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Analysis of Enteric Neural Crest Cell Migration Using Heterotopic Grafts of Embryonic Guts
采用胚胎内脏异位移植分析肠道神经嵴细胞迁移   

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Abstract

Hirschsprung disease (HSCR), also named aganglionic megacolon, is a severe congenital malformation characterized by a lack of enteric nervous system (ENS) in the terminal regions of the bowel (Bergeron et al., 2013). As the ENS notably regulates motility in the whole gastrointestinal track, the segment without neurons remains tonically contracted, resulting in functional intestinal obstruction and accumulation of fecal material (megacolon). HSCR occurs when enteric neural progenitors of vagal neural crest origin fail to fully colonize the developing intestines. These “enteric” neural crest cells (ENCCs) have to migrate in a rostro-caudal direction during a fixed temporal window, which is between embryonic day (e) 9.5 and e14.5 in the mouse (Obermayr et al., 2013). Recently, our group generated a new HSCR mouse model called Holstein in which migration of ENCCs is impaired because of increased collagen VI levels in their microenvironment (Soret et al., 2015). Here, we describe the method that allowed us to demonstrate the cell-autonomous nature of this migration defect. In this system adapted from a previously described heterotopic grafting approach (Breau et al., 2006), the donor tissue is a fully colonized segment of e12.5 midgut while the host tissue is an aneural segment of e12.5 hindgut. Extent of ENCC migration in host tissue is assessed after 24 h of culture and is greatly facilitated when donor tissue has a transgenic background such as the Gata4-RFP (Pilon et al., 2008) that allows endogenous labeling of ENCCs with fluorescence. Depending of the genetic background of donor and host tissues, this approach can allow evaluating both cell-autonomous and non-cell-autonomous defects of ENCC migration.

Keywords: Enteric nervous system(肠神经系统), Mouse(鼠标), Neural crest cells(神经嵴细胞), Cell migration(细胞迁移), ex vivo(体外)

Materials and Reagents

  1. Petri dishes (Corning, catalog number: 70165-102 )
  2. 24-well plate
  3. Nitrocellulose filter (Merck Millipore, catalog number: GSWP01300 )
  4. 8-chamber slides (ibiTreat μ-slide) (ibidi GmbH, catalog number: 80826 )
  5. Mature mice (≥ 2-month old)
  6. Isoflurane for inhaled anesthesia (Henry Schein Animal Health, catalog number: 050031 )
  7. 70% ethanol
  8. 1x phosphate-buffered saline (PBS)
  9. DMEM/Ham’s F-12 (WISENT, catalog number: 319-085-CL )
  10. Fetal Bovine Serum (WISENT, catalog number: 920-040 )
  11. Penicillin/streptomycin (WISENT, catalog number: 450-201-EL )

Equipment

  1. Dumont #5 dissection forceps (Fine Science Tools, catalog number: 11251-20 )
  2. Dumont #7 dissection forceps (Fine Science Tools, catalog number: 11274-20 )
  3. Dissection scissors (Moria Spring Scissors) (Fine Science Tools, catalog number: 15396-01 )
  4. Dissecting stereomicroscope (Leica Microsystems, model: M125 )
  5. CO2 cell culture incubator (Sanyo Scientific, model: MCO-18AIC )
    Note: This product has been discontinued.
  6. Infinity-2 camera (Lumenera Corporation) mounted on a fluorescent stereomicroscope (Leica Microsystems, model: M205FA )

Software

  1. ImageJ software

Procedure

  1. Mate mature mice (≥ 2-month old) overnight and check for the presence of a vaginal plug the next morning. Noon of the day a vaginal plug is observed is considered embryonic day (e) 0.5. A typical experiment requires one wildtype couple and one couple bearing the mutation to study.
  2. Twelve days later, euthanize isoflurane-anesthetized pregnant female(s) via CO2 inhalation.
  3. Use 70% ethanol to spray the mouse abdomen and open it with dissecting scissors to access the uterus.
  4. Remove the uterus into a glass petri dish containing 15 ml of sterile ice-cold PBS and cut it between individual embryos.
  5. Working on each embryo separately in another glass petri dish filled with 15 ml of ice-cold PBS, use fine forceps to remove the uterine muscle layers under a dissecting microscope (Figure 1 - step 1 and Video 1).
  6. Open the extra-embryonic membranes to access the embryo by taking care not to sever the developing intestines that are intertwined with the blood vessels connecting the embryo to the placenta/extra-embryonic membranes (Figure 1 - step 2 and Video 1).
  7. Cut embryo's head which can be used for PCR-based genotyping if necessary and open the abdominal cavity (Figure 1 - steps 3-4 and Video 1).
  8. After removing the liver, cut the esophagus and pull the whole gastrointestinal tract out of the abdominal cavity while being careful not to damage the colon still attached to the anus (Figure 1 - step 5 and Video 1).
  9. Cut at the anus to free the gastrointestinal tract. The stomach and cecum will allow keeping track of sample's orientation (Figure 1 - step 6 and Video 1). 

    Video 1. Whole dissection procedure
     
  10. Transfer each sample into a well of a 24-well plate containing 1 ml of sterile PBS and kept on ice. If PCR genotyping is required, be careful to match intestine and head numbers for each sample.
  11. Assemble each graft by depositing and juxtaposing donor (wildtype or mutant) and host (wildtype or mutant) tissues onto a small 13 mm nitrocellulose membrane in a glass petri dish containing 15 ml of sterile ice-cold PBS. Prepare each half of a graft in a sequential manner, so that the first half has already adhered to the filter before processing the second half. Take care to respect the normal rostro-caudal orientation (esophagus to anus) of both the donor and host tissues in the assembled graft. Host tissue from a selected sample is prepared by cutting a ~0.5 cm section of the most caudal hindgut whereas donor tissue is prepared from another sample by cutting a ~0.5 cm section of the midgut just upstream of the cecum.
  12. Transfer each graft-bearing filter in a chamber of an 8-chamber slide containing 250 µl of DMEM/F12 supplemented with 10% FBS and penicillin/streptomycin (Figure 2A).
  13. After 24 h of culture (37 °C, 5% CO2), colonization of aneural hindgut tissues by midgut-derived ENCCs is quantified by measuring the distance separating the most distal ENCC from the midgut-hindgut graft junction (Figure 2B).

Representative data



Figure 1. Key steps of the dissection procedure. Step 1. With fine forceps, remove the uterine muscle layers. Step 2. Open the extra-embryonic membrane to access the embryo, cut the blood vessels connecting the embryo to the placenta/extra-embryonic membranes. Step 3. Cut the embryo head and open the abdominal cavity. Step 4. Pull the whole gastrointestinal tract out of the abdominal cavity. Step 5. Remove the liver, cut the esophagus and release the digestive tract of the connective tissue. Step 6. Free the gastrointestinal tract by cutting it at the anus.


Figure 2. Overview of graft assembly and representative result. A. Schematic representation of a heterotopic e12.5 midgut-hindgut graft onto a nitrocellulose filter in a chamber of an 8-chamber slide. Red dots in midgut tissue represent ENCCs labeled by RFP (DsRed2) fluorescence provided by the G4-RFP transgene; B. Representative images of a graft (delineated by dotted lines) after 24 h of culture, showing the colonization of a previously aneural hindgut host tissue by fluorescently labeled ENCCs from midgut donor tissue. Pictures were taken using an Infinity-2 camera mounted on a Leica M205FA fluorescent stereomicroscope and images were analyzed using the ImageJ software. The white arrow points to the location of the migration front at the end of the culture period. Scale bar: 150 μm.

Notes

It is noteworthy that this method is greatly simplified when donor tissues are taken from embryos bearing a transgene such as the G4-RFP transgene (Pilon et al., 2008) that labels ENCCs with fluorescence. At step 10, this can allow the identification of mutant tissues (i.e., displaying delayed migration) by simple fluorescent microscopy instead of having to wait for genotyping results after graft assembly. Moreover, at step 13, such an intrinsic fluorescent labeling greatly facilitates the analysis of ENCC migration which otherwise requires immunofluorescence labeling using an antibody against a marker of undifferentiated enteric neural progenitors such as Sox10.

Acknowledgments

The Pilon laboratory is funded by grants from the Canadian Institute of Health Research (CIHR), the Natural Science and Engineering Research Council of Canada (NSERC) and the Fondation du grand défi Pierre Lavoie. RS holds a fellowship from the Fonds de la recherche du Québec Santé (FRQS) whereas NP is a FRQS Junior 2 Research Scholar as well as the recipient of a UQAM Research Chair on Rare Genetic Diseases. The authors thank the innovative work performed by Breau et al. (2006), on which this protocol was based.

References

  1. Bergeron, K. F., Silversides, D. W. and Pilon, N. (2013). The developmental genetics of Hirschsprung's disease. Clin Genet 83(1): 15-22.
  2. Breau, M. A., Pietri, T., Eder, O., Blanche, M., Brakebusch, C., Fassler, R., Thiery, J. P. and Dufour, S. (2006). Lack of β1 integrins in enteric neural crest cells leads to a Hirschsprung-like phenotype. Development 133(9): 1725-1734.
  3. Obermayr, F., Hotta, R., Enomoto, H. and Young, H. M. (2013). Development and developmental disorders of the enteric nervous system. Nat Rev Gastroenterol Hepatol 10(1): 43-57.
  4. Pilon, N., Raiwet, D., Viger, R. S. and Silversides, D. W. (2008). Novel pre- and post-gastrulation expression of Gata4 within cells of the inner cell mass and migratory neural crest cells. Dev Dyn 237(4): 1133-1143.
  5. Soret, R., Mennetrey, M., Bergeron, K. F., Dariel, A., Neunlist, M., Grunder, F., Faure, C., Silversides, D. W., Pilon, N. and Ente-Hirsch Study, G. (2015). A collagen VI-dependent pathogenic mechanism for Hirschsprung's disease. J Clin Invest 125(12): 4483-4496.

简介

Hirschsprung病(HSCR),也称为神经节巨结肠,是严重的先天性畸形,其特征在于在肠的末端区域缺乏肠神经系统(ENS)(Bergeron等人,2013)。由于ENS特别调节整个胃肠道的运动性,没有神经元的节段保持紧张收缩,导致功能性肠梗阻和粪便材料(巨结肠)的积累。 HSCR发生在迷走神经嵴来源的肠神经祖细胞不能完全殖民发育的肠道。这些"肠"神经嵴细胞(ENCC)必须在固定的时间窗口内沿着尾 - 尾方向迁移,其在小鼠的胚胎天(e)9.5和e14.5之间(Obermayr等, ,2013)。最近,我们的小组产生了称为荷斯坦的新的HSCR小鼠模型,其中ENCC的迁移受损,因为它们的微环境中胶原VI水平增加(Soret等人,2015)。在这里,我们描述了允许我们演示这种迁移缺陷的细胞自主性的方法。在从先前描述的异位移植方法改造的该系统中(Breau等人,2006),供体组织是e12.5中肠的完全定殖的区段,而宿主组织是e12的非耳部分.5后肠。在宿主组织中ENCC迁移的程度在培养24小时后评估,并且当供体组织具有转基因背景例如允许内源标记的Gata4-RFP(Pilon等人,2008)时极大地促进ENCC在宿主组织中的迁移的程度的ENCCs。根据供体和宿主组织的遗传背景,这种方法可以允许评估细胞自主和非细胞自主缺陷的ENCC迁移。

关键字:肠神经系统, 鼠标, 神经嵴细胞, 细胞迁移, 体外

材料和试剂

  1. 培养皿(Corning,目录号:70165-102)
  2. 24孔板
  3. 硝酸纤维素过滤器(Merck Millipore,目录号:GSWP01300)
  4. 8室玻片(ibidTreatμ-载玻片)(ibidi GmbH,目录号:80826)
  5. 成熟小鼠(≥2个月大)
  6. 用于吸入麻醉的异氟烷(Henry Schein Animal Health,目录号:050031)
  7. 70%乙醇
  8. 1×磷酸盐缓冲盐水(PBS)
  9. DMEM/Ham's F-12(WISENT,目录号:319-085-CL)
  10. 胎牛血清(WISENT,目录号:920-040)
  11. 青霉素/链霉素(WISENT,目录号:450-201-EL)

设备

  1. Dumont#5切开镊子(Fine Science Tools,目录号:11251-20)
  2. Dumont#7切开镊子(Fine Science Tools,目录号:11274-20)
  3. 解剖剪刀(Moria弹簧剪刀)(Fine Science Tools,目录号:15396-01)
  4. 解剖立体显微镜(Leica Microsystems,型号:M125)
  5. CO 2细胞培养孵化器(Sanyo Scientific,型号:MCO-18AIC)
    注意:此产品已停产。
  6. 安装在荧光立体显微镜(Leica Microsystems,型号:M205FA)上的Infinity-2照相机(Lumenera Corporation)

软件

  1. ImageJ软件

程序

  1. 使成熟的小鼠(≥2个月大)过夜,并检查第二天早上阴道塞的存在。 中午的阴道塞被观察到被认为是胚胎天(e)0.5。 一个典型的实验需要一个 野生型夫妇和一对携带突变的研究
  2. 十二天后,通过CO 2吸入安乐死异氟醚麻醉的怀孕女性。
  3. 使用70%乙醇喷洒小鼠腹部,并用解剖刀打开它访问子宫。
  4. 将子宫移入含有15ml无菌冰冷PBS的玻璃培养皿中,并在个体胚胎之间切开。
  5. 在另一个装有15ml冰冷PBS的玻璃培养皿中分别处理每个胚胎,在解剖显微镜下使用细镊子去除子宫肌层(图1-步骤1和视频1)。
  6. 打开胚外膜以访问胚胎,注意不要切断与连接胚胎到胎盘/超胚胎膜的血管缠结的发育中的肠(图1 - 步骤2和视频1)。
  7. 切割胚胎的头,可以用于基于PCR的基因分型,如果必要和打开腹腔(图1-步骤3-4和视频1)。
  8. 去除肝脏后,切开食管,将整个胃肠道从腹腔内拉出,同时小心不要损伤仍然附着在肛门上的结肠(图1-步骤5和视频1)。
  9. 在肛门切开以消除胃肠道。胃和盲肠将允许跟踪样品的方向(图1 - 步骤6和视频1)。 

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  10. 将每个样品转移到含有1ml无菌PBS的24孔板的孔中并保存在冰上。如果需要PCR基因分型,要小心匹配每个样品的肠和头数。
  11. 通过将供体(野生型或突变体)和宿主(野生型或突变体)组织沉积并并置在含有15ml无菌冰冷PBS的玻璃培养皿中的小13mm硝酸纤维素膜上,组装每个移植物。以连续的方式制备移植物的每一半,使得第一半在处理第二半之前已经粘附到过滤器。注意在组装的移植物中遵守供体和宿主组织的正常的罗斯特尾尾方向(食管到肛门)。来自选定样品的宿主组织通过切割大约尾部后肠的〜0.5cm部分来制备,而通过在盲肠上游切除中肠的〜0.5cm部分从另一样品制备供体组织。
  12. 转移每个移植物承载过滤器在8室幻灯片的室,其含有250μl补充有10%FBS和青霉素/链霉素的DMEM/F12(图2A)。
  13. 在培养24小时(37℃,5%CO 2)后,通过测量分离最远端ENCC与中肠 - 后肠移植物的距离来定量中肠衍生的ENCC的非中肠后肠组织的定植(图2B)。

代表数据



图1.解剖程序的关键步骤。步骤1.用细镊子取出子宫肌层。步骤2.打开胚胎外膜以进入胚胎,切割将胚胎连接到胎盘/胚胎外膜的血管。步骤3.切割胚胎头并打开腹腔。步骤4.将整个胃肠道拉出腹腔。步骤5.去除肝脏,切开食管并释放结缔组织的消化道。第6步。通过在肛门切除胃肠道

图2.移植物组装和代表性结果的概述。 A.在8室载玻片的室中,异位e12.5中肠 - 后肠移植物在硝酸纤维素滤膜上的示意图。中肠组织中的红点代表由G4-RFP转基因提供的RFP(DsRed2)荧光标记的ENCC; B.在培养24小时后的移植物的代表性图像(通过虚线描绘),显示通过来自中肠供体组织的荧光标记的ENCC的先前的未愈合的后肠宿主组织的定植。使用安装在Leica M205FA荧光立体显微镜上的Infinity-2照相机拍摄照片,并使用ImageJ软件分析图像。白色箭头指向培养期结束时迁移前沿的位置。比例尺:150μm。

笔记

值得注意的是,当从携带转基因的胚胎(例如,用荧光标记ENCC的G4-RFP转基因(Pilon等人,2008))获取供体组织时,该方法大大简化。在步骤10,这可以允许通过简单的荧光显微镜鉴定突变组织(即,显示延迟的迁移),而不是在移植物组装后等待基因分型结果。此外,在步骤13,这样的内在荧光标记极大地促进ENCC迁移的分析,否则需要使用针对未分化的肠神经祖细胞如Sox10的标记的抗体进行免疫荧光标记。

致谢

Pilon实验室由加拿大健康研究所(CIHR),加拿大自然科学和工程研究委员会(NSERC)和大学基金会皮埃尔·拉瓦耶(Fondation du granddéfiPierre Lavoie)资助。 RS拥有来自魁北克省魁北克省(FRQS)的研究金,而NP则是FRQS少年2研究学者,也是UQAM罕见遗传疾病研究主席的接受者。作者感谢Breau等人进行的创新工作。 (2006),本协议是基于。

参考文献

  1. Bergeron,KF,Silversides,DW和Pilon,N。(2013)。  Hirschsprung's disease的发育遗传学。 83(1):15-22。
  2. Breau,MA,Pietri,T.,Eder,O.,Blanche,M.,Brakebusch,C.,Fassler,R.,Thiery,JP和Dufour,S。(2006)。  在肠神经嵴细胞缺乏β1整合素导致Hirschsprung样表型。 133(9):1725-1734
  3. Obermayr,F.,Hotta,R.,Enomoto,H.and Young,HM(2013)。  肠道神经系统的发育和发育障碍。 Nat Rev Gastroenterol Hepatol 10(1):43-57。
  4. Pilon,N.,Raiwet,D.,Viger,RS和Silversides,DW(2008)。  在内细胞群和迁移性神经嵴细胞的细胞内的Gata4的新的前和后 - 胃泌腺表达。 Dev Dyn 237(4): 1133-1143。
  5. Soret,R.,Mennetrey,M.,Bergeron,KF,Dariel,A.,Neunlist,M.,Grunder,F.,Faure,C.,Silversides,DW,Pilon,N.and Ente-Hirsch Study, (2015)。  一种胶原VI依赖性致病机制Hirschsprung氏病。 125(12):4483-4496。
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用:Soret, R. and Pilon, N. (2016). Analysis of Enteric Neural Crest Cell Migration Using Heterotopic Grafts of Embryonic Guts. Bio-protocol 6(17): e1924. DOI: 10.21769/BioProtoc.1924.
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