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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.


Recombination pattern obtained with the Fgfr3-iCre-ERT2 mice.(A–E) Fgfr3-iCre-ERT2;Ai14(tdTomato) mice treated with tamoxifen at E13.5 and E14.5 show recombination in the organ of Corti and in the cochlear capsule, revealed by red fluorescence protein (RFP) immunohistochemistry on paraffin sections at E18.5. Recombination follows a base-to-apex gradient along the length of the cochlear duct. All Deiters' and pillar cells and OHCs in the basal (B) and lower medial coils (C) are recombined. A large part of these cells are recombined in the upper medial coil (D), but only rare recombined cells are found in the apex (E). Inner hair cells (asterisks in B–D) are not stained for RFP. The three OHC rows are numbered. Abbreviations: OHCs, outer hair cells; PCs, pillar cells; DCs, Deiters' cells; CC, cochlear capsule. Scale bar shown in E: A, 25 µm; B–E, 7 µm.
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f01: Recombination pattern obtained with the Fgfr3-iCre-ERT2 mice.(A–E) Fgfr3-iCre-ERT2;Ai14(tdTomato) mice treated with tamoxifen at E13.5 and E14.5 show recombination in the organ of Corti and in the cochlear capsule, revealed by red fluorescence protein (RFP) immunohistochemistry on paraffin sections at E18.5. Recombination follows a base-to-apex gradient along the length of the cochlear duct. All Deiters' and pillar cells and OHCs in the basal (B) and lower medial coils (C) are recombined. A large part of these cells are recombined in the upper medial coil (D), but only rare recombined cells are found in the apex (E). Inner hair cells (asterisks in B–D) are not stained for RFP. The three OHC rows are numbered. Abbreviations: OHCs, outer hair cells; PCs, pillar cells; DCs, Deiters' cells; CC, cochlear capsule. Scale bar shown in E: A, 25 µm; B–E, 7 µm.

Mentions: Cdc42 is widely expressed and has several roles in developing cells (Melendez et al., 2011). Consistently, Cdc42 knockout mice show early-embryonic lethality (Chen et al., 2000). As we wished to reveal the role of Cdc42 during cellular differentiation in the late-embryonic OC, a conditional and inducible approach was required to bypass the likely effects of Cdc42 on dividing progenitor cells of the early otocyst. We used the Fgfr3-iCre-ERT2 transgenic mouse line (Young et al., 2010) that was crossed with the Cdc42loxP/loxP mutant mice (Wu et al., 2006). We have previously shown that Fgfr3-iCre-ERT2 mice can be used for inducible, supporting cell-specific gene inactivation in the postnatal OC (Anttonen et al., 2012). To study recombination characteristics in the embryonic cochlea, Fgfr3-iCre-ERT2 mice were crossed with the ROSA26tm14(CAG-tdTomato) reporter mice to generate Fgfr3-iCre-ERT2;Ai14(tdTomato) mice. Recombined cells were recognized by RFP immunohistochemistry in paraffin-embedded cross-sections through the cochlea (Fig. 1A–E). Recombination was induced at E13.5 and E14.5, at the stages when Fgfr3 starts to be expressed in the presumptive OC (Hayashi et al., 2010). At this age, precursor cells have exited the cell cycle and are initiating differentiation into hair cells or supporting cells (Ruben, 1967; Chen and Segil, 1999). At E18.5, basal and medial coils of the cochlea of the Fgfr3-iCre-ERT2;Ai14(tdTomato) double-transgenic mice showed recombination in OHCs and in two types of supporting cells, the Deiters' and pillar cells (Fig. 1B–D). Importantly, the whole population of these cells showed recombination in the basal and medial coils, the regions of the cochlear duct analyzed in the present study. IHCs lacked RFP signal (asterisks in Fig. 1B–D). The apical cochlear coil showed recombination only in scattered sensory epithelial cells (Fig. 1E). This recombination pattern correlates well with the cell type-specific expression of Fgfr3 and with the dynamic Fgfr3 expression along the base-to-apex differentiation gradient in the developing cochlear duct (Hayashi et al., 2010). We conclude that Fgfr3-iCre-ERT2-mediated recombination efficiently targets OHCs, Deiters' cells and pillar cells of the late-embryonic OC.


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)

Recombination pattern obtained with the Fgfr3-iCre-ERT2 mice.(A–E) Fgfr3-iCre-ERT2;Ai14(tdTomato) mice treated with tamoxifen at E13.5 and E14.5 show recombination in the organ of Corti and in the cochlear capsule, revealed by red fluorescence protein (RFP) immunohistochemistry on paraffin sections at E18.5. Recombination follows a base-to-apex gradient along the length of the cochlear duct. All Deiters' and pillar cells and OHCs in the basal (B) and lower medial coils (C) are recombined. A large part of these cells are recombined in the upper medial coil (D), but only rare recombined cells are found in the apex (E). Inner hair cells (asterisks in B–D) are not stained for RFP. The three OHC rows are numbered. Abbreviations: OHCs, outer hair cells; PCs, pillar cells; DCs, Deiters' cells; CC, cochlear capsule. Scale bar shown in E: A, 25 µm; B–E, 7 µm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4400594&req=5

f01: Recombination pattern obtained with the Fgfr3-iCre-ERT2 mice.(A–E) Fgfr3-iCre-ERT2;Ai14(tdTomato) mice treated with tamoxifen at E13.5 and E14.5 show recombination in the organ of Corti and in the cochlear capsule, revealed by red fluorescence protein (RFP) immunohistochemistry on paraffin sections at E18.5. Recombination follows a base-to-apex gradient along the length of the cochlear duct. All Deiters' and pillar cells and OHCs in the basal (B) and lower medial coils (C) are recombined. A large part of these cells are recombined in the upper medial coil (D), but only rare recombined cells are found in the apex (E). Inner hair cells (asterisks in B–D) are not stained for RFP. The three OHC rows are numbered. Abbreviations: OHCs, outer hair cells; PCs, pillar cells; DCs, Deiters' cells; CC, cochlear capsule. Scale bar shown in E: A, 25 µm; B–E, 7 µm.
Mentions: Cdc42 is widely expressed and has several roles in developing cells (Melendez et al., 2011). Consistently, Cdc42 knockout mice show early-embryonic lethality (Chen et al., 2000). As we wished to reveal the role of Cdc42 during cellular differentiation in the late-embryonic OC, a conditional and inducible approach was required to bypass the likely effects of Cdc42 on dividing progenitor cells of the early otocyst. We used the Fgfr3-iCre-ERT2 transgenic mouse line (Young et al., 2010) that was crossed with the Cdc42loxP/loxP mutant mice (Wu et al., 2006). We have previously shown that Fgfr3-iCre-ERT2 mice can be used for inducible, supporting cell-specific gene inactivation in the postnatal OC (Anttonen et al., 2012). To study recombination characteristics in the embryonic cochlea, Fgfr3-iCre-ERT2 mice were crossed with the ROSA26tm14(CAG-tdTomato) reporter mice to generate Fgfr3-iCre-ERT2;Ai14(tdTomato) mice. Recombined cells were recognized by RFP immunohistochemistry in paraffin-embedded cross-sections through the cochlea (Fig. 1A–E). Recombination was induced at E13.5 and E14.5, at the stages when Fgfr3 starts to be expressed in the presumptive OC (Hayashi et al., 2010). At this age, precursor cells have exited the cell cycle and are initiating differentiation into hair cells or supporting cells (Ruben, 1967; Chen and Segil, 1999). At E18.5, basal and medial coils of the cochlea of the Fgfr3-iCre-ERT2;Ai14(tdTomato) double-transgenic mice showed recombination in OHCs and in two types of supporting cells, the Deiters' and pillar cells (Fig. 1B–D). Importantly, the whole population of these cells showed recombination in the basal and medial coils, the regions of the cochlear duct analyzed in the present study. IHCs lacked RFP signal (asterisks in Fig. 1B–D). The apical cochlear coil showed recombination only in scattered sensory epithelial cells (Fig. 1E). This recombination pattern correlates well with the cell type-specific expression of Fgfr3 and with the dynamic Fgfr3 expression along the base-to-apex differentiation gradient in the developing cochlear duct (Hayashi et al., 2010). We conclude that Fgfr3-iCre-ERT2-mediated recombination efficiently targets OHCs, Deiters' cells and pillar cells of the late-embryonic OC.

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.