Regulation of polarized extension and planar cell polarity in the cochlea by the vertebrate PCP pathway

Abstract

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 ...

Basic helix–loop–helix gene Hes6 delineates the sensory hair cell lineage in the inner ear

“…We and others have observed the onset of Math1 in columns of cells spanning the depth of the cochlear epithelium within the primordial organ of Corti (Chen et al, 2002;Kiernan et al, 2005a;Wang et al, 2005;Fig. 2B,C), and we have hypothesized that these columns of cells are the early hair cell precursors in a multi-cell layered primordial organ of Corti at E14.5. Subsequently, cellular intercalation movements, characteristic of convergent extension (Keller, 2002), lead to the extension of the organ of Corti and the formation of a single hair cell layer during terminal differentiation (Chen et al, 2002;Montcouquiol et al, 2003;McKenzie et al, 2004;Wang et al, 2005). At the basal region of the cochlea, the presumptive hair cell precursors marked by the expression of Math1 were seen as two columns of cells, presumably at the inner hair cell and the first row of outer hair cell locations (Fig.…”

“…Intriguingly, Math1 is expressed in a subpopulation of cells within the primordial organ of Corti as columns of cells spanning the depth of the cochlear epithelium at this stage ( Fig. 2; Chen et al, 2002;Kiernan et al, 2005a;Wang et al, 2005). We and others previously have shown that the longer and thinner mature organ of Corti is derived from a shorter and thicker postmitotic precursor domain (Ruben, 1967;Chen and Segil, 1999;Lowenheim et al, 1999), likely through cellular intercalation movements characteristic of convergent extension (Chen et al, 2002;McKenzie et al, 2004;Wang et al, 2005).…”

Role of chromatin remodeling gene Cecr2 in neurulation and inner ear development

“…Vangl2 mRNA shows similar expression in both locations at 14.5 dpc and 16.5 dpc (Montcouquiol et al, 2003). Celsr1, Dvl1, and Dvl2 are found in the statoacoustic ganglion (Wang et al, 2005). Unlike Cecr2, all the core PCP genes tested have also been found expressed in the sensory hair cells themselves (Wang et al, 2005;Jones and Chen, 2007;Seifert and Mlodzik, 2007).…”

“…Celsr1, Dvl1, and Dvl2 are found in the statoacoustic ganglion (Wang et al, 2005). Unlike Cecr2, all the core PCP genes tested have also been found expressed in the sensory hair cells themselves (Wang et al, 2005;Jones and Chen, 2007;Seifert and Mlodzik, 2007). However, Wnt5a expression also appears in the cochlear floor and not the hair cells (Qian et al, 2007).…”

“…Homologues in vertebrates include Vangl1/ 2, Frizzled3 and 6, Disheveled1-3, Celsr1, Prickle, Scribbled, PTK7, and Ankrd6 (Schwarz-Romond et al, 2002;Carreira-Barbosa et al, 2003;Curtin et al, 2003;Montcouquiol et al, 2003;Lu et al, 2004;Wang et al, 2006a,b;Torban et al, 2007). Examples of PCP can be observed throughout vertebrate embryogenesis, most notably in the convergent extension (CE) movements required to elongate and shape the neural epithelium during early neurulation, as well as the highly organized epithelium within the cochlear sensory structures (Park and Moon, 2002;Wang et al, 2005). PCP has also been implicated in the cell movements that form the eyelid (Murdoch et al, 2001;Wang and Nathans, 2007).…”