Detection of planar polarity proteins in mammalian cochlea

The "core genes" were identified as a group of genes believed to function as a conserved signaling cassette for the specification of planar polarity in Drosophila Melanogaster, and includes frizzled (fz), van gogh (vang) or strabismus (stbm), prickle (Pk), dishevelled (dsh), flamingo (fmi), and diego. The mutation of each of these genes not only causes the disruption of planar polarity within the wing or the eye of the animal, but also affects the localization of all the other protein members of the core group. These properties emphasize the importance of the interrelations between the proteins of this group. All of these core genes have homologs in vertebrates. Studies in Danio Rerio (zebrafish) and Xenopus laevis (frog) have uncovered other roles for some of these molecules in gastrulation and neurulation, during which the shape of a given tissue will undergo major transformation through cell movements. A disruption in these processes can lead to severe neural tube defects in diverse organisms, including humans. In fact, a large body of evidence suggests that planar polarity proteins are not involved in one specific cascade but in many different ones and many different mechanisms such as, but not limited to, hair or cilia orientation, asymmetric division, cellular movements, or neuronal migration. In mice cochleae, mutations in planar polarity genes lead to defects in the orientation of the stereociliary bundles at the apex of each hair cell. This phenotype established the cochlea as one of the clearest examples of planar polarity in mammals. Although significant progress has been made toward understanding the molecular basis required for the development of planar polarity in invertebrates, similar advances in vertebrates are more recent and rely mainly on the identification of a group of mammalian mutants that affect hair cell stereociliary bundle orientation. These include mutation of vangl2, scrb1, celsr1, PTK-7, dvl1-2, and more recently fz3 and fz6. In this chapter, we describe how to use the mammalian cochlea, which represents one of the best systems to study planar polarity in mammals, to identify planar polarity mutants, study protein distribution, do in vitro analysis, and perform Western blots to analyze putative planar polarity proteins.