Müller glia (MG) are the primary support cells in the vertebrate retina, regulating homeostasis in one of the most metabolically active tissues. In lower vertebrates such as fish, they respond to injury by proliferating and reprogramming to regenerate retinal neurons. In mammals, MG may also react to injury by proliferating, but they fail to initiate regeneration. The barriers to regeneration could be intrinsic to mammalian MG or the function of the niche that cannot support the MG reprogramming required for lineage conversion or both. Understanding these mechanisms in light of those being discovered in fish may lead to the formulation of strategies to unlock the neurogenic potential of MG and restore regeneration in the mammalian retina.
Recent progress in our understanding of brain development has significantly altered concepts for treating neurodegenerative diseases, including those that affect the retina to cause blindness. Contrary to previous thought, it is now recognized that neurons are generated in the sub ventricular zone (SVZ) of the lateral ventricle, and the sub-granular zone (SGZ) in the hippocampus from neural stem cells (NSC) of glial origin throughout life (1). Outside these discrete regions in the mammalian CNS, including the retina, active neurogenesis has not been reproducibly demonstrated under normal conditions (2). However, rare neurogenic changes are observed in the injured adult mammalian retina; the source of injury-induced neurogenesis is traced to Müller glia (MG) (3–5). Recent observations that mammalian MG possess NSC properties and are able to generate retinal neurons
To address this challenge, the neurogenic potentials of MG need to be evaluated against the backdrop of two models, with the understanding that examples from lower vertebrates would constitute a framework, but that solutions will be unique to the mammalian retina: (1) SVZ/SGZ model, where MG have NSC properties and thus the
The recent success of reprogramming to induce pluripotency in somatic cells and lineage-specific differentiation of pluripotent cells, whether through directed differentiation (SVZ/SGZ model) or trans-differentiation (Extra SVZ/SGZ model), is owed directly to the knowledge of developmental mechanisms (12). There is a significant knowledge gap in our understanding of how neurogenesis shifts to the generation of MG in the mammalian retina, information essential to navigate the cellular or molecular roadblocks to neuronal differentiation. For example, although we know that Notch signaling regulates the generation of MG, how it is incorporated into the gliogenic program and to what extent it is involved in suppressing neuronal differentiation remains rather unknown (4). Given the contextual role of Notch signaling (4), its complexity due to the oscillatory expression of its effector
Regardless of whether or not MG possesses dormant stem cell properties according to the aforementioned models they must initiate and complete an intertwined program of activation and neuronal conversion for successful regeneration (4). Given that injuries in general lead to proliferation of MG across species, but not to their neuronal differentiation in higher vertebrates, it is likely that the cross-talk between transcriptional networks sub-serving the activation and neuronal differentiation are either not connected to the process, or the network components are in place but are not epigenetically primed for optimal expression in the mammalian MG. These probabilities could be examined by a genome-wide screening of prospectively enriched MG in select animal models in quiescent and activated states (facilitated by lineage reporters and other enrichment protocols such as the side population (SP) cell profiling by microarrays, RNA seq, and ChIP seq analyses. A similar approach to characterizing MG in different states of activation and differentiation in controlled conditions
Regeneration takes place when homeostasis is disturbed. MG, the guardian of homeostasis in the retina, respond to injuries by proliferating and migrating out of the inner nuclear layer (5). However, despite proliferation and migration as in zebrafish retina, as mentioned, mammalian MG do not initiate regeneration effectively. This raises the possibility that, in addition to internal constraints discussed above, the environment in the mammalian retina might not be conducive for neurogenic conversion of MG. A niche-based approach to unlock neurogenic potential of MG will involve examining the relationship of MG with neighboring retinal cells, microglia, immigrant astrocytes, and endothelial cells in the context of signaling pathways and their capacity to engage the molecular axes involved in reprograming along neuronal lineage. For example, the understanding of the role of microglia, one of the first responders to injury, and which may mediate regeneration through inflammatory signals, is still evolving in the retina (29). Studies have demonstrated that Notch (8, 30), Wnt (5, 6), FGF (6, 8), insulin and insulin-like growth factor-1 (8), Shh (31), and cytokines such as TNF
This is fundamental information missing from studies of MG-dependent regeneration, without which the underlying mechanisms remain difficult to grasp and the therapeutic target rather illusory. Currently, the identity of the activated MG represents cells in the inner nuclear layer of the retina, which have incorporated BrdU in response to injury and express MG-specific markers. Given the transition of MG from a quiescent to a proliferative state and the generation of their progenies in different stages of development, the identity based on BrdU incorporation, though important, is of limited value. Attempts have been made to prospectively enrich activated MG by Hoechst dye efflux assays and to characterize them as SP cells, a phenotype shared by the majority of stem cells (4, 6). However, since SP cells are also heterogeneous, this functional approach has limitations in the absence of specific cellular markers. Although several signaling pathways (e.g. Notch signaling) and intrinsic factors (e.g., Ascl1) have been observed to regulate MG activation in fish, birds, and rodents (8), their status as markers of activated MG in different stages of regeneration remain unspecified. Progress in understanding the regenerative mechanisms in tissues like blood (34), intestine (35), and skin (36) has been facilitated by the identification and characterization of markers of the resident stem cells and their progenies. Within the CNS, insight into regeneration in the SVZ has come from the characterization of B1 quiescent and active progenitors and resulting neuroblasts (37). Therefore, to follow MG-mediated regeneration, spatially and temporally, and to identify the stage-specific intrinsic players, a concerted effort is needed to search for reliable and reproducible markers.
Currently, there is no consensus on reliable and reproducible mammalian models to study the activation and neurogenic potential of MG. Rodent models representing retinal injuries, ranging from those caused by the exposure to light, neurotoxins, and genetic mutations have been used. Given the observations that glia respond differently to different types and durations of injuries (2), that their levels of activation differ from species to species (Ahmad et al., unpublished observations), and that within the same species there are strain differences in responses (38), a case may be made for developing injury- and species/strain-specific models for reproducible and unambiguous examination of MG neurogenic potentials. These models will be valuable in incorporating transgenic technology for reliable lineage tracing, and more importantly for molecular characterization of activated MG. Characterization of these cells for gene regulatory network and epigenetic signature is essential to shed light on the status of their neurogenic potential and approaches to unlock it. Furthermore, cell type specific injury models would test the ability of MG to replace specific neuronal types, which will be helpful in designing approaches for MG-dependent therapeutic regeneration. For example, the loss of vision in two of the intractable blinding diseases, age-related macular degeneration (AMD) and glaucoma, is due to selective loss of photoreceptors and retinal ganglion cells (RGCs), respectively. The neurotoxin injury models where exposure of the retina to N-Methyl-N-nitrosourea (MNU) ablates photoreceptors (39) and NMDA causes the degeneration of RGCs (40) would reveal whether or not MG could differentiate along specific neuronal types. Also, it may shed light on if MG-dependent therapeutic regeneration would be a practical approach to address degenerative changes in AMD and/or glaucoma.
The discipline of MG-based regeneration is a recent one, particularly in mammals. With information on the nature of the activated MG and the molecular axes mediating their response to the niche, aided by reproducible animal models and lineage reporters, a pharmacological recruitment of these endogenous progenitors for therapeutic regeneration is a near possibility. In addition, information emerging from these studies will help us understand how the neurogenic potential in the adult mammalian brain is constrained relative to that of lower vertebrates, currently a significant knowledge gap.
This work was supported by Pearson Foundation and Nebraska Department of Health and Human Services (LB606). We are thankful to Dr. Andrew S. Yoo and Dr. George Q. Daley for the pLemir-9-124 and pMSCV-mLin28a plasmids, respectively.