May-Simera Helen
Cell and Matrix Biology, Institute of Zoology, Johannes Gutenberg-Universität Mainz;
J Vis Exp. 2016 Feb 21(108):53559. doi: 10.3791/53559.
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.
近年来，初级纤毛通过影响众多信号通路，已成为发育和疾病中的关键调节因子。最早被证明与纤毛功能相关的信号通路之一是非经典Wnt信号通路，也称为平面细胞极性（PCP）信号通路。在脊椎动物发育过程中，研究平面细胞极性（PCP）信号通路作用的最佳场所之一是哺乳动物的耳蜗。小鼠耳蜗中的PCP信号通路破坏会扰乱耳蜗的生长、细胞模式和毛细胞方向，所有这些都会受到纤毛功能障碍的影响。本方案的目的是通过表型分析、免疫组织化学和扫描电子显微镜描述发育中的哺乳动物耳蜗中PCP信号通路的分析。汇聚和延伸缺陷表现为耳蜗管缩短和/或细胞模式改变，在从发育中的小鼠突变体中解剖后可以进行量化。可以使用免疫荧光或扫描电子显微镜（SEM）观察和量化静纤毛束方向以及动纤毛长度或位置的变化。深入了解纤毛蛋白在细胞信号通路和其他生物学现象中的作用，对于我们理解细胞和发育生物学以及开发靶向治疗策略至关重要。
