Primary cilia influence various signaling pathways. The mammalian cochlea is ideal for examining planar cell polarity (PCP) signaling. Cilia dysfunction affects cochlear outgrowth, cellular patterning and hair cell orientation, readouts of PCP. Our goal is to analyze PCP signaling in mouse cochlea via phenotypic analysis, immunohistochemistry and scanning electron microscopy.
In recent years, primary cilia have emerged as key regulators in development and disease by influencing numerous signaling pathways. One of the earliest signaling pathways shown to be associated with ciliary function was the non-canonical Wnt signaling pathway, also referred to as planar cell polarity (PCP) signaling. One of the best places in which to study the effects of planar cell polarity (PCP) signaling during vertebrate development is the mammalian cochlea. PCP signaling disruption in the mouse cochlea disrupts cochlear outgrowth, cellular patterning and hair cell orientation, all of which are affected by cilia dysfunction. The goal of this protocol is to describe the analysis of PCP signaling in the developing mammalian cochlea via phenotypic analysis, immunohistochemistry and scanning electron microscopy. Defects in convergence and extension are manifested as a shortening of the cochlear duct and/or changes in cellular patterning, which can be quantified following dissection from developing mouse mutants. Changes in stereociliary bundle orientation and kinocilia length or positioning can be observed and quantitated using either immunofluorescence or scanning electron microscopy (SEM). A deeper insight into the role of ciliary proteins in cellular signaling pathways and other biological phenomena is crucial for our understanding of cellular and developmental biology, as well as for the development of targeted treatment strategies.
初级纤毛是长基于微管附属物,从大多数哺乳动物细胞的表面上延伸。初级纤毛经常被混淆与运动纤毛,其中总有每个细胞多,而其目的是在整个膜表面移动的液体。初级纤毛,与此相反,采用感觉角色并因此也被称为感觉纤毛。一旦早已被人遗忘,这种细胞器最近被“重新发现”它与人类遗传病1的众多协会的结果。理想的位置作为信号的细胞器,初级纤毛已显示调节众多信号通路,其中有许多是不仅在组织稳态和疾病重要的,但也发展2中。
示出一个将与纤毛功能障碍相关联的第一信号通路是非典型Wnt信号传导途径,也被称为平面细胞极性(PCP)途径<sup > 3。在果蝇中初步确定这信号级联反应,是胚胎发育的关键;特别是用于会聚和扩展过程和细胞在上皮4的平面中的正确方向。设置调节蛋白的核心的顺序信令转换方向提示这最终导致细胞骨架重排,并导致上皮细胞在平面5的协调极化。衔接和延伸的过程是绝对必需的蜗管伸长和正确的蜂窝图案6。由于这是通过PCP途径的激活调节,耳蜗PCP突变体的最显着的表型中的一个是一个缩短的蜗管与紊乱感觉上皮7。同样,小鼠突变,缺乏纤毛,也表现出这样的融合和扩展型8,9,但恰恰这是如何调控仍有待澄清。
ve_content“>由于收敛和扩展步骤是耳蜗管的生长,以及蜗管内的感觉上皮细胞图案化临界,显影耳蜗是其中脊椎动物发育过程中检查PCP信令理想器官。的器官尔蒂,给予该线耳蜗管,是由必须被均匀地取向为耳蜗的功能10非感官支持细胞和mechanosensory毛细胞的专门感觉上皮的术语。本mechanosensory毛细胞是所谓的由于从表皮板延伸纤毛束每个感觉毛细胞11(顶面)。这些充当机械感觉的,尽管其命名为纤毛主要传感器,实际上是由改性肌动蛋白基于灯丝的微绒毛。在每个字形的头发束,三排纤毛的以高度有序的和常规巴被组织ttern在楼梯状的方式。真实基于微管纤毛,称为kinocilia,需要用于该纤毛束 12的发展和方向。在每次毛细胞,一个单一的kinocilium被物理连接到纤毛束,位于中央相邻纤毛的最高行。该kinocilium的确切功能尚不清楚,一个假设是,因为他们从微绒毛12成熟kinocilium'拉'的纤毛成形。在脊椎动物中,在耳蜗kinocilia仅存在瞬时和从头发细胞之前听到11,13,14的发作缩回小鼠。在严重缩短耳蜗管显影耳蜗结果纤毛的完全丧失,错形成及误取向纤毛束,以及错误置位的基体8,9。一个功能纤毛不只是由纤毛轴丝的。纤毛相关的许多蛋白质功能发生在本地化的纤毛相关的子域复合物,如基体,过渡区,或纤毛轴丝15。基础体温,从中心体的中心粒母亲衍生,也是从纤毛向外延伸进入细胞体内微管微管组织中心并能调节细胞内贩卖和走私纤毛。睫状肌的过渡区是纤毛功能被组织纤毛化合物16的进口和出口方面管制的另一个区域。
多个研究已经确定纤毛和非经典Wnt(PCP信令)之间的连接,虽然确切机制尚不清楚17。睫状和PCP基因冗余和细胞极性的广义细胞异常的敏感性,使得难以突变直接链接到特定的PCP-赤字。一个PCP信令的读出出局的是基体和PRIMAR的定位Ÿ纤毛,因此分离主要由二次缺陷是具有挑战性的。在斑马鱼和小鼠突变一些研究表明信号18-20纤毛和Wnt之间没有联系。在数据差异可能反映物种,组织,或在朝向Wnt信号睫状捐款颞依赖性差异。此外,如果基体保持工作正常Wnt信号的响应可能会被保留。一个更深入的了解纤毛蛋白在细胞信号通路及其它生物现象的作用,是对我们的细胞和发育生物学的理解,以及为有针对性的治疗策略的发展至关重要。
当准备耳蜗组织进行分析,有要记住几个关键点。首先,在遗传背景的差异可以修改耳蜗的表型,因此有必要进行分析和比较只同窝对照。其次,需要彻底清除盖膜获得具有免疫最好的图像,是SEM至关重要。盖膜是不透明结构,并且可以直接模糊感觉上皮的细胞其下方,使得成像更具挑战性。偶尔在加工过程中,盖膜可能会退缩,露出毛细胞。即使在这些情况下,除去强烈建议。这一步需要耐心?…
The authors have nothing to disclose.
笔者要感谢马修·凯利,蒂齐阿纳Cogliati,杰西卡Gumerson,乌韦·沃尔夫鲁姆,Rivka Levron,中提琴克雷奇默尔和Zoe曼和手稿其关键的评价。这项工作是由Sofja Kovalevskaya奖(与洪堡基金会)和约翰内斯 – 古腾堡大学,美因茨,德国资助。
Tools/Equipment | |||
Silicone elastomere – Sylgard 184 | Sigma-Aldrich | 761028-5EA | See Note 2 |
Micro dissecting scissors-straight blade | Various | ||
Fine forceps (no. 5 and 55) and blunt forceps | Various | ||
Dissecting microscope. | Various | ||
Uncoated glass microscope slides | Various | ||
Microscope cover slips (22 mm × 40 mm × 0.15 mm) | Various | ||
Transfer pipettes | Various | ||
Minutien pins | Fine Science Tools | 26002-10 | |
SEM sample holder | tousimis | 8762 | |
Scanning electron microscopy studs | TED PELLA | 16111 | |
PELCO Tabs: Carbon adhesive | TED PELLA | 16084-3 | |
Fluorescent Microscope | Various | ||
Critical Point Dryer | Various | ||
Scanning Electron Microscope | Various | ||
Glass microscope slides | Various | ||
Glass coverslips | Various | ||
Kimwipe Tissue | Various | ||
Fine Paint Brush | |||
Reagents | |||
1× Phosphate buffered saline (PBS) | Gibco/Life Technologies | 10010023 | |
Paraformaldehyde (PFA) (EM Grade Required for EM) | Various | Prepare a 4% solution in 1× PBS made fresh each time. EM Grade Required for EM. | |
2.5% Glutaraldehyde Grade1 | Sigma-Aldrich | G5882 | |
Tris-HCl (pH 7.5) | Various | ||
NaCl | Various | ||
CaCl 2 | Various | ||
Triton X-100 | Various | ||
Normal Goat Serum | Various | ||
AffiniPure Fab Fragment Donkey Anti-Mouse IgG (H+L) | Jackson ImmunoResearch | 715-007-003 | |
Fluoromount-G Mounting media | SouthernBiotech | 0100-01 | |
10× Hanks’ Balanced Salt Solution (HBSS) | Gibco/Life Technologies | 14065 | |
Hepes | Gibco/Life Technologies | 15630-080 | |
Osmium tetroxide (OsO4 ) | Sigma-Aldrich/Fluka Analytical | 75632 | |
Tannic acid | Sigma-Aldrich | 403040 | |
Ethanol 200 proof | Various | ||
Antibodies | |||
anti Arl13b | Protein Tech | 17711-1-AP | Suggested concentration 1:1000 |
anti acetylated tubulin (611-B1) | Sigma-Aldrich | T6793 | Suggested concentration 1:800 |
anti gamma tubulin (GTU-88) | Sigma-Aldrich | T6557 | Suggested concentration 1:200 |
anti Zo_1 | Invitrogen | 40-2300 | Suggested concentration 1:500 |
Myosin VI | Proteus Biosciences | 25-6791 | Suggested concentration 1:1000 |
Myosin VIIa | Proteus Biosciences | 25-6790 | Suggested concentration 1:1000 |
anti Vangl2 | Merk Millipore | ABN373 | Suggested concentration 1:250 |
anti Gαi3 | Sigma-Aldrich | G4040 | Suggested concentration 1:250 |
Alexa Fluor® 488 Phalloidin | Invitrogen/Life Technologies | A12379 | Suggested concentration 1:300-1000 |
Alexa Fluor® 568 Phalloidin | Invitrogen/Life Technologies | A12380 | Suggested concentration 1:300-1000 |