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New Developments in Vision Research  |   July 2011
Müller Glia: A Promising Target for Therapeutic Regeneration
Author Affiliations & Notes
  • Iqbal Ahmad
    From the Department of Ophthalmology and Visual Sciences, University of Nebraska Medical Center, Omaha, Nebraska.
  • Carolina B. Del Debbio
    From the Department of Ophthalmology and Visual Sciences, University of Nebraska Medical Center, Omaha, Nebraska.
  • Ani V. Das
    From the Department of Ophthalmology and Visual Sciences, University of Nebraska Medical Center, Omaha, Nebraska.
  • Sowmya Parameswaran
    From the Department of Ophthalmology and Visual Sciences, University of Nebraska Medical Center, Omaha, Nebraska.
  • Corresponding author: Iqbal Ahmad, Department of Ophthalmology and Visual sciences, Durham Research Center 1, 98-5840 Nebraska Medical Center, Omaha, NE 68918-5840; iahmad@unmc.edu
Investigative Ophthalmology & Visual Science July 2011, Vol.52, 5758-5764. doi:10.1167/iovs.11-7308
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      Iqbal Ahmad, Carolina B. Del Debbio, Ani V. Das, Sowmya Parameswaran; Müller Glia: A Promising Target for Therapeutic Regeneration. Invest. Ophthalmol. Vis. Sci. 2011;52(8):5758-5764. doi: 10.1167/iovs.11-7308.

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      © ARVO (1962-2015); The Authors (2016-present)

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In the past 10 years, there has been a paradigm shift in our understanding of brain development and approaches to treat degenerative diseases, including those that affect the retina. The latest knowledge includes (1) the discovery that the adult brain harbors proliferating progenitors and that neurons are born throughout life, particularly in the subventricular zone (SVZ) of the lateral ventricle and the subgranular layer (SGL) of the dentate gyrus of the hippocampus 1 and (2) the observation that glia perform dual functions, providing homeostatic support and serving as the source of stem cells in the embryonic brain and the adult SVZ and SGL. 2 In contrast to the SVZ and SGL, active neurogenesis has not been detected in adult mammalian retina. However, neurogenic changes have been observed in injured retina, and the source of injury-induced neurogenesis is traced to Müller glia, the sole glial cells generated by multipotential retinal progenitors. Recent evidence that a subset of Müller glia possesses an evolutionarily conserved stem cell potential has posited these cells as a viable target for replacing degenerating neurons in diseases such as age-related macular degeneration, retinitis pigmentosa, and glaucoma, where vision loss is due to the degeneration of specific types of neurons. This approach has the potential to effectively address significant barriers, such as the lack of a renewable source of cells that are nonimmunogenic and nonteratogenic, which currently makes ex vivo cell therapy approach impractical. 
This review describes the recent progress made in our understanding of the stem cell properties and regenerative potential of Müller glia and includes a discussion of (1) the development of Müller glia; (2) the neurogenic potential of Müller glia across species; (3) the characterization of Müller glia as stem cells; (4) the facile activation of Müller glia; and (5) the molecular mechanisms underlying the stemness (i.e., stemlike properties) of Müller glia. This information is critical in making potential therapeutic approaches effective, efficient, predictable, and safe. This review does not include the homeostatic functions of Müller glia; readers are referred to an excellent review on the topic by Bringmann et al. 3  
Development of Müller Glia
In the developing retina, neurogenesis precedes gliogenesis, as elsewhere in the vertebrate central nervous system (CNS). For example, in the rat retina, Müller glia begin to differentiate in a relatively late stage of histogenesis, with the peak of generation of these cells at postnatal day (PN)4, trailing that of neurons (i.e., photoreceptors [PN1] and bipolar cells [PN3]), which develop during the same stage. 4 The mechanism underlying the neurogenic-to-gliogenic shift and hence the generation of Müller glia is not well defined. However, emerging evidence from the retina, 5 together with recent findings about the mechanism of astrocyte generation in the CNS, 6,7 suggests that this shift is likely to be orchestrated by complex interactions between the changing environment and cell-intrinsic machinery. 
In the retina, as elsewhere in the developing CNS, the environmental signals for gliogenesis are mediated by the interleukin (IL)-6 family of cytokines (e.g., ciliary neurotrophic factor [CNTF], leukemia inhibitory factors [LIFs], and cardiotrophin-1 [CT-1]) and the membrane-bound ligands, Jagged1 (Jag1) and Delta-like 1 (Dll1), acting through the JAK-STAT and Notch pathways, respectively. CNTF and LIF, as in astrocytes differentiation, 6 positively influence Müller glia differentiation through the JAK-STAT pathway. 8,9 Time-dependent activation of the Notch receptor ensures that progenitors remain uncommitted during early histogenesis and promotes glial differentiation during late histogenesis. Therefore, when Notch signaling is experimentally compromised during late histogenesis 5,9 or when the effectors of Notch signaling belonging to the Hes class are knocked out, 10 the generation of Müller glia is adversely affected. 
The mechanisms by which the JAK-STAT and Notch pathways collaborate with each other and with cell-intrinsic factors are not clear. The involvement of these pathways in astrocyte 7 and Müller glia 9 differentiation suggests the following scenario for Müller glia generation (Fig. 1). During early stages of retinal histogenesis, when gliogenesis is not favored, the expression of glial-specific genes is kept suppressed due to methylation of their regulatory regions. This epigenetic constraint explains why early retinal progenitors, when differentiated in vitro, generate fewer glia than late retinal progenitors under identical conditions. 11 Although Notch signaling can promote demethylation of glial-specific promoters, 12,13 a signaling threshold for the epigenetic change may exist that may not be reached by early retinal progenitors. As histogenesis progresses, relatively more neuroblasts expressing Notch ligands become committed, thereby stimulating Notch signaling in neighboring progenitors beyond the threshold to effectively inhibit the methylation of glial-specific genes. Around the same time during development, the concentration of cytokines secreted by neurons 7,14 and/or differentiating Müller glia 9 increases, activating hypomethylated glial specific genes. This premise is supported by the observation that CNTF alone is not as effective in promoting the differentiation of enriched late retinal progenitors into Müller glia as it is when Notch signaling is activated at the same time. 9 The cytokine-activated JAK-STAT pathway in turn has a feed-forward influence on Notch signaling that is likely to sustain the neurogenic–gliogenic switch at the late stage of histogenesis. 9,15 Another potential mechanism for a neurogenic–gliogenic switch may include the Lin28–Let7 pathway; however, the evidence for this in the retina is currently circumstantial (see below). 
Figure 1.
 
Mechanism underlying Müller gliogenesis. The retina as it enters the late stage of histogenesis (developing retina) contains early-born neurons (e.g., RGCs, cone photoreceptors, horizontal cells, and amacrine cells), nascent rod photoreceptors, progenitors (dark green), and neuroblasts (yellow). When neurogenesis is favored, progenitors receive sufficient Notch signaling from neighboring cells, including differentiating neuroblasts (box), which instruct the progenitors to remain uncommitted. At this stage Notch signaling is unable to inhibit the methylation of glial-specific genes, presumably because of its subthreshold levels, which therefore remain inactive. As development progresses through the late stage, the ever-increasing number of neuroblasts interacts with progenitors through Delta/Jagged (red bar)-Notch (blue bar) signaling as they migrate away, pushing the levels of Notch signaling beyond the threshold needed to inhibit the methylation of glial-specific genes. The differentiating neuroblasts elaborate cytokines (green dots), which now can activate glia-specific genes through the JAK-STAT pathway, thus paving the way for Müller glia differentiation and maturation (mature retina). The differentiating RX+ glioblasts (light green) may themselves contribute to gliogenesis by delivering Notch signaling in progenitors and elaborating cytokines. The evidence of the effects of Notch signaling on methylation and the involvement of Lin28 in Müller glia genesis is currently circumstantial and is therefore depicted by dashed boxes.
Figure 1.
 
Mechanism underlying Müller gliogenesis. The retina as it enters the late stage of histogenesis (developing retina) contains early-born neurons (e.g., RGCs, cone photoreceptors, horizontal cells, and amacrine cells), nascent rod photoreceptors, progenitors (dark green), and neuroblasts (yellow). When neurogenesis is favored, progenitors receive sufficient Notch signaling from neighboring cells, including differentiating neuroblasts (box), which instruct the progenitors to remain uncommitted. At this stage Notch signaling is unable to inhibit the methylation of glial-specific genes, presumably because of its subthreshold levels, which therefore remain inactive. As development progresses through the late stage, the ever-increasing number of neuroblasts interacts with progenitors through Delta/Jagged (red bar)-Notch (blue bar) signaling as they migrate away, pushing the levels of Notch signaling beyond the threshold needed to inhibit the methylation of glial-specific genes. The differentiating neuroblasts elaborate cytokines (green dots), which now can activate glia-specific genes through the JAK-STAT pathway, thus paving the way for Müller glia differentiation and maturation (mature retina). The differentiating RX+ glioblasts (light green) may themselves contribute to gliogenesis by delivering Notch signaling in progenitors and elaborating cytokines. The evidence of the effects of Notch signaling on methylation and the involvement of Lin28 in Müller glia genesis is currently circumstantial and is therefore depicted by dashed boxes.
Neurogenic Potential of Müller Glia
Anamniotes: Amphibians and Fish
In adult anamniotes, the retina grows continuously with the eyes, sustained largely by progenitors in the ciliary margin zone (CMZ), a germinal zone at the peripheral margin of the retina. 16 This constitutes the homeostatic development of the retina, allowing it to keep up with the normal growth of the eyes in the adult, which differs from regeneration of retinal cells that take place in response to injury. Active retinal regeneration is observed in adult anamniotes, but amphibians and fish appear to use different sources of cells to support their regeneration. The former uses the RPE cells, an extrinsic source, and the latter uses Müller glia, an intrinsic source. Both in urodele (newts and salamanders) and anuran (frogs) amphibians, the regeneration of retinal neurons is achieved through the transdifferentiation of RPE, which is mediated by the ERK pathways. 17 Such plasticity of RPE cells is observed in fish and higher vertebrates, but is restricted to the embryonic stages. 18 Therefore, regeneration through RPE transdifferentiation is not seen in adults, even in fish. 19  
The intrinsic progenitors for regeneration were discovered in embryonic and larval gold fish as groups of proliferating cells, called neurogenic clusters. 20 These clusters of cells radiate from the inner nuclear layer (INL) to the outer plexiform layer (OPL) and differentiate into rod photoreceptors during development. It was initially thought that these cells “seeded” rod precursors in the outer nuclear layer (ONL) during development and were later recruited in adults to generate rod photoreceptors for homeostatic development of the retina. 20,21 Later, Julian et al. 22 observed radial clusters of slow-cycling cells in adult rainbow trout, similar to the neurogenic clusters in larval fish. Using the sequential tagging of proliferating cells with nucleotide analogues BrdU and IdU, they demonstrated that the slow-cycling cells in the INL were the source of the previously discovered rapidly proliferating rod precursors in the ONL. This discovery led to the concept that intrinsic retinal progenitors sustain normal homoeostatic development and are also the source of regeneration in fish, observed in response to surgical removal of retinal cells, 23 as well as to chemical 24 and laser 25 injury. The location of these slow-cycling cells in the INL, where Müller cells reside, 22 and the observation that Müller cells re-enter the cell cycle and migrate into spaces vacated by dying photoreceptors in laser-damaged gold fish retina 25 suggest that Müller glia are the retinal progenitors in the neurogenic clusters that sustain regeneration. Using the optic nerve crush and mechanical injury models in transgenic Zebra fish, Fausett and Goldman 26 demonstrated that fate-mapped, green fluorescent protein (GFP)–tagged Müller glia are the source of cells involved in retinal regeneration. Further, they observed that these cells display properties of stem cells—that is, they activate the progenitor marker Pax6 in addition to α1T, a stem cell marker, and migrate to different retinal laminae, giving rise to different retinal cell types. Subsequently, Raymond et al. demonstrated that the progenitor properties of Zebra fish GFP-tagged Müller cells are not confined to their reaction to injury, but are also vital to the sustained growth of the retina in adults. 27 They proposed that the Pax6+ Müller glia in the INL respond to regulatory cues for the addition of new rod photoreceptors in the ever-growing retina by dividing asymmetrically to produce retinal progenitors. These progenitors cycle rapidly, move along the radial process of the daughter Müller glia to reach the ONL, where they eventually withdraw from the cell cycle and differentiate into rod photoreceptors. 
Amniotes: Chick and Mammals
The first evidence of the neurogenic potential of Müller glia was not obtained in amphibians and fish, but in chick retina. Fischer and Reh 28 observed cells in the INL that launch the proliferative response to injury after intraocular injection of N-methyl-d-aspartate (NMDA) in early postnatal chick retina. All the proliferating cells, 2 days after neurotoxin treatment, expressed glutamine synthetase (GS), an enzyme through which Müller glia convert glutamate into glutamine, suggesting that they represented Müller glia that had re-entered the cell cycle. The dividing cells expressed Chx10, Pax6, and Mash1, alluding to their progenitor nature. Furthermore, 7 days after injury, BrdU-positive cells were observed co-expressing markers of cell types of the inner retina, suggesting their neurogenic potential. In contrast to the observations in fish, 26,27 evidence for the neurogenic potential of Müller glia in chick remains rather indirect. 
Müller glia in mammals have long been known to be reactive in disease and injury, a response which, when includes the activation of the intermediate filament protein GFAP cellular hypertrophy, changes in ion transport properties, and proliferation are collectively known as reactive gliosis. 29 This reaction, which is common to glia in the CNS, ultimately ends up as a barrier to regeneration. Furthermore, not all injuries or diseases lead to the proliferation of Müller glia. Those causing rapid degeneration are usually accompanied by Müller glia proliferation, whereas in slow degeneration, as encountered in inherited dystrophy in the rds mouse, retina gliosis remains nonproliferative. 29 Given the observations that the initial stages of reactive gliosis are neuroprotective and regenerative, 29 the proliferative response of Müller glia in reactive gliosis was an early indication of their dormant and evolutionarily conserved regenerative potential. 
Ooto et al. 30 tested the regenerative potential of Müller glia in the adult rat, where the inner retina was damaged by intraocular injections of NMDA. Their activation results were almost identical with those observed in the chick, 28 where 100% of cells in the INL that incorporated BrdU expressed GS 2 days after neurotoxin injection. The number of BrdU+ cells decreased to 70% when the retina was examined 2 weeks later, suggesting that activated Müller glia differentiate into various cell types over time. This notion was supported by the presence of a small subset of BrdU+ cells in the injured retina that expressed markers corresponding to rod photoreceptors, bipolar cells, horizontal cells, and amacrine cells. Not only did this study demonstrate that the neurogenic potential of Müller cells is conserved in mammals, but it also suggested that the activated Müller glia are capable of generating a range of retinal neurons, including rod photoreceptors. Subsequently, different groups, using various approaches to activate Müller glia, such as through the Shh pathway 31 ; neurotoxic injury with the coincidental activation of ERK pathways 32 ; ONL injury mediated by N-methyl-N-nitrosourea 33 ; subtoxic doses of the l-glutamate analogue dl-α-aminoadipic acid 34 ; and through the Notch and Wnt pathways, 35 further demonstrated the neurogenic potential of Müller glia in mammals. 
Stem Cell Properties of Müller Glia
The evidence demonstrating that radial glia are neural stem cells 2 and the observations of the cross-species neurogenic potential of Müller glia, which share some morphologic, biochemical, and functional properties with radial glia, 36 raised the question of whether Müller glia are indeed stem cells. Das et al., 37 using the neurospheres assay, observed that a subset (0.018%) of retrospectively enriched rat Müller cells generates clonal neurospheres, consisting of proliferating cells that express neural progenitor markers. Immigrant astrocyte, microglia, and ciliary epithelial stem cell contamination was ruled out as a source of these neurospheres. Cells in neurospheres were multipotential, giving rise to both neurons and glia, and the resulting neurons displayed resting membrane potential and voltage-gated sodium currents, typical of neurons. They formed both secondary and tertiary neurospheres, containing cells that generated neurons in the same proportion as their parents, thus passing the test of self-renewal, the defining property of stem cells. Besides demonstrating that a subset of Müller glia possesses stem cell properties, which can be unmasked through the activation of Notch and Wnt signaling, this study provided direct evidence (see below) of the neurogenic potential of Müller glia in higher vertebrates (Fig. 2), compared with previous ones, 28,30 where the BrdU-based lineage-tracing approach raised the possibility that cells other than activated Müller cells could also be the source of the nascent neurons. In an alternate approach to obtaining evidence of neurogenic potential, activated Müller glia isolated from the fresh neurotoxin-damaged retina as a side-population (SP) of cells by the Hoechst dye efflux (HDE) assay 38 were transplanted directly into neonatal rat eyes. 37 The SP cell phenotype, which is due to the expression of an ATP class of transporter, ABCG2, is a characteristic of stem cells and progenitors in general, including those in the developing retina. 38 Cells that do not express ABCG2 (i.e., differentiated cells), segregate as non-SP (NSP) cells in an HDE assay. Two weeks after transplantation, CFDA-labeled Müller glia SP cells were observed to be integrated into different retinal layers. Those Müller glia SP cells in the ONL and GCL expressed markers corresponding to rod photoreceptors and RGCs, respectively, demonstrating the conversion of activated Müller glia into different retinal neurons in vivo. The stem cell nature of Müller glia was also demonstrated in transgenic Zebra fish 27 and in an immortalized human Müller cell line, 39 the former being significant for its in vivo approach and evidence. 
Figure 2.
 
Neurogenic potential of mammalian Müller glia. Intravitreal injection of neurotoxins (NMDA and kainite) and growth factors (FGF2 and insulin) causes a subset of Müller glia (curved arrow) to proliferate. The proliferating Müller glia express an ATP class of transporter ABCG2, which is the molecular basis of a method, the HDE assay (HDEA), by which the stem cells are enriched as SP cells by fluorescence-activated cell sorting (FACS). The activated Müller glia, prospectively enriched as Müller SP cells by HDE assay, proliferate as they incorporate BrdU and generate clonal neurospheres that express the neural progenitor markers musashi and nestin. Müller SP cells, labeled with CFDA and transplanted intravitreally in PN1 rat pups, are observed to incorporate in the different lamina of the host retina 2 weeks after transplantation. A subset of cells, incorporated in the ONL, expresses the rod photoreceptor-specific marker opsin, demonstrating the neurogenic conversion of activated Müller glia in vivo. Illustration adapted with permission from Das AV, Mallya KB, Zhao X, et al. Neural stem cell properties of Müller glia in the mammalian retina: regulation by Notch and Wnt signaling. Dev Biol. 2006;299:283–302.© Elsevier.
Figure 2.
 
Neurogenic potential of mammalian Müller glia. Intravitreal injection of neurotoxins (NMDA and kainite) and growth factors (FGF2 and insulin) causes a subset of Müller glia (curved arrow) to proliferate. The proliferating Müller glia express an ATP class of transporter ABCG2, which is the molecular basis of a method, the HDE assay (HDEA), by which the stem cells are enriched as SP cells by fluorescence-activated cell sorting (FACS). The activated Müller glia, prospectively enriched as Müller SP cells by HDE assay, proliferate as they incorporate BrdU and generate clonal neurospheres that express the neural progenitor markers musashi and nestin. Müller SP cells, labeled with CFDA and transplanted intravitreally in PN1 rat pups, are observed to incorporate in the different lamina of the host retina 2 weeks after transplantation. A subset of cells, incorporated in the ONL, expresses the rod photoreceptor-specific marker opsin, demonstrating the neurogenic conversion of activated Müller glia in vivo. Illustration adapted with permission from Das AV, Mallya KB, Zhao X, et al. Neural stem cell properties of Müller glia in the mammalian retina: regulation by Notch and Wnt signaling. Dev Biol. 2006;299:283–302.© Elsevier.
Facile Activation of Müller Glia
With the stem cell nature of mammalian Müller glia established, approaches are now being examined for activation of their neurogenic properties to sustain regeneration. An important question that is being addressed is whether Müller glia can be nudged into a regenerative mode without causing external injury to the retina. Emerging observations from different laboratories suggests that targeting specific signaling pathways, particularly those that regulate stem cells, may be sufficient to unmask the regenerative potential of Müller glia. Currently, these include the FGF, 40 Notch, 37,41 Wnt, 37,42 and Shh 31 pathways. Early evidence revealed that a single intraocular injection of Wnt2b can cause the activation of Müller glia in the uninjured adult rat retina. 37 Subsequently, Wan et al. 31 demonstrated that intraocularly injected Shh leads to Müller glia proliferation in the absence of injury and some of the BrdU+ cells expressed rod photoreceptor-specific markers. Del Debbio et al. 35 have demonstrated that the activation of Wnt and Notch signaling pathways is sufficient to stimulate Müller glia without resorting to neurotoxin-mediated retinal injury. The activated Müller glia in this study, both in vitro and in S334ter rats, an animal model of photoreceptor degeneration, differentiated along the rod photoreceptor lineage. 
The lineage conversion of activated Müller glia was demonstrated using different approaches, based on the premise that an activated Müller glial cell may not completely erase its parental property before acquiring a neuronal lineage. For example, 2 weeks after the activation of Notch and Wnt signaling in vivo, BrdU+GS+ cells were observed in the ONL expressing the rod photoreceptor-specific marker opsin. In a direct lineage-tracing experiment where activated Müller glia were fate-mapped by GS-GFP lentivirus transduction, GFP+opsin+ cells were observed in the ONL in the same time frame. Last, with the HDE assay, it was demonstrated that the activated Müller glia (BrdU+GS+ cells) reside exclusively in the SP compartment after the activation of Notch and Wnt signaling, as expected of stem cells. However, after 2 weeks of differentiation, the entire population of BrdU+GS+ cells shifted from the SP compartment to the non-SP (NSP) compartment, where differentiated cells reside, presumably owing to the decrease in the expression of ABCG2. A subset of BrdU+GS+ cells in the NSP compartment expressed opsin, thus reconfirming their conversion along a rod photoreceptor lineage. A recovery of light perception was also observed in S334 rats in which Notch and Wnt signaling was activated, correlating with the number of activated Müller glia expressing opsin, thus suggesting that Müller glia–mediated therapeutic regeneration. However, this study left two important questions unanswered in this regard. First, do converted Müller cells function as photoreceptors while retaining some of the parental features (i.e., expression of GS)? Second, is the functional recovery in light perception observed due to Müller glia conversion, or survival of host photoreceptors, or both? Similar approaches in different animal models of photoreceptor degeneration are necessary to confirm the proof of principle of therapeutic regeneration via Müller glia. 
Mechanisms Underlying the Neurogenic Potential of Müller Glia
While the regenerative properties of Müller glia are evolutionarily conserved, it appears that, in contrast to lower vertebrates, mammals have lost the ability to regenerate retinal neurons, perhaps because of constraints of the nonneurogenic environment of the adult retina and/or mammalian Müller glia may be intrinsically different than those of their fish counterparts. Currently, evidence favors the former because mammalian Müller glia share the expression of neurogenic regulators with those in fish, 37,43,44 and they can generate retinal neurons with better efficiency when they are cultured in a simulated environment of the developing retina, implying the existence of inhibitory properties in the non-neurogenic environment of the adult retina. 37 A similar constraint of the environment on neurogenic potential of activated astrocytes has been observed elsewhere in the adult CNS. For example, when astrocytes are activated in adult cerebral cortex in response to stab wounds, no neurons could be traced back to fate-mapped astrocytes. 45 However, when these astrocytes from injured cerebral cortex were isolated and cultured, they readily generated neurons. 
Extrinsic and intrinsic factors most likely cooperate to regulate the neurogenic potential of Müller glia (Fig. 3). Extrinsic regulation includes signaling mediated by the Notch-, Wnt-, Shh-, and FGF pathways. Activation of these pathways, either due to injury or exogenous ligands, moves a subset of Müller glia into the G1–S phase of the cell cycle. A critical molecular event for this transition and a link to intrinsic regulation is likely to be the attenuation of the expression of p27kip1, a CDK inhibitor, with expression that is characteristically high in Müller glia. 48 The expression of p27kip1 in Müller glia may have evolved to keep their proclivity to proliferate in check and is likely to be negatively regulated by Notch/Wnt/FGF signaling, thus making cell-cycle regulation of Müller glia sensitive to changes in their external environment. 37 Therefore, a transient activation of these pathways in response to injury may prompt Müller glia to divide and give rise to intermediate progenitor cells (IPCs), similar to those generated by radial glia during neurogenesis. 2 The Müller IPCs, which express the retinal progenitor markers Pax6, Rx, and Chx10, 37,43 migrate out of the INL and into the site of injury. Along the way, the epigenetic status of Müller IPCs is progressively altered, allowing the modulation of gene expression involved in the regeneration pathway. Genes that may be involved in this process include regulators of progenitor proliferation and neurogliogenesis (e.g., Lin28) and proneural bHLH transcription factors (e.g., Mash1). 
Figure 3.
 
Mechanism underlying stemness and neurogenic potential of Müller glia. A subset of Müller glia (dark green color), presumably those with dormant stem cell properties and proximity to blood vessels (red ellipse), responds to injury (i.e., the loss of rod photoreceptors) by activating the key stem cell regulatory pathways Notch and Wnt signaling. The activation is demonstrated by upregulation of Hes1, and Lef1, the intracellular effectors of Notch and Wnt signaling, respectively. Other signaling pathways known to be recruited by Müller glia include the FGF and Shh pathways. One of the intrinsic events these pathways may regulate is the expression of p27Kip1 to move Müller glia into the G1–S phase of the cell cycle. Proliferating Müller glia generate intermediate progenitor cells (IPCs), which migrate out of the inner nuclear layer (INL). These IPCs display the properties of retinal progenitors during histogenesis, in that they express Pax6, Rx, and Chx10. Within INL, or concomitant with their migration from the INL, there is a change in the expression of a network of genes, presumably due to epigenetic modulation of these cells. The neurogenic network of genes may also include Lin28, whose expression is pre-activated (consequently, decreasing the expression of Let7) to endow progenitor properties, Mash1, whose expression is also upregulated to confer neurogenic properties, and a candidate gene REST, whose expression is attenuated, presumably to remove the brake on neuronal gene expression for regenerating dead photoreceptors. A speculative and rather simple linear regulatory relationship between these genes is shown based on the works of Ryback et al. 46 and Ramachandran et al. 47
Figure 3.
 
Mechanism underlying stemness and neurogenic potential of Müller glia. A subset of Müller glia (dark green color), presumably those with dormant stem cell properties and proximity to blood vessels (red ellipse), responds to injury (i.e., the loss of rod photoreceptors) by activating the key stem cell regulatory pathways Notch and Wnt signaling. The activation is demonstrated by upregulation of Hes1, and Lef1, the intracellular effectors of Notch and Wnt signaling, respectively. Other signaling pathways known to be recruited by Müller glia include the FGF and Shh pathways. One of the intrinsic events these pathways may regulate is the expression of p27Kip1 to move Müller glia into the G1–S phase of the cell cycle. Proliferating Müller glia generate intermediate progenitor cells (IPCs), which migrate out of the inner nuclear layer (INL). These IPCs display the properties of retinal progenitors during histogenesis, in that they express Pax6, Rx, and Chx10. Within INL, or concomitant with their migration from the INL, there is a change in the expression of a network of genes, presumably due to epigenetic modulation of these cells. The neurogenic network of genes may also include Lin28, whose expression is pre-activated (consequently, decreasing the expression of Let7) to endow progenitor properties, Mash1, whose expression is also upregulated to confer neurogenic properties, and a candidate gene REST, whose expression is attenuated, presumably to remove the brake on neuronal gene expression for regenerating dead photoreceptors. A speculative and rather simple linear regulatory relationship between these genes is shown based on the works of Ryback et al. 46 and Ramachandran et al. 47
Lin28, which encodes an RNA-binding protein, was discovered in Caenorhabditis elegans as a regulator of developmental timing. 49 In vertebrates, it is expressed during early embryonic development and histogenesis of a variety of tissues, including the CNS. 50,51 Recent evidence suggests that in the developing CNS, Lin28 may play out its evolutionarily conserved role of a heterochronic gene, regulating the timing of neurogliogenesis. 52 This may also be the case in the developing retina, where Lin28 is expressed 50,51 and levels of its transcript correlate with two stages of histogenesis (Ahmad et al., unpublished observations, 2011); higher expression in early retinal progenitors that preferentially generate neurons and lower expression in late retinal progenitors from which Müller glia are derived. 
One of the lin28-based mechanisms of neurogliogenesis involves the heterochronic micro RNA (miRNA) let-7 (Mirlet7), which promotes differentiation. 52,53 Lin28 blocks the processing of Let-7, thus inhibiting differentiation temporally. 46,54 An increase in Lin28 expression in injury-activated Müller glia will move these cells closer to the molecular status of retinal progenitors and inhibit Let7 expression, destabilizing their glial phenotype for regenerative purposes. This premise is recently supported by work by Ramachandran et al., 47 in which an increase and decrease in the levels of expression of Lin28 and Let7, respectively, were observed in injury-induced Müller glia in Zebra fish retina. 
The pro-neural bHLH transcription factor, Mash1, which is expressed in activated Zebra fish 55 and chick 28 Müller glia, may also play an important role in retinal regeneration. It has been observed that proneural bHLH transcription factors promote neuronal differentiation but inhibit gliogenesis simultaneously in developing retina, 56 as elsewhere in the CNS. 57 The onset of Mash1 expression in activated Müller glia may represent another mechanism to endow these cells with the potential to differentiate along neuronal lineages. 
It is unlikely that these genes work in isolation; more likely, they act in concert as a regenerative gene network. For example, Mash1 may be upregulated in activated Müller glia, presumably in response to attenuation in the expression of a gene whose function may be to prevent premature neuronal differentiation. The identity of this gene is unknown, but REST, which is a global repressor of neuronal genes 58 and is expressed in retinal progenitors, 59 may be a likely candidate. The expression of Lin28, which was recently shown to be a target of Mash1, 47 may increase in response to upregulation of Mash1. Furthermore, the Lin28-dependent decrease in Let7 may remove the negative feedback inhibition of Lin28 by Let7, 46 thus sustaining the expression of Lin28. Together, these changes may promote activated Müller glia plasticity and regeneration. 
Summary
In the past 5 years, evidence has emerged from work on different species, in which a variety of approaches have been used, that Müller glia represent a latent stem cell population in the adult retina and are therefore a valid target for therapeutic regeneration. While the stemness of Müller glia is evolutionarily conserved, the efficiency with which they regenerate retinal neurons in vivo is not. Compared with fish, the ability of these cells to differentiate along neuronal lineages in mammals is remarkably constrained. This remains the major barrier to Müller glia-based therapeutic regeneration. There are several outstanding questions that must be answered in this nascent field to circumvent this barrier. For example: Do all Müller glia possesses stem cell potential? If stemness is associated with a subset of Müller glia, as it currently appears, is it a dormant property that is unmasked or is it acquired de novo (dedifferentiation)? What is the nature and contribution of the microniche around this special class of cells? What are the signaling pathways after injury that activate these cells, and how do they cross-talk with each other and with the intrinsic cellular machinery in altering the epigenetic status of cells that ultimately suppress gliogenesis and encourage neurogenesis? Answers to these questions will identify molecular targets for facile activation of Müller glia, moving them efficiently and predictably along a desired neuronal lineage for a variety of potential therapeutic purposes. 
Footnotes
 Supported by the Lincy Foundation.
Footnotes
 Disclosure: I. Ahmad, None; C.B. Del Debbio, None; A.V. Das, None; S. Parameswaran, None
The authors thank Jerry Chader, Vijay Sarthy, and Gregory Bennett for critical reading of the manuscript and invaluable suggestions. 
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Figure 1.
 
Mechanism underlying Müller gliogenesis. The retina as it enters the late stage of histogenesis (developing retina) contains early-born neurons (e.g., RGCs, cone photoreceptors, horizontal cells, and amacrine cells), nascent rod photoreceptors, progenitors (dark green), and neuroblasts (yellow). When neurogenesis is favored, progenitors receive sufficient Notch signaling from neighboring cells, including differentiating neuroblasts (box), which instruct the progenitors to remain uncommitted. At this stage Notch signaling is unable to inhibit the methylation of glial-specific genes, presumably because of its subthreshold levels, which therefore remain inactive. As development progresses through the late stage, the ever-increasing number of neuroblasts interacts with progenitors through Delta/Jagged (red bar)-Notch (blue bar) signaling as they migrate away, pushing the levels of Notch signaling beyond the threshold needed to inhibit the methylation of glial-specific genes. The differentiating neuroblasts elaborate cytokines (green dots), which now can activate glia-specific genes through the JAK-STAT pathway, thus paving the way for Müller glia differentiation and maturation (mature retina). The differentiating RX+ glioblasts (light green) may themselves contribute to gliogenesis by delivering Notch signaling in progenitors and elaborating cytokines. The evidence of the effects of Notch signaling on methylation and the involvement of Lin28 in Müller glia genesis is currently circumstantial and is therefore depicted by dashed boxes.
Figure 1.
 
Mechanism underlying Müller gliogenesis. The retina as it enters the late stage of histogenesis (developing retina) contains early-born neurons (e.g., RGCs, cone photoreceptors, horizontal cells, and amacrine cells), nascent rod photoreceptors, progenitors (dark green), and neuroblasts (yellow). When neurogenesis is favored, progenitors receive sufficient Notch signaling from neighboring cells, including differentiating neuroblasts (box), which instruct the progenitors to remain uncommitted. At this stage Notch signaling is unable to inhibit the methylation of glial-specific genes, presumably because of its subthreshold levels, which therefore remain inactive. As development progresses through the late stage, the ever-increasing number of neuroblasts interacts with progenitors through Delta/Jagged (red bar)-Notch (blue bar) signaling as they migrate away, pushing the levels of Notch signaling beyond the threshold needed to inhibit the methylation of glial-specific genes. The differentiating neuroblasts elaborate cytokines (green dots), which now can activate glia-specific genes through the JAK-STAT pathway, thus paving the way for Müller glia differentiation and maturation (mature retina). The differentiating RX+ glioblasts (light green) may themselves contribute to gliogenesis by delivering Notch signaling in progenitors and elaborating cytokines. The evidence of the effects of Notch signaling on methylation and the involvement of Lin28 in Müller glia genesis is currently circumstantial and is therefore depicted by dashed boxes.
Figure 2.
 
Neurogenic potential of mammalian Müller glia. Intravitreal injection of neurotoxins (NMDA and kainite) and growth factors (FGF2 and insulin) causes a subset of Müller glia (curved arrow) to proliferate. The proliferating Müller glia express an ATP class of transporter ABCG2, which is the molecular basis of a method, the HDE assay (HDEA), by which the stem cells are enriched as SP cells by fluorescence-activated cell sorting (FACS). The activated Müller glia, prospectively enriched as Müller SP cells by HDE assay, proliferate as they incorporate BrdU and generate clonal neurospheres that express the neural progenitor markers musashi and nestin. Müller SP cells, labeled with CFDA and transplanted intravitreally in PN1 rat pups, are observed to incorporate in the different lamina of the host retina 2 weeks after transplantation. A subset of cells, incorporated in the ONL, expresses the rod photoreceptor-specific marker opsin, demonstrating the neurogenic conversion of activated Müller glia in vivo. Illustration adapted with permission from Das AV, Mallya KB, Zhao X, et al. Neural stem cell properties of Müller glia in the mammalian retina: regulation by Notch and Wnt signaling. Dev Biol. 2006;299:283–302.© Elsevier.
Figure 2.
 
Neurogenic potential of mammalian Müller glia. Intravitreal injection of neurotoxins (NMDA and kainite) and growth factors (FGF2 and insulin) causes a subset of Müller glia (curved arrow) to proliferate. The proliferating Müller glia express an ATP class of transporter ABCG2, which is the molecular basis of a method, the HDE assay (HDEA), by which the stem cells are enriched as SP cells by fluorescence-activated cell sorting (FACS). The activated Müller glia, prospectively enriched as Müller SP cells by HDE assay, proliferate as they incorporate BrdU and generate clonal neurospheres that express the neural progenitor markers musashi and nestin. Müller SP cells, labeled with CFDA and transplanted intravitreally in PN1 rat pups, are observed to incorporate in the different lamina of the host retina 2 weeks after transplantation. A subset of cells, incorporated in the ONL, expresses the rod photoreceptor-specific marker opsin, demonstrating the neurogenic conversion of activated Müller glia in vivo. Illustration adapted with permission from Das AV, Mallya KB, Zhao X, et al. Neural stem cell properties of Müller glia in the mammalian retina: regulation by Notch and Wnt signaling. Dev Biol. 2006;299:283–302.© Elsevier.
Figure 3.
 
Mechanism underlying stemness and neurogenic potential of Müller glia. A subset of Müller glia (dark green color), presumably those with dormant stem cell properties and proximity to blood vessels (red ellipse), responds to injury (i.e., the loss of rod photoreceptors) by activating the key stem cell regulatory pathways Notch and Wnt signaling. The activation is demonstrated by upregulation of Hes1, and Lef1, the intracellular effectors of Notch and Wnt signaling, respectively. Other signaling pathways known to be recruited by Müller glia include the FGF and Shh pathways. One of the intrinsic events these pathways may regulate is the expression of p27Kip1 to move Müller glia into the G1–S phase of the cell cycle. Proliferating Müller glia generate intermediate progenitor cells (IPCs), which migrate out of the inner nuclear layer (INL). These IPCs display the properties of retinal progenitors during histogenesis, in that they express Pax6, Rx, and Chx10. Within INL, or concomitant with their migration from the INL, there is a change in the expression of a network of genes, presumably due to epigenetic modulation of these cells. The neurogenic network of genes may also include Lin28, whose expression is pre-activated (consequently, decreasing the expression of Let7) to endow progenitor properties, Mash1, whose expression is also upregulated to confer neurogenic properties, and a candidate gene REST, whose expression is attenuated, presumably to remove the brake on neuronal gene expression for regenerating dead photoreceptors. A speculative and rather simple linear regulatory relationship between these genes is shown based on the works of Ryback et al. 46 and Ramachandran et al. 47
Figure 3.
 
Mechanism underlying stemness and neurogenic potential of Müller glia. A subset of Müller glia (dark green color), presumably those with dormant stem cell properties and proximity to blood vessels (red ellipse), responds to injury (i.e., the loss of rod photoreceptors) by activating the key stem cell regulatory pathways Notch and Wnt signaling. The activation is demonstrated by upregulation of Hes1, and Lef1, the intracellular effectors of Notch and Wnt signaling, respectively. Other signaling pathways known to be recruited by Müller glia include the FGF and Shh pathways. One of the intrinsic events these pathways may regulate is the expression of p27Kip1 to move Müller glia into the G1–S phase of the cell cycle. Proliferating Müller glia generate intermediate progenitor cells (IPCs), which migrate out of the inner nuclear layer (INL). These IPCs display the properties of retinal progenitors during histogenesis, in that they express Pax6, Rx, and Chx10. Within INL, or concomitant with their migration from the INL, there is a change in the expression of a network of genes, presumably due to epigenetic modulation of these cells. The neurogenic network of genes may also include Lin28, whose expression is pre-activated (consequently, decreasing the expression of Let7) to endow progenitor properties, Mash1, whose expression is also upregulated to confer neurogenic properties, and a candidate gene REST, whose expression is attenuated, presumably to remove the brake on neuronal gene expression for regenerating dead photoreceptors. A speculative and rather simple linear regulatory relationship between these genes is shown based on the works of Ryback et al. 46 and Ramachandran et al. 47
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