By E18

By E18.5, the membrane crescent of Fz3 was not detectable in the mutant (Fig. V-shaped stereociliary bundle (or hair bundle) with intrinsic structural polarity: the actin-based stereocilia are organized into rows of graded heights, forming a staircase-like pattern. Furthermore, auditory hair bundles are uniformly ONX-0914 oriented across the OC, with the V pointing toward the outer (lateral) border of the cochlear duct. These features are essential for the correct perception of sound and are established by an intricate gene network during development (Frolenkov et al., 2004;Petit and Richardson, 2009). In the mouse, hair bundle development and maturation proceed in two perpendicular gradients, from the base to the apex and from the medial to lateral side of the cochlea over a period from late embryogenesis to the first two postnatal weeks (Frolenkov et al., 2004;Petit and Richardson, 2009). During the initial phase, a single tubulin-based kinocilium, derived from the primary cilium, migrates from the center to the lateral edge of the hair cell apex. Concomitant with this migration, microvilli around the kinocilium elongate to form stereocilia of graded heights. By the end of embryogenesis, nascent hair bundles exhibit a crescent shape with the kinocilium centered next to the tallest stereocilia. Next, during the first postnatal week, stereocilia undergo further row-specific, differential outgrowth, eventually forming a hair bundle with a staircase organization. Finally, the kinocilium retracts around postnatal day 10. Tremendous progress has been made toward understanding the molecular mechanisms that regulate hair bundle morphogenesis. In particular, genetic analysis and cloning of deafness mutations in humans and mice have identified a number of structural and regulatory proteins of the actin cytoskeleton (El-Amraoui and Petit, 2005;Leibovici et al., 2008;Petit and Richardson, 2009). In addition, a Wnt/planar cell polarity (PCP) pathway, with both evolutionarily conserved (such as the Frizzled and Dishevelled proteins) and novel (such as Scrb1 and PTK7) components, plays an important role in regulating cochlear elongation and hair bundle orientation (Rida and Chen, 2009). Despite the importance of the actin cytoskeleton in hair cell morphogenesis, the roles of Rho GTPases, central regulators of actin dynamics, in this process remain poorly comprehended. Rho family members, including Rho, Rac, and Cdc42, have been shown to influence cell migration, cellcell adhesion, microtubule dynamics, ONX-0914 cell proliferation, apoptosis, and gene transcription (Jaffe and Hall, 2005). Thus, Rho GTPases could potentially regulate many aspects of cochlear epithelial morphogenesis. In this study, we show that the small GTPase Rac1 plays a critical role in the morphogenesis of the OC and the auditory hair bundles. Furthermore, we provide evidence that Rac1 regulates interactions between the kinocilium and stereocilia via p21-activated kinase (PAK), which is required for the cohesion of the developing hair bundle. These results establish Rac1 as a key regulator of auditory hair cell morphogenesis. == Materials and Methods == == == == == == Mice. == TheRac1conditional allele,Foxg1-Cremice, andPax2-Cremice were previously described (Hbert and McConnell, 2000;Glogauer et al., 2003;Ohyama and Groves, 2004). All strains were maintained on a mixed genetic background.LtapLpmice were obtained from the Jackson Laboratory and CD1 mice from Charles River.Foxg1-Cre; Rabbit polyclonal to AADACL2 Rac1KO/+males were bred withRac1CO/COfemales to generateFoxg1-Cre; Rac1KO/COmutants and littermate controls.Pax2-Cre; Rac1CO/+females were bred withRac1CO/COmales to generatePax2-Cre; Rac1CO/COmutants and littermate controls. PCR genotyping of theRac1alleles was performed as described previously (Glogauer et al., 2003). Mice were genotyped for Cre using the following primers: 5-AGAACCTGAAGATGTTCGCG-3 and 5-GGCTATACGTAACAGGGTGT-3. For timed pregnancies, the morning of the plug was designated as embryonic day 0.5 (E0.5), and the day of birth postnatal day 0 (P0). Animal care and use was in accordance with National Institutes of Health guidelines and ONX-0914 was approved by the Animal Care.