β-Catenin Inactivation Is a Pre-Requisite for Chick Retina Regeneration

Citation: Zhu J, Luz-Madrigal A, Haynes T, Zavada J, Burke AK, et al. ( b-Catenin Inactivation Is a Pre-Requisite for Chick Retina Regeneration Jie Zhu 0 Agustin Luz-Madrigal 0 Tracy Haynes 0 Julia Zavada 0 Amy K. Burke 0 Katia Del Rio-Tsonis 0 Amit Singh, University of Dayton, United States of America 0 Department of Biology, Miami University , Oxford, Ohio , United States of America In the present study we explored the role of b-catenin in mediating chick retina regeneration. The chick can regenerate its retina by activating stem/progenitor cells present in the ciliary margin (CM) of the eye or via transdifferentiation of the retinal pigmented epithelium (RPE). Both modes require fibroblast growth factor 2 (FGF2). We observed, by immunohistochemistry, dynamic changes of nuclear b-catenin in the CM and RPE after injury (retinectomy). b-catenin nuclear accumulation was transiently lost in cells of the CM in response to injury alone, while the loss of nuclear b-catenin was maintained as long as FGF2 was present. However, nuclear b-catenin positive cells remained in the RPE in response to injury and were BrdU-/p27+, suggesting that nuclear b-catenin prevents those cells from entering the cell cycle. If FGF2 is present, the RPE undergoes dedifferentiation and proliferation concomitant with loss of nuclear b-catenin. Moreover, retinectomy followed by disruption of active b-catenin by using a signaling inhibitor (XAV939) or over-expressing a dominant negative form of Lef-1 induces regeneration from both the CM and RPE in the absence of FGF2. Our results imply that b-catenin protects cells of the CM and RPE from entering the cell cycle in the developing eye, and specifically for the RPE during injury. Thus inactivation of b-catenin is a pre-requisite for chick retina regeneration. - Funding: This work was supported by NIA grant AG024937 and NEI grant EY017319 to KDRT; Sigma Xi grant to JZ; CONACYT 162930 and 142523 to ALM; and USS and DUOS scholarship from Miami University to Julia Z and Amy KB, respectively. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Retina regeneration studies have been conducted in different animal models for many years, however, the molecular mechanism underlying regeneration via different cellular sources is still under rigorous investigation [1,2,3,4,5]. At Embryonic day 4 (E4, HH stage 2224) [6] chick eyes can regenerate a complete retina upon retinectomy, as long as there is a source of growth factors present in the eye [7,8,9,10,11]. The embryonic chick can regenerate its retina via two different mechanisms: by the activation of stem/ progenitor cells present in the CM and by RPE transdifferentiation. During transdifferentiation, the RPE dedifferentiates, proliferates and forms a neuroepithelium that eventually differentiates into retinal cells [8,11]. Several signaling pathways including FGF, Sonic Hedgehog (Shh) and Bone Morphogenetic Protein (BMP), as well as inflammation molecules C3a, C5a and IL-6 have been shown to be involved in chick retina regeneration [7,8,9,10,11, 12,13]; however the molecular mechanism underlying both regeneration processes still needs to be further explored. b-catenin is a dual function protein, playing a critical role in cell-cell adhesion as well as mediating gene transcription through Wnt signaling [14,15]. Overexpression of active b-catenin during early chick eye development (E1.5; HH stage 10) promotes retinal cells to change their fates and gain peripheral identity [16]. bcatenin is also required for RPE specification during avian and mammalian eye development by directly regulating two RPEspecific genes, Microphthalmia-associated transcription factor (Mitf) and orthodenticle homolog 2 (Otx2). Disruption of bcatenin causes the RPE to lose its phenotype and to start to express retinal progenitor markers, while the overexpression of b-catenin and Otx2 is sufficient to induce Mitf expression in retinal progenitors [17,18,19]. The transcriptional functions of b-catenin can be regulated by Wnt signaling which controls b-catenin levels in the cytoplasm by modulating phosphorylation events. In the absence of Wnt signaling, the b-catenin destruction complex continuously phosphorylates b-catenin protein at Ser 33, 37, 45 and Thr 41 to target b-catenin for degradation via an ubiquitin-dependent pathway [20,21,22]. Upon stimulation, Wnt ligands bind to membrane receptor Frizzled and co-receptor LRP5/6, triggering a downstream signaling cascade leading to the inactivation of the destruction complex and the stabilization of b-catenin which is no longer phosphorylated at the same residues. The stabilized bcatenin is then targeted for nuclear translocation [23]. It has been shown that in chick neural cells, phosphorylation of b-catenin at tyrosine 489 (Y489) targets it to the nucleus where it binds to its partner(s) TCF/Lef1 and acts as a co-activator of transcription [24]. In this complex, b-catenin functions as a transcriptional coactivator to facilitate the binding of the complex to chromatin and also to recruit components to promote chromatin remodeling [25,26]. Since b-catenin has been reported to be important for the development or maintenance of the RPE and CM, we sought to explore the role of b-catenin in these tissues during retina regeneration. Here, we report that active b-catenin is associated with cells having a low proliferative status in the CM and RPE and Figure 1. Nuclear b-catenin presence during chick eye development. (A) Schematic diagram of the eye showing the anterior (A) and posterior (P) region of the eye as well as the location of the ciliary margin (CM), retina (R), retinal pigmented epithelium (RPE) and Lens (L). The orientation of the eye in the diagram applies to all images in this paper. (BF) Immunohistochemistry showing the location of nuclear b-catenin (Nu b-cat) in the developing optic vesicles at E1.5 (HH 10) (BD) and E2 (HH 13) (EF). (GP) Nu b-cat presence in the developing CM and RPE at E4 (HH 2224) (GK) and E5 (HH 2728) (LP). DAPI stains the nuclei of the cells in (B, D, F, G, I, K, L, N, P). B, G and L are the corresponding negative controls for E 1.5 (B), E4 (G) and E5 (L). SE: surface Ectoderm; NE: neuroepithelium; NPE: non-pigmented epithelium; PE: pigmented epithelium. OCL: optic cup lip. The scale bar in (B) represents 100 mm and applies to all panels. doi:10.1371/journal.pone.0101748.g001 that blocking the transcriptional activity of b-catenin is a necessary step in retina regeneration. Materials and Methods Chick embryos Fertilized Specific Pathogen Free (SPF) chicken eggs (Charles River Laboratories, Wilmington, MA, USA) were incubated in a humidified rotating incubator at 38uC. Surgical procedures Retinectomies were performed in eyes of chick embryos at E4 (HH stage 2224) using fine forceps as previously d (...truncated)