研究Wnt信号传导过程中的航空公司图案
Summary
利用耦合到整装报告小鼠和切片染色，显微镜和体内试验方便的呼吸道的正常图案形成底层机制的分析。在这里，我们将介绍这些技术如何促进Wnt信号的气管发育过程中的分析。
Abstract
Wnt signaling pathways play critical roles during development of the respiratory tract. Defining precise mechanisms of differentiation and morphogenesis controlled by Wnt signaling is required to understand how tissues are patterned during normal development. This knowledge is also critical to determine the etiology of birth defects such as lung hypoplasia and tracheobronchomalacia. Analysis of earliest stages of development of respiratory tract imposes challenges, as the limited amount of tissue prevents the performance of standard protocols better suited for postnatal studies. In this paper, we discuss methodologies to study cell differentiation and proliferation in the respiratory tract. We describe techniques such as whole mount staining, processing of the tissue for confocal microscopy and immunofluorescence in paraffin sections applied to developing tracheal lung. We also discuss methodologies for the study of tracheal mesenchyme differentiation, in particular cartilage formation. Approaches and techniques discussed in the current paper circumvent the limitation of material while working with embryonic tissue, allowing for a better understanding of the patterning process of developing conducting airways.
Introduction
呼吸道发展是通过用Nkx2.1阳性细胞中的腹侧内胚层前肠1,2-外观胚胎9天（E9）来启动。食管-气管套管分离将E11.5解决将管可以区分为不同的实体，分别由间质组织3所包围。 Wnt信号起着呼吸道作为WNT2和WNT2B缺失的说明书中的关键作用，通过从内胚呼吸道上皮内脏间质和β连环蛋白的缺失表达将导致肺发育不全4,5。我们先前的研究确定删除WLS，所有的Wnt配体的受体货物介导的分泌，从肺发育不良的内胚呼吸道结果，肺血管发展和气管间质6,7的错误图案的缺陷。这些数据支持这样的上皮 – 间质CRO的重要性SS交谈在细胞分化和说明书中，因为它也已在其它研究8,9所示。
肺发育的最初阶段的研究依赖于遗传， 在使我们能够更好地理解机制驾驶呼吸身份10-16 体外和体外技术。在空气液体相间全肺外植体培养已被广泛用来研究肺分支形态10,17,18的早期阶段的生长因子的作用。虽然这种方法被用作形态学变化，如分支形态和基因表达的调制读出，它是有限的发育过程的早期阶段的研究中，作为培养本身不支持脉管17的发展。气管软骨的发展需要更长的孵化时间，可能与此培养技术不兼容。
要analyzÈ呼吸道形成过程中的Wnt信号传导的作用，我们已经适应的标准技术，以满足我们的胚胎的研究的需要。我们已经修改卷，染色次，石蜡包埋和定时气管，肺组织清算处理循环。优化在本研究中所描述的技术的主要目的是分析气管发展的早期阶段中所发生从E11到E14.5小鼠。使用报告小鼠线Axin2LacZ在发展中国家间质气管的Wnt我们/β-catenin的活动的精确确定地点。我们还适合整装气管组织凝集素染色的过程。因此，我们能够想象的间充质凝结和预测网站，软骨会发生。整装和WlsShhCre小鼠，加上先进的显微技术获得的胚胎组织切片，染色使我们能够推出由TRA所产生的Wnt配体的作用CHEAL上皮气管图案。
Protocol
Representative Results
Discussion
事件呼吸道的形态底层不完全了解，特别是用于传导气道的图案化所需的过程。先前的研究已利用离体技术，其特征在于显影外植体在空气-液体相间培养或嵌入在基质胶21,22。这些研究已经表明生长因子如何影响显影气管的构图和气管软骨的形成。一个限制到这些研究是该组织的结构没有正确保持，因此，他们可能不太概括软骨的体内过程。
图案形成<em…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
我们承认麦克Muntifering和马特Kofron激光共聚焦成像和盖尔马科与组织程序的帮助。这项工作是部分由卫生NHLBI全国学院（K01HL115447到DS）的支持。
Materials
| Anti Sox9 ab. | Millipore | AB5535 | 1:400 , rabbit |
| Anti Sox9 ab. | Santa Cruz | Sc-20095 | 1:50, rabbit |
| Anti Smooth Muscle Actin ab. | Sigma | A5228 | 1:2k, mouse |
| Anti NKX2.1 ab. | Seven Hills | n/a | 1:100, guinea pig |
| Anti NKX2.1 ab. | Seven Hills | n/a | 1:400, mouse |
| Anti Brdu ab. | Abcam | AB1893 | 1:200, sheep |
| Anti Brdu ab. | Santa Cruz | Sc-32323 | 1:4k, mouse |
| PNA Lectin | Sigma | L 7381 | |
| Secondary antibodies | Life technologies | Alexa fluor Molecular probes | |
| K3Fe(CN)6 | Sigma | P8131 | |
| K4Fe(CN)6 | Sigma-Aldrich | P3289 | |
| MgCl2 | Sigma-Aldrich | M9272 | |
| NaDOC | Life Technologies | 89905 | |
| NP4O | Life Technologies | 85124 | |
| Alcian Blue 8GX | Sigma | A-3157 | |
| Fisher brand super-frost plus | Fisher | 12-550-15 | |
| PFA (16%) | EMS | 15710 | |
| PBS | Gibco | 70011-044 | |
| Fetal Calf Serum | Sigma | 11K413 | |
| Blocking reagent | Invitrogen | Component of TSA kit #2 ( T20932) | |
| BrDu | Sigma | B5002-5g | |
| Vectashield mounting medium | Vector labs | H-1000 | |
| Permount | Fisher | SP15-500 | |
| Tissue-loc cassettes Histoscreen | Fisher | C-0250-GR | |
| Biopsy cassettes | Premiere | BC0109 | Available in different colors |
| Nuclear fast red Kernechtrot 0.1% | Sigma | N3020 | |
| Citric acid | Sigma | C1909-500G | |
| Sodium citrate tribasic dihydrate | Sigma | S4641-1Kg | |
| Trizma hydrochloride | Sigma | T5941-500G | |
| Xylene | Pharmco-AAPER | 399000000 | |
| Ethanol | Pharmco-AAPER | 111000200 | |
| Micro knives | FST | 10318-14 | |
| Dumont #5 ceramic coated | FST | 11252-50 | |
| Dumont #5CO | FST | 11295-20 | |
| Dumont # 5 | FST | 91150-20 | |
| Thermo/Shandon Excelsior ES | Thermo Fisher | ||
| Microtome | Leica | RM2135 | |
| Nikon i90 | Nikon | Wide field microscope | |
| NikonA1Rsi | Nikon | Confocal microscopy. Settings:NikonA1 plus camera, scanner: Galvano, detector:DU4. Optics Plan Apo lambda 10x. Modality: Widefield fluorescence laser confocal. | |
| Leica MS 16 FA | Leica | Fluorescence Dissecting microscope | |
| Zeiss | Zeiss | Automated fluorescence microscope | |
| Leica Application suite | Leica | Leica imaging software | |
| NIS | Nikon | Nikon imaging software | |
| IMARIS | Bitplane | Imaging processing software |
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