Levi Todd

A comparative analysis of Muller glia-mediated regeneration in the vertebrate retina.

This article reviews the current state of knowledge regarding the potential of Müller glia to become neuronal progenitor cells in the avian retina. We compare and contrast the remarkable proliferative and neurogenic capacity of Müller glia in the fish retina to the limited capacity of Müller glia in avian and rodent retinas. We summarize recent findings regarding the secreted factors, signaling pathways and cell intrinsic factors that have been implicated in the formation of Müller glia-derived progenitors. We discuss several key similarities and differences between the fish, rodent and chick model systems, highlighting several of the key transcription factors and signaling pathways that regulate the formation of Müller glia-derived progenitors.

Reactive microglia and macrophage facilitate the formation of Müller glia‐derived retinal progenitors

In retinas where Müller glia have been stimulated to become progenitor cells, reactive microglia are always present. Thus, we investigated how the activation or ablation of microglia/macrophage influences the formation of Müller glia‐derived progenitor cells (MGPCs) in the retina in vivo. Intraocular injections of the Interleukin‐6 (IL6) stimulated the reactivity of microglia/macrophage, whereas other types of retinal glia appear largely unaffected. In acutely damaged retinas where all of the retinal microglia/macrophage were ablated, the formation of proliferating MGPCs was greatly diminished. With the microglia ablated in damaged retinas, levels of Notch and related genes were unchanged or increased, whereas levels of ascl1a, TNFα, IL1β, complement component 3 (C3) and C3a receptor were significantly reduced. In the absence of retinal damage, the combination of insulin and Fibroblast growth factor 2 (FGF2) failed to stimulate the formation of MGPCs when the microglia/macrophage were ablated. In addition, intraocular injections of IL6 and FGF2 stimulated the formation of MGPCs in the absence of retinal damage, and this generation of MGPCs was blocked when the microglia/macrophage were absent. We conclude that the activation of microglia and/or infiltrating macrophage contributes to the formation of proliferating MGPCs, and these effects may be mediated by components of the complement system and inflammatory cytokines. GLIA 2014;62:1608–1628

Hedgehog signaling stimulates the formation of proliferating Müller glia-derived progenitor cells in the chick retina

Müller glia can be stimulated to de-differentiate and become proliferating progenitor cells that regenerate neurons in the retina. The signaling pathways that regulate the formation of proliferating Müller glia-derived progenitor cells (MGPCs) are beginning to be revealed. The purpose of this study was to investigate whether Hedgehog (Hh) signaling influences the formation of MGPCs in the chick retina. We find that Hh signaling is increased in damaged retinas where MGPCs are known to form. Sonic Hedgehog (Shh) is normally present in the axons of ganglion cells, but becomes associated with Müller glia and MGPCs following retinal damage. Activation of Hh signaling with recombinant human SHH (rhShh) or smoothened agonist (SAG) increased levels of Ptch1, Gli1, Gli2, Gli3, Hes1 and Hes5, and stimulated the formation of proliferating MGPCs in damaged retinas. In undamaged retinas, SAG or rhShh had no apparent effect upon the Müller glia. However, SAG combined with FGF2 potentiated the formation of MGPCs, whereas SAG combined with IGF1 stimulated the nuclear migration of Müller glia, but not the formation of MGPCs. Conversely, inhibition of Hh signaling with KAAD-cyclopamine, Gli antagonists or antibody to Shh reduced numbers of proliferating MGPCs in damaged and FGF2-treated retinas. Hh signaling potentiates Pax6, Klf4 and cFos expression in Müller glia during the formation of MGPCs. We find that FGF2/MAPK signaling recruits Hh signaling into the signaling network that drives the formation of proliferating MGPCs. Our findings implicate Hh signaling as a key component of the network of signaling pathways that promote the de-differentiation of Müller glia and proliferation of MGPCs. Summary: In the chick retina, Hedgehog signaling contributes to retinal repair by promoting the de-differentiation of Muller glia and the proliferation of Muller glia-derived progenitor cells.

Heparin-binding EGF-like growth factor (HB-EGF) stimulates the proliferation of Muller glia-derived progenitor cells in avian and murine retinas.

Müller glia can be stimulated to de-differentiate, proliferate and form Müller glia-derived progenitor cells (MGPCs) that regenerate retinal neurons. In the zebrafish retina, heparin-binding EGF-like growth factor (HB-EGF) may be one of the key factors that stimulate the formation of proliferating MGPCs. Currently nothing is known about the influence of HB-EGF on the proliferative potential of Müller glia in retinas of birds and rodents. In the chick retina, we found that levels of both hb-egf and egf-receptor are rapidly and transiently up-regulated following NMDA-induced damage. Although intraocular injections of HB-EGF failed to stimulate cell-signaling or proliferation of Müller glia in normal retinas, HB-EGF stimulated proliferation of MGPCs in damaged retinas. By comparison, inhibition of the EGF-receptor (EGFR) decreased the proliferation of MGPCs in damaged retinas. HB-EGF failed to act synergistically with FGF2 to stimulate the formation of MGPCs in the undamaged retina and inhibition of EGF-receptor did not suppress FGF2-mediated formation of MGPCs. In the mouse retina, HB-EGF stimulated the proliferation of Müller glia following NMDA-induced damage. Furthermore, HB-EGF not only stimulated MAPK-signaling in Müller glia/MGPCs, but also activated mTor- and Jak/Stat-signaling. We propose that levels of expression of EGFR are rate-limiting to the responses of Müller glia to HB-EGF and the expression of EGFR can be induced by retinal damage, but not by FGF2-treatment. We conclude that HB-EGF is mitogenic to Müller glia in both chick and mouse retinas, and HB-EGF is an important player in the formation of MGPCs in damaged retinas.

Jak/Stat signaling regulates the proliferation and neurogenic potential of Müller glia-derived progenitor cells in the avian retina

Müller glia are capable of de-differentiating and proliferating to become Müller glia-derived progenitor cells (MGPCs) with the ability to regenerate retinal neurons. One of the cell-signaling pathways that drives the reprogramming of Müller glia into MGPCs in the zebrafish retina is the Jak/Stat-pathway. However, nothing is known about the influence of Jak/Stat-signaling during the formation of MGPCs in the retinas of warm-blooded vertebrates. Accordingly, we examined whether Jak/Stat-signaling influences the formation of MGPCs and differentiation of progeny in the avian retina. We found that Jak/Stat-signaling is activated in Müller glia in response to NMDA-induced retinal damage or by CNTF or FGF2 in the absence of retinal damage. Inhibition of gp130, Jak2, or Stat3 suppressed the formation of proliferating MGPCs in NMDA-damaged and FGF2-treated retinas. Additionally, CNTF combined with FGF2 enhanced the formation of proliferating MGPCs in the absence of retinal damage. In contrast to the zebrafish model, where activation of gp130/Jak/Stat is sufficient to drive neural regeneration from MGPCs, signaling through gp130 inhibits the neurogenic potential of MGPCs and promotes glial differentiation. We conclude that gp130/Jak/Stat-signaling plays an important role in the network of pathways that drives the formation of proliferating MGPCs; however, this pathway inhibits the neural differentiation of the progeny.

mTor signaling is required for the formation of proliferating Müller glia-derived progenitor cells in the chick retina

We investigate the roles of mTor signaling in the formation of Müller glia-derived progenitor cells (MGPCs) in the chick retina. During embryonic development, pS6 (a readout of active mTor signaling) is present in early-stage retinal progenitors, differentiating amacrine and ganglion cells, and late-stage progenitors or maturing Müller glia. By contrast, pS6 is present at low levels in a few scattered cell types in mature, healthy retina. Following retinal damage, in which MGPCs are known to form, mTor signaling is rapidly activated in Müller glia. Inhibition of mTor in damaged retinas prevented the accumulation of pS6 in Müller glia and reduced numbers of proliferating MGPCs. Inhibition of mTor had no effect on MAPK signaling or on upregulation of the stem cell factor Klf4, whereas Pax6 upregulation was significantly reduced. Inhibition of mTor potently blocked the MGPC-promoting effects of Hedgehog, Wnt and glucocorticoid signaling in damaged retinas. In the absence of retinal damage, insulin, IGF1 and FGF2 induced pS6 in Müller glia, and this was blocked by mTor inhibitor. In FGF2-treated retinas, in which MGPCs are known to form, inhibition of mTor blocked the accumulation of pS6, the upregulation of Pax6 and the formation of proliferating MGPCs. We conclude that mTor signaling is required, but not sufficient, to stimulate Müller glia to give rise to proliferating progenitors, and the network of signaling pathways that drive the formation of MGPCs requires activation of mTor. Highlighted article: Inhibition of mTor signaling in vivo prevents the proliferation of Müller glia cells and blocks the regenerative response in the chick retina.

Comparative analysis of glucagonergic cells, glia, and the circumferential marginal zone in the reptilian retina

Retinal progenitors in the circumferential marginal zone (CMZ) and Müller glia‐derived progenitors have been well described for the eyes of fish, amphibians, and birds. However, there is no information regarding a CMZ and the nature of retinal glia in species phylogenetically bridging amphibians and birds. The purpose of this study was to examine the retinal glia and investigate whether a CMZ is present in the eyes of reptilian species. We used immunohistochemical analyses to study retinal glia, neurons that could influence CMZ progenitors, the retinal margin, and the nonpigmented epithelium of ciliary body of garter snakes, queen snakes, anole lizards, snapping turtles, and painted turtles. We compare our observations on reptile eyes to the CMZ and glia of fish, amphibians, and birds. In all species, Sox9, Pax6, and the glucocorticoid receptor are expressed by Müller glia and cells at the retinal margin. However, proliferating cells were found only in the CMZ of turtles and not in the eyes of anoles and snakes. Similar to eyes of chickens, the retinal margin in turtles contains accumulations of GLP1/glucagonergic neurites. We find that filamentous proteins, vimentin and GFAP, are expressed by Müller glia, but have different patterns of subcellular localization in the different species of reptiles. We provide evidence that the reptile retina may contain nonastrocytic inner retinal glial cells, similar to those described in the avian retina. We conclude that the retinal glia, glucagonergic neurons, and CMZ of turtles appear to be most similar to those of fish, amphibians, and birds. J. Comp. Neurol. 524:74–89, 2016.

Wnt/β‐catenin‐signaling and the formation of Müller glia‐derived progenitors in the chick retina

Müller glia can be stimulated to de‐differentiate, proliferate, and form Müller glia‐derived progenitor cells (MGPCs) that are capable of producing retinal neurons. The signaling pathways that influence the de‐differentiation of mature Müller glia and proliferation of MGPCs may include the Wnt‐pathway. The purpose of this study was to investigate how Wnt‐signaling influences the formation of MGPCs in the chick retina in vivo. In NMDA‐damaged retinas where MGPCs are known to form, we find dynamic changes in retinal levels of potential readouts of Wnt‐signaling, including dkk1, dkk3, axin2, c‐myc, tcf‐1, and cd44. We find accumulations of nuclear β‐catenin in MGPCs that peaks at 3 days and rapidly declines by 5 days after NMDA‐treatment. Inhibition of Wnt‐signaling with XAV939 in damaged retinas suppressed the formation of MGPCs, increased expression of ascl1a and decreased hes5, but had no effect upon the differentiation of progeny produced by MGPCs. Activation of Wnt‐signaling, with GSK3β‐inhibitors, in the absence of retinal damage, failed to stimulate the formation of MGPCs, whereas activation of Wnt‐signaling in damaged retinas stimulated the formation of MGPCs. In the absence of retinal damage, FGF2/MAPK‐signaling stimulated the formation of MGPCs by activating a signaling network that includes Wnt/β‐catenin. In FGF2‐treated retinas, inhibition of Wnt‐signaling reduced numbers of proliferating MGPCs, whereas activation of Wnt‐signaling failed to influence the formation of proliferating MGPCs. Our findings indicate that Wnt‐signaling is part of a network initiated by FGF2/MAPK or retinal damage, and activation of canonical Wnt‐signaling is required for the formation of proliferating MGPCs.

Wnt/βcatenin‐signaling and the formation of Müller glia‐derived progenitors in the chick retina

Müller glia can be stimulated to de‐differentiate, proliferate, and form Müller glia‐derived progenitor cells (MGPCs) that are capable of producing retinal neurons. The signaling pathways that influence the de‐differentiation of mature Müller glia and proliferation of MGPCs may include the Wnt‐pathway. The purpose of this study was to investigate how Wnt‐signaling influences the formation of MGPCs in the chick retina in vivo. In NMDA‐damaged retinas where MGPCs are known to form, we find dynamic changes in retinal levels of potential readouts of Wnt‐signaling, including dkk1, dkk3, axin2, c‐myc, tcf‐1, and cd44. We find accumulations of nuclear β‐catenin in MGPCs that peaks at 3 days and rapidly declines by 5 days after NMDA‐treatment. Inhibition of Wnt‐signaling with XAV939 in damaged retinas suppressed the formation of MGPCs, increased expression of ascl1a and decreased hes5, but had no effect upon the differentiation of progeny produced by MGPCs. Activation of Wnt‐signaling, with GSK3β‐inhibitors, in the absence of retinal damage, failed to stimulate the formation of MGPCs, whereas activation of Wnt‐signaling in damaged retinas stimulated the formation of MGPCs. In the absence of retinal damage, FGF2/MAPK‐signaling stimulated the formation of MGPCs by activating a signaling network that includes Wnt/β‐catenin. In FGF2‐treated retinas, inhibition of Wnt‐signaling reduced numbers of proliferating MGPCs, whereas activation of Wnt‐signaling failed to influence the formation of proliferating MGPCs. Our findings indicate that Wnt‐signaling is part of a network initiated by FGF2/MAPK or retinal damage, and activation of canonical Wnt‐signaling is required for the formation of proliferating MGPCs.

BMP‐ and TGFβ‐signaling regulate the formation of Müller glia‐derived progenitor cells in the avian retina

Müller glia‐derived progenitor cells (MGPCs) have the capability to regenerate neurons in the retinas of different vertebrate orders. The formation of MGPCs is regulated by a network of cell‐signaling pathways. The purpose of this study was to investigate how BMP/Smad1/5/8‐ and TGFβ/Smad2/3‐signaling are coordinated to influence the formation of MGPCs in the chick model system. We find that pSmad1/5/8 is selectively up‐regulated in the nuclei of Müller glia following treatment with BMP4, FGF2, or NMDA‐induced damage, and this up‐regulation is blocked by a dorsomorphin analogue DMH1. By comparison, Smad2/3 is found in the nuclei of Müller glia in untreated retinas, and becomes localized to the cytoplasm following NMDA‐ or FGF2‐treatment. These findings suggest a decrease in TGFβ‐ and increase in BMP‐signaling when MGPCs are known to form. In both NMDA‐damaged and FGF2‐treated retinas, inhibition of BMP‐signaling suppressed the proliferation of MGPCs, whereas inhibition of TGFβ‐signaling stimulated the proliferation of MGPCs. Consistent with these findings, TGFβ2 suppressed the formation of MGPCs in NMDA‐damaged retinas. Our findings indicate that BMP/TGFβ/Smad‐signaling is recruited into the network of signaling pathways that controls the formation of proliferating MGPCs. We conclude that signaling through BMP4/Smad1/5/8 promotes the formation of MGPCs, whereas signaling through TGFβ/Smad2/3 suppresses the formation of MGPCs.