Pigmented epithelium to retinal transdifferentiation and Pax6 expression in larval Xenopus laevis

Abstract

This study examines the retinal transdifferentiation (TD) of retinal pigmented epithelium (RPE) fragments dissected from Xenopus laevis larvae and implanted into the vitreous chamber of non-lentectomized host eyes. In these experimental conditions, most RPE implants transformed into polarized vesicles in which the side adjacent to the lens maintained the RPE phenotype, while the side adjacent to the host retina transformed into a laminar retina with the photoreceptor layer facing the cavity of the vesicle and with the ganglionar cell layer facing the host retina. The formation of a new retina with a laminar organization is the result of depigmentation, proliferation and differentiation of progenitor cells under the influence of inductive factors from the host retina. The phases of the TD process were followed using BrdU labelling as a marker of the proliferation phase and using a monoclonal antibody (mAbHP1) as a definitive indicator of retina formation. Pigmented RPE cells do not express Pax6. In the early phase of RPE to retinal TD, all depigmented and proliferating progenitor cells expressed Pax6. Changes in the Pax6 expression pattern became apparent in the early phase of differentiation, when Pax6 expression decreased in the presumptive outer nuclear layer (ONL) of the new-forming retina. Finally, during the late differentiation phase, the ONL, which contains photoreceptors, no longer expressed Pax6, Pax6 expression being confined to the ganglion cell layer and the inner nuclear layer. It is well known that larval and adult newts are able to regenerate the neural retina (NR) from the retinal pigmented epithelium (RPE) through a transdifferentiation (TD) process (Stone, '50; Hasegawa, '58; Reyer, '77; Stroeva and Mitashov, '83) and that the RPE of larval and adult frogs transdifferentiates into NR when implanted into the vitreous chamber of a host tadpole (Lopashov and Sologub, '72; Sologub, '77). The regeneration of NR via TD from RPE also occurs in the chick and rat embryos, but this capacity is restricted to early stages of ocular development prior to the irreversible commitment of the RPE to the pigmented cell fate (Park and Hollenberg, '89; Pittack et al., '91; Zhao et al., '95). Some recent studies have begun to investigate the molecular nature of factors involved in this TD process. The basic fibroblast growth factor (FGF2) has been demonstrated to be a potent factor capable of triggering the retinal TD of the embryonic chick RPE both in vivo and in vitro (Park and Hollenberg, '89; Pittack et al., '91; Araki et al., '98), as well as triggering the retinal TD of the embryonic rat RPE cultured in vitro (Zhao et al., '95). Sakaguchi et al. ('97) demonstrated that FGF2 also promoted retinal TD of larval Xenopus laevis RPE cultured in vitro, supporting the idea that retinal TD undergone by X. laevis RPE fragments implanted into the vitreous chamber of host tadpoles is the result of retinal-derived FGF2 accumulated into the vitreous chamber. However, the retina regeneration in newts is a u...

Retinal stem/progenitor cells in the ciliary marginal zone complete retinal regeneration: A study of retinal regeneration in a novel animal model

“…Transdifferentiation from RPE cells to retinal cells is a primary feature of retinal regeneration in the adult newt (Mitashov, 1997). This process also plays a crucial role in regeneration in X. laevis as previously reported (Arresta et al, 2005;Araki, 2014). The present observations in X. tropicalis retinal regeneration indicate that this process differs substantially in these two animals and the CMZ plays a major role.…”

Complete reconstruction of the retinal laminar structure from a cultured retinal pigment epithelium is triggered by altered tissue interaction and promoted by overlaid extracellular matrices

“…Several studies reported the expression of Pax6, a key transcription factor gene for regeneration (Ashery-Padan and Gruss, 2001), during retinal regeneration of the newt, although it is still unknown what factors induce Pax6 expression (Kaneko et al, 1998;Arresta et al, 2005). We, therefore, examined the early expression pattern of Pax6 under a culture condition and obtained interesting results that the initial upregulation of Pax6 is dependent on the alteration in the tissue interaction, but not on FGF2 signaling.…”

Retinal pigment epithelium development, plasticity, and tissue homeostasis

“…On the other hand, Sox2 overexpression directly induces RPE depigmentation, decreases expression of RPE genes such as Mitf and Otx2, and promotes proliferation and differentiation into ganglion cell, amacrine cells and/or photoreceptors (Ishii et al, 2009; Ma et al, 2009). Rx and Pax6 are upregulated and required at the stage of transdifferentiation when RPE cells begin to acquire retinal progenitor characteristics (Arresta et al, 2005; Azuma et al, 2005; Ishii et al, 2009; Kuriyama et al, 2009; Martinez-De Luna et al, 2011; Nabeshima et al, 2013). Since Pax6 exerts also RPE-promoting functions under certain conditions (see above(Bharti et al,2012)), it is possible that a high expression level of ectopic Pax6 expression in the RPE is critical for the transdifferentiation effect.…”