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Retinal development: Communication helps you see the light
Dispatch
R433
Retinal development: Communication helps you see the light Robert J. Wechsler-Reya* and Barbara A. Barres†
signals that are transmitted along the optic nerve to the brain, where they are processed and interpreted to form a visual image. But how do retinal neurons, glia and endothelial cells come together in the first place? And how are their numbers and locations precisely matched to form an intact, functional retina? Although the process is not completely understood, several recent studies suggest that communication between neurons, astrocytes and endothelial cells early in development is critical for the generation of retinal architecture.
Recent studies suggest that interactions between neurons, glial cells and endothelial cells are critical in determining the structure of the retina and the optic nerve. Dysregulation of these interactions can lead to disruption of retinal architecture and impairment of vision. Addresses: Departments of *Developmental Biology and †Neurobiology, Stanford University School of Medicine, Stanford, California 94305, USA. Current Biology 1997, 7:R433–R436 http://biomednet.com/elecref/09609822007R0433
One of the first clues to the nature of this communication came from the observation, made more than 30 years ago, that the development of blood vessels in the retina is regulated by oxygen [1]. Exposure of animals to high levels of oxygen during early development led to obliteration of newly formed blood vessels in their retinas, while reduction of oxygen below normal levels (hypoxia) stimulated growth of new vessels. The mechanism underlying this effect remained a mystery until the early 1990s, when two key discoveries were made [2]. The first was the identification of vascular endothelial growth factor (VEGF) as an important inducer of endothelial cell growth and blood vessel development. The second was the observation that production of VEGF can be stimulated by
© Current Biology Ltd ISSN 0960-9822
In introductory biology courses, the retina is often described as a blank screen upon which an image of the outside world is projected. But this simple metaphor belies the extraordinary structural and functional complexity of the retina. Far from being a flat, homogeneous surface, the retina is a three-dimensional mosaic consisting of six types of neuron, two types of glial cell, and a network of endothelial cells that make up the retinal capillaries (see Figure 1). In a fully-developed retina, these cells cooperate with one another to carry out the initial steps of vision, converting light into electrochemical Figure 1 Architecture of the retina. When light enters the eye, it must filter through a complex network of blood vessels, glial cells (astrocytes and Müller glia) and neurons before reaching the photoreceptors at the back of the retina. The photoreceptors (rods and cones) convert the light into electrochemical signals, and these are integrated and transmitted by interneurons (horizontal, bipolar, and amacrine cells) to the retinal ganglion cells (RGCs) at the front of the retina. The axons of retinal ganglion cells converge to form the optic nerve, which exits through the back of the eye and carries the visual information to the brain.
Blood vessel Astrocyte Electrical signals to the brain
Ganglion cell layer
Ganglion cell (RGC) Blood vessels
Amacrine cell Müller cell
Inner nuclear layer
Bipolar cell Horizontal cell
Optic nerve Retina Outer nuclear layer
Photoreceptor Cone
Pigment layer
© 1997 Current Biology
Choroid layer Sclera
Rod
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Figure 2
Optic nerve astrocytes
Increased oxygen supply
Hypoxia
bFGF
PDGF
Optic nerve (RGC axons)
RGCs
VEGF
Retinal astrocytes
© 1997 Current Biology
hypoxia. These findings raised the possibility that VEGF might mediate the effects of oxygen on retinal blood vessel development. A number of studies supported the notion that VEGF levels in the retina can be elevated by subjecting animals to hypoxia [3,4]. But in 1995, Jonathan Stone, Eli Keshet and their co-workers [5] provided strong evidence that VEGF plays a role in normal retinal development. They performed a detailed analysis of VEGF production in the retinas of developing rats and cats, and found that the growth factor was produced in precisely the regions where blood vessels were being generated. In these regions, VEGF was made by glial cells — astrocytes and Müller glia — just before blood vessel formation. Moreover, the newly formed vessels expressed high-affinity VEGF receptors, indicating that they are capable of responding to the growth factor. Finally, secretion of VEGF by retinal astrocytes increased in response to hypoxia and decreased in response to high levels of oxygen, suggesting that astrocyte-derived VEGF could indeed mediate the effects of oxygen on the retinal vasculature. On the basis of these observations, Stone et al. [5] proposed a model for how retinal blood vessels normally develop (see Figure 2). According to this model, as retinal neurons become metabolically active, they use up oxygen in their environment, causing local hypoxia. This ‘physiological’ hypoxia causes retinal astrocytes to secrete VEGF, which promotes growth of endothelial cells and formation of retinal blood vessels. Then, when the blood vessels open up and carry oxygenated blood to the area, the hypoxia is relieved, and VEGF production by astrocytes decreases to baseline levels. In this manner, blood vessel formation is matched to oxygen demand.
Retinal blood vessels
Regulation of retinal architecture by cell–cell communication. During normal development, retinal ganglion cells (RGCs) secrete basic fibroblast growth factor (bFGF) and plateletderived growth factor (PDGF), which stimulate proliferation of astrocytes in the retina and along the optic nerve. Metabolic activity of retinal ganglion cells (and other neurons) may deplete oxygen in the area, causing ‘physiological’ hypoxia. Retinal astrocytes respond to this hypoxia by secreting vascular endothelial growth factor (VEGF), which stimulates growth of endothelial cells and generation of retinal blood vessels. These blood vessels carry oxygen to the area, relieving the hypoxia and shutting off production of VEGF by astrocytes. Exposure to high oxygen levels, or dysregulation of PDGF secretion, can lead to abnormal astrocyte and blood vessel development, altered retinal architecture and impairment of vision.
This model not only helped explain the process of normal blood vessel development in the retina, but also shed light on the cause of a human disease called ‘retinopathy of prematurity’. In this disease, premature infants who are exposed to high levels of oxygen (to help them breathe during a period when their lungs are not fully formed) develop severe retinopathy that can lead to blindness. The late stages of retinopathy of prematurity are characterized by aberrant growth of blood vessels (neovascularization) in the retina, and a number of studies suggested that VEGF might be involved in this process [3,4]. But the precise role of VEGF in the pathogenesis of retinopathy of prematurity was unclear. Once again, Stone and Keshet and their colleagues [6] collaborated to investigate this issue. Their studies suggested that, when newborn animals (or premature infants) are exposed to high levels of oxygen, their retinal astrocytes stop producing VEGF, and this causes newly formed blood vessels, which depend on the growth factor, to die. Then, when animals are returned to normal air, which has much less oxygen, the astrocytes turn back on and make large amounts of VEGF. This sudden increase in VEGF levels stimulates growth of new blood vessels, but these vessels are weak and abnormally leaky — a side-effect of excessive VEGF, which also acts to increase vascular permeability — and there are often so many of them that they disrupt retinal architecture and cause loss of vision. Previous studies had indicated that blocking VEGF — or providing supplemental oxygen to suppress its production — during the second phase of retinopathy of prematurity can help prevent neovascularization and improve vision [7,8]. But on the basis of their findings, Alon et al. [6] suggested that it might be more useful to intervene during
Dispatch
the first phase of retinopathy of prematurity, when normal blood vessels are being destroyed. They and others [9] have demonstrated that, by supplying exogenous VEGF to animals before they are exposed to high oxygen, newlyformed vessels can be preserved and the severity of retinopathy can be reduced. The studies of Stone, Keshet and colleagues [5,6] make it clear that astrocytes are critical in establishing the correct number of blood vessels in the retina. But what determines the number of astrocytes? Recent work by two groups suggests that astrocyte growth is dependent on signals from retinal neurons. The first group, led by William Richardson, had previously found that retinal ganglion cells, the neurons that carry signals out of the retina, secrete plateletderived growth factor (PDGF), while retinal astrocytes express the PDGF receptor [10]. This raised the possibility that PDGF was somehow involved in communication between retinal ganglion cells and astrocytes. To determine the nature of this communication, Richardson and colleagues [11] set out to alter PDGF levels in the developing retina. First, they generated cells that secreted a soluble form of the PDGF receptor, which would bind any PDGF secreted by the retinal ganglion cells and prevent it from reaching astrocytes. They implanted these cells into the eyes of newborn rats, and examined the effects on astrocyte development five days later. In control animals that received no cells (or cells that did not make soluble receptor), astrocytes and their processes had spread across the whole inner surface of the retina to form a rich, highly branched network. In contrast, in animals implanted with soluble PDGF receptor-secreting cells, the retinal astrocyte network was perturbed: astrocytes migrated only half as far, and their processes were much less branched than those of control animals. Similar results were obtained when animals were injected with antibodies against the PDGF receptor, which prevented astrocytes from responding to PDGF. These results indicated that the development of the retinal astrocyte network was dependent on PDGF from retinal ganglion cells. To clarify how PDGF was acting, the investigators artificially increased the amount of PDGF available to developing astrocytes by generating transgenic mice that expressed abnormally high levels of PDGF in their retinal ganglion cells. When they examined the retinas of these animals, they observed a dramatic overgrowth of astrocytes. This suggested that PDGF worked by promoting astrocyte proliferation. To examine this directly, the authors isolated retinas from wild-type and transgenic animals, and counted the number of astrocytes in each retina and the amount of DNA in each cell. As expected, the transgenic animals had two to four times as many astrocytes as controls. Moreover, these astrocytes contained more DNA than control cells, indicating that more of them had entered the cell cycle and
R435
begun to divide. In other words, PDGF was inducing proliferation of retinal astrocytes. The consequences of this proliferation became apparent when the researchers examined the vasculature of transgenic mice. As discussed above, astrocytes control the development of retinal blood vessels by secreting VEGF. In transgenic animals, the hyperproliferation of astrocytes was accompanied by an overproduction of capillaries in the retina. Early in life, many of these animals displayed retinal bleeding. By adulthood, both astrocytes and blood vessels had penetrated into the deeper layers of the retina (in normal animals, they are found primarily on the inner retinal surface), and the animals showed severely disrupted retinal structure. The authors suggested that PDGF, like VEGF, might play a role in some forms of human retinopathy. Interestingly, the ability of retinal ganglion cells to control astrocyte proliferation is not restricted to the retina. The axons of retinal ganglion cells extend out of the retina to form the optic nerve, and the optic nerve, like the retina, is lined with astrocytes. The second study, by Burne and Raff [12], demonstrates that the proliferation of these astrocytes is also regulated by retinal ganglion cells. The first indication of this came from earlier studies of transgenic mice that expressed high levels of the survival-promoting gene bcl-2 in their retinal ganglion cells [13]. These mice not only had increased numbers of retinal ganglion cells, but increased numbers of optic nerve astrocytes as well. This suggested that the development or proliferation of optic nerve astrocytes might depend on signals from retinal ganglion cell axons. To test this notion, the authors [12] studied the effects of cutting the optic nerve, which causes degeneration of retinal ganglion cell axons. When they examined astrocytes from cut optic nerves, they found a dramatic decrease in proliferation compared to that seen in astrocytes from normal optic nerves, indicating that signals from the axons are necessary for normal astrocyte proliferation. Interestingly, a similar drop in proliferation was observed when the experiment was performed in mutant mice whose optic nerves do not degenerate following transection. This suggested that, even if retinal ganglion cell axons were intact, they could not stimulate astrocyte proliferation unless they were physically connected to the cell bodies of retinal ganglion cells. One possible reason that the axons had to be intact was so that they could conduct electrical activity. In fact, previous studies [14] had shown that oligodendrocyte precursors in the optic nerve depend on electrical activity in retinal ganglion cell axons for their proliferation. However, activity turned out not to be required for the mitogenic effect of retinal ganglion cells on astrocytes, as injection of
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tetrodotoxin, which blocks electrical activity, did not have the same effect as transection. In contrast, injection of colchicine, which prevents transport down the axon, was able to mimic the effects of transection. From these observations, the authors concluded that molecules transported down retinal ganglion cell axons are required for the mitogenic effect on astrocytes. To explore the basis of this effect, the investigators cultured optic nerve astrocytes in vitro in the presence or absence of retinal ganglion cells. In the presence of retinal ganglion cells, they found that the proliferation of astrocytes increased 20-fold. The investigators attempted to mimic the mitogenic effect of retinal ganglion cells using a variety of growth factors known to be secreted by these cells, and found that only one, basic fibroblast growth factor (bFGF), was effective. Notably, PDGF, which promotes the proliferation of astrocytes within the retina, was unable to stimulate astrocytes from the optic nerve. This is particularly interesting, as retinal astrocytes are thought to originate in the optic nerve [15,16]. These results indicated that retinal ganglion cells control the development of astrocytes in both the retina and the optic nerve, albeit by different mechanisms. The work of Stone, Keshet and colleagues [5,6], Fruttiger et al. [11] and Burne and Raff [12] clearly demonstrates that assembly of the retina and the optic nerve is regulated by a series of intricate cell–cell interactions, and that interference with any of these interactions can lead to disruption of retinal architecture and impairment of vision. But while these studies provide a clearer picture of how the elements of the visual system are put together, a number of important questions remain. For example, Stone et al. [5] postulate that neuronal activity in the developing retina results in local hypoxia, which induces astrocytes to secrete VEGF; although this is a plausible hypothesis, there is no direct evidence that such physiological hypoxia occurs, or that it is in fact the stimulus for VEGF production. Assuming that it is, the mechanism by which astrocytes sense changes in local oxygen levels, and the signaling pathways that allow them to respond by secreting VEGF, still remain to be elucidated. Similarly, the studies of Fruttiger et al. [11] and Burne and Raff [12] leave little doubt that retinal ganglion cells can control the proliferation of astrocytes by secreting soluble factors, such as PDGF and bFGF. But what triggers the secretion of these factors? And perhaps more importantly, what shuts them off so that astrocyte proliferation and vascularization do not continue unchecked? The approaches these investigators have used to map out the circuitry thus far — detailed analysis of cell–cell interactions and specific manipulation of these interactions in vitro and in vivo — will undoubtedly shed light on these issues in the near future.
References 1. Ashton N: Oxygen and the growth and development of retinal vessels. Am J Ophthal 1966, 62:412–435. 2. Klagsbrun M, Soker S: VEGF/VPF: the angiogenesis factor found? Curr Biol 1993, 3:699–702. 3. Miller JW, Adamis AP, Shima DT, D’Amore PA, Moulton RS, O’Reilly MS, Folkman J, Dvorak HF, Brown LF, Berse B, et al.: Vascular endothelial growth factor/vascular permeability factor is temporally and spatially correlated with ocular angiogenesis in a primate model. Am J Pathol 1994, 145:574–584. 4. Pierce EA, Avery RL, Foley ED, Aiello LP, Smith LE: Vascular endothelial growth factor/vascular permeability factor expression in a mouse model of retinal neovascularization. Proc Natl Acad Sci USA 1995, 92:905–909. 5. Stone J, Itin A, Alon T, Pe’er J, Gnessin H, Chan-Ling T, Keshet E: Development of retinal vasculature is mediated by hypoxiainduced vascular endothelial growth factor (VEGF) expression by neuroglia. J Neurosci 1995, 15:4738–4747. 6. Alon T, Hemo I, Itin A, Pe’er J, Stone J, Keshet E: Vascular endothelial growth factor acts as a survival factor for newly formed retinal vessels and has implications for retinopathy of prematurity. Nature Med 1995, 1:1024–1028. 7. Aiello LP, Pierce EA, Foley ED, Takagi H, Chen H, Riddle L, Ferrara N, King GL, Smith LE: Suppression of retinal neovascularization in vivo by inhibition of vascular endothelial growth factor (VEGF) using soluble VEGF-receptor chimeric proteins. Proc Natl Acad Sci USA 1995, 92:10457–10461. 8. Robinson GS, Pierce EA, Rook SL, Foley E, Webb R, Smith LE: Oligodeoxynucleotides inhibit retinal neovascularization in a murine model of proliferative retinopathy. Proc Natl Acad Sci USA 1996, 93:4851–4856. 9. Pierce EA, Foley ED, Smith LEH: Regulation of vascular endothelial growth factor by oxygen in a model of retinopathy of prematurity. Arch Ophthalmol 1996, 114:1219–1228. 10. Mudhar HS, Pollock RA, Wang C, Stiles CD, Richardson WD: PDGF and its receptors in the developing rodent retina and optic nerve. Development 1993, 118: 539–552. 11. Fruttiger M, Calver AR, Krüger WH, Mudhar HS, Michalovich D, Takakura N, Nishikawa SI, Richardson WD: PDGF mediates a neuron-astrocyte interaction in the developing retina. Neuron 1996, 17:1117–1131. 12. Burne J, Raff MC: Retinal ganglion cell axons drive the proliferation of astrocytes in the developing rodent optic nerve. Neuron 1997, 18:223–230. 13. Burne J, Staple J, Raff M: Glial cells are increased proportionally in transgenic optic nerves with increased numbers of axons. J Neurosci 1996, 16:2064–2073. 14. Barres BA, Raff MC: Proliferation of oligodendrocyte precursor cells depends on electrical activity in axons. Nature 1993, 361:258–260. 15. Watanabe T, Raff MC: Retinal astrocytes are immigrants from the optic nerve. Nature 1988, 332:834–837. 16. Ling T, Mitrofanis J, Stone J: Origin of retinal astrocytes in the rat: evidence of migration from the optic nerve. J Comp Neurol 286:345–352.
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Retinal development: Communication helps you see the light Robert J. Wechsler-Reya* and Barbara A. Barres†
signals that are transmitted along the optic nerve to the brain, where they are processed and interpreted to form a visual image. But how do retinal neurons, glia and endothelial cells come together in the first place? And how are their numbers and locations precisely matched to form an intact, functional retina? Although the process is not completely understood, several recent studies suggest that communication between neurons, astrocytes and endothelial cells early in development is critical for the generation of retinal architecture.
Recent studies suggest that interactions between neurons, glial cells and endothelial cells are critical in determining the structure of the retina and the optic nerve. Dysregulation of these interactions can lead to disruption of retinal architecture and impairment of vision. Addresses: Departments of *Developmental Biology and †Neurobiology, Stanford University School of Medicine, Stanford, California 94305, USA. Current Biology 1997, 7:R433–R436 http://biomednet.com/elecref/09609822007R0433
One of the first clues to the nature of this communication came from the observation, made more than 30 years ago, that the development of blood vessels in the retina is regulated by oxygen [1]. Exposure of animals to high levels of oxygen during early development led to obliteration of newly formed blood vessels in their retinas, while reduction of oxygen below normal levels (hypoxia) stimulated growth of new vessels. The mechanism underlying this effect remained a mystery until the early 1990s, when two key discoveries were made [2]. The first was the identification of vascular endothelial growth factor (VEGF) as an important inducer of endothelial cell growth and blood vessel development. The second was the observation that production of VEGF can be stimulated by
© Current Biology Ltd ISSN 0960-9822
In introductory biology courses, the retina is often described as a blank screen upon which an image of the outside world is projected. But this simple metaphor belies the extraordinary structural and functional complexity of the retina. Far from being a flat, homogeneous surface, the retina is a three-dimensional mosaic consisting of six types of neuron, two types of glial cell, and a network of endothelial cells that make up the retinal capillaries (see Figure 1). In a fully-developed retina, these cells cooperate with one another to carry out the initial steps of vision, converting light into electrochemical Figure 1 Architecture of the retina. When light enters the eye, it must filter through a complex network of blood vessels, glial cells (astrocytes and Müller glia) and neurons before reaching the photoreceptors at the back of the retina. The photoreceptors (rods and cones) convert the light into electrochemical signals, and these are integrated and transmitted by interneurons (horizontal, bipolar, and amacrine cells) to the retinal ganglion cells (RGCs) at the front of the retina. The axons of retinal ganglion cells converge to form the optic nerve, which exits through the back of the eye and carries the visual information to the brain.
Blood vessel Astrocyte Electrical signals to the brain
Ganglion cell layer
Ganglion cell (RGC) Blood vessels
Amacrine cell Müller cell
Inner nuclear layer
Bipolar cell Horizontal cell
Optic nerve Retina Outer nuclear layer
Photoreceptor Cone
Pigment layer
© 1997 Current Biology
Choroid layer Sclera
Rod
R434
Current Biology, Vol 7 No 7
Figure 2
Optic nerve astrocytes
Increased oxygen supply
Hypoxia
bFGF
PDGF
Optic nerve (RGC axons)
RGCs
VEGF
Retinal astrocytes
© 1997 Current Biology
hypoxia. These findings raised the possibility that VEGF might mediate the effects of oxygen on retinal blood vessel development. A number of studies supported the notion that VEGF levels in the retina can be elevated by subjecting animals to hypoxia [3,4]. But in 1995, Jonathan Stone, Eli Keshet and their co-workers [5] provided strong evidence that VEGF plays a role in normal retinal development. They performed a detailed analysis of VEGF production in the retinas of developing rats and cats, and found that the growth factor was produced in precisely the regions where blood vessels were being generated. In these regions, VEGF was made by glial cells — astrocytes and Müller glia — just before blood vessel formation. Moreover, the newly formed vessels expressed high-affinity VEGF receptors, indicating that they are capable of responding to the growth factor. Finally, secretion of VEGF by retinal astrocytes increased in response to hypoxia and decreased in response to high levels of oxygen, suggesting that astrocyte-derived VEGF could indeed mediate the effects of oxygen on the retinal vasculature. On the basis of these observations, Stone et al. [5] proposed a model for how retinal blood vessels normally develop (see Figure 2). According to this model, as retinal neurons become metabolically active, they use up oxygen in their environment, causing local hypoxia. This ‘physiological’ hypoxia causes retinal astrocytes to secrete VEGF, which promotes growth of endothelial cells and formation of retinal blood vessels. Then, when the blood vessels open up and carry oxygenated blood to the area, the hypoxia is relieved, and VEGF production by astrocytes decreases to baseline levels. In this manner, blood vessel formation is matched to oxygen demand.
Retinal blood vessels
Regulation of retinal architecture by cell–cell communication. During normal development, retinal ganglion cells (RGCs) secrete basic fibroblast growth factor (bFGF) and plateletderived growth factor (PDGF), which stimulate proliferation of astrocytes in the retina and along the optic nerve. Metabolic activity of retinal ganglion cells (and other neurons) may deplete oxygen in the area, causing ‘physiological’ hypoxia. Retinal astrocytes respond to this hypoxia by secreting vascular endothelial growth factor (VEGF), which stimulates growth of endothelial cells and generation of retinal blood vessels. These blood vessels carry oxygen to the area, relieving the hypoxia and shutting off production of VEGF by astrocytes. Exposure to high oxygen levels, or dysregulation of PDGF secretion, can lead to abnormal astrocyte and blood vessel development, altered retinal architecture and impairment of vision.
This model not only helped explain the process of normal blood vessel development in the retina, but also shed light on the cause of a human disease called ‘retinopathy of prematurity’. In this disease, premature infants who are exposed to high levels of oxygen (to help them breathe during a period when their lungs are not fully formed) develop severe retinopathy that can lead to blindness. The late stages of retinopathy of prematurity are characterized by aberrant growth of blood vessels (neovascularization) in the retina, and a number of studies suggested that VEGF might be involved in this process [3,4]. But the precise role of VEGF in the pathogenesis of retinopathy of prematurity was unclear. Once again, Stone and Keshet and their colleagues [6] collaborated to investigate this issue. Their studies suggested that, when newborn animals (or premature infants) are exposed to high levels of oxygen, their retinal astrocytes stop producing VEGF, and this causes newly formed blood vessels, which depend on the growth factor, to die. Then, when animals are returned to normal air, which has much less oxygen, the astrocytes turn back on and make large amounts of VEGF. This sudden increase in VEGF levels stimulates growth of new blood vessels, but these vessels are weak and abnormally leaky — a side-effect of excessive VEGF, which also acts to increase vascular permeability — and there are often so many of them that they disrupt retinal architecture and cause loss of vision. Previous studies had indicated that blocking VEGF — or providing supplemental oxygen to suppress its production — during the second phase of retinopathy of prematurity can help prevent neovascularization and improve vision [7,8]. But on the basis of their findings, Alon et al. [6] suggested that it might be more useful to intervene during
Dispatch
the first phase of retinopathy of prematurity, when normal blood vessels are being destroyed. They and others [9] have demonstrated that, by supplying exogenous VEGF to animals before they are exposed to high oxygen, newlyformed vessels can be preserved and the severity of retinopathy can be reduced. The studies of Stone, Keshet and colleagues [5,6] make it clear that astrocytes are critical in establishing the correct number of blood vessels in the retina. But what determines the number of astrocytes? Recent work by two groups suggests that astrocyte growth is dependent on signals from retinal neurons. The first group, led by William Richardson, had previously found that retinal ganglion cells, the neurons that carry signals out of the retina, secrete plateletderived growth factor (PDGF), while retinal astrocytes express the PDGF receptor [10]. This raised the possibility that PDGF was somehow involved in communication between retinal ganglion cells and astrocytes. To determine the nature of this communication, Richardson and colleagues [11] set out to alter PDGF levels in the developing retina. First, they generated cells that secreted a soluble form of the PDGF receptor, which would bind any PDGF secreted by the retinal ganglion cells and prevent it from reaching astrocytes. They implanted these cells into the eyes of newborn rats, and examined the effects on astrocyte development five days later. In control animals that received no cells (or cells that did not make soluble receptor), astrocytes and their processes had spread across the whole inner surface of the retina to form a rich, highly branched network. In contrast, in animals implanted with soluble PDGF receptor-secreting cells, the retinal astrocyte network was perturbed: astrocytes migrated only half as far, and their processes were much less branched than those of control animals. Similar results were obtained when animals were injected with antibodies against the PDGF receptor, which prevented astrocytes from responding to PDGF. These results indicated that the development of the retinal astrocyte network was dependent on PDGF from retinal ganglion cells. To clarify how PDGF was acting, the investigators artificially increased the amount of PDGF available to developing astrocytes by generating transgenic mice that expressed abnormally high levels of PDGF in their retinal ganglion cells. When they examined the retinas of these animals, they observed a dramatic overgrowth of astrocytes. This suggested that PDGF worked by promoting astrocyte proliferation. To examine this directly, the authors isolated retinas from wild-type and transgenic animals, and counted the number of astrocytes in each retina and the amount of DNA in each cell. As expected, the transgenic animals had two to four times as many astrocytes as controls. Moreover, these astrocytes contained more DNA than control cells, indicating that more of them had entered the cell cycle and
R435
begun to divide. In other words, PDGF was inducing proliferation of retinal astrocytes. The consequences of this proliferation became apparent when the researchers examined the vasculature of transgenic mice. As discussed above, astrocytes control the development of retinal blood vessels by secreting VEGF. In transgenic animals, the hyperproliferation of astrocytes was accompanied by an overproduction of capillaries in the retina. Early in life, many of these animals displayed retinal bleeding. By adulthood, both astrocytes and blood vessels had penetrated into the deeper layers of the retina (in normal animals, they are found primarily on the inner retinal surface), and the animals showed severely disrupted retinal structure. The authors suggested that PDGF, like VEGF, might play a role in some forms of human retinopathy. Interestingly, the ability of retinal ganglion cells to control astrocyte proliferation is not restricted to the retina. The axons of retinal ganglion cells extend out of the retina to form the optic nerve, and the optic nerve, like the retina, is lined with astrocytes. The second study, by Burne and Raff [12], demonstrates that the proliferation of these astrocytes is also regulated by retinal ganglion cells. The first indication of this came from earlier studies of transgenic mice that expressed high levels of the survival-promoting gene bcl-2 in their retinal ganglion cells [13]. These mice not only had increased numbers of retinal ganglion cells, but increased numbers of optic nerve astrocytes as well. This suggested that the development or proliferation of optic nerve astrocytes might depend on signals from retinal ganglion cell axons. To test this notion, the authors [12] studied the effects of cutting the optic nerve, which causes degeneration of retinal ganglion cell axons. When they examined astrocytes from cut optic nerves, they found a dramatic decrease in proliferation compared to that seen in astrocytes from normal optic nerves, indicating that signals from the axons are necessary for normal astrocyte proliferation. Interestingly, a similar drop in proliferation was observed when the experiment was performed in mutant mice whose optic nerves do not degenerate following transection. This suggested that, even if retinal ganglion cell axons were intact, they could not stimulate astrocyte proliferation unless they were physically connected to the cell bodies of retinal ganglion cells. One possible reason that the axons had to be intact was so that they could conduct electrical activity. In fact, previous studies [14] had shown that oligodendrocyte precursors in the optic nerve depend on electrical activity in retinal ganglion cell axons for their proliferation. However, activity turned out not to be required for the mitogenic effect of retinal ganglion cells on astrocytes, as injection of
R436
Current Biology, Vol 7 No 7
tetrodotoxin, which blocks electrical activity, did not have the same effect as transection. In contrast, injection of colchicine, which prevents transport down the axon, was able to mimic the effects of transection. From these observations, the authors concluded that molecules transported down retinal ganglion cell axons are required for the mitogenic effect on astrocytes. To explore the basis of this effect, the investigators cultured optic nerve astrocytes in vitro in the presence or absence of retinal ganglion cells. In the presence of retinal ganglion cells, they found that the proliferation of astrocytes increased 20-fold. The investigators attempted to mimic the mitogenic effect of retinal ganglion cells using a variety of growth factors known to be secreted by these cells, and found that only one, basic fibroblast growth factor (bFGF), was effective. Notably, PDGF, which promotes the proliferation of astrocytes within the retina, was unable to stimulate astrocytes from the optic nerve. This is particularly interesting, as retinal astrocytes are thought to originate in the optic nerve [15,16]. These results indicated that retinal ganglion cells control the development of astrocytes in both the retina and the optic nerve, albeit by different mechanisms. The work of Stone, Keshet and colleagues [5,6], Fruttiger et al. [11] and Burne and Raff [12] clearly demonstrates that assembly of the retina and the optic nerve is regulated by a series of intricate cell–cell interactions, and that interference with any of these interactions can lead to disruption of retinal architecture and impairment of vision. But while these studies provide a clearer picture of how the elements of the visual system are put together, a number of important questions remain. For example, Stone et al. [5] postulate that neuronal activity in the developing retina results in local hypoxia, which induces astrocytes to secrete VEGF; although this is a plausible hypothesis, there is no direct evidence that such physiological hypoxia occurs, or that it is in fact the stimulus for VEGF production. Assuming that it is, the mechanism by which astrocytes sense changes in local oxygen levels, and the signaling pathways that allow them to respond by secreting VEGF, still remain to be elucidated. Similarly, the studies of Fruttiger et al. [11] and Burne and Raff [12] leave little doubt that retinal ganglion cells can control the proliferation of astrocytes by secreting soluble factors, such as PDGF and bFGF. But what triggers the secretion of these factors? And perhaps more importantly, what shuts them off so that astrocyte proliferation and vascularization do not continue unchecked? The approaches these investigators have used to map out the circuitry thus far — detailed analysis of cell–cell interactions and specific manipulation of these interactions in vitro and in vivo — will undoubtedly shed light on these issues in the near future.
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