V-ATPase Regulates Retinal Progenitor Cell Proliferation During Eye Regrowth in Xenopus.

Purpose: The induction of retinal progenitor cell (RPC) proliferation is a strategy that holds promise for alleviating retinal degeneration. However, the mechanisms that can stimulate RPC proliferation during repair remain unclear. Xenopus tailbud embryos successfully regrow functional eyes within 5 days after ablation, and this process requires increased RPC proliferation. This model facilitates identification of mechanisms that can drive in vivo reparative RPC proliferation. This study assesses the role of the essential H+ pump, V-ATPase, in promoting stem cell proliferation. Methods: Pharmacological and molecular loss of function studies were performed to determine the requirement for V-ATPase during embryonic eye regrowth. The resultant eye phenotypes were examined using histology and antibody markers. Misexpression of a yeast H+ pump was used to test whether the requirement for V-ATPase in regrowth is dependent on its H+ pump function. Results: V-ATPase inhibition blocked eye regrowth. Regrowth-incompetent eyes resulting from V-ATPase inhibition contained the normal complement of tissues but were much smaller. V-ATPase inhibition caused a significant reduction in reparative RPC proliferation but did not alter differentiation and patterning. Modulation of V-ATPase activity did not affect apoptosis, a process known to be required for eye regrowth. Finally, increasing H+ pump activity was sufficient to induce regrowth. Conclusions: V-ATPase is required for eye regrowth. These results reveal a key role for V-ATPase in activating regenerative RPC proliferation and expansion during successful eye regrowth.

From Cell Death to Regeneration: Rebuilding After Injury.

The ability to regrow lost or damaged tissues is widespread, but highly variable among animals. Understanding this variation remains a challenge in regeneration biology. Numerous studies from Hydra to mouse have shown that apoptosis acts as a potent and necessary mechanism in regeneration. Much is known about the involvement of apoptosis during normal development in regulating the number and type of cells in the body. In the context of regeneration, apoptosis also regulates cell number and proliferation in tissue remodeling. Apoptosis acts both early in the process to stimulate regeneration and later to regulate regenerative patterning. Multiple studies indicate that apoptosis acts as a signal to stimulate proliferation within the regenerative tissues, producing the cells needed for full regeneration. The conservation of apoptosis as a regenerative mechanism demonstrated across species highlights its importance and motivates the continued investigation of this important facet of programmed cell death. This review summarizes what is known about the roles of apoptosis during regeneration, and compares regenerative apoptosis with the mechanisms and function of apoptosis in development. Defining the complexity of regenerative apoptosis will contribute to new knowledge and perspectives for understanding mechanisms of apoptosis induction and regulation.

Studying In Vivo Retinal Progenitor Cell Proliferation in Xenopus laevis.

The efficient generation and maintenance of retinal progenitor cells (RPCs) are key goals needed for developing strategies for productive eye repair. Although vertebrate eye development and retinogenesis are well characterized, the mechanisms that can initiate RPC proliferation following injury-induced regrowth and repair remain unknown. This is partly because endogenous RPC proliferation typically occurs during embryogenesis while studies of retinal regeneration have largely utilized adult (or mature) models. We found that embryos of the African clawed frog, Xenopus laevis, successfully regrew functional eyes after ablation. The initiation of regrowth induced a robust RPC proliferative response with a concomitant delay of the endogenous RPC differentiation program. During eye regrowth, overall embryonic development proceeded normally. Here, we provide a protocol to study regrowth-dependent RPC proliferation in vivo. This system represents a robust and low-cost strategy to rapidly define fundamental mechanisms that regulate regrowth-initiated RPC proliferation, which will facilitate progress in identifying promising strategies for productive eye repair.

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

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.

A model for investigating developmental eye repair in Xenopus laevis.

Vertebrate eye development is complex and requires early interactions between neuroectoderm and surface ectoderm during embryogenesis. In the African clawed frog, Xenopus laevis, individual eye tissues such as the retina and lens can undergo regeneration. However, it has been reported that removal of either the specified eye field at the neurula stage or the eye during tadpole stage does not induce replacement. Here we describe a model for investigating Xenopus developmental eye repair. We found that tailbud embryos can readily regrow eyes after surgical removal of over 83% of the specified eye and lens tissues. The regrown eye reached a comparable size to the contralateral control by 5 days and overall animal development was normal. It contained the expected complement of eye cell types (including the pigmented epithelium, retina and lens), and is connected to the brain. Our data also demonstrate that apoptosis, an early mechanism that regulates appendage regeneration, is also required for eye regrowth. Treatment with apoptosis inhibitors (M50054 or NS3694) blocked eye regrowth by inhibiting caspase activation. Together, our findings indicate that frog embryos can undergo successful eye repair after considerable tissue loss and reveals a required role for apoptosis in this process. Furthermore, this Xenopus model allows for rapid comparisons of productive eye repair and developmental pathways. It can also facilitate the molecular dissection of signaling mechanisms necessary for initiating repair.