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Keywords:

  • Müller glia;
  • retina;
  • Notch;
  • Hes;
  • p27Kip1;
  • p27Xic1

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. NOTCH AND MÜLLER GLIOGENESIS
  5. EFFECTORS OF THE NOTCH PATHWAY IN THE RETINA
  6. CYCLIN DEPENDENT KINASE INHIBITORS IN THE RETINA
  7. REGULATION OF REACTIVE GLIOSIS OF MÜLLER CELLS
  8. REGULATION OF MÜLLER GLIAL DIFFERENTIATION BY CYCLIN DEPENDENT KINASE INHIBITORS
  9. Acknowledgements
  10. REFERENCES

During development of the vertebrate neural retina, multipotent stem cells give rise to retinal neurons as well as to Müller cells, the principal glial population in the retina. Recent studies have shed light upon the extracellular and intracellular signaling pathways that regulate Müller glial cell genesis. Emerging evidence demonstrates that activation of the Notch signaling pathway can play a role in regulating Müller cell development as well as gliogenesis in other parts of the central nervous system. Cyclin dependent kinase (CDK) inhibitors of the Cip/Kip subfamily are cell cycle regulators that can regulate progenitor proliferation during retinal development, but also regulate the proliferation of Müller glia when they become activated in response to stress or injury. Surprisingly this class of proteins can also promote the development of Müller glia. In this review we discuss the role of both Notch and the CDK inhibitors in regulating Müller cell development. © 2001 Wiley-Liss, Inc.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. NOTCH AND MÜLLER GLIOGENESIS
  5. EFFECTORS OF THE NOTCH PATHWAY IN THE RETINA
  6. CYCLIN DEPENDENT KINASE INHIBITORS IN THE RETINA
  7. REGULATION OF REACTIVE GLIOSIS OF MÜLLER CELLS
  8. REGULATION OF MÜLLER GLIAL DIFFERENTIATION BY CYCLIN DEPENDENT KINASE INHIBITORS
  9. Acknowledgements
  10. REFERENCES

A central issue in neurobiology has been how structural and functional distinctions between neurons and glia arise during embryonic development. The vertebrate neural retina has long been used as a relatively simple, accessible model for investigating cell type specification during development. All vertebrate retinas contain six classes of neuronal cells and one glial cell type, the Müller glial cell (Cepko et al., 1996; Harris, 1997). While these cells have become specialized for distinct functions, they all are derived from a common retinal stem cell (Turner and Cepko, 1987). During retinal development, stem cells either remain as multipotent progenitors, differentiate as neurons, or differentiate as glial cells. Significant progress has been made in elucidating the extrinsic and intrinsic factors responsible for generating the various types of retinal neurons. For example, positively acting basic helix-loop-helix (bHLH) genes and other transcription factors have been shown to regulate neuronal fate in the retina (reviewed in Cepko, 1999).

In contrast, there has been little investigation into how the Müller cell, the principal glial cell in the retina, is generated from retinal stem cells. Some of the molecular signals that influence the generation of these major support cells for retinal neurons have been identified, including signaling factors such as the epidermal growth factor receptor (EGFR) (Lillien, 1995). Yet for the most part, the extracellular signals and intracellular mediators of those signals that determine glial fates within the retina have been largely undefined. Over the past year, there have been important advances in our understanding of some of the molecular mechanisms involved in Müller fate determination. In this review we will examine evidence implicating the Notch signaling pathway in Müller gliogenesis and evidence that suggests that cell cycle proteins may be involved in Müller glia cell development.

One of the problems with glial-promoting substances identified in culture was that most were thought to act at long range and it was difficult to imagine how they might be influencing the choice of retinal cell fate within a field of multipotent retinal progenitors. Recently, several studies have demonstrated that signaling by Notch, a contact-mediated system, is a potent inducer of glial differentiation, including Müller glia. The Notch receptors are a family of conserved transmembrane proteins that together with multiple ligands participate in diverse cell fate decisions throughout embryonic development, including the differentiation of neural stem cells. Generally, activation of Notch signaling has two distinct results: cells are either kept in an undifferentiated state or different cell types are specified (Artavanis-Tsakonas et al., 1999). It is not completely understood how Notch activation leads to these two apparently contradictory outcomes. However, the documented role of Notch signaling in mediating interactions that partition fates, make it an attractive mechanism for regulating the competency of a retinal progenitor cell to respond to differentiation signals.

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Figure 1. p27Kip1/Xic1 and Notch pathway components promote Müller glial cell differentiation. This model incorporates findings from studies in the retina as well as other CNS tissues. Retinal neuron differentiation is regulated by proneural basic helix-loop-helix (bHLH) transcription factors that promote the expression of Delta, a ligand for the Notch receptor. Activation of Notch on neighboring retinal progenitor cells can lead to expression of the transcription factors Hes1/Hes5, which inhibit expression of proneural bHLH genes. Activation of Notch can also promote expression of glial-specific genes, such as glial fibrillary acidic protein (GFAP), potentially through Hes1/Hes5. Since Hes1/Hes5 are transcriptional repressors, additional downstream components are likely required. Notch expression can be regulated by the paired-type homeodomain transcription factor rax. The cell cycle regulators 27Xic1/Kip1 can also promote Müller glial differentiation through unknown mechanisms. They may promote the expression of glial-specific genes, or potentially regulate the competence of progenitor cells to respond to other glial-promoting signals.

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NOTCH AND MÜLLER GLIOGENESIS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. NOTCH AND MÜLLER GLIOGENESIS
  5. EFFECTORS OF THE NOTCH PATHWAY IN THE RETINA
  6. CYCLIN DEPENDENT KINASE INHIBITORS IN THE RETINA
  7. REGULATION OF REACTIVE GLIOSIS OF MÜLLER CELLS
  8. REGULATION OF MÜLLER GLIAL DIFFERENTIATION BY CYCLIN DEPENDENT KINASE INHIBITORS
  9. Acknowledgements
  10. REFERENCES

Although Notch is known to inhibit neurogenesis in many systems (reviewed by Artavanis-Tsakonas et al., 1999), only recently has there been evidence that it plays an instructive role in gliogenesis (reviewed in Lowell, 2000; Wang and Barres, 2000). In murine P19 carcinoma cells, forced expression of a constitutively active form of Notch suppressed neuronal and muscle differentiation, but not gliogenesis (Nye et al., 1994). In the peripheral nervous system (PNS) and central nervous system (CNS), Notch activation promotes the differentiation of Schwann cells from neural crest stem cells (Morrison et al., 2000) and radial glia in the mouse forebrain respectively (Gaiano et al., 2000). More recently, Tanigaki et al. (2001) demonstrated that both Notch1 and Notch3 direct commitment of multipotent rat hippocampus-derived progenitor cells to the astroglial fate. Clonal analysis indicated that Notch1 and Notch3 act instructively in the choice of astroglial fate.

In the vertebrate retina, Notch is expressed by retinal progenitor cells and is then downregulated in differentiating and mature neurons. Notch expression is upregulated in differentiating Müller glia in Xenopus laevis, rat, and mouse, consistent with a possible role in Müller development (Dorsky et al., 1995; Tomita et al., 1996a; Bao and Cepko, 1997; Perron et al., 1998; Furukawa et al., 2000). It had been previously observed that Notch might permit gliogenesis in the retina in addition to its well-documented role in blocking neurogenesis. In rat, expression of a constitutively active form of Notch was inhibitory to neurons, but permissive for Müller glial differentiation (Bao and Cepko, 1997). A more instructive role for Notch in gliogenesis was demonstrated by Furukawa et al. (2000) who used retroviral vectors to infect rat retinal progenitor cells with a constitutively active form of the Notch receptor. Over 90% of precursor cells infected with the Notch retrovirus differentiated as Müller glia, in contrast to 8% of control virus-infected cells. The Müller glial cells were identified not only by morphological criteria, but also by immunostaining with antisera to three Müller glia markers. Since numbers of later-born neuronal cell types (bipolar cells and rods) were decreased in the retrovirus-infected progenitors, it appears as though Notch can specifically promote progenitors to generate glia (Furukawa et al., 2000). Based on both retinal and other studies in the central nervous system (CNS), it appears that Notch instructively directs progenitors to a Müller glial fate; however, it is difficult to completely rule out the possibility that Notch simply prevents the cells from differentiating until very late in development so that they adopt the late Müller glial fate. The fact that increases in the numbers of other later-born cell types such as bipolar cells or rods were not observed in Notch-transfected retinas argues that Notch is not simply retaining progenitors in a progenitor state (Furukawa et al., 2000), but the ability to distinguish instruction from permission is hampered by the fact that Müller glia are the last retinal cells to differentiate. To further complicate matters, the transfected/infected cells are in a complex, changing environment of intrinsic and extrinsic signaling molecules. Experiments performed on isolated cells would be useful in distinguishing between these two models.

Although the evidence in rodent systems suggests that Notch plays a role in Müller gliogenesis, the situation is more ambiguous in the amphibian system. In Xenopus, similar to the rat retina, Müller glia are the last cells to be generated and they are the last cells to express Notch (Dorsky et al., 1995). However, when progenitor cells were transfected in vivo with activated Notch, neither cell division nor Müller glial fates were promoted, instead differentiation was blocked (Dorsky et al., 1995). Although the authors did not rule out the possibility that the transfected cells were immature glial cells, this result is not consistent with Notch actively promoting gliogenesis. It is possible that differences in experimental approach could explain the different effects of Notch activation in rodents and amphibians. For example, different target cells or groups of progenitor cells may have been transfected, or there may be differences in gene expression levels that affected the experimental results. Alternatively, the different outcomes may be due to the use of different Notch constructs.

One potential reason for Notch's failure to induce Müller glia in Xenopus is that overexpression approaches in Xenopus target early progenitor cells, and progenitor cells at these early stages may not be competent to respond to the Notch signal to make glia. This hypothesis is consistent with the model proposed by Cepko et al. (1996), which predicts that mitotic retinal cells change over time in their ability to respond to the retinal environment (Cepko et al., 1996). Since the effects of Notch activation may also change over the course of retinal development (Wang and Barres, 2000), it would be interesting to examine the effects of transfecting Notch later in Xenopus retinal development with the prediction that progenitors at later stages would have the ability to differentiate as Müller glia. Conversely, Notch may play a more permissive role in determining glial fates in the amphibian retina. Dorsky et al.(1997) tested the effects of expressing high levels of DeltaSTU, a truncated Notch ligand (Chitnis et al., 1995) that interferes with activation of the Notch pathway in retinal progenitors. Cells misexpressing DeltaSTU never differentiated as Müller glia, but adopted earlier neuronal fates (Dorsky et al., 1997). These results suggest that even if Notch signaling does not play an instructive role in Müller gliogenesis in Xenopus, activation of this pathway may be important for controlling the response of progenitor cells to differentiation signals, including those that promote Müller glial fate. Further experiments will be necessary to determine whether the role of Notch in regulating Müller gliogenesis is truly instructive and if its role is conserved across species.

EFFECTORS OF THE NOTCH PATHWAY IN THE RETINA

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. NOTCH AND MÜLLER GLIOGENESIS
  5. EFFECTORS OF THE NOTCH PATHWAY IN THE RETINA
  6. CYCLIN DEPENDENT KINASE INHIBITORS IN THE RETINA
  7. REGULATION OF REACTIVE GLIOSIS OF MÜLLER CELLS
  8. REGULATION OF MÜLLER GLIAL DIFFERENTIATION BY CYCLIN DEPENDENT KINASE INHIBITORS
  9. Acknowledgements
  10. REFERENCES

It remains to be determined precisely how Notch instructs progenitors to specify Müller glial fate in the rodent retina. One possible mechanism involves the activation of negatively acting basic helix-loop-helix factors related to Drosophila hairy and Enhancer of split such as Hes genes (Fig. 1). Consistent with this hypothesis, Furukawa et al. (2000) demonstrated that Hes1 is expressed in retinal progenitor cells and downregulated in differentiated neurons, while its expression is maintained in Müller glia. Differentiating glial cells in the mouse retina also express Hes5 (Hojo et al., 2000). Notch activation has been shown to induce the expression of both Hes1 and Hes5 in mammals (Nishimura et al., 1998; Ohtsuka et al., 1999); however, this has not been demonstrated in the context of retinal development.

In mouse, Tomita et al. (1996a) misexpressed Hes1 in cultured E17 mouse retinas and concluded that Hes1-transfected cells did not resemble any mature retinal cells. Instead Hes1 appeared to keep cells in an undifferentiated precursor stage (Tomita et al., 1996a). Although analysis of the Müller glia phenotype was not specifically addressed in this study using glial-specific markers, Hes1 appeared to inhibit both glial and neuronal differentiation. A later analysis using retroviral vectors and a number of Müller cell-specific antibodies was performed by Furukawa et al. (2000) who demonstrated that forced misexpression of Hes1 in P0 rat retinas resulted in increased numbers of Müller glia. Conversely, when Notch signaling was blocked using a dominant negative form of Hes1, there were fewer Müller glia (Furukawa et al., 2000). This latter study is consistent with Hes1 mediating the effects of Notch activation. Differences between these two studies might be explained by use of different retroviral constructs that might lead to differences in gene expression levels. In addition, Hes1 was transfected into retinal cultures harvested at slightly different developmental times, one from E17.5-P0 retinas, and the other from only P0 postnatal retinas. It is possible that progenitors from different stages respond differently to Hes1 (Cepko et al., 1996; Wang and Barres, 2000) The effects also might depend upon the presence or absence of other factors in these retinal cultures, again consistent with the model proposed by Cepko et al. (1996).

Another Notch effector, Hes5 is also a good candidate for mediating the Notch signal during Müller gliogenesis. When misexpressed in the developing mouse retina, Hes5 has the ability to increase the number of Müller glia while inhibiting generation of rod photoreceptors, a late-born neuronal cell type (Hojo et al., 2000). This effect appears to be the result of conversion of precursors to the glial fate at the expense of neuronal fate. In mice embryos deficient for Hes5, the numbers of retinal Müller glia are reduced. The increases in Müller glia in cells misexpressing either Hes1 or Hes5, combined with the corresponding decreases in Müller glia in cells expressing a dominant negative Hes1 or lacking Hes5 suggest that the Hes genes are important for mediating the effects of Notch in the regulation of Müller gliogenesis. Whether Notch is activating one or both Hes genes in vivo during retinal development has yet to be determined.

Recently, a gene related to the Hes genes, hairy/Enhancer of split [E(spl)] homolog 2 (hesr2) was found to promote gliogenesis in retinal explant cultures from mouse embryos and neonates (Satow et al., 2001). hesr2 did not affect proliferation or cell death, suggesting that hesr2 acts instructively to promote the glial fate. hesr2 had a greater effect when misexpressed in postnatal cultures than prenatal culture, again supporting a model in which the competence of progenitors to respond to an environmental signal can change over the course of retinal development (Cepko et al., 1996). It has not been shown whether activation of hesr2 is Notch-dependent.

The mechanism(s) by which these Notch effectors specify glial fates is not clear. Gliogenesis may occur as a result of direct Hes or hesr2-mediated activation of a glial-specific pathway. It is possible that these genes may upregulate transcription factors that induce gliogenesis. In support of this idea, Tanigaki et al. (2001) demonstrated that both Notch1 and Notch3 promote astrocyte differentiation of multipotent rat hippocampus-derived progenitor cells and also promote the activation of the promoter for glial fibrillary acidic protein (GFAP), a glial-specific gene that is also expressed in Müller glia. Similar mechanisms may be acting in the retina. Alternatively, the glial fate may be a state that can only be attained when neuronal-promoting factors are repressed and the environment is biased toward promoting glial fates. In support of this hypothesis, Müller glial cells are repressed by positively acting bHLH genes (Kanekar et al., 1997; Brown et al., 1998; Morrow et al., 1999) and are increased in the retinas of Mash1 and NeuroD-deficient mice (Tomita et al., 1996b; Morrow et al., 1999). Correlations between increased numbers of Müller glial and the upregulation of hesr2 were observed in retinas of Mash1-Math3 double knockout mice, suggesting that Mash1 and Math3 could normally repress gliogenesis by inhibiting hesr2 (Satow et al., 2001). Hes1 and Hes5 may repress the expression of proneural bHLH genes such as NeuroD and Mash1 that normally function to promote retinal neuronal cell fates (Fig. 1). Hes1 has been shown to repress Mash1 activity and expression (Sasai et al., 1992; Ishibashi et al., 1994; Ishibashi et al., 1995). There is no direct evidence for Hes5 repression of positively acting bHLH factors in the retina; however, Mash1 expression is upregulated in regions lacking Hes5 in Notch mutant mice (de la Pompa et al., 1997). Thus, the key step for Müller gliogenesis may be the loss of neurogenic capacity, which then allows other factors to promote or allow glial differentiation. This model is consistent with recent studies demonstrating that elsewhere in the CNS, proneural bHLH genes regulate the neuronal versus glial fate decision by both promoting neurogenesis and inhibiting gliogenesis. Using murine cortical cultures, Sun et al. (2001) showed that the bHLH factor neurogenin1 has the ability to promote neural fate by functioning as a transcriptional activator, while inhibiting astrocyte differentiation by sequestering a transcriptional coactivator, CBP/p300, required for the activation of glial differentiation genes. A second study examined the role of neurogenin2 and Mash1 in cultures from murine cortical progenitors (Nieto et al, 2001). Since a population of ngn2;Mash1 mutant progenitor cells changed fates, shifting from neuronal to glial, this strongly argues that neurogenin2 and Mash1 normally bias cells toward a neuronal fate. It is reasonable to postulate that similar bHLH factors may control the fate of retinal neural and Müller glial progenitors directly by activating neurogenic transcription and repressing gliogenesis, and/or by indirectly regulating the competence of progenitors to respond to extrinsic neurogenic and gliogenic signals such as Notch.

Taken together, these studies provide us with an initial basis for understanding how Müller glial cells are generated. The role of Notch in the process of Müller gliogenesis is consistent with its emerging role in gliogenesis in both the peripheral nervous system and central nervous system. Although there is good evidence that Notch signaling and the Hes genes promote Müller glial cell formation, we have only begun to tease out the components involved in Notch-mediated gliogenesis in the retina. The observations that Müller glia are still generated in Hes5 null mice and that Notch overexpression promotes progenitor proliferation in the retina, while Hes1 does not, suggest that the Notch signal may not always act via the Hes genes (Furukawa et al., 2000; Hojo et al., 2000). Notch may activate other genes such as Deltex that can inhibit differentiation genes independently from the Hes genes (Matsuno et al., 1998) or it may activate the hesr2 gene (Satow et al., 2001). The outcome of Notch signaling during retinogenesis also might be dependent upon the presence or absence of extra- or intra-cellular signals. One potential candidate is the epidermal growth factor receptor (EGFR) that has been implicated in Müller glial cell genesis (Lillien, 1995; Lillien and Wancio, 1998). Notch has been shown to regulate some cells' response to EGF family members (Wang and Sternberg, 1999). What is regulating the ability of the cell to respond to ligands for Notch? Furakawa et. al (2000) demonstrated that overexpression of the rax homeobox gene leads to upregulation of Notch in retinal cells and can promote Müller glial cell differentiation, suggesting that Rax can regulate the expression of Notch in progenitors (Fig. 1). However, additional details regarding the upstream regulation of Notch signaling in the retina remain for the most part unresolved.

Müller cell fates, along with the fates of specific retinal neurons, are likely to be acquired in response to extrinsic signals such as Notch through the activation and repression of unique combinations of positively- and negatively acting bHLH factors, homeobox genes, and downstream targets. The focus now needs to be on identifying the other transcription factors that act downstream or in collaboration with the Hes genes, and the target genes that function to repress neurogenesis or to promote gliogenesis in the retina.

CYCLIN DEPENDENT KINASE INHIBITORS IN THE RETINA

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. NOTCH AND MÜLLER GLIOGENESIS
  5. EFFECTORS OF THE NOTCH PATHWAY IN THE RETINA
  6. CYCLIN DEPENDENT KINASE INHIBITORS IN THE RETINA
  7. REGULATION OF REACTIVE GLIOSIS OF MÜLLER CELLS
  8. REGULATION OF MÜLLER GLIAL DIFFERENTIATION BY CYCLIN DEPENDENT KINASE INHIBITORS
  9. Acknowledgements
  10. REFERENCES

Retinal progenitors have been proposed to respond to extracellular cues, such as signaling through the Notch pathway or activation of the epidermal growth factor receptor (EGFR) (Lillien, 1995; Lillien and Wancio, 1998), to help determine when they will exit the cell cycle and what phenotype they will adopt. Therefore, regulation of cell cycle control is intimately coupled to the control of retinal cell differentiation. Cell cycle progression is controlled by the activity of cyclin-dependent kinases (CDKs), which are negatively regulated through interaction with cyclin-dependent kinase inhibitors (CKIs) (reviewed in Nakayama and Nakayama, 1998 and Sherr and Roberts, 1999). CKIs respond to growth inhibitory signals in the extracellular environment by promoting arrest of the cell cycle in G1. CKIs therefore play an important role in regulating the proliferation of progenitor cells in response to extracellular signals. Members of the Cip/Kip subfamily of CKIs include three proteins in mammals, p21Cip1, p27Kip1 and p57Kip2. All three of these proteins contain a cyclin/cdk binding domain in their amino terminus which is required for inhibition of cyclin-dependent kinase activity. Several recent studies have suggested that some members of this subfamily of CKIs regulate Müller glial cell proliferation, and also play a role in Müller glial development.

The CKI protein p27Kip1 shows two phases of expression in the mouse retina, suggesting a role at more than one stage of eye development. There is an early phase of p27Kip1 expression in early postmitotic retinal cells as they exit the cell cycle, consistent with a role in regulating cell cycle withdrawal in retinal progenitors (Levine et al., 2000). Indeed, Levine et al. (Levine et al., 2000) showed that overexpression of p27Kip1 in retinal progenitor cells in culture was sufficient to promote cell cycle withdrawal, even in the presence of mitogen. However, in addition to its early expression during retinal development, there is also persistent expression of p27Kip1 in differentiated Müller glia of the mature retina, suggesting that p27Kip1 may regulate some aspect of the mature phenotype of these cells (Dyer and Cepko, 2000; Levine et al., 2000).

REGULATION OF REACTIVE GLIOSIS OF MÜLLER CELLS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. NOTCH AND MÜLLER GLIOGENESIS
  5. EFFECTORS OF THE NOTCH PATHWAY IN THE RETINA
  6. CYCLIN DEPENDENT KINASE INHIBITORS IN THE RETINA
  7. REGULATION OF REACTIVE GLIOSIS OF MÜLLER CELLS
  8. REGULATION OF MÜLLER GLIAL DIFFERENTIATION BY CYCLIN DEPENDENT KINASE INHIBITORS
  9. Acknowledgements
  10. REFERENCES

Targeted disruption of p27Kip1 in mice results in increased proliferation and multi-organ hyperplasia, including effects on the central nervous system (Nakayama et al., 1996). The p27Kip1 knockout mice also display a retinal dysplasia phenotype that upon closer examination appears to be due to alterations in the Müller glia rather than due to generalized effects on progenitor proliferation. Levine et al. (2000) found that the window of time during which retinal cells proliferate was extended in p27Kip1 knockout mice, but p27Kip1 was not ultimately required for retinal progenitors to exit the cell cycle. However, in the p27Kip1 knockout mice the Müller glia were disorganized, proliferated beyond the normal period of Müller cell genesis and expressed glial fibrillary acidic protein (GFAP), which is normally expressed in these cells in response to acute retinal stress or injury. Dyer and Cepko (Dyer and Cepko, 2000) documented a 10-20 fold induction of GFAP-immunoreactive Müller glial cells in p27Kip1 knockout mice. The reactive Müller glia disrupted the outer limiting membrane of the retina and caused displacement of photoreceptor cell bodies and Müller glia into the region normally containing outer segments, thus giving rise to the retinal dysplasia phenotype originally observed (Dyer and Cepko, 2000; Levine et al., 2000).

To demonstrate that the retinal dysplasia phenotype was due to activation of the Müller glial cells, Dyer and Cepko (2000) induced reactive gliosis in the retina using ouabain. They found a dramatic increase in the number of GFAP-positive Müller cells, coupled with downregulation of p27Kip1 and activation of proliferation in these cells. In addition, inducing reactive gliosis in this manner was sufficient to cause disruption of the outer limiting membrane leading to retinal dysplasia similar to that seen in the p27Kip1 knockout mice. To explain why reactive Müller glial cells do not continue to proliferate unchecked in the absence of p27Kip1 function, Dyer and Cepko (2000) showed that there was downregulation of the positive cell cycle regulator cyclin D3 within 24 hours after activation of reactive gliosis. In the oligodendrocyte lineage of the optic nerve final growth arrest in vivo has similarly been linked to specific downregulation of cyclin E levels (Casaccia-Bonnefil et al., 1999). Thus, regulation of cyclin levels may be an important compensatory mechanism that may ensure that cell cycle control is not strictly dependent upon the activity of the CKI proteins such as p27Kip1. p27Kip1 thus appears to play an important role in regulating initiation of reactive gliosis in the retina, likely by controlling the re-entry of Müller glial cells into the cell cycle. One issue that would be interesting to explore is whether cell cycle re-entry is sufficient to initiate the reactive glial phenotype or whether p27Kip1 has a more direct role in regulating the reactive phenotype in Müller glia.

REGULATION OF MÜLLER GLIAL DIFFERENTIATION BY CYCLIN DEPENDENT KINASE INHIBITORS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. NOTCH AND MÜLLER GLIOGENESIS
  5. EFFECTORS OF THE NOTCH PATHWAY IN THE RETINA
  6. CYCLIN DEPENDENT KINASE INHIBITORS IN THE RETINA
  7. REGULATION OF REACTIVE GLIOSIS OF MÜLLER CELLS
  8. REGULATION OF MÜLLER GLIAL DIFFERENTIATION BY CYCLIN DEPENDENT KINASE INHIBITORS
  9. Acknowledgements
  10. REFERENCES

No effects on the ratio of differentiated cells types in the retina were observed in p27Kip1 knockout mice, suggesting that p27Kip1 in mice does not play a role in normal retinal histogenesis (Dyer and Cepko, 2000; Levine et al., 2000). However, contrasting results were obtained in Xenopus using p27Xic1. This protein is a member of the Cip/Kip family of CKIs that shows homology to both Cip1 and Kip1 (Su et al., 1995), and is also expressed in retinal cells as they begin differentiation (Ohnuma et al., 1999). At later stages, expression of p27Xic1 in the mature cells of the central retina decreases, but persists at the margins, where retinal histogenesis continues throughout the life of the animal. Ohnuma et al. (Ohnuma et al., 1999) showed that overexpression of p27Xic1 in retinal progenitors of the Xenopus retina caused a dramatic increase in the representation of Müller glia at the expense of bipolar cells. There was early cell cycle arrest in response to p27Xic1 overexpression, consistent with what was observed with p27Kip1 overexpression. However, cell cycle arrest alone was not sufficient to induce the formation of Müller glia since hydroxyurea treatment or overexpression of dominant-negative forms of Cdk2 and cdc2 did not induce Müller glia. These results argue that p27Xic1 has an instructive role in Müller glial cell development (Fig. 1).

Using deletion analysis of the p27Xic1 protein Ohnuma et al. (1999) were able to show that the ability of p27Xic1 to promote Müller glial cell differentiation was separable from the CDK inhibitory activity of the protein. A region in the N-terminus of p27Xic1 known as the CDK/cyclin-binding region was required for both functions, although a short stretch of five amino acids was required for Müller cell inducing activity but not for CDK inhibitory activity. The CDK/cyclin binding region is conserved in all members of the Cip/Kip subfamily of CKIs, consistent with the finding that mammalian p27Kip1 and p21Cip1 also have Müller glial promoting activity when overexpressed (Ohnuma et al., 1999). A mutant form of p21Cip1 that had diminished ability to inhibit cell cycle progression was still able to efficiently promote Müller glial cell differentiation. These data provide striking and surprising evidence that p27Xic1 and related molecules can regulate Müller glia genesis during normal development, and that this function is independent of the cell cycle regulatory activity of the CKIs. The mechanism underlying this ability to promote Müller glial cell differentiation is unclear. Coexpressing cyclin A2 and Cdk2 with p27Kip1 inhibited its Müller glial promoting activity, perhaps by binding to p27Kip1 and titrating it, or sterically blocking its Müller glial cell-inducing domain, as suggested by Ohnuma et al. (1999). Since a very small region of the p27Xic1 protein has been specifically implicated in the ability to promote Müller glial cell differentiation there is a starting point for chasing potential interacting components. Overexpression of p27Xic1 in the rodent retina can cause a modest increase in Müller glial cell genesis at the expense of bipolar cells, similar to what was observed in Xenopus. However, the same effect was not observed with p27Kip1 (M.A. Dyer and C.L. Cepko, personal communication). Further experiments, potentially using in vitro culture assays will be necessary to define the components of the Cip/Kip proteins that are important for regulating Müller glial cell genesis.

Is p27Xic1 required for normal Müller glial cell genesis in the Xenopus retina? Ohnuma et al. (1999) reduced endogenous p27Xic1 expression in the developing retina using antisense constructs and found a 50% reduction in the representation of Müller glia. In contrast, there was no loss of Müller glia in the p27Kip1 knockout mouse, but rather the glia became abnormally reactive (Dyer and Cepko, 2000; Levine et al., 2000). How can these apparently disparate findings be reconciled? p27Xic1 from Xenopus and p27Kip1 from mouse are not strict homologues, thus although the activity of p27Xic1 may be required for Müller glial cell genesis in vivo, the same may not be true for p27Kip1. This function could be provided by another related molecule in mouse. This is supported by the observation that p27Xic1 but not p27Kip1 can promote Müller glial cell differentiation in the rodent retina. There may also be differences in the basic mechanisms used to generate Müller glia in such widely divergent species as amphibians and rodents. It is also possible that compensatory mechanisms come into play in the p27Kip1 mouse knockout to ensure that normal numbers of Müller glia are generated. Since the expression of antisense constructs in the Xenopus retina affects only a small subset of cells, similar compensatory mechanisms may not be called into action. Levine et al. (2000) also raise the possibility that in the Xenopus antisense experiments the glia may be present but simply are abnormally reactive, thus precluding their identification using laminar position and morphology as criteria. Further experiments will be necessary to determine whether members of the Kip/Cip subfamily of CKIs play a conserved requisite role in regulating Müller glial cells genesis.

An important function of the CKIs is to respond to extracellular signals to regulate cell cycle progression. Therefore it is intriguing to consider the possibility that this class of proteins may also integrate information from extrinsic cues to regulate Müller glial cell genesis. Several extrinsic signaling pathways have been shown to influence Müller glia cell genesis. As discussed above, activation of the Notch pathway can promote Müller glial cell differentiation. Interestingly Ohnuma et al. (1999) found that Notch could synergize with p27Xic1 in the regulation of Müller glial differentiation. Overexpression of the two together caused an even larger proportion of cells to differentiate as Müller glia than overexpression of p27Xic1 alone. It has not yet been determined whether other extracellular signaling pathways also synergize with CDK inhibitor proteins to regulate Müller glial differentiation. One candidate to be considered is the EGFR signaling pathway. Enhancement of signaling by overexpressing high levels of EGFR in retinal progenitor cells increases the proportion of cells that differentiate into Müller glia (Lillien, 1995). Therefore, signaling through the EGFR could have differential effects on CDK inhibitor proteins such as p27Kip1 to regulate either proliferation or Müller glial cell differentiation. High levels of EGFR signaling in retinal progenitors do not always promote the Müller glial phenotype. Rather this outcome depends upon the developmental stage of the environment in which the progenitors are placed (Lillien and Wancio, 1998). Therefore other as yet unidentified signaling pathways must also be important in regulating the Müller glial phenotype.

Taken together these recent studies suggest that the CDK inhibitor proteins can regulate Müller glial cell proliferation during reactive gliosis in the retina as well as the genesis of Müller glial cells during development. The challenge now is to determine how general these functions may be. The vertebrate neural retina has long served as a model system for understanding development of the CNS in general. The role of Notch in regulating glial differentiation appears to be conserved between the retina and other parts of the CNS. Therefore it is interesting to consider the possibility that the CDK inhibitor proteins also regulate reactive gliosis or gliogenesis in other parts of the nervous system as well.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. NOTCH AND MÜLLER GLIOGENESIS
  5. EFFECTORS OF THE NOTCH PATHWAY IN THE RETINA
  6. CYCLIN DEPENDENT KINASE INHIBITORS IN THE RETINA
  7. REGULATION OF REACTIVE GLIOSIS OF MÜLLER CELLS
  8. REGULATION OF MÜLLER GLIAL DIFFERENTIATION BY CYCLIN DEPENDENT KINASE INHIBITORS
  9. Acknowledgements
  10. REFERENCES

We would like to thank A.V. Maricq and E. Levine for comments on the manuscript. M.L.V. is supported by NIH grant number EY12274 and by the Pew Scholars Program in the Biomedical Sciences sponsored by the Pew Charitable Trusts. K.B.M. is supported by NIH grant number EY10096.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. NOTCH AND MÜLLER GLIOGENESIS
  5. EFFECTORS OF THE NOTCH PATHWAY IN THE RETINA
  6. CYCLIN DEPENDENT KINASE INHIBITORS IN THE RETINA
  7. REGULATION OF REACTIVE GLIOSIS OF MÜLLER CELLS
  8. REGULATION OF MÜLLER GLIAL DIFFERENTIATION BY CYCLIN DEPENDENT KINASE INHIBITORS
  9. Acknowledgements
  10. REFERENCES