) the abdomen is rinsed with 70% ethanol (see Note 2).
    3. To collect uterus, grasp the skin with Graefe forceps and make an incision with Lahey scissors and manually open the incision. Open the peritoneal cavity, externalize the uterine structures containing embryo’s and surgically remove both uterine horns.
    4. Transfer the uterus to a 50 ml conical tube filled with cold PBS supplemented with P/S and rinse to remove blood.
    5. Transfer the uterus to a Petri dish filled with PBS supplemented with P/S and visualize using a stereo microscope with bright field fiber-optic illumination. Remove embryos from the uterus by manually opening the uterine wall with a pair of Dumont forceps. Collect and transfer the embryos with a perforated spoon to a new Petri dish kept on ice.
    6. Switch the microscope to the fluorescent mode and use GFP channel; from now on the dissection will be under fluorescent channels.
    7. Place each embryo on its left flank and visualize by epifluorescence using a 488 nm (GFP) filter (Figure 1A). Brain and limbs will be visible by auto-fluorescence. Use one pair of forceps to stabilize the embryo while gently removing skin tissue with a second pair of forceps. Remove the right forelimb first, then open the thorax along the right flank from the neck to abdomen (Figure 1B). The exposed lung and esophagus will show clear GFP fluorescence that is visible above background auto-fluorescence. The heart and the liver are removed to fully expose the lung and GI tract, all of which will are marked by GFP (Figures 1C-1D). Stomach and distal GI structures are removed and remaining GFP-positive tissue (lung and esophagus) removed using forceps to gently separate from surrounding connective tissue. Any remaining adventitial tissue should be carefully removed from the isolated lung (Figures 1E and 1F).
    8. Isolated lung tissue is transferred to individual wells of a 12-well plate containing embryonic lung culture medium with a sterile transfer pipet.


      Figure 1. Dissection of E12.5 embryonic lungs from ShhCre/+; RosamTmG/+ mice by fluorescence microscopy. A. Whole embryo view of E12.5 ShhCre/+; RosamTmG/+ mice with dual detection of GFP (green) and RFP (red) fluorescence. B. View after removing the right forelimb and exposure of the thoracic cavity along the right flank. C-D. View of lung and GI tract after removal of heart and liver. E. Lung and esophagus. F. Lung after removal of the esophagus. Scale bars = 500 µm.

  2. Embryonic lung culture
    Note: From now on, every step should ideally be performed in a sterile environment such as a tissue culture hood.
    1. Add 2 ml of embryonic lung culture medium per well.
    2. Place a nuclepore polycarbonate (smooth side down) track-etch membrane suspended above media in each well (see Note 3).
    3. Use a sharp sterile razor blade to remove approximately 1.5 cm of a 200 μl pipet tip and use the resulting wide bore to gently capture one embryonic lung and transfer to the upper surface of the nuclepore polycarbonate track-etch membrane.
    4. Ensure that the lung is flat on the polycarbonate membrane by visualization under an inverted light microscope; gently adjust using fine forceps to ensure that it is flat.
    5. Image the lung as day 0 (Figure 2).
    6. Any treatment, including inhibitors, growth factors, siRNA etc., should be included in culture media at this time.
    7. Transfer the 12-well plate to a humidified cell culture incubator at 37 °C and monitor the experiment by imaging at the desired time points (Figure 2).


      Figure 2. Potentiation of the hedgehog pathway by culturing in the presence of the smoothened receptor agonist SAG, induces mesodermal proliferation and blocks endoderm branching. Panels show whole-mount fluorescence images of embryonic lung endoderm (green) and mesoderm (red) cultured for 0-3 days in the presence of media containing DMSO vehicle (upper 4 panels) or media supplemented with 10 µM SAG (lower 4 panels). SAG treatment induces loss of branching and abnormal growth of mesodermal cells (red) indicative of roles played by Sonic Hedgehog signaling in the appropriate regulation of lung branching.

Data analysis

Images were taken and processed using Zen blue software, statistics analysis was performed using PRISM software version 7.

Notes

  1. Any animal work should follow federal and local regulation.
  2. To check how deep is the anesthetization, use a pair of forceps to pinch toes and check if there is any reflection, do not proceed until there is totally no reflection.
  3. Make sure the shiny smooth side of the nuclepore polycarbonate track-etch membrane face down towards the media and the rough side face up. The lungs would be transferred with a drop of media surrounded on top of the rough side without slipping away.

Recipes

  1. PBS with P/S
    1x phosphate buffered saline (PBS), liquid, without calcium and magnesium
    50 U/ml of penicillin-streptomycin
  2. Embryonic lung culture medium
    DMEM/F-12, GlutaMAXTM (Nutrient Mix F-12 (1x), liquid, 1:1, Contains GlutaMAX, but no HEPES buffer)
    50 U/ml of penicillin-streptomycin (P/S)
    10% BenchMark fetal bovine serum

Acknowledgments

This work was funded by the National Institutes of Health 1T32HL134637-01, R01HL135163-01; California Institution of Regenerative Medicine CIRM LA1-06915. The protocol described here is adapted from Yao et al. (2017). All authors declared that no competing interest exists.

References

  1. Carraro, G., del Moral, P. M. and Warburton, D. (2010). Mouse embryonic lung culture, a system to evaluate the molecular mechanisms of branching. J Vis Exp (40).
  2. Del Moral, P. M. and Warburton, D. (2010). Explant culture of mouse embryonic whole lung, isolated epithelium, or mesenchyme under chemically defined conditions as a system to evaluate the molecular mechanism of branching morphogenesis and cellular differentiation. Methods Mol Biol 633: 71-79.
  3. Goss, A. M., Tian, Y., Tsukiyama, T., Cohen, E. D., Zhou, D., Lu, M. M., Yamaguchi, T. P. and Morrisey, E. E. (2009). Wnt2/2b and β-catenin signaling are necessary and sufficient to specify lung progenitors in the foregut. Dev Cell 17(2): 290-298.
  4. Metzger, R. J., Klein, O. D., Martin, G. R. and Krasnow, M. A. (2008). The branching programme of mouse lung development. Nature 453(7196): 745-750.
  5. Minoo, P. and King, R. J. (1994). Epithelial-mesenchymal interactions in lung development. Annu Rev Physiol 56: 13-45.
  6. Montgomery, R. L., Davis, C. A., Potthoff, M. J., Haberland, M., Fielitz, J., Qi, X., Hill, J. A., Richardson, J. A. and Olson, E. N. (2007). Histone deacetylases 1 and 2 redundantly regulate cardiac morphogenesis, growth, and contractility. Genes Dev 21(14): 1790-1802.
  7. Yao, C., Carraro, G., Konda, B., Guan, X., Mizuno, T., Chiba, N., Kostelny, M., Kurkciyan, A., David, G., McQualter, J. L. and Stripp, B. R. (2017). Sin3a regulates epithelial progenitor cell fate during lung development. Development 144(14): 2618-2628.

简介

早期鼠肺发育,包括原基的规范,早期内胚层的构图和区域祖细胞命运的确定,受到严格的调控。 培养移植胚胎肺组织的能力提供了一种易处理的模型来研究调节这些过程的细胞相互作用和旁分泌因子。 我们提供最新的协议,以建立这种文化模式及其应用来研究肺部发育中的刺猬信号。

【背景】小鼠肺发育起始于前肠前内胚层的内胚层憩室(E9.5),随后关闭近端气管食管中隔以形成不同的气管和食道管(Minoo和King,1994)。原始内胚层管的随后分支通过E12.5产生平面肺结构,随后正交分支产生成熟肺的三维结构特征(Metzger等人,2008)。在E12.5之前分离的肺遗传的平面结构适合于在空气液体界面进行体外培养(Carraro等人,2010; Del Moral和Warburton, 2010)。胚胎肺通过解剖使用立体显微镜在亮场照明下或通过与谱系追踪和荧光报道分子偶联时的荧光照明进行分离。在这里,我们描述了使用Shh Cre / Rosa mTmG报告小鼠,其允许Cre介导的从约E8.75起在前部前肠内胚层内的膜定位的GFP的激活(Montgomery <等人,2007;高斯等人,2009; Yao等人,2017)。因此,肺内胚层通过红色荧光由绿色荧光和周围组织显现,从而允许清晰识别和显微切割包括肺在内的发育内胚层结构,并在体外培养期间成像。

关键字:小鼠胚胎肺, 体外, 外植体培养

材料和试剂

  1. 整个胚胎肺分离
    1. BD 1毫升TB注射器26克(BD,目录号:309625)
    2. 50ml锥形管(Denville Scientific,目录号:C1062-P(1000799))
    3. 培养皿(Greiner Bio One International,目录号:663161)
    4. Shh Cre小鼠(THE JACKSON LABORATORY,目录号:005622)
    5. Rosa mTmG / +小鼠(THE JACKSON LABORATORY,目录号:007576)
    6. 来自Shh Cre / +,Rosa mTmG / +小鼠的小鼠胚胎肺
      注:小鼠胚胎肺来自Shh Cre / + ;在E10.5和E12.5之间收获Rosa mTmG / + 小鼠。阴道塞检测当天被认为是E0.5
    7. 全身麻醉:氯胺酮(VET 1,NDC 13985-702-10)和甲苯噻嗪(AnaSed Injection,NDC 59339-110-20)
    8. 70%乙醇(Fisher Scientific,目录号:BP8201500)
    9. 磷酸盐缓冲液(PBS)(1x),液体,不含钙和镁(Corning,目录号:21-040-CV)
    10. 青霉素 - 链霉素(Thermo Fisher Scientific,Gibco TM,目录号:15070063)
    11. PBS与P / S(见食谱)

  2. 整个胚胎肺文化
    1. 12孔板(Denville Scientific,目录号:T1012)
    2. 一次性移液器,无菌(VWR,目录号:414004-036)
    3. 剃须刀片(VWR,目录号:55411-050)
    4. Nuclepore Polycarbonate Track-Etch膜(13 mm,8μm)(GE Healthcare,目录号:150446)
    5. Dulbecco改良的Eagle培养基:营养混合物F-12(DMEM / F12)(1x),液体,1:1含有GlutaMAX但不含HEPES缓冲液(Thermo Fisher Scientific,Gibco TM,目录号:10565042 )
    6. 青霉素 - 链霉素(P / S)(Thermo Fisher Scientific,Gibco TM,目录号:15070063)
    7. BenchMark胎牛血清(Gemini Bio-Products,目录号:100-106)
    8. 胚胎肺培养基(见食谱)

设备

  1. 手术器械包括:
    1. 2 Dumont#5镊子(精细科学工具,目录号:11295-10)
    2. Moria 勺(穿孔)(Fine Science Tools,目录号:10370-17)
  2. 小鼠尸检仪器包括:
    1. Metzenbaum(Lahey)剪刀(Roboz手术器械,目录号:RS-6950)
    2. 微型解剖剪刀4“Blunt(Roboz手术器械,目录号:RS-5980)
    3. Graefe镊子直(Roboz手术器械,目录号:RS-5130)

    4. Graefe镊子弯曲(Roboz手术器械,目录号:RS-5135)
  3. CO 2培养箱(Thermo Fisher Scientific,型号:Heracell TM 150i)。
  4. 荧光立体显微镜(Carl Zeiss,型号:Zeiss SteREO Discovery.V8,放大8倍),配备5 MP,36位,珀耳帖冷却蔡司Axiocam MRc5相机(卡尔蔡司,型号:AxioCam MRc 5)

软件

  1. 禅蓝色软件(卡尔蔡司)
  2. PRISM软件版本7

程序

  1. 胚胎肺隔离
    1. 按照机构动物护理和使用方法,通过腹腔注射氯胺酮/甲苯噻嗪(氯胺酮100mg / kg,赛拉嗪10mg / kg)和30G针,将定时怀孕小鼠(在交配后11.5-12.5天)置于全身麻醉下委员会批准的协议(见注1)。
    2. 一旦进入手术平面(通过缺乏脚趾捏和眨眼反射来确定),腹部用70%乙醇冲洗(见注2)。
    3. 为了收集子宫,用Graefe钳子抓住皮肤并用Lahey剪刀切开并手动打开切口。打开腹膜腔,将包含胚胎的子宫结构外化并通过手术去除两个子宫角。
    4. 将子宫转移到装有补充有P / S的冷PBS并冲洗以除去血液的50ml锥形管中。
    5. 将子宫转移到填充有补充有P / S的PBS的培养皿中,并使用具有明场光纤照明的体视显微镜进行可视化。通过用一对杜蒙钳手动打开子宫壁去除子宫的胚胎。
      使用穿孔勺将胚胎收集并转移到保存在冰上的新培养皿中。
    6. 将显微镜切换到荧光模式并使用GFP通道;从现在起解剖将在荧光通道下。
    7. 将每个胚胎放在其左侧,并使用488nm(GFP)过滤器通过落射荧光显影(图1A)。通过自动荧光可以看到大脑和四肢。使用一对镊子来稳定胚胎,同时用第二对镊子轻轻地去除皮肤组织。首先取下右前肢,然后沿右侧从脖子向腹部打开胸部(图1B)。暴露的肺和食道将显示清晰的GFP荧光,其在背景自动荧光之上可见。将心脏和肝脏移除以完全暴露肺和胃肠道,所有这些都将由GFP标记(图1C-1D)。取出胃和远端胃肠道结构,用镊子将剩余的GFP阳性组织(肺和食管)切除,从周围的结缔组织中轻轻地分离。
      任何剩余的外膜组织都应仔细从分离的肺部取出(图1E和1F)。
    8. 使用无菌移液管将分离的肺组织转移至含有胚胎肺培养基的12孔板的各个孔中。


      图1.从Shh Cre / +中分离E12.5胚胎肺; Rosa mTmG / +小鼠的荧光显微镜观察.A.1 E12.5 Shh Cre / +的整个胚胎视图; Rosa mTmG / +小鼠,其具有GFP(绿色)和RFP(红色)荧光的双重检测。 B.取下右前肢并沿右侧暴露胸腔后观察。光盘。去除心脏和肝脏后的肺和胃肠道视图。 E.肺和食管。 F.去除食道后的肺。

  2. 胚胎肺文化
    注意:从现在开始,每个步骤应该在无菌环境中进行,如组织培养罩。

    1. 每孔加入2ml胚胎肺培养基。

    2. 在每个孔中放置悬浮在介质上方的核孔聚碳酸酯(光面朝下)轨迹蚀刻膜(参见注释3)。
    3. 使用锋利的无菌剃须刀片去除大约1.5厘米的200微升移液枪头,并使用最终的大口径轻轻捕获一个胚胎肺,并转移到核孔聚碳酸酯轨迹蚀刻膜的上表面。
    4. 通过在倒置光学显微镜下观察,确保聚碳酸酯膜上的肺是平坦的;用细镊子轻轻调整,以确保它是平的。
    5. 将第二天的肺图像化(图2)。

    6. 任何治疗,包括抑制剂,生长因子,siRNA等等都应包括在培养基中。
    7. 将12孔板转移到37°C的潮湿细胞培养箱中,通过在所需时间点成像监测实验(图2)。


      图2.通过在平滑受体激动剂SAG存在下培养刺激hedgehog途径的增强,诱导中胚层增殖并阻断内胚层分支。图显示胚胎肺内胚层的全贴装荧光图像(绿色)和在含有DMSO媒介物的培养基(上4个小图)或补充有10μMSAG的培养基(下4个小图)的存在下培养0-3天的中胚层(红色)。 SAG处理诱导中分裂细胞的分支丢失和异常生长(红色),表明Sonic Hedgehog信号传导在适当调节肺部分支中发挥的作用。

数据分析

使用Zen Blue软件拍摄和处理图像,使用PRISM软件版本7进行统计分析。

笔记

  1. 任何动物工作都应遵循联邦和地方法规。
  2. 要检查麻醉深度,请使用一对镊子夹住脚趾,检查是否有任何反射,直到完全没有反射时才进行。
  3. 确保核磁聚碳酸酯轨道蚀刻膜的光滑平滑面朝下并朝向介质,粗糙面朝上。









食谱

  1. PBS与P / S
    1x磷酸盐缓冲液(PBS),液体,不含钙和镁
    50U / ml青霉素 - 链霉素
  2. 胚胎肺脏培养基
    DMEM / F-12,GlutaMAX TM(营养混合物F-12(1x),液体,1:1,含有GlutaMAX,但不含HEPES缓冲液)
    50U / ml青霉素 - 链霉素(P / S)
    10%BenchMark胎牛血清

致谢

这项工作由国立卫生研究院1T32HL134637-01,R01HL135163-01;加利福尼亚再生医学研究所CIRM LA1-06915。这里描述的协议是从Yao et。(2017)改编的。所有作者声明不存在竞争利益。

参考

  1. Carraro,G.,del Moral,P.M。和Warburton,D。(2010)。 小鼠胚胎肺文化,一种评估分支机制的系统 J Vis Exp (40)。
  2. Del Moral,P.M。和Warburton,D。(2010)。 小鼠胚胎全肺,分离的上皮或间充质在化学定义的条件下作为系统的外植体培养物评估分支形态发生和细胞分化的分子机制。方法分子生物学 633:71-79。
  3. Goss,A.M.,Tian,Y.,Tsukiyama,T.,Cohen,E.D。,Zhou,D.,Lu,M.M.,Yamaguchi,T.P.and Morrisey,E.E。(2009)。 Wnt2 / 2b和β-catenin信号传导对于确定前肠中的肺祖细胞是必需的和足够的。 / a> Dev Cell 17(2):290-298。
  4. Metzger,R.J.,Klein,O.D。,Martin,G.R。和Krasnow,M.A。(2008)。 小鼠肺部发育的分支计划 Nature 453 (7196):745-750。
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引用:Yao, C., Carraro, G. and Stripp, B. R. (2018). In vitro Explant Cultures to Interrogate Signaling Pathways that Regulate Mouse Lung Development. Bio-protocol 8(10): e2852. DOI: 10.21769/BioProtoc.2852.
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