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The Rho GTPase Cdc42 regulates hair cell planar polarity and cellular patterning in the developing cochlea.

Kirjavainen A, Laos M, Anttonen T, Pirvola U - Biol Open (2015)

Bottom Line: Atypical protein kinase C (aPKC), a putative Cdc42 effector, colocalized with Cdc42 at the hair cell apex, and aPKC expression was altered upon Cdc42 depletion.Our data suggest that Cdc42 together with aPKC is part of the machinery establishing hair cell planar polarity and that Cdc42 acts on polarity through the cell-surface microtubule network.In addition, our data demonstrates that Cdc42 is required for stereociliogenesis in the immature cochlea.

View Article: PubMed Central - PubMed

Affiliation: Department of Biosciences, Viikinkaari 1, 00014 University of Helsinki, Finland.

No MeSH data available.


Misorientation of stereociliary bundles of outer hair cells of Cdc42loxP/loxP;Fgfr3-iCre-ERT2 mice.Confocal images of whole mount specimens at E18.5. (A,A′) Shown by double-labeling, the medial coil of the control cochlea displays uniform orientation of phalloidin-labeled hair bundles and their laterally pointing vertices. The kinocilia, positive for acetylated tubulin, are located at vertices. (B,B′) The medial coil of the mutant cochlea shows several OHCs with misoriented bundles. (A″,B″) Schematic representations of hair bundle orientations. (C) Quantification (see materials and methods) shows the distribution of randomly oriented bundles in the medial coil of mutant cochleas. (D) Phalloidin labeling shows normal hair bundle orientation in the basal coil of the control cochlea. (E) The basal coil of the mutant specimen shows occasional OHCs with misoriented bundles (arrows), but hair cell rows are normally organized. (F–G) Triple-labeling for F-actin, Vangl2 and acetylated tubulin (kinocilia pseudocolored in yellow) shows Vangl2 expression in the contact sites between the OHC's medial wall and Deiters' cells in the control specimen. (H,H) In the medial coil of the mutant cochlea, Vangl2 expression pattern is influenced by cellular disorganization. (I) In the basal coil of the mutant cochlea where cellular disorganization is absent, Vangl2 expression domain is similar as in the control specimen (compare to Fig. 5G). Abbreviations: OHC, outer hair cell; IHC, inner hair cell. Scale bar shown in I: A–E, 6 µm; F–H′, 5 µm; G,I, 2 µm.
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f05: Misorientation of stereociliary bundles of outer hair cells of Cdc42loxP/loxP;Fgfr3-iCre-ERT2 mice.Confocal images of whole mount specimens at E18.5. (A,A′) Shown by double-labeling, the medial coil of the control cochlea displays uniform orientation of phalloidin-labeled hair bundles and their laterally pointing vertices. The kinocilia, positive for acetylated tubulin, are located at vertices. (B,B′) The medial coil of the mutant cochlea shows several OHCs with misoriented bundles. (A″,B″) Schematic representations of hair bundle orientations. (C) Quantification (see materials and methods) shows the distribution of randomly oriented bundles in the medial coil of mutant cochleas. (D) Phalloidin labeling shows normal hair bundle orientation in the basal coil of the control cochlea. (E) The basal coil of the mutant specimen shows occasional OHCs with misoriented bundles (arrows), but hair cell rows are normally organized. (F–G) Triple-labeling for F-actin, Vangl2 and acetylated tubulin (kinocilia pseudocolored in yellow) shows Vangl2 expression in the contact sites between the OHC's medial wall and Deiters' cells in the control specimen. (H,H) In the medial coil of the mutant cochlea, Vangl2 expression pattern is influenced by cellular disorganization. (I) In the basal coil of the mutant cochlea where cellular disorganization is absent, Vangl2 expression domain is similar as in the control specimen (compare to Fig. 5G). Abbreviations: OHC, outer hair cell; IHC, inner hair cell. Scale bar shown in I: A–E, 6 µm; F–H′, 5 µm; G,I, 2 µm.

Mentions: The read-out of PCP in the OC is the uniform orientation of hair bundles in the plane parallel to the epithelial surface. In cochlear whole mounts prepared from control and mutant mice at E18.5, phalloidin labeling was used to visualize the F-actin-rich stereocilia. Acetylated tubulin was used to mark kinocilia (Fig. 5A–E). In control specimens, hair bundles were uniformly oriented across the epithelium and kinocilia were positioned at the vertex of the bundles (Fig. 5A–A″). Cochleas of mutant mice showed OHC bundles with random orientation, vertices pointing to many directions, demonstrating an abnormal planar polarity phenotype. Unrecombined IHCs showed unaltered bundle orientation (Fig. 5A–E). Similar to patterning and shape abnormalities, bundle orientation defects were concentrated, but not restricted to the medial coil (Fig. 5B–B″). Quantification performed in the medial coil revealed a clear difference in OHC bundle orientations between mutant and control specimens (Fig. 5C). In the medial coil, the same OHC often displayed all abnormalities. In contrast, the basal coil showed OHCs with misoriented bundles lacking patterning and shape defects (Fig. 5D,E). Thus, bundle misorientation can occur independently of the defects at the level of the cell body. This was also found in experiments where induction of recombination was shifted from E13.5 and E14.5 to E15.5 and E16.5, assuming that this later onset of Cdc42 inactivation does not cause patterning defects in the medial coil. Despite the fact that there was a lack of patterning and shape defects, OHCs still showed a planar polarity defect when analyzed at E18.5 (data not shown).


The Rho GTPase Cdc42 regulates hair cell planar polarity and cellular patterning in the developing cochlea.

Kirjavainen A, Laos M, Anttonen T, Pirvola U - Biol Open (2015)

Misorientation of stereociliary bundles of outer hair cells of Cdc42loxP/loxP;Fgfr3-iCre-ERT2 mice.Confocal images of whole mount specimens at E18.5. (A,A′) Shown by double-labeling, the medial coil of the control cochlea displays uniform orientation of phalloidin-labeled hair bundles and their laterally pointing vertices. The kinocilia, positive for acetylated tubulin, are located at vertices. (B,B′) The medial coil of the mutant cochlea shows several OHCs with misoriented bundles. (A″,B″) Schematic representations of hair bundle orientations. (C) Quantification (see materials and methods) shows the distribution of randomly oriented bundles in the medial coil of mutant cochleas. (D) Phalloidin labeling shows normal hair bundle orientation in the basal coil of the control cochlea. (E) The basal coil of the mutant specimen shows occasional OHCs with misoriented bundles (arrows), but hair cell rows are normally organized. (F–G) Triple-labeling for F-actin, Vangl2 and acetylated tubulin (kinocilia pseudocolored in yellow) shows Vangl2 expression in the contact sites between the OHC's medial wall and Deiters' cells in the control specimen. (H,H) In the medial coil of the mutant cochlea, Vangl2 expression pattern is influenced by cellular disorganization. (I) In the basal coil of the mutant cochlea where cellular disorganization is absent, Vangl2 expression domain is similar as in the control specimen (compare to Fig. 5G). Abbreviations: OHC, outer hair cell; IHC, inner hair cell. Scale bar shown in I: A–E, 6 µm; F–H′, 5 µm; G,I, 2 µm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4400594&req=5

f05: Misorientation of stereociliary bundles of outer hair cells of Cdc42loxP/loxP;Fgfr3-iCre-ERT2 mice.Confocal images of whole mount specimens at E18.5. (A,A′) Shown by double-labeling, the medial coil of the control cochlea displays uniform orientation of phalloidin-labeled hair bundles and their laterally pointing vertices. The kinocilia, positive for acetylated tubulin, are located at vertices. (B,B′) The medial coil of the mutant cochlea shows several OHCs with misoriented bundles. (A″,B″) Schematic representations of hair bundle orientations. (C) Quantification (see materials and methods) shows the distribution of randomly oriented bundles in the medial coil of mutant cochleas. (D) Phalloidin labeling shows normal hair bundle orientation in the basal coil of the control cochlea. (E) The basal coil of the mutant specimen shows occasional OHCs with misoriented bundles (arrows), but hair cell rows are normally organized. (F–G) Triple-labeling for F-actin, Vangl2 and acetylated tubulin (kinocilia pseudocolored in yellow) shows Vangl2 expression in the contact sites between the OHC's medial wall and Deiters' cells in the control specimen. (H,H) In the medial coil of the mutant cochlea, Vangl2 expression pattern is influenced by cellular disorganization. (I) In the basal coil of the mutant cochlea where cellular disorganization is absent, Vangl2 expression domain is similar as in the control specimen (compare to Fig. 5G). Abbreviations: OHC, outer hair cell; IHC, inner hair cell. Scale bar shown in I: A–E, 6 µm; F–H′, 5 µm; G,I, 2 µm.
Mentions: The read-out of PCP in the OC is the uniform orientation of hair bundles in the plane parallel to the epithelial surface. In cochlear whole mounts prepared from control and mutant mice at E18.5, phalloidin labeling was used to visualize the F-actin-rich stereocilia. Acetylated tubulin was used to mark kinocilia (Fig. 5A–E). In control specimens, hair bundles were uniformly oriented across the epithelium and kinocilia were positioned at the vertex of the bundles (Fig. 5A–A″). Cochleas of mutant mice showed OHC bundles with random orientation, vertices pointing to many directions, demonstrating an abnormal planar polarity phenotype. Unrecombined IHCs showed unaltered bundle orientation (Fig. 5A–E). Similar to patterning and shape abnormalities, bundle orientation defects were concentrated, but not restricted to the medial coil (Fig. 5B–B″). Quantification performed in the medial coil revealed a clear difference in OHC bundle orientations between mutant and control specimens (Fig. 5C). In the medial coil, the same OHC often displayed all abnormalities. In contrast, the basal coil showed OHCs with misoriented bundles lacking patterning and shape defects (Fig. 5D,E). Thus, bundle misorientation can occur independently of the defects at the level of the cell body. This was also found in experiments where induction of recombination was shifted from E13.5 and E14.5 to E15.5 and E16.5, assuming that this later onset of Cdc42 inactivation does not cause patterning defects in the medial coil. Despite the fact that there was a lack of patterning and shape defects, OHCs still showed a planar polarity defect when analyzed at E18.5 (data not shown).

Bottom Line: Atypical protein kinase C (aPKC), a putative Cdc42 effector, colocalized with Cdc42 at the hair cell apex, and aPKC expression was altered upon Cdc42 depletion.Our data suggest that Cdc42 together with aPKC is part of the machinery establishing hair cell planar polarity and that Cdc42 acts on polarity through the cell-surface microtubule network.In addition, our data demonstrates that Cdc42 is required for stereociliogenesis in the immature cochlea.

View Article: PubMed Central - PubMed

Affiliation: Department of Biosciences, Viikinkaari 1, 00014 University of Helsinki, Finland.

No MeSH data available.