Conditional ablation of the Notch2 receptor in the ocular lens. Dev Biol

Laboratory of Molecular and Developmental Biology, National Eye Institute, National Institutes of Health, Bethesda, MD 20892, USA.
Developmental Biology (Impact Factor: 3.55). 11/2011; 362(2):219-29. DOI: 10.1016/j.ydbio.2011.11.011
Source: PubMed


Notch signaling is essential for proper lens development, however the specific requirements of individual Notch receptors have not been investigated. Here we report the lens phenotypes of Notch2 conditionally mutant mice, which exhibited severe microphthalmia, reduced pupillary openings, disrupted fiber cell morphology, eventual loss of the anterior epithelium, fiber cell dysgenesis, denucleation defects, and cataracts. Notch2 mutants also had persistent lens stalks as early as E11.5, and aberrant DNA synthesis in the fiber cell compartment by E14.5. Gene expression analyses showed that upon loss of Notch2, there were elevated levels of the cell cycle regulators Cdkn1a (p21Cip1), Ccnd2 (CyclinD2), and Trp63 (p63) that negatively regulates Wnt signaling, plus down-regulation of Cdh1 (E-Cadherin). Removal of Notch2 also resulted in an increased proportion of fiber cells, as was found in Rbpj and Jag1 conditional mutant lenses. However, Notch2 is not required for AEL proliferation, suggesting that a different receptor regulates this process. We found that Notch2 normally blocks lens progenitor cell death. Overall, we conclude that Notch2-mediated signaling regulates lens morphogenesis, apoptosis, cell cycle withdrawal, and secondary fiber cell differentiation.

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Available from: Chunqiao Liu, Sep 22, 2014
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    • "Importantly, several homeodomain-containing proteins that act as transcription factors were identified and characterized as regulators of cell proliferation and cell differentiation in the developing lens. In contrast, few studies described the roles of basic-helix-loop-helix (bHLH) transcription factors in lens development [2]–[5]. Some recent studies addressed how these transcriptional networks functionally interact in vivo to regulate cell proliferation during lens ontogenesis [6], [7]. "
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    ABSTRACT: Myc protooncogenes play important roles in the regulation of cell proliferation, growth, differentiation and survival during development. In various developing organs, c-myc has been shown to control the expression of cell cycle regulators and its misregulated expression is detected in many human tumors. Here, we show that c-myc gene (Myc) is highly expressed in developing mouse lens. Targeted deletion of c-myc gene from head surface ectoderm dramatically impaired ocular organogenesis, resulting in severe microphtalmia, defective anterior segment development, formation of a lens stalk and/or aphakia. In particular, lenses lacking c-myc presented thinner epithelial cell layer and growth impairment that was detectable soon after its inactivation. Defective development of c-myc-null lens was not caused by increased cell death of lens progenitor cells. Instead, c-myc loss reduced cell proliferation, what was associated with an ectopic expression of Prox1 and p27(Kip1) proteins within epithelial cells. Interestingly, a sharp decrease in the expression of the forkhead box transcription factor Foxe3 was also observed following c-myc inactivation. These data represent the first description of the physiological roles played by a Myc family member in mouse lens development. Our findings support the conclusion that c-myc regulates the proliferation of lens epithelial cells in vivo and may, directly or indirectly, modulate the expression of classical cell cycle regulators in developing mouse lens.
    Full-text · Article · Feb 2014 · PLoS ONE
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    • "When FGF-treated explants are cultured in the presence of the gamma secretase inhibitor, DAPT, which is widely used to inhibit Notch signaling, fiber elongation and Jag-1 expression is diminished (Fig. 8). This is consistent with the previously reported inhibition of Jag-1 and fiber differentiation marker expression that occurs following the application of DAPT to lens explant cultures (Saravanamuthu et al., 2012). Moreover, in relation to the role of Notch signaling in maintaining a proliferating epithelial phenotype, we also show that in the presence of the DAPT inhibitor the FGF-treated cells have an EdU incorporation rate of 9.35%70.23 that is significantly reduced from the 54.26% 71.83 incorporation rate in the epithelial islands of explants treated with FGF alone (p r0.05, 2-tailed t-test). "
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    ABSTRACT: How tissues and organs develop and maintain their characteristic three-dimensional cellular architecture is often a poorly understood part of their developmental program; yet, as is clearly the case for the eye lens, precise regulation of these features can be critical for function. During lens morphogenesis cells become organized into a polarized, spheroidal structure with a monolayer of epithelial cells overlying the apical tips of elongated fiber cells. Epithelial cells proliferate and progeny that shift below the lens equator differentiate into new fibers that are progressively added to the fiber mass. It is now known that FGF induces epithelial to fiber differentiation; however, it is not fully understood how these two forms of cells assemble into their characteristic polarized arrangement. Here we show that in FGF-treated epithelial explants, elongating fibers become polarized/oriented towards islands of epithelial cells and mimic their polarized arrangement in vivo. Epithelial explants secrete Wnt5 into the culture medium and we show that Wnt5 can promote directed behaviour of lens cells. We also show that these explants replicate aspects of the Notch/Jagged signaling activity that has been shown to regulate proliferation of epithelial cells in vivo. Thus, our in vitro study identifies a novel mechanism, intrinsic to the two forms of lens cells, that facilitates self-assembly into the polarized arrangement characteristic of the lens in vivo. In this way the lens, with its relatively simple cellular composition, serves as a useful model to highlight the importance of such intrinsic self-assembly mechanisms in tissue developmental and regenerative processes.
    Preview · Article · Nov 2013 · Developmental Biology
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    • "Our data not only confirm the essential engagement of canonical Hippo-Yap signaling in the crucial decision making process in lens progenitor cells, but also suggest mechanistic collaboration with additional regulatory pathways. The near-complete loss of the lens seen in early neonatal Yap CKO mice is reminiscent of reported phenotypes mediated by decreased proliferation in several mouse mutants including Pax6, β-Catenin, Notch, Jagged1, FoxE3, FGFR2 and β-integrin (Ashery-Padan and Gruss, 2001; Blixt et al., 2000; Cain et al., 2008; Garcia et al., 2005; Le et al., 2009; Saravanamuthu et al., 2011). This suggests a possible higher-order interaction at both extra-and intracellular levels (Graw, 2010). "
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    ABSTRACT: Hippo-Yap signaling has been implicated in organ size determination via its regulation of cell proliferation, growth and apoptosis (Pan, 2007). The vertebrate lens comprises only two major cell types, lens progenitors and differentiated fiber cells, thereby providing a relatively simple system for studying size-controlling mechanisms. In order to investigate the role of Hippo-Yap signaling in lens size regulation, we conditionally ablated Yap in the developing mouse lens. Lens progenitor-specific deletion of Yap led to near obliteration of the lens primarily due to hypocellularity in the lens epithelium (LE) and accompanying lens fiber (LF) defects. A significantly reduced LE progenitor pool resulted mainly from failed self-renewal and increased apoptosis. Additionally, Yap-deficient lens progenitor cells precociously exited the cell cycle and expressed the LF marker, β-Crystallin. The mutant progenitor cells also exhibited multiple cellular and subcellular alterations including cell and nuclear shape change, organellar polarity disruption, and disorganized apical polarity complex and junction proteins such as Crumbs, Pals1, Par3 and ZO-1. Yap-deficient LF cells failed to anchor to the overlying LE layer, impairing their normal elongation and packaging. Furthermore, our localization study results suggest that, in the developing LE, Yap participates in the cell context-dependent transition from the proliferative to differentiation-competent state by integrating cell density information. Taken together, our results shed new light on Yap's indispensable and novel organizing role in mammalian organ size control by coordinating multiple events including cell proliferation, differentiation, and polarity.
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