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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.


Related in: MedlinePlus

Cdc42 in situ hybridization and immunostaining in the organ of Corti at E18.5.(A) Schematic representation of the cytoarchitecture of the organ of Corti at E18.5. (B,C) The antisense (as) probe shows ubiquitous Cdc42 mRNA expression in the sensory epithelium (B). The Cdc42 sense (s) probe reveals the background level. Asterisk marks IHC. The three OHC rows are numbered. (C). (D–F) Confocal images of cochlear whole mount specimens stained for antibodies against Cdc42. Strong expression is seen in the stereocilia of hair bundles and weaker expression around centrioles (arrows), lateral to the bundle (D). Double-labeling shows the absence of Cdc42 immunostaining in the Cdc42loxP/loxP;Fgfr3-iCre-ERT2 mice (E), while nectin 3 marks the junctions between OHCs and Deiters' cells (E′). Control animals show weak Cdc42 staining in the medial surface domain of OHCs. Contours of an OHC are outlined and a line is drawn to separate the medial (m) and lateral (l) surface domains. Staining is also found at the level of adherens junctions between OHCs and Deiters' cells (G). (F) Schematic picture shows the planes and areas covered in the immunofluorescence views. The red dot marks the site of the kinocilium and basal body. Abbreviations: IHC, inner hair cell; OHCs, outer hair cells; DCs, Deiters' cells; PCs, pillar cells. Scale bar shown in G: A–C, 20 µm; D, 3 µm; E–G, 5 µm.
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f02: Cdc42 in situ hybridization and immunostaining in the organ of Corti at E18.5.(A) Schematic representation of the cytoarchitecture of the organ of Corti at E18.5. (B,C) The antisense (as) probe shows ubiquitous Cdc42 mRNA expression in the sensory epithelium (B). The Cdc42 sense (s) probe reveals the background level. Asterisk marks IHC. The three OHC rows are numbered. (C). (D–F) Confocal images of cochlear whole mount specimens stained for antibodies against Cdc42. Strong expression is seen in the stereocilia of hair bundles and weaker expression around centrioles (arrows), lateral to the bundle (D). Double-labeling shows the absence of Cdc42 immunostaining in the Cdc42loxP/loxP;Fgfr3-iCre-ERT2 mice (E), while nectin 3 marks the junctions between OHCs and Deiters' cells (E′). Control animals show weak Cdc42 staining in the medial surface domain of OHCs. Contours of an OHC are outlined and a line is drawn to separate the medial (m) and lateral (l) surface domains. Staining is also found at the level of adherens junctions between OHCs and Deiters' cells (G). (F) Schematic picture shows the planes and areas covered in the immunofluorescence views. The red dot marks the site of the kinocilium and basal body. Abbreviations: IHC, inner hair cell; OHCs, outer hair cells; DCs, Deiters' cells; PCs, pillar cells. Scale bar shown in G: A–C, 20 µm; D, 3 µm; E–G, 5 µm.

Mentions: To study Cdc42 mRNA expression in the developing cochlea, we used radioactive in situ hybridization on paraffin sections. As analyzed at E13.5, E15.5, E18.5 and P0, Cdc42 was ubiquitously expressed in the presumptive and, later, in differentiating hair cells and supporting cells (Fig. 2A,B; data not shown). Signal obtained by the Cdc42 antisense probe clearly surpassed the background signal obtained with the Cdc42 sense probe (Fig. 2B,C). Upon tamoxifen-induced recombination, the level of Cdc42 antisense signal in the OC of the Cdc42loxP/loxP;Fgfr3-iCre-ERT2 mice (hereafter termed as the mutant mice) was comparable to background signal, consistent with prior studies (data not shown; Anttonen et al., 2012).


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)

Cdc42 in situ hybridization and immunostaining in the organ of Corti at E18.5.(A) Schematic representation of the cytoarchitecture of the organ of Corti at E18.5. (B,C) The antisense (as) probe shows ubiquitous Cdc42 mRNA expression in the sensory epithelium (B). The Cdc42 sense (s) probe reveals the background level. Asterisk marks IHC. The three OHC rows are numbered. (C). (D–F) Confocal images of cochlear whole mount specimens stained for antibodies against Cdc42. Strong expression is seen in the stereocilia of hair bundles and weaker expression around centrioles (arrows), lateral to the bundle (D). Double-labeling shows the absence of Cdc42 immunostaining in the Cdc42loxP/loxP;Fgfr3-iCre-ERT2 mice (E), while nectin 3 marks the junctions between OHCs and Deiters' cells (E′). Control animals show weak Cdc42 staining in the medial surface domain of OHCs. Contours of an OHC are outlined and a line is drawn to separate the medial (m) and lateral (l) surface domains. Staining is also found at the level of adherens junctions between OHCs and Deiters' cells (G). (F) Schematic picture shows the planes and areas covered in the immunofluorescence views. The red dot marks the site of the kinocilium and basal body. Abbreviations: IHC, inner hair cell; OHCs, outer hair cells; DCs, Deiters' cells; PCs, pillar cells. Scale bar shown in G: A–C, 20 µm; D, 3 µm; E–G, 5 µm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f02: Cdc42 in situ hybridization and immunostaining in the organ of Corti at E18.5.(A) Schematic representation of the cytoarchitecture of the organ of Corti at E18.5. (B,C) The antisense (as) probe shows ubiquitous Cdc42 mRNA expression in the sensory epithelium (B). The Cdc42 sense (s) probe reveals the background level. Asterisk marks IHC. The three OHC rows are numbered. (C). (D–F) Confocal images of cochlear whole mount specimens stained for antibodies against Cdc42. Strong expression is seen in the stereocilia of hair bundles and weaker expression around centrioles (arrows), lateral to the bundle (D). Double-labeling shows the absence of Cdc42 immunostaining in the Cdc42loxP/loxP;Fgfr3-iCre-ERT2 mice (E), while nectin 3 marks the junctions between OHCs and Deiters' cells (E′). Control animals show weak Cdc42 staining in the medial surface domain of OHCs. Contours of an OHC are outlined and a line is drawn to separate the medial (m) and lateral (l) surface domains. Staining is also found at the level of adherens junctions between OHCs and Deiters' cells (G). (F) Schematic picture shows the planes and areas covered in the immunofluorescence views. The red dot marks the site of the kinocilium and basal body. Abbreviations: IHC, inner hair cell; OHCs, outer hair cells; DCs, Deiters' cells; PCs, pillar cells. Scale bar shown in G: A–C, 20 µm; D, 3 µm; E–G, 5 µm.
Mentions: To study Cdc42 mRNA expression in the developing cochlea, we used radioactive in situ hybridization on paraffin sections. As analyzed at E13.5, E15.5, E18.5 and P0, Cdc42 was ubiquitously expressed in the presumptive and, later, in differentiating hair cells and supporting cells (Fig. 2A,B; data not shown). Signal obtained by the Cdc42 antisense probe clearly surpassed the background signal obtained with the Cdc42 sense probe (Fig. 2B,C). Upon tamoxifen-induced recombination, the level of Cdc42 antisense signal in the OC of the Cdc42loxP/loxP;Fgfr3-iCre-ERT2 mice (hereafter termed as the mutant mice) was comparable to background signal, consistent with prior studies (data not shown; Anttonen et al., 2012).

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.


Related in: MedlinePlus