Planar cell polarity (PCP) refers to the polarization of cells within the plane of a cell sheet. A distinctive epithelial PCP in vertebrates is the uniform orientation of stereociliary bundles of the sensory hair cells in the mammalian cochlea. In addition to establishing epithelial PCP, planar polarization is also required for convergent extension (CE); a polarized cellular movement that occurs during neural tube closure and cochlear extension. Studies in Drosophila and vertebrates have revealed a conserved PCP pathway, including Frizzled (Fz) receptors. Here we use the cochlea as a model system to explore the involvement of known ligands of Fz, Wnt morphogens, in PCP regulation. We show that Wnt5a forms a reciprocal expression pattern with a Wnt antagonist, the secreted frizzled-related protein 3 (Sfrp3 or Frzb), along the axis of planar polarization in the cochlear epithelium. We further demonstrate that Wnt5a antagonizes Frzb in regulating cochlear extension and stereociliary bundle orientation in vitro, and that Wnt5a(-/-) animals have a shortened and widened cochlea. Finally, we show that Wnt5a is required for proper subcellular distribution of a PCP protein, Ltap/Vangl2, and that Wnt5a interacts genetically with Ltap/Vangl2 for uniform orientation of stereocilia, cochlear extension, and neural tube closure. Together, these findings demonstrate that Wnt5a functions in PCP regulation in mice.

Planar cell polarity (PCP) refers to coordinated polarization of cells within the plane of a cell sheet. A conserved signaling pathway is required for the establishment of PCP in epithelial tissues and for polarized cellular rearrangements known as convergent extension. During PCP signaling, core PCP proteins are sorted asymmetrically along the polarization axis; this sorting is thought to direct coordinated downstream morphogenetic changes across the entire tissue. Here, we show that a gene encoding a ciliary protein (a 'ciliary gene'), Ift88, also known as Polaris, is required for establishing epithelial PCP and for convergent extension of the cochlear duct of Mus musculus. We also show that the proper positioning of ciliary basal bodies and the formation of polarized cellular structures are disrupted in mice with mutant ciliary proteins ('ciliary mutants'), whereas core PCP proteins are partitioned normally along the polarization axis. Thus, our data uncover a distinct requirement for ciliary genes in basal body positioning and morphological polarization during PCP regulation.

The mammalian auditory sensory organ, the organ of Corti, consists of sensory hair cells with uniformly oriented stereocilia on the apical surfaces, displaying a distinct planar cell polarity (PCP) parallel to the sensory epithelium 1-3 . It is not clear how this polarity is achieved during differentiation 4-5 . Here we show that the organ of Corti is formed from a thicker and shorter postmitotic primordium through unidirectional extension, characteristic of cellular intercalation known as convergent extension 6 . Mutations in the PCP pathway interfere with this extension, resulting a shortened and widened cochlea, as well as misorientation of stereocilia. Furthermore, parallel to the homologous pathway in Drosophila 7,8 , a mammalian PCP component Dishevelled2 displays PCPdependent polarized subcellular localization across the organ of Corti. Together, these data suggest a conserved molecular mechanism for PCP pathways in invertebrates and vertebrates, and indicate that the mammalian PCP pathway might directly couple cellular intercalations to PCP establishment in the cochlea.Between embryonic day 13 (E13) and E14, precursors for the organ of Corti can be identified as a zone of non-proliferating cells within the cochlear duct using BrdU pulse-labeling (Fig. 1c, brackets) 4,9-10 . Subsequently, a gradient of cell differentiation within the precursor domain initiates near the base of the cochlea and leads to the patterning of one row of inner and three rows of outer hair cells (Fig. 1d , 1e) along the entire length of the cochlea by E18.5 10 , with uniformly oriented stereocilia on the apical surface of hair cells (Fig. 1e, green). From E14.5 to E18.5, the length of the cochlea is increased approximately 2 fold ( Fig. 1a-b). As viewed in cross sections, the developing organ of Corti in the same period is thinned from a 4-5 cell layered primordium (Fig. 1g) to only two layers of cells (Fig. 1h). One layer of hair cells lies above a layer of supporting cell nuclei whose cytoplasmic phalangeal processes reach the apexes of the hair cells, separating them from each other (Fig. 1h). Based on these observations, we have previously proposed that the longer and thinner mature organ is formed from a defined number of postmitotic precursor cells through integrated cellular intercalation movements 4 . BrdU chasing from E14 to E18 further indicated that there was no detectable cell mixing between the primordial organ of Corti and the surrounding cells (Fig. 1d) the hair cells at E14.5 marked by red fluorescent protein (Math1/RFP) were multi-cell layered ( Fig. 1f) within the sensory primordium (Fig. 1g). The thinning of the sensory primordium is accomplished by E18.5 without detectable cell death ( Fig. 1g-h).To further test our hypothesis, we bisected E14.5 cochlear epithelia into apical and basal portions, cultured them in a defined media. Using the cutting wound sites as reference points, we monitored the extension of the organ of Corti from apical-to-basal or basal-to-apical directions for the apical cultures ...

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.

ORCID IDs: 0000-0002-6213-9791 (D.Q.); 0000-0002-5178-0700 (Y.X.)Functional divergence in paralogs is an important genetic source of evolutionary innovation. Actin-depolymerizing factors (ADFs) are among the most important actin binding proteins and are involved in generating and remodeling actin cytoskeletal architecture via their conserved F-actin severing or depolymerizing activity. In plants, ADFs coevolved with actin, but their biochemical properties are diverse. Unfortunately, the biochemical function of most plant ADFs and the potential mechanisms of their functional divergence remain unclear. Here, in vitro biochemical analyses demonstrated that all 11 ADF genes in Arabidopsis thaliana exhibit opposing biochemical properties. Subclass III ADFs evolved F-actin bundling (B-type) function from conserved F-actin depolymerizing (D-type) function, and subclass I ADFs have enhanced D-type function. By tracking historical mutation sites on ancestral proteins, several fundamental amino acid residues affecting the biochemical functions of these proteins were identified in Arabidopsis and various plants, suggesting that the biochemical divergence of ADFs has been conserved during the evolution of angiosperm plants. Importantly, N-terminal extensions on subclass III ADFs that arose from intron-sliding events are indispensable for the alteration of D-type to B-type function. We conclude that the evolution of these N-terminal extensions and several conserved mutations produced the diverse biochemical functions of plant ADFs from a putative ancestor.