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Hearing impairment due to the loss of sensory hair cells is permanent in humans. Considerable interest targets the hair cell differentiation factor Atoh1 as a potential tool with which to promote hair cell regeneration. We generated a novel mouse model to direct the expression of Atoh1 in a spatially and temporally specific manner in the postnatal mammalian cochlea to determine the competency of various types of cochlear epithelial cells for hair cell differentiation. Atoh1 can generate cells in young animals with morphological, molecular, and physiological properties reminiscent of hair cells. This competency is cell type specific and progressively restricted with age. Significantly, Atoh1 induces ectopic sensory patches through Notch signaling to form a cellular mosaic similar to the endogenous sensory epithelia and expansion of the sensory mosaic through the conversion of supporting cells and nonautonomous supporting cell production. Furthermore, Atoh1 also activates proliferation within the normally postmitotic cochlear epithelium. These results provide insight into the potential and limitations of Atoh1-mediated hair cell regeneration.

The planar cell polarity (PCP) pathway is responsible for polarizing and orienting cochlear hair cells during development through movement of a primary cilium, the kinocilium. GPSM2/LGN, a mitotic spindle-orienting protein associated with deafness in humans, is a PCP effector involved in kinocilium migration. Here, we link human and mouse truncating mutations in the GPSM2/LGN gene, both leading to hearing loss. The human variant, p.(Trp326*), was identified by targeted genomic enrichment of genes associated with deafness, followed by massively parallel sequencing. Lgn (ΔC) mice, with a targeted deletion truncating the C-terminal GoLoco motifs, are profoundly deaf and show misorientation of the hair bundle and severe malformations in stereocilia shape that deteriorates over time. Full-length protein levels are greatly reduced in mutant mice, with upregulated mRNA levels. The truncated Lgn (ΔC) allele is translated in vitro, suggesting that mutant mice may have partially functioning Lgn. Gαi and aPKC, known to function in the same pathway as Lgn, are dependent on Lgn for proper localization. The polarization of core PCP proteins is not affected in Lgn mutants; however, Lgn and Gαi are misoriented in a PCP mutant, supporting the role of Lgn as a PCP effector. The kinocilium, previously shown to be dependent on Lgn for robust localization, is essential for proper localization of Lgn, as well as Gαi and aPKC, suggesting that cilium function plays a role in positioning of apical proteins. Taken together, our data provide a mechanism for the loss of hearing found in human patients with GPSM2/LGN variants.

The vertebrate planar cell polarity (PCP) pathway shares molecular components with the β-catenin–mediated canonical Wnt pathway but acts through membrane complexes containing Vang or Frizzled to orient neighboring cells coordinately. The molecular interactions underlying the action of Vang in PCP signaling and specification, however, are yet to be delineated. Here, we report the identification of Rack1 as an interacting protein of a vertebrate Vang protein, Vangl2. We demonstrate that Rack1 is required in zebrafish for PCP-regulated processes, including oriented cell division, cellular polarization, and convergent extension during gastrulation. We further show that the knockdown of Rack1 affects membrane localization of Vangl2 and that the Vangl2-interacting domain of Rack1 has a dominant-negative effect on Vangl2 localization and gastrulation. Moreover, Rack1 antagonizes canonical Wnt signaling. Together, our data suggest that Rack1 regulates the localization of an essential PCP protein and acts as a molecular switch to promote PCP signaling.

The N-(E)-fluorobutenyl-3beta-(para-halo-phenyl)nortropanes 9-12 were synthesized as ligands of the dopamine transporter (DAT) for use as (18)F-labeled positron emission tomography (PET) imaging agents. In vitro competition binding assays demonstrated that compounds 9-12 have a high affinity for the DAT and are selective for the DAT compared to the serotonin and norepinephrine transporters. MicroPET imaging with [(18)F]9-[(18)F]11 in anesthetized cynomolgus monkeys showed high uptake in the putamen with lesser uptake in the caudate, but significant washout of the radiotracer was only observed for [(18)F]9. PET imaging with [(18)F]9 in an awake rhesus monkey showed high and nearly equal uptake in both the putamen and caudate with peak uptake achieved after 20 min followed by a leveling-off for about 10 min and then a steady washout and attainment of a quasi-equilibrium. During the time period 40-80 min postinjection of [(18)F]9, the ratio of uptake in the putamen and caudate vs cerebellum uptake wasor = 4.

Mutations in genes coding for connexin26 (Cx26) and/or Cx30 are linked to approximately half of all cases of human autosomal nonsyndromic prelingual deafness. Cx26 and Cx30 are the two major Cx isoforms found in the cochlea, and they coassemble to form hybrid (heteromeric and heterotypic) gap junctions (GJs). This molecular arrangement implies that homomeric GJs would remain in the cochlea if one of the coassembly partners were mutated resulting in null expression. We generated mice in which extra copies of the Cx26 gene were transgenically expressed from a modified bacterial artificial chromosome in a Cx30 −/− background. In the absence of the Cx30 gene, Cx26 expressed from extra alleles completely restored hearing sensitivity and prevented hair cell death in deaf Cx30 −/− mice. The results indicated that hybrid GJs consisting of Cx26 and Cx30 were not essential for normal hearing in mice and suggested that up-regulation of Cx26 or slowing down its protein degradation might be a therapeutic strategy to prevent and treat deafness caused by Cx30 mutations.

The vertebrate planar cell polarity (PCP) pathway consists of conserved PCP and ciliary genes. During development, the PCP pathway regulates convergent extension (CE) and uniform orientation of sensory hair cells in the cochlea. It is not clear how these diverse morphogenetic processes are regulated by a common set of PCP genes. Here, we show that cellular contacts and geometry change drastically and that the dynamic expression of N-cadherin and E-cadherin demarcates sharp boundaries during cochlear extension. The conditional knockout of a component of the adherens junctions, p120-catenin, leads to the reduction of E-cadherin and N-cadherin and to characteristic cochlear CE defects but not misorientation of hair cells. The specific CE defects in p120-catenin mutants are in contrast to associated CE and hair cell misorientation defects observed in common PCP gene mutants. Moreover, the loss-of-function of a conserved PCP gene, Vangl2, alters the dynamic distribution of N-cadherin and E-cadherin in the cochlea and causes similar abnormalities in cellular morphology to those found in p120-catenin mutants. Conversely, we found that Pcdh15 interacts genetically with PCP genes to regulate the formation of polar hair bundles, but not CE defects in the cochlea. Together, these results indicate that the vertebrate PCP pathway regulates CE and hair cell polarity independently and that a p120-catenin-dependent mechanism regulates CE of the cochlea.

Background Wnt proteins play roles in many biological processes, including axon guidance and cell migration. In the mammalian hindbrain, facial branchiomotor (FBM) neurons undergo a striking rostral to caudal migration, yet little is known of the underlying molecular mechanisms. In this study, we investigated a possible role of Wnts and the planar cell polarity (PCP) pathway in this process. Results Here we demonstrate a novel role for Wnt proteins in guiding FBM neurons during their rostral to caudal migration in the hindbrain. We found that Wnt5a is expressed in a caudalhigh to rostrallow gradient in the hindbrain. Wnt-coated beads chemoattracted FBM neurons to ectopic positions in an explant migration assay. The rostrocaudal FBM migration was moderately perturbed in Wnt5a mutant embryos and severely disrupted in Frizzled3 mutant mouse embryos, and was aberrant following inhibition of Wnt function by secreted Frizzled-related proteins. We also show the involvement of the Wnt/PCP pathway in mammalian FBM neuron migration. Thus, mutations in two PCP genes, Vangl2 and Scribble, caused severe defects in FBM migration. Inhibition of JNK and ROCK kinases strongly and specifically reduced the FBM migration, as well as blocked the chemoattractant effects of ectopic Wnt proteins. Conclusion These results provide in vivo evidence that Wnts chemoattract mammalian FBM neurons and that Wnt5a is a candidate to mediate this process. Molecules of the PCP pathway and the JNK and ROCK kinases also play a role in the FBM migration and are likely mediators of Wnt signalling.

The inner ear sensory organs possess extraordinary structural features necessary to conduct mechanosensory transduction for hearing and balance. Their structural beauty has fascinated scientists since the dawn of modern science and ensured a rigorous pursuit of the understanding of mechanotransduction. Sensory cells of the inner ear display unique structural features that underlie their mechanosensitivity and resolution, and represent perhaps the most distinctive form of a type of cellular polarity, known as planar cell polarity (PCP). Until recently, however, it was not known how the precise PCP of the inner ear sensory organs was achieved during development. Here, we review the PCP of the inner ear and recent advances in the quest for an understanding of its formation.

Background During mouse development, the precursor cells that give rise to the auditory sensory organ, the organ of Corti, are specified prior to embryonic day 14.5 (E14.5). Subsequently, the sensory domain is patterned precisely into one row of inner and three rows of outer sensory hair cells interdigitated with supporting cells. Both the restriction of the sensory domain and the patterning of the sensory mosaic of the organ of Corti involve Notch-mediated lateral inhibition and cellular rearrangement characteristic of convergent extension. This study explores the expression and function of a putative Notch target gene. Results We report that a putative Notch target gene, hairy-related basic helix-loop-helix (bHLH) transcriptional factor Hey2, is expressed in the cochlear epithelium prior to terminal differentiation. Its expression is subsequently restricted to supporting cells, overlapping with the expression domains of two known Notch target genes, Hairy and enhancer of split homolog genes Hes1 and Hes5. In combination with the loss of Hes1 or Hes5, genetic inactivation of Hey2 leads to increased numbers of mis-patterned inner or outer hair cells, respectively. Surprisingly, the ectopic hair cells in Hey2 mutants are accompanied by ectopic supporting cells. Furthermore, Hey2 -/- ;Hes1 -/- and Hey2 -/- ;Hes1 +/- mutants show a complete penetrance of early embryonic lethality. Conclusion Our results indicate that Hey2 functions in parallel with Hes1 and Hes5 in patterning the organ of Corti, and interacts genetically with Hes1 for early embryonic development and survival. Our data implicates expansion of the progenitor pool and/or the boundaries of the developing sensory organ to account for patterning defects observed in Hey2 mutants.