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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.


Cell shape changes in the organ of Corti of Cdc42loxP/loxP;Fgfr3-iCre-ERT2 mice at E18.5.(A–H) Phalloidin-labeled whole mount specimens from the medial coil of the cochlea viewed under confocal microscopy. Views from the apical level of OHCs, just beneath the stereociliary bundles (A,B; the level defined in the inset), and from the level above the nuclei (C,D; the level defined in the inset). OHCs in the control specimen show uniform diameters, as opposed to OHCs in the mutant specimen. Arrow in D points to an OHC with a small, squeezed cell body. Variation in cell shapes in mutants can also be seen in y-z plane across one OHC row (E,G). In x-y (transverse) plane, DAPI-labeled OHC nuclei (asterisks) in the control cochlea are located at the same level, in contrast to the nuclei in the mutant cochlea (F,H). Note the squeezed cell body of the OHC with an abnormally positioned nucleus (H). (I,J) Immunohistochemistry on paraffin sections shows fodrin expression in the cuticular plates and lateral walls in the apical portion of OHCs of both genotypes. OHCs of the three rows are marked (1–3). In OHCs of mutant mice with a squeezed cell body, cortical fodrin expression extends basally (arrow). Abbreviations: OHC, outer hair cell; IHC, inner hair cell; cp, cuticular plate. Scale bar shown in J: A–J, 5 µm.
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f04: Cell shape changes in the organ of Corti of Cdc42loxP/loxP;Fgfr3-iCre-ERT2 mice at E18.5.(A–H) Phalloidin-labeled whole mount specimens from the medial coil of the cochlea viewed under confocal microscopy. Views from the apical level of OHCs, just beneath the stereociliary bundles (A,B; the level defined in the inset), and from the level above the nuclei (C,D; the level defined in the inset). OHCs in the control specimen show uniform diameters, as opposed to OHCs in the mutant specimen. Arrow in D points to an OHC with a small, squeezed cell body. Variation in cell shapes in mutants can also be seen in y-z plane across one OHC row (E,G). In x-y (transverse) plane, DAPI-labeled OHC nuclei (asterisks) in the control cochlea are located at the same level, in contrast to the nuclei in the mutant cochlea (F,H). Note the squeezed cell body of the OHC with an abnormally positioned nucleus (H). (I,J) Immunohistochemistry on paraffin sections shows fodrin expression in the cuticular plates and lateral walls in the apical portion of OHCs of both genotypes. OHCs of the three rows are marked (1–3). In OHCs of mutant mice with a squeezed cell body, cortical fodrin expression extends basally (arrow). Abbreviations: OHC, outer hair cell; IHC, inner hair cell; cp, cuticular plate. Scale bar shown in J: A–J, 5 µm.

Mentions: In addition to patterning defects, cochleas of mutant mice showed abnormally shaped OHCs, revealed in phalloidin-labeled whole mount specimens at birth (Fig. 4A–H). OHCs with squeezed morphology were found (Fig. 4E,G) and their nuclei were positioned at variable heights in the apico-basal axis of the sensory epithelium, in contrast to OHCs of control animals with uniformly positioned nuclei (Fig. 4F,H). Similar to patterning defects, OHCs with abnormal morphology were concentrated to the medial coil and were intermingled with OHCs with a normal morphology. To find out whether the atypical OHC shape was linked with changes in the expression of actin-interacting-proteins, paraffin sections were immunolabeled for alpha-fodrin (non-erythroid spectrin) that cross-links actin filaments. Fodrin is a constituent of the cuticular plate and cortical lattice of hair cells (Ylikoski et al., 1992). In agreement, control specimens showed fodrin expression in these regions in the apical portion of hair cells (Fig. 4I,J). In the Cdc42 mutant mice, the cuticular plate staining was unaltered, but fodrin as well as phalloidin labeling in the cortex extended towards the basal side of the squeezed OHCs. This was best seen in top-down views showing F-actin accumulation at the most shrunken level of the atypical, flask-shaped OHCs (Fig. 4D). These data suggest that Cdc42 regulates the cortical actin cytoskeleton and that defects in this regulation lead to cell shape abnormalities.


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)

Cell shape changes in the organ of Corti of Cdc42loxP/loxP;Fgfr3-iCre-ERT2 mice at E18.5.(A–H) Phalloidin-labeled whole mount specimens from the medial coil of the cochlea viewed under confocal microscopy. Views from the apical level of OHCs, just beneath the stereociliary bundles (A,B; the level defined in the inset), and from the level above the nuclei (C,D; the level defined in the inset). OHCs in the control specimen show uniform diameters, as opposed to OHCs in the mutant specimen. Arrow in D points to an OHC with a small, squeezed cell body. Variation in cell shapes in mutants can also be seen in y-z plane across one OHC row (E,G). In x-y (transverse) plane, DAPI-labeled OHC nuclei (asterisks) in the control cochlea are located at the same level, in contrast to the nuclei in the mutant cochlea (F,H). Note the squeezed cell body of the OHC with an abnormally positioned nucleus (H). (I,J) Immunohistochemistry on paraffin sections shows fodrin expression in the cuticular plates and lateral walls in the apical portion of OHCs of both genotypes. OHCs of the three rows are marked (1–3). In OHCs of mutant mice with a squeezed cell body, cortical fodrin expression extends basally (arrow). Abbreviations: OHC, outer hair cell; IHC, inner hair cell; cp, cuticular plate. Scale bar shown in J: A–J, 5 µm.
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Related In: Results  -  Collection

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f04: Cell shape changes in the organ of Corti of Cdc42loxP/loxP;Fgfr3-iCre-ERT2 mice at E18.5.(A–H) Phalloidin-labeled whole mount specimens from the medial coil of the cochlea viewed under confocal microscopy. Views from the apical level of OHCs, just beneath the stereociliary bundles (A,B; the level defined in the inset), and from the level above the nuclei (C,D; the level defined in the inset). OHCs in the control specimen show uniform diameters, as opposed to OHCs in the mutant specimen. Arrow in D points to an OHC with a small, squeezed cell body. Variation in cell shapes in mutants can also be seen in y-z plane across one OHC row (E,G). In x-y (transverse) plane, DAPI-labeled OHC nuclei (asterisks) in the control cochlea are located at the same level, in contrast to the nuclei in the mutant cochlea (F,H). Note the squeezed cell body of the OHC with an abnormally positioned nucleus (H). (I,J) Immunohistochemistry on paraffin sections shows fodrin expression in the cuticular plates and lateral walls in the apical portion of OHCs of both genotypes. OHCs of the three rows are marked (1–3). In OHCs of mutant mice with a squeezed cell body, cortical fodrin expression extends basally (arrow). Abbreviations: OHC, outer hair cell; IHC, inner hair cell; cp, cuticular plate. Scale bar shown in J: A–J, 5 µm.
Mentions: In addition to patterning defects, cochleas of mutant mice showed abnormally shaped OHCs, revealed in phalloidin-labeled whole mount specimens at birth (Fig. 4A–H). OHCs with squeezed morphology were found (Fig. 4E,G) and their nuclei were positioned at variable heights in the apico-basal axis of the sensory epithelium, in contrast to OHCs of control animals with uniformly positioned nuclei (Fig. 4F,H). Similar to patterning defects, OHCs with abnormal morphology were concentrated to the medial coil and were intermingled with OHCs with a normal morphology. To find out whether the atypical OHC shape was linked with changes in the expression of actin-interacting-proteins, paraffin sections were immunolabeled for alpha-fodrin (non-erythroid spectrin) that cross-links actin filaments. Fodrin is a constituent of the cuticular plate and cortical lattice of hair cells (Ylikoski et al., 1992). In agreement, control specimens showed fodrin expression in these regions in the apical portion of hair cells (Fig. 4I,J). In the Cdc42 mutant mice, the cuticular plate staining was unaltered, but fodrin as well as phalloidin labeling in the cortex extended towards the basal side of the squeezed OHCs. This was best seen in top-down views showing F-actin accumulation at the most shrunken level of the atypical, flask-shaped OHCs (Fig. 4D). These data suggest that Cdc42 regulates the cortical actin cytoskeleton and that defects in this regulation lead to cell shape abnormalities.

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.