Regulation of polarized extension and planar cell polarity in the cochlea by the vertebrate PCP pathway - PubMed (original) (raw)

Comparative Study

doi: 10.1038/ng1622. Epub 2005 Aug 14.

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Comparative Study

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

Jianbo Wang et al. Nat Genet. 2005 Sep.

Abstract

The mammalian auditory sensory organ, the organ of Corti, consists of sensory hair cells with uniformly oriented stereocilia on the apical surfaces and has a distinct planar cell polarity (PCP) parallel to the sensory epithelium. It is not certain how this polarity is achieved during differentiation. 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. Mutations in the PCP pathway interfere with this extension, resulting a shorter and wider cochlea as well as misorientation of stereocilia. Furthermore, parallel to the homologous pathway in Drosophila melanogaster, a mammalian PCP component Dishevelled2 shows PCP-dependent polarized subcellular localization across the organ of Corti. Taken together, these data suggest that there is 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.

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Figures

Fig. 1

Fig. 1

Differentiation of the organ of Corti from its postmitotic precursor domain. a-b: Paint-filled inner ears at E14.5 (a) and E18.5 (b). Arrows indicate the base of the cochlea. Scale: 200 μm. c-e: Confocal scans of E14.5 (c) or E18.5 (d) cochlear whole-mounts pulse-labeled for 6 hours or chased with BrdU for 4 days, respectively, and double-stained for BrdU(red) and myosinVI (green). The bracket in (c) indicates the non-proliferating primordium, within which a row of inner hair cells (indicated by an arrowhead) is seen at the medial edge (c). By E18.5 (d), no BrdU+ cells were detected in the organ of Corti. The organ of Corti from a P1 animal (e) displays uniformly oriented stereocilia (green). The vortexes with a single kinocilium (red) of the V-shaped stereocilia point to the lateral side. Scale: 50 μm (c-d); 10 μm (e). f-h: The expression of Math1/RFP (f) marks the earliest hair cells in a multi-layered column (arrowheads) at the medial edge of the precursor domain (bracket), which is demarcated by Isl1 expression in E14.5 cross sections (g). By E18.5 (h), the organ of Corti has one layer of hair cells (red) above the layer of supporting cell nuclei (indicated by an arrow). No activated-caspase3+ cells (green in f and g, marked by asterisks) were detected within the organ of Corti from E14.5 to E18.5. Scale: 50 μm. Inner and outer hair cells were indicated by arrowheads and brackets (d, e, h), respectively. The magenta arrowhead (e) indicates the pillar cell region separating inner from outer hair cells.

Fig. 2

Fig. 2

Unidirectional extension of the organ of Corti in vitro. a-b: bisected basal (a) and apical (b) portions of the same cochlear duct. Nuclei-localized GFP is expressed under the control of Math1 enhancers, marking the hair cells in live cultures. The bisection sites were indicated by arrows. Both the basal (basal culture) and apical (apical culture) portions of the cochlea were used for in vitro cultures (c-e). Note that the direction of prospective extensions beyond the wound sites for the basal or the apical cultures will be basal-to-apical or apical-to-basal, respectively. c-e: both the basal (basal culture) and apical (apical culture) portions of the same cochlea (a-b) were cultured and extensions were monitored using the cutting wound sites (indicated by arrows) as reference points (c-e). Hair cells (GFP+) differentiated normally in the apical culture after 4 DIV (c). However, the rows of hair cells did not pass the bisection site (c), indicating no apical-to-basal extension in the apical culture. The rows of hair cells in the basal cultures (d and e) extended past the bisection sites (d, e) from basal-to-apical direction. No BrdU incorporation in the organ of Corti was detected in extended basal cultures (e). The rows of hair cells were bracketed with a pair of arrowheads, and the orientation of the explants was indicated in (c-e).The bottom panels (f-j) are corresponding phase images of the upper panels.

Fig. 3

Fig. 3

Presence of a functional PCP pathway in the developing organ of Corti. a-c: in situ hybridization of Celsr1 (a) and Dvl1 (b) at E16.5, and DVL2-EGFP signals (c) at E18.5. The brackets in (a-b) indicate the organ of Corti. d-g: Confocal scans of the E17.5 organ of Corti at 80% (d), 75% (e), 50% (f), and 25% (g) from the base of the cochlear duct. At the base (g), hair cells are differentiated and stereocilia (red) are visible. In the medial region (f), the stereocilia are less developed and not visible (red). However, both inner and outer hair cells are differentiated (blue) and the kinocilia (green) are eccentric, indicating a polarized region. At the apical region (d-e), myosinVI expression (blue) has yet to complete in the outer hair cell region and the kinocilia (green) are centric, indicating a transition stage prior to the polarization process. h-m: Confocal scanning of DVL2-EGFP (green) in a whole mount organ of Corti at E17.5 from the corresponding apical (h), medial (i), and basal (j) regions as shown in d-g. Counter-staining with hair cell membrane (l, red) and DVL2-EGFP (k) at E18.5 indicated DVL2-EGFP signal at the lateral side of hair cells (m) o-p: DVL2-EGFP in wild-type (o) and Lp/Lp (p) littermates at E18.5. Note the loss of the wild-type (o) subcellular localization of DVL2-EGFP in the Lp/Lp mutant (p, the asterisks mark hair cells with DVL2-EGFP at the medial side). Arrowheads and brackets (c-p) mark the inner and outer hair cells, respectively. Scale: 50 μm (a-m).

Fig. 4

Fig. 4

The stereocilia of the organ of Corti displayed abnormal polarity in Dvl1−/−;Dvl2−/− (DvlDKO), Lp/Lp, and Dvl2−/−;Lp/+ embryos. a-i: Confocal surface scanning of organs of Corti stained for stereocilia (red) and kinocilia (green) from E18.5 wild-type (a-c), Lp/Lp (d-f), DvlDKO(g-i) and Dvl2−/−; Lp/+ (m-o) embryos. Note that the stereocilia were readily stained in the basal and medial regions of the cochlea at this stage in the control (b-c), while they were less developed in the apical region (c). j-k: Scanning EM of the organs of Corti at comparable medial regions from E18.5 control Dvl1−/−;Dvl2+/− (j) and DvlDKO embryos (k). l: Higher magnification of SEM image for the two cells circled in (k). Note the relative positions of kinocilium (arrowhead) and stereocilia were maintained while the orientation of the stereocilia was altered in one of the cells. The outer and inner hair cell regions (a-k) are indicated by brackets and arrowheads, respectively. Scale bars: 50 μm (a-i); 10 μm (j and k); 952 nm (l).

Fig. 5

Fig. 5

Shortened and widened cochlear duct and its sensory organ, and defective extension in PCP mutants. a: inner ears from PCP mutants, DvlDKO, Lp/Lp, Dvl2−/−; Lp/+, and control embryos at E18.5. The brackets indicate the cochlea portion of the inner ear. The remainder is vestibule. Scale: 500 μm. b-c: dissected cochlear ducts (b) from a control (upper) and DvlDKO (lower) E18.5 embryos. Comparable regions from both samples were shown (c). The lines outline the diameters of the cochlear ducts at comparable regions (b and c). Scale: 200 μm. d-e: confocal images of the apical region of the organ of Corti from the control (d) and DvlDKO (e) E18.5 littermates. MyosinVI (green) for hair cells and acetylated α-tubulin (red) for kinocilia stainings were shown. Notice the organized patterning of both hair cells and kinocilia in the control samples (d) vs. increased hair cell rows and displacement of kinocilia in the mutant sample (e). Scale: 25 μm. f-h: overlays of myosin VI (green) and phase images of the organ of Corti at the apical-most (f), one fourth from the apex (g), and the basal-medial (h) regions of the cochlea from E18.5 DvlDKO mutants. The asterisks mark the cells that are yet to complete the thinning process. Scale: 25 μm.

Fig. 6

Fig. 6

Direct requirement of PCP pathway in extension and planar cell polarity of the organ of Corti. a-b: inner ears from control and Lp/Lp littermates at E14.5. Brackets mark the cochlear portion of the inner ear. c-f: intact cochlear ducts from control (c and e) and Lp/Lp (d and f) cultured 6 DIV. Diameters of the cochlear ducts at the comparable apical regions were outlined (e and f). Hair cells were visualized by Math1-EGFP. g-k: confocal images stereocilia (phalloidin) for control (g-i) and Lp/Lp (j-k) cultures at the apical (g and j), medial (h and k), and basal (i) regions. Scale: 25 μm. l-q: bisected basal regions of the cochlear ducts from wild type (l-m) and Lp/Lp (n-o) cultured for 6 DIV, and the confocal images of stereocilia near the bisection sites for the wildtype (p) and Lp/Lp (q). Arrows in l-o indicate the bisection sites. Arrowheads and brackets in (g-k, p-q) mark the inner and outer hair cell regions, respectively, and magenta arrowheads mark the pillar cell region that was stained with p75 (h, k, p,magenta).

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