Kha CX et al. (2019), Using the Xenopus Developmental Eye Regrowth Sy...

XB-ART-55978

Front Physiol 2019 Jan 01;10:502. doi: 10.3389/fphys.2019.00502.

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Using the Xenopus Developmental Eye Regrowth System to Distinguish the Role of Developmental Versus Regenerative Mechanisms.

Kha CX , Guerin DJ , Tseng KA .

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A longstanding challenge in regeneration biology is to understand the role of developmental mechanisms in restoring lost or damaged tissues and organs. As these body structures were built during embryogenesis, it is not surprising that a number of developmental mechanisms are also active during regeneration. However, it remains unclear whether developmental mechanisms act similarly or differently during regeneration as compared to development. Since regeneration is studied in the context of mature, differentiated tissues, it is difficult to evaluate comparative studies with developmental processes due to the latter's highly proliferative environment. We have taken a more direct approach to study regeneration in a developmental context (regrowth). Xenopus laevis, the African clawed frog, is a well-established model for both embryology and regeneration studies, especially for the eye. Xenopus eye development is well-defined. Xenopus is also an established model for retinal and lens regeneration studies. Previously, we demonstrated that Xenopus tailbud embryo can successfully regrow a functional eye that is morphologically indistinguishable from an age-matched control eye. In this study, we assessed the temporal regulation of retinal differentiation and patterning restoration during eye regrowth. Our findings showed that during regrowth, cellular patterning and retinal layer formation was delayed by approximately 1 day but was restored by 3 days when compared to eye development. An assessment of the differentiation of ganglion cells, photoreceptor cells, and Müller glia indicated that the retinal birth order generated during regrowth was consistent with that observed for eye development. Thus, retina differentiation and patterning during regrowth is similar to endogenous eye development. We used this eye regrowth model to assess the role of known mechanisms in development versus regrowth. Loss-of-function studies showed that Pax6 was required for both eye development and regrowth whereas apoptosis was only required for regrowth. Together, these results revealed that the mechanisms required for both development and regrowth can be distinguished from regrowth-specific ones. Our study highlights this developmental model of eye regrowth as a robust platform to systematically and efficiently define the molecular mechanisms that are required for regeneration versus development.

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Species referenced: Xenopus laevis
Genes referenced: calb1 isl1 pax6 rho rpe rpe65
GO keywords: regeneration
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	FIGURE 1. Regrown eyes regain cellular patterning by 3 dps. Images shown are immunostained, transverse sections at three developmental timepoints corresponding to 1, 2, and 3 days post surgery (dps). The top schematic is a diagram of a section through a mature, differentiated tadpole eye. (A,B) Regrowing eyes display retinal patterning comparable to the contralateral control eyes (unoperated) by 3 days. White dashed lines delineate each eye. (A’,B’) Representative images shown in panels A’ and B’ correspond to the region shown in the inset box in panel A4 for the corresponding A or B panel at high magnification. Blue color indicates nuclear staining (TO-PRO-3). Green color indicates the basal lamina (anti-Laminin), which is expressed in all basement membranes and outlines the optic vesicle. Magenta color indicates neural tissues (Xen1). Sample sizes: 1 day, n = 6; 2 days, n = 5; and 3 days, n = 5. (A,B, A’,B’) Up = dorsal, down = ventral, lens is on the left. Scale bar: A,B = 100 μm and A’,B’ = 50 μm.
	FIGURE 2. Regrown eyes regain retinal differentiation by 3 dps. Images shown are immunostained, transverse sections at three developmental timepoints corresponding to 1, 2, and 3 days post surgery (dps). (A,B) The contralateral control eyes (unoperated) complete retinogenesis by st. 41. By 1 dps, RPE is already visible in the regrowing eye as shown by anti-RPE65 signal (retinal pigmented epithelium; green). By 3 dps, Islet1 expression (identifying subpopulations of retinal ganglion cells and subsets of amacrine cells, bipolar cells, and horizontal cells; green) show expected retinal patterning of a mature eye. White dashed lines delineate each eye. (A’,B’) Images shown in panels A’ and B’ correspond to the region shown in the inset box in panel A4 for the corresponding (A or B) panel at high magnification. Blue color indicates nuclear staining (TO-PRO-3). Sample sizes: 1 day, n = 5; 2 days, n = 7; and 3 days, n = 6. (A,B, A’,B’) Up = dorsal, down = ventral, lens is on the left. Scale bar: (A,B) = 100 μm and (A’,B’) = 50 μm.
	FIGURE 3. Regrown eyes regain cone differentiation by 3 dps. Images shown are immunostained, transverse sections at three developmental timepoints corresponding to 1, 2, and 3 days post surgery (dps). (A,B) Differentiation of cone photoreceptor cells is delayed during 1, 2 dps but regains patterning that is comparable to contralateral control eyes (unoperated) by 3 days. White dashed lines delineate each eye. (A’,B’) Images shown in panels A’ and B’ correspond to the region shown in the inset box in panel A4 for the corresponding A or B panel at high magnification. Blue color indicates nuclear staining (TO-PRO-3). Green color indicates anti-Calbindin signal (cone photoreceptors). Sample sizes: 1 day, n = 6; 2 days, n = 5; and 3 days, n = 6. (A,B, A’,B’) Up = dorsal, down = ventral, lens is on the left. Scale bar: A,B = 100 μm and A’,B’ = 50 μm.
	FIGURE 4. Regrown eyes regain rod differentiation by 3 dps. Images shown are immunostained, transverse sections at three developmental timepoints corresponding to 1, 2, and 3 days post surgery (dps). (A,B) Differentiation of rod photoreceptor cells is delayed during 1, 2 dps but regains patterning that is comparable to contralateral control eyes (unoperated) by 3 days. White dashed lines delineate each eye. (A’,B’) Images shown in panels A’ and B’ correspond to the region shown in the inset box in panel A4 for the corresponding A or B panel at high magnification. Blue color indicates nuclear staining (TO-PRO-3). Green color indicates anti-Rhodopsin signal (rod photoreceptors). Sample sizes: 1 day, n = 5; 2 days, n = 7; and 3 days, n = 7. (A,B, A’,B’) Up = dorsal, down = ventral, lens is on the left. Scale bar: A,B = 100 μm and A’,B’ = 50 μm. (C) Quantification of rod photoreceptor cells in the regrowing eye structure at three developmental timepoints corresponding to 1, 2, and 3 dps. The number of rod photoreceptors per 60 μm section in the regrown eye is comparable to number of rod photoreceptor cells in the contralateral control eyes by 3 dps. ∗denotes p < 0.05 (n > 5 per timepoint). Data are means ± SEM. (D) Rod photoreceptor cells expression pattern was measured and compared to the overall circumference of the retinal layer from one end of the ciliary margin zone (CMZ) to the end of the opposite CMZ in both regrowing and contralateral eyes. The ratio of rhodopsin expression in the retinal layer over the retinal layer circumference measurements is shown. By 3 dps, the rod photoreceptor cell expression is comparable to the contralateral control eye. ∗denotes p < 0.05 (n > 6 per timepoint). Data are means ± SEM.
	FIGURE 5. Regrown eyes regain Müller glia differentiation by 4 dps. Images shown are immunostained, transverse sections at four developmental timepoints corresponding to 1, 2, 3, and 4 days post surgery (dps). (A,B) Regrown eyes show differentiation of Müller glial cells beginning at 2 dps. However, proper patterning of Müller glial cells is delayed in the regrowing eyes when compared to the contralateral control eyes until 4 dps. White dashed lines delineate each eye. (A’,B’) Images shown in panels A’ and B’ correspond to the region shown in the inset box in panel A5 for the corresponding A or B panel at high magnification. Blue color indicates nuclear staining (TO-PRO-3). Green color indicates anti-Glutamine Synthetase (identifies Müller glial). Sample sizes: 1 day, n = 5; 2 days, n = 5; and 3 days, n = 5. (A,B, A’,B’) Up = dorsal, down = ventral, lens is on the left. Scale bar: A,B = 100 μm and A’-B’ = 50 μm.
	FIGURE 6. Regrown eyes regain Pax6 patterning by 3 dps. Images shown are immunostained, transverse sections at three developmental timepoints corresponding to 1, 2, and 3 days post surgery (dps). (A,B) Pax6 expression in the regrowing eye is less organized at 1 dps but regains patterning similar to contralateral control eyes (unoperated) by 3 dps. White dashed lines delineate each regrowing eye. (A’,B’) Images shown in panels A’ and B’ correspond to the region shown in the inset box in panel A4 for the corresponding A or B panel at high magnification. Blue color indicates nuclear staining (TO-PRO-3). Green color indicates anti-Pax6 signal. Sample sizes: 1 day, n = 5; 2 days, n = 7; and 3 days, n = 6. (A,B, A’,B’) Up = dorsal, down = ventral, lens is on the left. Scale bar: A,B = 100 μm and A’,B’ = 50 μm.
	FIGURE 7. Development and regrowth require Pax6, but only regrowth requires apoptosis. (A) Comparison of developmental eye defects percentage from embryos injected at the 4-cell stage (1 blastomere) with either the control or Pax6 morpholino or treated with DMSO control or M50054 at st. 10. A zero denotes no abnormal phenotype in the control by st. 27. ∗denotes p < 0.05 (n > 90). Data are means ± SEM. (B) Graphical representation of tadpoles achieving full eye regrowth at 5 dps (st. 46) with morpholino injection or apoptosis inhibitor treatment. ∗∗denotes p < 0.01 (n > 20). Data are means ± SEM. (C,D) Comparison of requirements for eye development and regrowth. (C) Pax6 morpholino injected tadpoles show reduced eyes when compared to the control by st. 46 in development. Apoptosis inhibitor show no effect on eye development (n > 30 per condition). Closed yellow arrowhead indicates eye of control, untreated tadpole. Open yellow arrowhead indicates eye of treated tadpole. (D) Pax6 morpholino and apoptosis inhibitor affects eye regrowth (n > 30). Closed yellow arrowhead indicates the eye of a control, untreated tadpole. Open yellow arrowhead indicates the eye of an inhibitor treated tadpole. (C,D) Up = anterior, down = posterior. Scale bar: C,D = 500 μm.
	FIGURE 8. Summary. (A) A summary of retinal cell differentiation during regrowth as compared to development. The stages correspond to 1, 2, 3, and 4 days post surgery (dps), respectively. (B) Summary of required processes identified in eye development and regrowth.

References [+] :

Araki, Regeneration of the amphibian retina: role of tissue interaction and related signaling molecules on RPE transdifferentiation. 2007, Pubmed, Xenbase