c-myc Regulates Cell Proliferation during Lens Development Gabriel R. Cavalheiro., Gabriel E. Matos-Rodrigues., Anielle L. Gomes, Paulo M. G. Rodrigues, Rodrigo A. P. Martins* Programa de Biologia Celular e do Desenvolvimento, Instituto de Cieˆncias Biome´dicas, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Rio de Janeiro, Brazil 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 p27Kip1 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. Citation: Cavalheiro GR, Matos-Rodrigues GE, Gomes AL, Rodrigues PMG, Martins RAP (2014) c-myc Regulates Cell Proliferation during Lens Development. PLoS ONE 9(2): e87182. doi:10.1371/journal.pone.0087182 Editor: Amit Singh, University of Dayton, United States of America Received April 19, 2013; Accepted December 20, 2013; Published February 4, 2014 Copyright: ß 2014 Cavalheiro et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: International Brain Research Organization (IBRO); International Retinal Research Foundation (IRRF); Fundac¸a˜o Carlos Chagas Filho de Amparo a` Pesquisa do Estado do Rio de Janeiro (FAPERJ); Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq). GRC, ALG and PMRG received undergrad student fellowships from the PIBIC program (CNPq/UFRJ) or from FAPERJ. The funders had no role in study design, data collection and analysis, decisionto publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: [email protected] . These authors contributed equally to this work. Introduction The refined architecture and well characterized stages of the developing vertebrate lens makes it an excellent model to study Complex developmental processes must be carefully orches- how these basic cellular processes are regulated in coordination. trated for the correct formation of the vertebrate eye. Even During embryonic development of the mouse, the contact between though optic-cup morphogenesis was recently reproduced in vitro the optic vesicle and the head surface ectoderm occurs around by the use of three-dimensional stem cell culture systems [1], the embryonic day 9 (E9) and triggers the thickening and invagination molecular mechanisms driving eye organogenesis in vivo are still a of the ectoderm, forming the lens vesicle. Then, cells located on major question in developmental biology. Concomitant with the the posterior end of the vesicle exit cell cycle and elongate to coordinated growth of the developing lens and retina, the terminally differentiate into primary lens fiber cells, while anterior formation of a functionally mature eye depends on the precise cells continue proliferating and form the lens epithelia. At later coordination of cell proliferation, cell cycle exit and cell stages, cell proliferation continues in the germinative zone from differentiation within these structures. In the past several years, where cells migrate towards lens equator, exit cell cycle and start a lot has been learned about the mechanisms that regulate these the secondary differentiation process. Through adulthood, epithe- events, including the cell-extrinsic cues, such as growth factors, lial cells exit the cell cycle and differentiate into fiber cells at the and cell-intrinsic factors, including cell cycle proteins and equatorial region of the lens. The adult lens is composed of post transcriptional regulators. Importantly, several homeodomain- mitotic terminally differentiated fiber cells and a layer of cuboidal containing proteins that act as transcription factors were epithelial cells in its anterior region. Some of these epithelial cells identified and characterized as regulators of cell proliferation remain as proliferative stem/progenitor cells [8]. Proper lens and cell differentiation in the developing lens. In contrast, few organogenesis requires the maintenance of the epithelial identity studies described the roles of basic-helix-loop-helix (bHLH) and cell proliferation by lens epithelial cells in precise coordination transcription factors in lens development [2–5]. Some recent with cell cycle exit and cell differentiation. Many secreted growth studies addressed how these transcriptional networks functionally interact in vivo to regulate cell proliferation during lens factors, such as FGF, BMP and others [9–12], are known ontogenesis [6,7]. regulators of these events. PLOS ONE | www.plosone.org 1 February 2014 | Volume 9 | Issue 2 | e87182 c-myc Drives Cell Proliferation in Developing Lens The molecular mechanisms driving cell proliferation in Materials and Methods developing lens have been extensively studied [13]. Proper cell cycle exit and terminal differentiation of fiber cells critically Mice depends on the Rb pathway. In Rb-null lens, cells in the transition Experimental procedures with animals were approved by the zone fail to exit cell cycle [14]. A similar phenotype of Committee of Ethics in Animal Use (CEUA) of the Health hyperproliferation, followed by apoptotic cell death, was observed Science Center (CCS) based on the currently accepted interna- when CDK inhibitors p27Kip1 and p57Kip2 were both inactivated, tional rules. tm2Fwa suggesting that these CKIs functionally cooperate as upstream The c-myc floxed [32] (Myc , MGI id:2178233) and the regulators of Rb pathway during lens terminal differentiation. Lens-Cre [33] (Tg(Pax6-cre,GFP)1Pgr, MGI id:3045749) mice These CDK inhibitors p27Kip1 and p57Kip2 are expressed in the were previously generated and kindly shared. The control group Ctrl +/+ +/F F/F fibers and their upregulation in the context of cell cycle exit (c-myc ) was composed of c-myc , c-myc and c-myc . Mice depends on the transcription factor Prox1 (prospero-related with homozygous inactivation of c-myc specifically in the lens were Le-Cre F/F +/2 homeobox 1) [15]. Regulation of cell cycle exit by Prox1 was identified as c-myc = c-myc ; Le-Cre and mice with Het previously shown in several developing tissues and in cancer [16– heterozygosis of c-myc in the lens were identified as c-myc = +/F +/2 18] and evidence that Prox1 may regulate p27Kip1 transcription by c-myc ; Le-Cre . To ensure that the offspring would inherit directly binding to its promoter was also observed [19]. Little is only one copy of the Cre transgene, Cre-positive animals +/2 F/F known about how Prox1 expression is regulated during lens (Le-Cre ; c-myc ) were always mated to Cre-negative animals 2/2 F/F 2/2 +/F development [17–18], specially how its expression gets restricted to (Le-Cre ; c-myc or Le-Cre ; c-myc ). and maintained in early differentiating cells following terminal differentiation [20–22]. RNA extraction, cDNA synthesis, and real-time RT-PCR Previous studies have described that Myc transcription factors analysis are expressed in developing lens of various vertebrates [23,24]. Dissected lenses were obtained from staged embryonic (E12.5, The Myc family of proto-oncogenes includes c-myc (Myc), N-myc E14.5, E17.5) and postnatal (P0, P3, P11, adult) C57BL/6 mice. (Mycn) and L-myc (Myc1) that encode transcription factors RNA extraction and cDNA synthesis were performed as containing basic-helix-loop-helix leucine zipper (bHLHZ) motifs previously described [29,34]. Real time RT-PCR reactions were and are known to regulate gene expression through a variety of performed in an ABI7500 machine (Applied Biosystems) using mechanisms, including transcriptional activation through the TaqManH probes synthesized with 59-FAM and 39-BHQ for c- formation of a heterodimer with Max as well as Max-independent myc (Myc), Foxe3, p27Kip1 (Cdkn1b), actin (Actb) and GAPDH mechanisms of c-myc-mediated transcriptional repression [25–28]. (Gapd). Primers used were listed in Table S1. Data analysis and Myc proto-oncogenes have been shown to regulate cell survival, normalization were performed as previously described [29,34]. size, differentiation and specially cell proliferation in several developing organs, explaining why these