Cell Adhesion Underlies The Role For The Development Of The Mammalian Hearing Organ

Objects Planar cell polarity signaling regulates cochlear extension and coordinated orientation of sensory hair cells in the inner ear.In this research,we characterized the correlation of cell shape and geometry change with adhesion molecules,and explored the role of cell adhesion in the planar cell polarity signaling pathway during terminal differentiation of the mammalian hearing organ.Methods The experimental animals for this research are control,pl20 conditional knockout(CKO)mice,and looptail mutant mice.Using whole mount cochelar preparations and immunostaining,we analyzed the epithelial morphology changes in Embryos E14.5 cochlear duct from apex to base.We characterized the relative subcellular localization of F-actin,PCP proteins Vangl2 and Fzd3,and adhesion molecules aPKC,P120 and E-cadherin in the wholemount cochlear epithelia from E14.5 to E18.5.We analyzed and compared the localization of E-cadherin and planar cell polarity(PCP) proteins(Vangl2,Fzd3)in P120-catenin CKO mice,PCP mutant looptail mice and wild-type control mice.Results①The cellular shape and orientation change drastically during terminal differentiation of the mammalian hearing organ.One feature of the morphological changes occurred is the alternation of the long axis of cells. In the apex,the long axis oriented at medial-to-lateral direction, conversely,in the base,the long axis oriented at a perpendicular direction. Ovall,it is apparent that the developing hearing organ is much narrower at the base at E14.5,as a consequence of a gradient of terminal differentiation that initiates from the base of the cochlear.Cellular shape and geometry changes as well as the overall width and length changes of the developing hearing organ support hat the hearing organ undergoes convergent extension(CE)during terminal differentiation.②P120 protein colocalizes with E-cadherin and PCP protein Vangl2 and Fzd3.③Loss of P120 leads to characteristic CE defects.At E14.5,the developing hearing organ(marked by Sox2 expression) from P120 CKO is significantly widened.At E18.5,the cochleae from the P120 CKO are much shorter than the ones from the control animals.In addition,the hearing organ in the P120 CKO embryo is widened in comparison to the wild-type control animal,evidenced by extra rows of inner hair cells(IHCs)and outer hair cells(OHCs).Surprisingly,the orientation of hair cells in P120 CKO,however,is not apparently affected.④E-cadherin and PCP proteins at cellular junctions are differentiatlly affected in P120 CKO mice.In P120 CKO mice,E-cadherin is undetectable while PCP proteins Vangl2 and Fzd3 still show asymmetric subcellular localization, albeit at reduced levels.⑤Defects in PCP signaling affects asymmetric membrane association of PCP proteins and E-cadherin at cellular junctions. In PCP mutant looptial(lp/lp)mice,membrane association of Fzd3 is not detected and E-cadherin is lost at some cellular junctions.In addition,the remaining junction-localized E-cadherin appears to be more apical in comparison to its localization in the control.The levels of P120 appear to be slightly reduced.Conclusions①Cell-cell contacts and cell geometry change drastically in the developing organ of Corti during terminal differentiation,consistent with cellular behavior of convergent extension.Cell adhesion molecules colocalize with membrane-associated PCP proteins,suggesting an interplay between PCP proteins and adhesive molecules.②In p120 mutants,diminished levels of E-cadherin and reduced membrane-association of PCP proteins likely account for the CE defects observed.③In PCP mutants,asymmetric membrane association of PCP proteins is defective,causing defects in both CE and hair cell orientation.④Together,our data suggest that the membrane-associated PCP proteins,through polarized and asymmetric subcellular localization, provide directions to coordinately orient hair cells and to direct remodeling of cell adhesion for CE.Conversely,cell adhesion junction complexes help to recruit and stabilize PCP complexes at the junctions.In PCP mutants, polarized membrane association of PCP proteins is not achieved,and both CE and hair cell polarity are affected.In cell adhesion mutants,PCP complexes are sorting but are less stable due to the weakening at cellular junctions. The loss of junctional complexes leads to a failure to execute directions from membrane-associated PCP complexes to drive CE.