操纵小鼠泪腺
Summary
泪腺（LG电子）是一家生产所必需的眼泪水成分对维持视力和眼睛健康的分支机关。在这里，我们描述了小鼠LG解剖和体外培养技术破译的信号参与了LG的发展道路。
Abstract
泪腺（LG）的分泌所必需的维持角膜的结构和功能，一个透明组织为视力必不可少水性眼泪。在人单LG驻留在上面的每个眼睛通过3输送泪到眼表面的侧端的轨道 – 5管道。该鼠标有三对大眼腺，研究得最多的，其中是exorbital泪腺（LG电子）位于前部和腹侧的耳朵。类似于其他腺体器官，LG的发展，通过上皮分支形态，其中内稠间质单个上皮芽经历多轮芽和管形成的，以形成分泌腺泡和导管的一个复杂的互连网络的过程。这个复杂的过程已经在文献中许多其它上皮器官如胰腺，唾液腺。不过，LG一直不太探索和形态控制的机制是不好下站了起来。我们怀疑这代表性不足作为一个模型系统是与发现，解剖和培养了LG的困难造成的。因此，在这里我们描述和解剖技术收获胚胎和产后LG为组织的体外培养方法。
Introduction
泪腺（LG）是负责水性泪液分泌为视力和健康，维护和保护眼睛表面的细胞是至关重要的。 LG的功能障碍的结果中最常见的和衰弱的眼部疾病之一：水缺乏干眼疾病，其特点是由眼部刺激，光敏感性和视力下降1。在人对LG驻留在轨道上方的眼睛的侧端，其中3 – 5排泄管道存款泪到眼表面。鼠标有三对大眼腺体，研究最多的，其中是泪腺（LG）位于前部和腹侧耳（exorbital）流着泪，通过一个单一的排泄管行驶到眼睛。类似于其他腺体器官，对LG通过上皮分支形态发生的过程，其中内稠间质单个上皮芽经历多轮芽和管道形成为开发米分泌腺泡和导管的复杂互连网络（ 图1）2。在开发过程中的上皮细胞成为血管化以及大量从颈上神经节3支配由翼腭神经节的副交感神经，并在较小程度上是由交感神经。这些细胞类型，即神经元细胞，上皮细胞，内皮细胞和间充质细胞之间的相互作用，是必不可少的功能和维持成人组织中。然而，协调LG的发育和再生以及如何小区间型通信引导这些过程的分子机制仍不清楚。
胚胎体外培养技术的出现，使发育和再生途径中的多个分支机关4鉴定。培养体外使研究人员操纵机构（我的能力械，遗传或化学的）所定义的条件下，以及表征实时器官发育和细胞 – 细胞相互作用。鼠标的exorbital LG非常适合于这种技术，最近的研究已经定义了信令规范其发展2.5系统。然而，尽管有必要了解分子线索支撑LG的发育和再生，但目前仍然是充分研究，很有可能是由于在分离的器官的技术困难。在本文中，我们将介绍如何隔离并进行胚胎小鼠LG的体外培养，以明确的发展计划。
Protocol
在严格按照指南中的美国国立卫生研究院实验动物的护理和使用的建议进行的所有动物的工作。该协议被批准的机构动物护理和使用委员会加州大学（IACUC），旧金山。 1.小鼠胚胎泪腺（LG）：收获和显微切割继批准的机构动物科学研究伦理委员会的程序，安乐死定时怀孕的CD-1（或转基因）的女性不断上升的二氧化碳吸入，然后确认颈椎脱位收获胚胎在适当的…
Representative Results
LG的发展，通过上皮分支形态发生的过程。解剖在E14，E15，E16，E17，和P2 LG的胚胎的明图片说明此事件。 图1. LG开发通过上皮分支形态的过程。胚胎LG的新鲜切开的E14，E15，E16，E17和P2的明场图像。 <a href="https://www.jove.com/files/ftp_upload/51970/51970fig1highres.jpg…
Discussion
多功能性文化和操纵LG 体外为研究其发展显著的优势。这包括在哪个速度研究者能够验证假设和许多可被执行，以评估上皮细胞，神经细胞，内皮细胞和mesencyhmal细胞相互作用以形成器官扰动。但是，使用这种模式时，有一些需要注意的地方。首先，由于其隔离的腺不再连接到外围血管或神经系统，这可能会影响信号转导途径被测试，如果他们在协同工作与由神经或血管9,10-传递信?…
Declarações
The authors have nothing to disclose.
Acknowledgements
作者要感谢由加州大学旧金山分校的资源分配计划提供了资金。
Materials
| Name | Catalog #. | Company | Comments/Description |
| DMEM/F12 without HEPES | SH3027101 | Thermo Scientific | |
| Penicillin-Streptomycin | P0781 | Sigma-Aldrich | |
| Albumin solution from bovine serum | A9576 | Sigma-Aldrich | |
| Paraformaldehyde 16% Solution | 15710 | Electron Microscopy Sciences | Diluted to 4% in 1X PBS |
| Phosphate Buffered Saline 10X | BP665-1 | Fisher Scientific | |
| Phosphate Buffered Saline 1X | Prepared from 10X stock | ||
| holo-Transferrin bovine | T1283 | Sigma-Aldrich | 25 mg/ml stock solution in water. Freeze single-use aliquots at -20C. Add to DMEM/F12 media to a final concentration of 50 ug/mL. |
| L- Ascorbic acid (Vitamin C) | A4544 | Sigma-Aldrich | 25 mg/ml stock solution in water. Freeze single-use aliquots at -20C. Add to DMEM/F12 media to a final concentration of 50 ug/mL. |
| BioLite 100mm Tissue Culture Treated Dishes | 130182 | Thermo Scientific | Non-tissue culture-treated plates can also be used. |
| Stereo Microscope with substage iluminator | Stemi 2000 | Carl Zeiss | Any stereo dissecting microscope can be used that has a transmitted light base. |
| Substage Illuminator Base for Stereo Microscope | TLB 4000 | Diagnostics Instruments, Inc. | |
| Falcon 35mm Tissue Culture Treated Dishes | 353001 | Corning | Non-tissue culture-treated plates can also be used. |
| 50mm uncoated glass bottom dishes | P50G-1.5-14-F | MatTek Corporation | |
| Widefield fluorescence microscope | Axio Observer Z1 | Carl Zeiss | Any fluorescence microscope (upright, inverted or stereo dissecting microscope) can be used to monitor GFP expression at low magnification with an attached digital camera. |
| Confocal Microscope | TCS SP5 | Leica Microsystems | Confocal microscopy is necessary to see detailed cell structures. Any confocal microscope can be used. |
| Ideal for harvesting glands from embryos | |||
| SWISS Micro-Fine Forceps, #5 (11.2cm) | 17-305X | Integra LifeSciences Corporation | Fine tips are required for removing mesenchyme from epithelium. Tungsten needles can also be used. |
| Dumont Standard Tip Forceps, #5 (11cm) | 91150-20 | Fine Science Tools (USA), Inc. | Ideal for harvesting glands from embryos |
| Reusable Plastic Surgical Knife Handles, style no. 3. | 4-30 | Integra LifeSciences Corporation | |
| Stainless Steel Sterile Surgical Blades, no. 10 | 4-310 | Integra LifeSciences Corporation | |
| RNAqueous-Micro Total RNA Isolation Kit | AM1931 | Life Technologies | |
| Cultrex 3D Culture Matrix Laminin I | 3446-005-01 | Trevigen | 6 mg/ml stock diluted 1:1 with DMEM/F12 |
| Timed-pregnant Crl:CD1(ICR) mice | Charles River Labs | Embryos are harvested on day 14 (with day of plug discovery designated as day 0). | |
| Nucleopore Track-Etched Hydrophilic Membranes, 0.1 um pre size, 13mm | 110405 | Whatman | |
| Timed-pregnant Pax6-Cre,GFP mice | Tg(Pax-Cre, GFP)1Pgr | The Jackson Laboratory | Optional mice for learning how to locate the LG. |
Referências
- Liu, K. C., Huynh, K., Grubbs, J., Davis, R. M. Autoimmunity in the pathogenesis and treatment of keratoconjunctivitis sicca. Current allergy and asthma reports. 14, 403 (2014).
- Makarenkova, H. P., et al. FGF10 is an inducer and Pax6 a competence factor for lacrimal gland development. Development. 127, 2563-2572 (2000).
- Dartt, D. A. Neural regulation of lacrimal gland secretory processes: relevance in dry eye diseases. Progress in retinal and eye research. 28, 155-177 (2009).
- Nigam, S. K. Concise review: can the intrinsic power of branching morphogenesis be used for engineering epithelial tissues and organs. Stem cells translational medicine. 2, 993-1000 (2013).
- Qu, X., et al. Glycosaminoglycan-dependent restriction of FGF diffusion is necessary for lacrimal gland development. Development. 139, 2730-2739 (2012).
- Rebustini, I. T., et al. MT2-MMP-dependent release of collagen IV NC1 domains regulates submandibular gland branching morphogenesis. Developmental cell. 17, 482-493 (2009).
- Hoffman, M. P., et al. Gene Expression Profiles of Mouse Submandibular Gland Development: FGFR1 Regulates Branching Morphogenesis in Vitro Through BMP- and FGF-Dependent Mechanisms. Development. 129, 24-5778 (2002).
- Steinberg, Z., et al. FGFR2b signaling regulates ex vivo submandibular gland epithelial cell proliferation and branching morphogenesis. Development. 132, 1223-1234 (2005).
- Knox, S. M., et al. . Parasympathetic innervation maintains epithelial progenitor cells during salivary organogenesis. 329, 1645-1647 (2010).
- Magenheim, J., et al. Blood vessels restrain pancreas branching, differentiation and growth. Development. 138, 4743-4752 (2011).
- Jaskoll, T., et al. FGF10/FGFR2b signaling plays essential roles during in vivo embryonic submandibular salivary gland morphogenesis. BMC developmental biology. 5, 11 (2005).
- Takasato, M., et al. Directing human embryonic stem cell differentiation towards a renal lineage generates a self-organizing kidney. Nature cell biology. 16, 118-126 (2014).
- Knox, S. M., et al. Parasympathetic stimulation improves epithelial organ regeneration. Nature communications. 4, 1494 (2013).
Citar este artigo
Finley, J. K., Farmer, D., Emmerson, E., Cruz Pacheco, N., Knox, S. M. Manipulating the Murine Lacrimal Gland. J. Vis. Exp. (93), e51970, doi:10.3791/51970 (2014).