直立成像<em>果蝇</em>蛋钱伯斯
Summary
The upright imaging method described in this protocol allows for the detailed visualization of the poles of a developing Drosophila melanogaster egg. This end-on view provides a new perspective into the arrangements and morphologies of multiple cell types in the follicular epithelium.
Abstract
Drosophila melanogaster oogenesis provides an ideal context for studying varied developmental processes since the ovary is relatively simple in architecture, is well-characterized, and is amenable to genetic analysis. Each egg chamber consists of germ-line cells surrounded by a single epithelial layer of somatic follicle cells. Subsets of follicle cells undergo differentiation during specific stages to become several different cell types. Standard techniques primarily allow for a lateral view of egg chambers, and therefore a limited view of follicle cell organization and identity. The upright imaging protocol describes a mounting technique that enables a novel, vertical view of egg chambers with a standard confocal microscope. Samples are first mounted between two layers of glycerin jelly in a lateral (horizontal) position on a glass microscope slide. The jelly with encased egg chambers is then cut into blocks, transferred to a coverslip, and flipped to position egg chambers upright. Mounted egg chambers can be imaged on either an upright or an inverted confocal microscope. This technique enables the study of follicle cell specification, organization, molecular markers, and egg development with new detail and from a new perspective.
Introduction
研究果蝇提供了巨大的见解的各种现象的遗传调控。特别是，出现了对蛋发展广泛的研究，因为卵子发生提供了一种易于处理的方式来调查许多不同的发育过程，包括组织图案化，细胞极性的变化，细胞周期的开关，和翻译调控1,2,3,4。卵子发生过程中一个重要的事件形态是一组细胞称为边缘细胞（5审查）的规格，收购蠕动，和迁移。因为细胞迁移是动物形态的一个主要特点，并且因为这个过程的基因调节是非常保守，在果蝇确定的机制很可能是在其他情况下非常重要的。因此，我们正在调查边境细胞迁移的分子控制。对于我们的研究，我们已经开发出一种新的方法来观察开发鸡蛋的前极，其中，边缘细胞产生，检查他们详细介绍了如何发展。
内卵巢，卵腔室中的不同发育阶段沿链称为卵巢管，其包封在薄鞘（ 图1A）存在。每个鸡蛋室将继续形成一个鸡蛋。在卵子发生，几种不同类型的细胞必须协调发展的方式。 D.在果蝇卵室由16个种系细胞，包括1卵母细胞，由单层滤泡上皮6包围。出现在上皮的前部和后部磁极少数特殊细胞，称为极性细胞。 6-8边缘细胞起源于卵室的前上皮细胞，诱导细胞极性5,7。在中期卵子发生（9级），边界细胞从他们的邻居和分离的滋养细胞之间迁移的卵室5,7的后到达卵母细胞。这个动作必须完成而边缘细胞停留在周边两个非能动极性细胞集群，使之成为一个类型的集体细胞迁移。边界小区集群的成功迁移确保蛋壳的珠孔，这是必要的施肥适当发展。
前极性细胞指示边境细胞命运通过激活的信号传导级联。极性细胞分泌细胞因子，不成对（UPD），其结合跨膜受体，无圆顶（DOME），对卵母细胞发育的8,9级8中相邻的毛囊细胞。 UPD的结合导致的Janus酪氨酸激酶（JAK）磷酸化转录（STAT）8,10,11的信号转导和激活。 STAT然后移动到细胞核来激活转录。慢边缘细胞（SLBO）是一种转录因子是STAT的直接转录目标，也需要边境细胞迁移12。蛋室外侧观点表明吨帽子STAT活性调节横跨前壁上皮8,11,13-阿松香三烯梯度。最接近极性细胞毛囊细胞具有活化的STAT的最高水平，因而它们变得边缘细胞和侵入相邻的种系组织。
理解边界细胞是如何内指定，并从上皮脱落，我们需要观察如何组织的组织结构。如果我们把鸡蛋室来自前上的角度来看，我们希望在周边极地细胞的毛囊细胞STAT活动的径向对称。一个端视图还更精确地之前和脱离比不同的焦平面相比较的细胞中表现出的膜蛋白和细胞间的接口的差异。因为蛋室是长方形和彼此附接由柄细胞，它们定居到载玻片横向，从而难以观察前架构。因此，有关的细胞在卵腔的磁极多的信息具有被推断，从横向的看法。虽然一些信息可以通过光的部分，光散射，光漂白算法的3-D重建，并在Z轴分辨率较差限制得到使该信息少在没有像超分辨率显微镜14昂贵的技术详细和可靠。其他种类的部分的基于成像（例如，电子显微镜或薄样切片机的切片）需要大量的操作的组织，包括脱水，增加了对工件的可能性。因此，我们开发了一种新的方法来形象D.果蝇卵室，而直立。这种方法已经被证明非常有用在阐明如何游动细胞注定（见代表性的成果和曼宁等人，正在审查 ），并且很可能是更广泛的有价值的卵子等方面的研究。
Protocol
Representative Results
Discussion
在这里，我们描述的方法来安装和图像小，从终端的角度上制定的蛋室。成像蛋室常见的技术横向的意见进行了优化，主要允许内 – 外滤泡细胞的精确的可视化，当用荧光抗体染色。在观看多个焦平面使用的Z-堆栈或三维重建助剂，但仍有不足之处为亚细胞的椭圆卵腔室（ 图2D）的极点的分辨率。虽然这可以部分地用诸如超分辨率成像的最新的显微技术来克服，这些方法是昂贵的并且…
Disclosures
The authors have nothing to disclose.
Acknowledgements
We appreciate assistance from members of the fly community, particularly Dr. Denise Montell, Dr. Lynn Cooley, and Dr. Pernille Rorth, for reagents. We thank Flybase, the Bloomington Drosophila Stock Center, and Developmental Studies Hybridoma Bank for information and providing fly stocks and antibodies, respectively. LM is supported by the Department of Education Grant, Graduate Assistance in the Areas of National Need (GAANN) training fellowship (P200A120017) and by a NIGMS Initiative for Maximizing Student Development Grant (2 R25-GM55036). A portion of the microscopy work was supported by NSF MRI grant DBI-0722569 and the Keith R. Porter Core Imaging Facility. Research was supported in part by a NSF CAREER Award (1054422) and a Basil O’Connor Starter Scholar Award from the March of Dimes, both awarded to MSG.
Materials
| Name of Reagent/ Equipment | Company | Catalog Number | Comments/Description |
| 0.1 M Potassium phosphate Buffer (KPO4 Buffer) | Add 3.1 g of NaH2PO4•H2O and 10.9 g of Na2HPO4 (anhydrous) to distilled H2O to make a volume of 1 L. The pH of the final solution will be 7.4. This buffer can be stored for up to 1 mo at 4°C. | ||
| 16% Paraformaldehyde aqueous solution, EM grade | Electron Microscopy Sciences | 15710 | methanol-free to preserve GFP fluorescence |
| 30G x 1/2 inch needle | VWR | BD305106 | Regular bevel |
| Active Dry Yeast | Genesee Scientific | 62-103 | To fatten female flies |
| Bovine Serum Albumin | PAA-cell culture company | A15-701 | Used in NP40 Wash Buffer |
| Dumont #5 Forceps | Fine Science Tools | 11295-10 | Dumostar alloy, biologie tip; sharp tips are essential for ovariole dissection |
| DAPI (4′,6-Diamidino-2-phenylindole dihydrochloride) | Sigma Aldrich | D9542 | 5mg/ml stock solution |
| Fetal Bovine Serum (FBS) | Life Technologies | 16140-071 | Added to supplement dissection medium (10%) |
| Glass culture tube | VWR | 47729-576 | 14mL |
| Glass Depression Slides | VWR | 470019-020 | 1.2 mm Thick, Double Cavity |
| Glycerin Jelly | Electron Microsocpy Sciences | 17998-10 | Mounting media |
| Glycerol | IBI Scientific | IB15760 | 70% in PBS |
| IGEPAL CA-630 | Sigma Aldrich | I3021-500ML | (interchangable for Nonidet P-40) Used in NP40 Wash Buffer |
| Leica Fluorescent Stereoscope | Leica Microsystems | ||
| Leica SP5 Confocal Microscope | Leica Microsystems | 40x/0.55NA dry objective | |
| Micro spatula | VWR | 82027-518 | Stainless steel |
| Microknife | Roboz Surgical Instrument Company | 37-7546 | 45ᵒ angle |
| Microscope Slide | VWR | 16004-368 | 75x25x1 mm |
| NP40 Wash Buffer | 50mM TRIS-HCL, 150mM NaCl, 0.5% Ipegal, 1mg/ml BSA, and 0.02% sodium azide | ||
| Penicillin-Streptomycin-Glutamine | Life Technologies | 10378-016 | Added to supplement dissection medium (0.6X) |
| Petridish- Polysterine, sterile | VWR | 82050-548 | 60Wx15H mm |
| Phosphate Buffer Saline | Sigma Aldrich | P3813-10PAK | 10 packs of Powder |
| Potassium phosphate dibasic | Sigma Aldrich | P3786-500G | Used in 0.1M KPO4 Buffer |
| Potassium phosphate monobasic | Sigma Aldrich | P9791-500G | Used in 0.1M KPO4 Buffer |
| Schneider’s Insect Medium | Life Technologies | 11720018 |
With L-glutamine and sodium bicarbonate |
| Sodium azide | Sigma Aldrich | S2002-25G | Used in fluorescent antibody staining |
| Sodium chloride | Sigma Aldrich | S3014-500G | Used in NP40 Wash Buffer |
| TRIS-HCL | IBI Scientific | IB70144 | 1M TRIS-HCL, pH 7.4 |
| Volocity 3D Image Analysis Software | PerkinElmer | For processing confocal Z-stacks |
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