利用共聚焦分析<em>非洲爪蟾</em>调查的Wnt和Shh形态发生素梯度的调节剂
Summary
该手稿这里提供一组简单的用于分析的在爪蟾荧光标记的配体的分泌和扩散的方法。这提供了一个上下文用于测试的其他蛋白质的修改配体分布的能力，并允许实验，可能会给洞察调节形态发生素梯度的机制。
Abstract
这个协议描述了一种方法来可视化分布在细胞的场配位体。表达外源蛋白，用自己的细胞在早期胚胎中大尺寸在一起的易用性，使 非洲爪蟾 的可视化GFP标记的配体的一个有用的模型。的合成mRNAs注射后能有效地转化为早期爪 蟾胚胎 ，和注射剂可以针对单个小区。当与谱系示踪剂组合如膜拴RFP，注入的细胞（和它的后代）所产生的过表达的蛋白质可以容易地遵循。这个协议描述了生产的方法的荧光从注入的mRNA标记的Wnt和Shh配体。该方法涉及英里CRO清扫外胚层植（动物帽）和配体扩散的多个样品中分析。通过使用共焦成像，可以获得关于配体分泌和扩散过的细胞的字段信息。共焦图象的统计分析提供关于配体梯度的形状的量化数据。这些方法可能是有用的研究人员谁想要测试的可能调节形态发生梯度的形状因素的影响。
Introduction
在早期胚胎发育，细胞被逐步致力于遵循分化的特定谱系：这意味着一组全能性（或多潜能）细胞变得逐渐限制到建立确定为产生一种细胞类型的祖细胞群体的。细胞 – 细胞信号传导是至关重要的谱系说明书的胚胎发育过程中的调节。这些信号的操作将被要求直接干细胞向特定的命运，以支持新的医疗方法。
相当一小部分的信号通路的发展过程中重申，包括路径响应TGF超家族（nodals和骨形成蛋白）1-2的FGFs 3，Wnts 4，和刺猬5。这些分泌的蛋白质结合存在于细胞膜，激活的信号转导，从而改变基因的表达和/或细胞行为受体。细胞标志的严格监管阿灵是细胞谱系的规范和正常发展的必要条件。而这些通路中的串扰是在确定细胞命运的重要，一个单一的配位体可以本身引起不同浓度不同的反应。形态发生素梯度被描述在100多年前作为一个理论来解释如何不同类型的细胞可以从细胞中6场获得。信令由一个小区组产生的分子可以扩散在一定范围内，减少在浓度与来自源更大的距离。暴露于信号的细胞将响应的局部浓度在其在电池领域位置，与细胞在不同的位置响应不同，以不同的水平的信号。用于形态发生素的存在的证据来自早期果蝇胚胎7和翼盘 8，以及脊椎动物肢9和神经管10的研究。
方法都需要在vestigate怎么形态发生素梯度成立，并确定在调节这些梯度其他重要分子。使用免疫组织化学来可视化内源性蛋白在体内的不同遗传背景的上下文优雅实验已经用于研究形态发生素梯度11-12然而，好的抗体和特定的突变体不总是可用的，所以我们在这里描述使用爪蟾荧光配体的过表达，是提供一种替代方案中，简单的方法来剖析外源基因产物如何影响跨越细胞的场配位体的分布的协议。 非洲爪蟾提供了一个很好的系统，其胚胎外部发展，使他们在最初阶段访问承接这些类型的实验。他们的大尺寸（直径1-1.5mm）简化了显微注射和手术操作和囊胚阶段的细胞很容易的图像，因为它们仍然是比较大的（约2081，M对面）。在爪蟾表达研究是简单的事：基因注入胚胎早期可以有针对性的特殊细胞，并有效地翻译。
使用PCS2 Wnt8a-HA 13，PCS2 Wnt11b-HA 14和绿色荧光蛋白生成的荧光标记Wnt8a / Wnt11b-HA-EGFP结构。的HA肽是重要的，包括，不仅提供额外的分子标记，而且还因为它被认为是作为一个隔离物分离的Wnt和eGFP的蛋白质允许两个基因产物的功能。用于Shh的可视化的构建体是以前用于产生转基因小鼠表达的Shh-EGFP融合蛋白15;这是好心由安迪·麦克马洪提供。重要的是，对于所有的构建体的GFP标签克隆的3'到信号序列，这样它被处理后保留。它也必须确保最终蛋白质包括所需的修饰序列，例如所述additioÑ脂质作为是亚克隆到被合成mRNA的脾淋巴细胞优化PCS2 +表达载体Shh和Wnt信号ligands.The的cDNA的情况下它包含一个SP6启动子和多聚腺苷酸化信号（http://sitemaker.umich.edu/dlturner.vectors）。
这里介绍的工作提供了一个简单的协议，用于比较的分泌和荧光扩散标记的Wnt和Shh标记的配体。通过注入合成的mRNA的确定量，协议会绕开可变表达使用不同启动子的不同向量有关的任何问题。这些方法最近已应用到调查硫酸乙酰肝素endosulfatase Sulf1上的Shh-EGFP和Wnt8a / Wnt11b-HA-eGFP的分泌和扩散在爪蟾16-17的影响。
Protocol
Representative Results
Discussion
此协议的一个重要部分是产生了通常处理的，分泌的，并能够引发所述接收单元在响应中，尽管具有连接荧光部分生物活性配体。关键是要建立的荧光标记的基因产物是生物学活性使用适当的测定法。为的Shh-GFP，以激活PTC1的表达的能力，确认16。 Wnt8a具有显着有效的能力以诱导次级轴时在单个卵裂球腹20-21表达，Wnt8a-HA-eGFP的显示出保留这种生物活性。 Wnt11b抑制活化处理外?…
Declarações
The authors have nothing to disclose.
Acknowledgements
这个工作是由一个BBSRC授予环保部（BB / H010297 / 1），一个BBSRC助学金名额特区，以及MRC助学金为SWF。
Materials
| Agarose | Melford | MB 1200 | |
| Ammonium acetate | Ambion | From Megascript SP6 Kit AM 1330 | |
| Bicarbonate | VWR International | RC-091 | |
| Calcium nitrate | Sigma | C13961 | |
| Cap analog (m7G(5')) | Applied Biosystems | AM 8050 | |
| Chloroform | Sigma | C 2432 | |
| L-Cysteine hydrochloride monohydrate | Sigma | C7880 | |
| dNTPs | Invitrogen | 18427-013 | |
| Ethylenediaminetetraacetic acid (EDTA) | Sigma | O3690 | |
| Ethanol | VWR International | 20821.33 | |
| Ficoll 400 | Sigma | F 4375 | |
| Fiji image J software | N/A | N/A | Free download http://fiji.sc/Fiji |
| Gentamycin | Melford | G 0124 | |
| Glacial acetic acid | Fisher Scientific | A/0400/PB17 | |
| Glass cover slips, No.1.5 | Scientific Laboratory Supplies | 22X22-SGJ3015. 22X50-SGJ3030 | |
| Glass needle puller | Narishige | Narishige PC -10 | |
| Glass pull needles | Drummond Scientific | 3-000-203-G/X | |
| Human chronic gonadotropin (HCG) | Intervet | ||
| Isopropanol | Fisher Scientific | P/7500/PB17 | |
| Lithium chloride (LiCl) | Sigma | L-7026 | |
| LSM710 and Zen software (2008-2010) | Carl Zeiss | ||
| Matlab software | Mathworks | http://uk.mathworks.com/ | |
| Molecular grade water | Fisher Scientific | BP 2819-10 | |
| Nail varnish | Boots | Bar code 3600530 373048 | |
| Spectrophotometer | Lab.tech International | ND-1000 / ND8000 | |
| Petri dish (55mm) | VWR International | 391-0865 | |
| Phenol-chloroform | Sigma | P3803 | |
| Photoshop software | Adobe | N/A | http://www.photoshop.com/products |
| High fidelity DNA polymerase and buffers | Biolabs | M0530S Buffer – M0531S | |
| Potassium chloride (KCl) | Fisher Scientific | P/4280/53 | |
| PVC insulation tape | Onecall | SH5006MPK | |
| Gel extraction kit | Qiagen | S28704 | |
| Restriction enzymes buffers | Roche | SuRE/CUT Buffer Set 11082 035 001 | |
| RNAse-free DNAse | Promega | ME10A | |
| Steel back single edge blades | Personna | 66-0403-0000 | |
| Sodium chloride (NaCl) | Fisher Scientific | 27810.364 | |
| SP6 transcription kit | Ambion | AM1330 | |
| Glass slides | Thermo Fisher | SHE 2505 | |
| Tris base | Invitrogen | 15504-020 | |
| Tungsten needles | N/A | N/A | homemade |
| Zen lite software | Carl Zeiss | N/A | Free download http://www.zeiss.co.uk/microscopy/en_gb/downloads/zen.html |
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