Purpose Connexins 46 (Cx46) and 50 (Cx50) support lens development and homeostasis. Knockout (KO) of Cx50, but not Cx46, causes defects in lens fiber organization, F-actin enrichment, gap junction (GJ) size, ball-and-socket (BS) maturation, and GJ-associated protein distributions. To further determine the unique roles of Cx50 and Cx46, we investigated whether these defects persisted in Cx46 knock-in (Ki) lenses. Ki mice had Cx46 knocked-in to their Cx50 loci, where it was expressed under endogenous Cx50 promoters. Methods Fiber cell morphology and the distribution of lens membrane/cytoskeleton proteins from wild-type (WT), Ki, and Cx50 KO mice were visualized by immunofluorescent labeling and confocal microscopy. Results Cx46 Ki partially rescued Cx50 KO lens fiber defects. Three-week-old Ki lens fibers had typical F-actin distributions but were nonuniformly sized and disorganized. The Cx-associated proteins zonula occludens-1 (ZO-1) and β-dystroglycan (βDys) no longer localized to the nuclei but remained absent from GJs. BS formed, but this occurred with lower than WT frequency. BS appeared with greater frequency in 8-week-old Ki lenses, but so did aberrant balloon-like structures similar to those in Cx50 KO lenses. Unexpectedly, 8-week-old Cx50 KO and Ki cortical lens fibers were no longer disorganized. Conclusions Cx identity is important for some aspects of fiber development (organization, Cx association with ZO-1 and βDys) but not others (F-actin enrichment). Either Cx supports BS maturation, but the sparsity of BS and presence of balloon-like structures in Ki lenses suggest that Cx50 is more capable of doing so. The partial rescue of BS structures may support the rapid growth of cortical fibers to the improved growth of Ki lenses compared to Cx50 KO lenses at young ages. Neither absence of Cx50 nor presence of Ki Cx46 affects cortical fiber cell organization by the age of 8 weeks.
