Detection of Planar Polarity Proteins in Mammalian Cochlea
Methods in Molecular Biology. 2008-01-01; : 207-219
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Montcouquiol M(1), Jones JM, Sans N.
(1)Equipe Avenir, Development Neurosciences, INSERM U862, Institut Francois Magendie, Université Bordeaux II, France.
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.