Retinal regeneration in the Xenopus laevis tadpole: a new model system

Abstract

Purpose: Retinal regeneration research holds potential for providing new avenues for the treatment of degenerative diseases of the retina. Various animal models have been used to study retinal regeneration over the years, providing insights into different aspects of this process. However the mechanisms that drive this important phenomenon remain to be fully elucidated. In the present study, we introduce and characterize a new model system for retinal regeneration research that uses the tadpole of the African clawed frog, Xenopus laevis.

Methods: The neural retina was surgically removed from Xenopus laevis tadpoles at stages 51-54, and a heparin-coated bead soaked in fibroblast growth factor 2 (FGF-2) was introduced in the eyes to induce regeneration. Histological and immunohistochemical analyses as well as DiI tracing were performed to characterize the regenerate. A similar surgical approach but with concomitant removal of the anterior portion of the eye was used to assess the capacity of the retinal pigmented epithelium (RPE) to regenerate a retina. Immunohistochemistry for FGF receptors 1 and 2 and phosphorylated extracellular signal-regulated protein kinase (pERK) was performed to start elucidating the intracellular mechanisms involved in this process. The role of the mitogen activated protein kinase (MAPK) pathway was confirmed through a pharmacological approach using the MAPK kinase (MEK) inhibitor U0126.

Results: We observed that Xenopus laevis tadpoles were able to regenerate a neural retina upon induction with FGF-2 in vivo. The regenerated tissue has the characteristics of a differentiated retina, as assessed by the presence and distribution of different retinal cell markers, and DiI tracing indicated that it is able to form an optic nerve. We also showed that retinal regeneration in this system could take place independently of the presence of the anterior eye tissues. Finally, we demonstrated that FGF-2 treatment induces ERK phosphorylation in the pigmented epithelia 10 days after retinectomy, and that inhibition of the MAPK pathway significantly decreases the amount of retina regenerated at 30 days post-operation.

Conclusions: Regeneration of a complete neural retina can be achieved in larval Xenopus laevis through activation of the MAPK signaling pathway by administering exogenous FGF-2. This mechanism is conserved in other animal models, which can regenerate their retina via pigmented epithelium transdifferentiation. Our results provide an alternative approach to retinal regeneration studies, capitalizing on the advantages of the Xenopus laevis tadpole as a model system.

Figure 1

FGF-2 induces retinal regeneration in Xenopus laevis in vivo after complete retinectomy. Shown are sections of retinectomized tadpole eyes in which a control bead (A-D) or an FGF-2-soaked bead (E-J) was introduced at day 0. The eyes were collected at postoperative days 0 (A and E), 10 (B and F), 20 (C, G, I), and 30 (D, H, J), and stained with hematoxylin and eosin. Notice the absence of nonpigmented tissues inside the eye at the earlier time points. At 20 days, a layered neural retina was evident only in the eyes treated with FGF-2 (G); this retina was larger by 30 days (H). I and J are close up images of the regenerated retinas observed in G and H respectively. Abbreviations: optic nerve (ON); cornea (C); regenerated retina (RR); regenerated lens (RL). Asterisks indicate control or FGF-2-soaked bead. Scale bars represent 100 μm (scale bar in H applies to A-H and scale bar in J applies also to I).

Figure 2

Regenerated retina induced by FGF-2 expresses markers of normal retinal cells. Intact eyes (A, B, E, F, I, J, M, N, Q, R) or eyes 30 days postretinectomy with FGF-2 administration (C, D, G, H, K, L, O, P, S, T) were immunolabeled for different retinal cell markers. B, F, J, N, R, D, H, L, P, and T are close up images of the retinas shown in A, E, I, M, Q, C, G, K, O, and S, respectively. A-D: AP2α was used as a marker for amacrine cells. E-H: Islet-1 was used as a marker of ganglion cells but could also detect some subpopulations of amacrine, bipolar, and horizontal cells. I-L: Xap-2 was used to mark rod photoreceptors. M-P: Recoverin was used to detect photoreceptors and midget cone bipolar cells. Q-T: Vimentin was used as a marker of Müller glia. Red arrows point at dark-colored Müller glia processes labeled with the vimentin antibody. There was a general light brown background throughout the sections, whereas the staining of the antibody was actually dark brown. All the markers tested were expressed in both the intact and regenerated retinas. Scale bars represent 100 μm (Scale bars in O and S apply to A, E, I, M, Q, C, G, K, O, S; scale bars in P and T apply to B, F, J, N, R, D, H, L, P, T).

Figure 3

The regenerated retina is able to form an optic nerve. Intact eyes (A, D) and eyes 30 days postretinectomy exposed to either an FGF-2 bead (B, E) or a control bead (C, F) were injected with DiI to label cell membranes. Ten days later, the ganglion cell axons could be seen projecting through the optic nerve in both intact and FGF-2 exposed eyes (D and E). Control retinectomized eyes did not regenerate a retina and therefore did not project their axons through what remained of the optic nerve (F). D, E, and F correspond to fluorescent views (DiI labeling) of the bright-field images shown in A, B, and C respectively. Abbreviations: eyeball (E); optic nerve (ON).

Figure 4

The RPE is a likely source of retinal regeneration. The anterior third of the eye was dissected out, and the neural retina was removed from the posterior eyecup of Xenopus laevis tadpoles, at which point either an FGF-2-soaked bead (A, D), a control bead (B, E), or no bead (C, F) was introduced in eyecups. The panel shows histological sections of eyes collected 30 days postsurgery and stained with hematoxylin and eosin. D-F are higher magnification images of A-C respectively. Robust retinal regeneration was observed in all eyes treated with FGF-2 (A, D), whereas there was no retinal regeneration in any case of eyes exposed to control beads (B, E) or no bead at all (C, F). Abbreviations: choroid layer (Ch); retinal pigmented epithelium (RPE); regenerated retina (RR), cornea (C). Asterisks indicate beads. Scale bars represent 100 μm (scale bar in C applies to A-C and scale bar in F applies to D-F).

Figure 5

FGF receptors 1 and 2 expression and phosphorylated extracellular signal-regulated protein kinase (pERK) are upregulated during regeneration. A-F: Immunohistochemistry for FGF receptor 1 (flg, A-C) and FGF receptor 2 (bek, D-F) was performed on intact eyes (A, D), as well as on eyes exposed to a control bead soaked in PBS or an FGF-2 soaked bead and collected 10 days postretinectomy (B, C, E, F). C and F are a close up images of B and E respectively. Notice that FGF receptors (red) were detected in the neural retina and not in the pigmented tissues of the intact eye, whereas after retina removal, expression of these receptors was evident in the RPE of eyes exposed to control or FGF-2 beads (arrows). G-J: Immunohistochemistry for pERK (red) at 10 days postretinectomy in eyes treated with control (G, I) or FGF-2-soaked beads (H, J). I and J are a close up views of G and H respectively. Only the pigmented epithelium of eyes exposed to FGF-2 beads was labeled by the pERK antibody. Arrows point to the pigmented epithelium. Asterisks indicate control or FGF-2 soaked beads. Abbreviations: lens (L); neural retina (NR); retinal pigmented epithelium (RPE); iris (I); pigmented ciliary body (PCB); cornea (C). Scale bars represent 100 μm. Scale bar in E applies to A, B, D, and E; scale bar in F applies to C and F; scale bars in H and J apply to G, H, and I, J respectively.

Figure 6

Inhibition of the MAPK pathway decreases FGF-induced retinal regeneration in Xenopus laevis. U0126, a potent inhibitor of MEK, was used for inhibition studies at concentrations of 100 µM and 1 mM. Tadpoles were retinectomized. Both an FGF-2-soaked heparin-coated bead and an affigel blue bead soaked in either the inhibitor or in DMSO for control were introduced in their eyes. The pictures show representative sections of eyes collected at 30 days postsurgery and stained with hematoxylin and eosin. A: Normal retinal regeneration was evident in the eyes that were treated with FGF-2 plus a control affigel blue bead. B: Eyes treated with FGF-2 and 100 µM U0126 affigel blue beads showed severe reduction of regeneration. C: No regeneration of the retina was detected in eyes treated with an FGF-2 bead and a 1 mM U0126 affigel blue bead. Arrows point to regenerated neural retina. Asterisks indicate FGF-2-soaked heparin beads. Abbreviations: affigel blue bead, soaked in the inhibitor or control (ab); cornea (C). Scale bar in C represents 100 μm and applies to all panels.