Distinct roles for N‐Cadherin linked c‐Src and fyn kinases in lens development

Abstract

Background Src family tyrosine kinases (SFKs) are often coincidently expressed but few studies have dissected their individual functions in the same cell during development. Using the classical embryonic lens as our model we investigated SFK signaling in the regulation of both differentiation initiation and morphogenesis, and the distinct functions of c-Src and Fyn in these processes. Results Blocking SFK activity with the highly specific inhibitor PP1 induced initiation of the lens differentiation program but blocked lens fiber cell elongation and organization into mini lens-like structures called lentoids. These dichotomous roles for SFK signaling were discovered to reflect distinct functions of c-Src and Fyn and their differentiation-state-specific recruitment to and action at N-cadherin junctions. c-Src was highly associated with the nascent N-cadherin junctions of undifferentiated lens epithelial cells. Its siRNA knockdown promoted N-cadherin junctional maturation, blocked proliferation, and induced lens cell differentiation. In contrast, Fyn was recruited to mature N-cadherin junctions of differentiating lens cells and siRNA knockdown suppressed differentiation-specific gene expression and blocked morphogenesis. Conclusions Through inhibition of N-cadherin junction maturation c-Src promotes lens epithelial cell proliferation and the maintenance of the lens epithelial cell undifferentiated state, while Fyn, signaling downstream of mature N-cadherin junctions, promotes lens fiber cell morphogenesis.

EphA2 and Src regulate equatorial cell morphogenesis during lens development

“…It might be important to study other molecules that are involved in the restricted activation of Src and cortactin that establish organized meridional rows prior to fiber cell elongation and differentiation. It has recently been reported that Fyn, another member of the Src family, is utilized to regulate N-cadherin junctions in the chick lens fiber cell (Leonard et al, 2013). Since the disruption of Src −/− lens fiber cells is less severe than that of Epha2 −/− lens fiber cells (Fig.…”

“…Studies in chick and quail lens explant models suggest that Src might be involved in posterior capsular cataracts and cortical cataracts (Leonard et al, 2013;Walker et al, 2007;Zhou et al, 2007;Zhou and Menko, 2004). However, Src has not been reported to be directly associated with human cataracts.…”

“…Interestingly, in contrast to Epha2 −/− lenses that have normal lens weight but severely disrupted fiber cell organization, Src −/− lenses 4243 RESEARCH ARTICLE EphA2 in lens cell morphogenesis show a ~15% reduction in lens weight with mild fiber cell alterations (data not shown). The phenotypic variation between Epha2 −/− and Src −/− lens fiber cells suggests that other RTKs are likely to control Src activation to regulate the proliferation of lens epithelial cells and lens size; other members of the Src family, such as Fyn, may act as downstream targets of EphA2/ephrin signaling in the regulation of lens fiber cell organization by affecting Ncadherin localization (Jun et al, 2009;Leonard et al, 2013). This work is consistent with a recent study revealing disorganized lens cells and altered lens refractive index in Epha2 −/− lenses (Shi et al, 2012).…”

Signaling and Gene Regulatory Networks in Mammalian Lens Development

“…E- and N-cadherins are localized at the apical and lateral sides of the lens epithelial cells to maintain their polarity and adhesion to each other, which also requires the RapGAP protein Sipa1l3 and ephrin-A5 signaling [154, 222–225]. In the lens fiber cells, N-cadherin promotes cell elongation and morphogenesis by activating the Src family kinase Fyn, intersecting with EphA2 receptor tyrosine kinases that induce phosphorylation of the Src and F-actin binding protein cortactin to control actin assembly and fiber cell alignment [226, 227]. Also at the lateral side of the lens fiber cells, the gap junction protein Cx50 (Gja8) not only forms channels for ion and metabolite exchange, but also negatively regulates E3 ubiquitin ligase Skp2 to prevent degradation of the cell cycle inhibitor Cdkn1b/p27, promoting cell cycle exit and differentiation [228].…”

Establishment of a Clinically Relevant <em>Ex Vivo</em> Mock Cataract Surgery Model for Investigating Epithelial Wound Repair in a Native Microenvironment

“…3. Study the separated OAZ and CMZ fractions for molecular analysis such as RNA-Seq 24 or biochemical analysis such as western blot or coimmunoprecipitation 23,25 .…”