Ординатура / Офтальмология / Английские материалы / Eye, Retina, and Visual System of the Mouse_Chalupa, Williams_2008

Figure 32.5 Important techniques for studying RGC projections in the developing mouse visual system. A, DT and VT retinal explants plated near a border of 0.5 μg/mL ephrin-B2 (red). Axons from DT explants project into the ephrin-B2 region, while VT axons are clearly inhibited. B, Time-lapse imaging can be used to study RGC growth cone behavior in real time as they encounter a border, in this case ephrin-B2. Arrowheads highlight two growth cones, one of which projects into the ephrin-B2 region and the other of which is repelled and travels parallel to the ephrin-B2

excellent model in which to study retinal specification and axon guidance. Guidance cues and regulatory genes have been uncovered in the retina, optic nerve, chiasm, and tract whose combined interactions help to guide RGC axons from the retina toward their targets. Perturbation of these molecules can result in a wide variety of projection errors at the chiasm, ranging from an increase or decrease in the size of ipsilateral projections to axon defasciculation and ectopic projections.

Significant progress has been made in identifying molecular mechanisms that guide RGC axon divergence at the optic chiasm, an essential step for the development of binocular vision. Zic2 appears to regulate EphB1 in VT retina, which directs ipsilateral projections via interaction with ephrin-B2 at the chiasm midline. At later stages, Islet2 and

border. C, Diagram depicting in utero retinal electroporation. Briefly, DNA is injected into embryonic retina. Current is passed through electroporation paddles, and embryos are allowed to survive for several days. Embryos are perfused, the retina is collected, and heads are cryosectioned. In this case, GFP-positive cells can be seen in the retina, and many GFP-positive RGC projections are visible through the optic nerve, chiasm, and tract. Red denotes neurofilament. See color plate 22. (All images courtesy of T. Petros and C. Mason.)

Nr-CAM are required for crossed projections from VT retina. It will be of interest to test these mice mutants for changes in visual acuity, to link RGC projection errors with functional consequences.

Despite this progress, there are still many questions that remain unanswered. What are the molecular mechanisms that guide crossed projections from non-VT retina—the bulk of the crossed projection? What is the role of the pioneer axons? How do axon-axon interactions help direct RGC projections? And are these findings in mice relevant to the human visual system? Although there is new evidence that ephrin-B2 expression is analogous in humans and mice (Lambot et al., 2005), further studies are needed to answer these questions and identify how many developmental principles in mice can be extended to the human visual pathway.

acknowledgments Work was supported by NIH grant nos. EY012736, EY015290, and F31 NS051008. We are grateful to members of the Mason Lab, both past and present, for their comments on this manuscript and for contributions to this body of work.

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JEFFREY L. GOLDBERG

How do retinal ganglion cell (RGC) axons grow during development, and why do they fail to regrow when injured? RGCs extend long axons carrying all of the visual information through the optic nerve, across the optic chiasm, and into the brain. Our understanding of the essential regulators of this process has advanced considerably in recent years. Investigating the regulation of axon growth is crucial to understanding why axons fail to regenerate in the central nervous system (CNS) after injury or in disease, and rodent model systems have proved invaluable to expanding our understanding of axon growth and regeneration.

What signals induce axon growth?

Do Neurons Need Specific Extrinsic Signals for Axon

Growth? Because the same signals that induce axon growth are generally critical for neuronal survival, these two functions have been difficult to disentangle—remove putative axon growth signals in vitro or in vivo, and the neurons die. The development of genetic tools to block apoptosis in neurons made it possible to address this question (Goldberg and Barres, 2000). For example, RGCs can be purified and cultured in the complete absence of glial cells; elevating the expression of the antiapoptotic protein Bcl-2 maintains neuronal survival after all trophic signals are withdrawn (Goldberg et al., 2002a). Despite high levels of survival in the absence of exogenous signals, Bcl-2-overexpressing RGCs fail to elaborate axons or dendrites unless axon growthinducing signals are present, clearly demonstrating that axon growth is not a default function of a surviving neuron but must be specifically signaled (figure 33.1).

RGCs are similar to other neurons in this requirement for extrinsic signals to signal axon growth. For example, when developing dorsal root ganglion (DRG) sensory neurons from transgenic mice that lack the pro-apoptotic protein Bax are cultured in vitro, withdrawal of neurotrophic factors does not induce apoptosis. These Bax−/− DRG sensory neurons grow rudimentary axons, but neurotrophins greatly increase axon outgrowth in vitro (Lentz et al., 1999). Even embryonic peripheral neurons that survive in culture without added trophic factors fail to extend neurites (Lindsay et al., 1985), and adult DRG neurons that do not appear to depend

on neurotrophic factors for survival still respond to such factors by increasing axon outgrowth (Lindsay, 1988). Taken together, these findings indicate that surviving neurons do not constitutively extend axons and that axon growth must be specifically signaled by extracellular signals.

What Are the Extracellular Signals That Induce Axon

Growth? A great variety of extracellular signals have been found to induce axon growth. The most potent signals are peptide trophic factors. Many different families have been studied intensively and have widespread roles in regulating the developing and adult nervous system. Different neurons are frequently responsive to different trophic factors, and multiple trophic factors can often combine to induce even greater axon growth.

The best studied peptide trophic factors for RGC axon growth in vitro (and regeneration in vivo) are brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), insulin-like growth factor (IGF), basic fibroblast growth factor (bFGF), and glial cell line–derived neurotrophic factor (GDNF). Interestingly, each of these belongs to a different family, acts on different receptors, and activates different downstream signaling molecules, but all appear to converge on similar regulators of axon growth (table 33.1).

RGCs extend axons well in response to BDNF and CNTF, but both together induce more axon growth than either does alone (Goldberg et al., 2002a; Jo et al., 1999; Logan et al., 2006; Loh et al., 2001). Interestingly, in studies that dissociate survival from axon growth, the peptides that most strongly promote cell survival are the same ones that most strongly promote axon growth (Mansour-Robaey et al., 1994; Mey and Thanos, 1993). This is even more remarkable in light of data suggesting that the survival and axon growth responses to trophic factors diverge inside the neuron, using different intracellular signaling pathways (Atwal et al., 2000; Goldberg et al., 2002a). Recent evidence has extended the list of trophic signaling molecules beyond these traditional families. For example, a small, macrophage-derived, calcium-binding protein named oncomodulin binds RGCs and strongly promotes RGC axon growth in vitro and in vivo (Yin et al., 2006).

Are extracellular signals other than peptide trophic factors sufficient to induce axon elongation? Extracellular matrix molecules such as laminin and heparin sulfate proteoglycans, and cell adhesion molecules such as L1 and N- cadherin, are expressed in the visual pathway (Lafont et al., 1992; Reichardt and Tomaselli, 1991) and help promote axon growth in vitro and in vivo. Eliminating N-cadherin expression, for example, clearly decreases RGC axon growth in vivo (Riehl et al., 1996). Although these signaling interactions can potentiate axon outgrowth in purified CNS neurons if soluble glial or peptide trophic signals are present, so far they do not appear to be sufficient to induce axon growth on their own (Goldberg et al., 2002a). Rather, they appear to provide a critical substrate along which axons extend,

Figure 33.1 RGCs require extrinsic signals to extend axons. A and B, Purified rat RGCs were infected acutely with a virus carrying the antiapoptotic gene Bcl-2 and cultured in media without (A) or with (B) peptide trophic factors. Scale bar = 30 μm. C, Population data showing the percent of purified RGCs extending axons more than 15 μm from the cell body (mean ± SEM). D, Population data as in C, for Bcl-2-expressing RGCs first cultured 1 week in minimal media lacking peptide trophic factors and then returned to media with or without trophic factors, as marked. (Reprinted from Goldberg, J. L., Espinosa, J. S., Xu, Y., Davidson, N., Kovacs, G. T., Barres, B. A. [2002]. Retinal ganglion cells do not extend axons by default: Promotion by neurotrophic signaling and electrical activity. Neuron 33(5):689–702. Copyright 2002, with permission from Elsevier.)

thereby strongly potentiating axon outgrowth. Other adhesion and guidance molecules that normally direct RGC axons as they steer through the visual pathway to their targets in the brain (Oster et al., 2004) may also contribute to the signaling of axon elongation. For example, netrin-1, a well-described axon guidance molecule homologous to laminin that guides early RGC axons to the optic nerve head as they exit the retina (de la Torre et al., 1997), may also stimulate elongation (Serafini et al., 1994), although it may not be as effective in stimulating RGC axon growth on its own (Goldberg et al., 2002a). As axon guidance molecules activate the same growth cone mechanisms discussed earlier, it is not surprising that their effects may overlap with effects on elongation as well. Finally, other extracellular signals as simple as purine nucleotides may be critical to inducing axon growth, although the mechanism of this activity is still mysterious (Benowitz et al., 1998). Therefore, peptide trophic factors appear to most strongly induce axon outgrowth, but adhesion molecules can greatly potentiate this growth, and molecules thought to be primarily involved in growth cone guidance may also have some elongation-promoting activity.

Where Do Extracellular Signals Act to Induce Axon

Elongation? Where do trophic peptides act, at the cell body or on the axons themselves? Local application of neurotrophins to a growth cone is sufficient to enhance the rate of growth cone extension in vitro (Letourneau, 1978) and in vivo (Tucker et al., 2001). The definitive answer, however, has come from experiments using Campenot chambers, a powerful technique in which neurons are cultured with their cell bodies in one compartment while their axons grow under a wall into a separate compartment, with the culture media in each compartment kept separated. Classic experiments using cultures of peripheral neurons in these chambers demonstrated the necessity for trophic peptides to signal at the axons and growth cones themselves for the axons to survive and grow (figure 33.2) (Campenot, 1994). Trophic peptides in the cell body compartment keep the neurons alive, but the axons in the peripheral compartment die back. Amazingly, in these experiments, the

Figure 33.2 Campenot chambers have proven invaluable in dissecting the functions important to axons from those important to the cell body. In these experiments, a wall separates the culture dish into two or more compartments, and although the axons can grow under the wall into the distal compartment, the contents of the culture media do not diffuse from one side to the other. In this way, factors can be added exclusively to the axons or cell bodies to the exclusion of the other. For example, in the classic experiments dissociating cellular survival from axon growth, neurotrophins such as NGF added to the axon or cell body compartments supported neuronal survival, but the neurotrophin had to be added specifically to the axon compartment to support the survival and growth of the axons themselves (see the text). (Reprinted from Goldberg, J. L. [2003]. How does an axon grow? Genes and Development 17(8):941–958. Copyright 2003, with permission from Cold Spring Harbor Press.)

presence of trophic peptides only in the peripheral (axon) compartment is sufficient to induce survival of the cell body. Therefore, the provision of proper growth signals to the axons is as important as providing survival signals to the cell body and points to the importance of providing developing neurons with axon growth signals all along the growth pathway. For example, RGC axon growth is stimulated by soluble signals from the retina, optic nerve, and superior colliculus (Goldberg et al., 2002b), and these are likely to stimulate passing axons in vivo.

Although trophic factors may act solely at the axon, to induce axon elongation they must also activate processes at the cell body. How do growth signals at the axon get passed along to the distant cell body, where gene transcription and translation are induced to support survival and axon growth? Neurotrophin-mediated activation of tyrosine kinase (Trk) receptors leads to endocytosis of activated, ligand-bound receptors into clathrin-coated vesicles. These signaling endosomes carry with them the machinery of the ras-raf-MAP kinase signaling cascades and can continue to activate these pathways once inside the cell (Howe et al., 2001). Neurotrophin signaling endosomes may then be retrogradely transported along microtubules back to the cell body, where they can activate transcriptional regulators and induce new gene expression (Riccio et al., 1997; Watson et al., 1999). In

addition, there may be retrograde signaling of neurotrophinmediated survival signals without retrograde transport of NGF-containing endosomes (MacInnis and Campenot, 2002), raising an interesting hypothesis that survival signals and axon growth or other signals may be carried along separate but complementary pathways. It is not yet known whether the retrograde transport of signals from the growth cone is necessary for axon growth if similar signals are already acting at the cell body, as is likely to happen in vivo. Retrograde carriage of neurotrophic signals may modify the signal to carry more information, for example by changing signaling components in different cellular compartments, and may provide crucial signals along the way, for example for maintenance of axonal structure along its length, or for suppression or support of axon branching. Experiments tackling the relationship between vesicle localization and signaling effects will help shed light on these important questions.

How Do Extracellular Signals Induce Axon Elongation? We know surprisingly little about the intracellular mechanisms by which neurotrophic signals elicit axon growth. Two relevant questions can be addressed in this domain: what are the intermediate signaling pathways that receptors activate to elicit growth, and what are the specific aspects of axon growth that this signaling pathway regulates? A few models have been particularly fruitful in defining which signaling cascades are important for axon growth. Campenot chamber experiments similar to those described earlier showed that for peripheral neurons, both ras-raf-MAP kinase and PI3-kinase-Akt signaling pathways downstream of neurotrophin receptors contribute to axon outgrowth (Atwal et al., 2000). In recent years, significant progress has been made in identifying how these two signaling pathways are activated by neurotrophin receptors; details of these experiments have been reviewed extensively elsewhere (Kaplan and Miller, 2000). A second type of experiment takes advantage of neurons in which apoptosis is blocked by knocking out Bax or overexpressing Bcl-2. In Bcl-2- overexpressing RGCs, pharmacologic inhibition of either MAP kinase or PI3 kinase partially reduces axon outgrowth, but inhibiting both together is necessary to block axon elongation altogether (Goldberg et al., 2002a). Although the specifics may vary from neuron to neuron, these data hint at a fascinating complexity of the regulation of these different parameters of axon growth that remains to be more fully studied.

How does the activation of these signaling pathways actually regulate axon growth? Regulation of the transcription and translation of genes needed for general cellular growth, axonal cytoskeletal dynamics, supply and insertion of membrane and cytoplasmic building blocks, translation of axonally localized mRNAs, and proteosome activity have all

been implicated and have been reviewed in depth elsewhere (Goldberg, 2003). Much of this work has been initiated in models outside the visual system, but early indications are that many of these regulatory pathways are shared by all neurons. Interestingly, in purified RGC cultures, both BDNF and CNTF induce similar rates of axon growth, but the two together induce more than either does alone, raising the hypothesis that different trophic factors may be responsible for different facets of axon growth (Goldberg et al., 2002a).

Control of Neuronal Responsiveness to Trophic

Peptides An interesting difference between the ability of trophic peptides to promote axon growth by CNS neurons such as RGCs and by peripheral nervous system (PNS) neurons has been identified. Peptide trophic factors such as neurotrophins are sufficient to induce axon growth by purified PNS neurons in culture. In contrast, to elicit axon growth from CNS neurons in culture, peptide trophic signals alone are insufficient. For instance, RGCs fail to survive in the presence of such trophic signals as BDNF or CNTF unless their cAMP levels are elevated, either pharmacologically or by depolarization (Meyer-Franke et al., 1995). Cyclic AMP elevation and depolarization do not promote axon growth on their own. Similarly, RGCs kept alive with Bcl-2 overexpression extend axons only poorly in response to BDNF, but this axon growth is greatly potentiated by cAMP elevation or by physiological levels of electrical activity, either from endogenous retinal activity or from direct electrical stimulation when cultured on a silicon chip (Goldberg et al., 2002a) (figure 33.3).

Do neurons have to be electrically active to respond to trophic activities for axon growth? Experiments in vivo suggest that electrical activity is not absolutely required: injecting tetrodotoxin into the eye to block action potentials (Shatz and Stryker, 1988) or studying the munc-18 knockout mouse in which synaptic release of neurotransmitters is essentially eliminated (Verhage et al., 2000) both reveal that RGCs and other CNS neurons successfully elongate their axons to their targets. Recent studies, however, also highlight the possible effects of electrical activity in sculpting axonal morphology. Activity regulates growth cone responsiveness to guidance cues, enhancing netrin responsiveness and inhibiting myelin repulsion (Ming et al., 2001). In addition, activity shapes the specificity of local connectivity of axons, for example in the selection and elimination of cortical innervation targets (Catalano and Shatz, 1998; Kalil et al., 1986; Katz and Shatz, 1996). Data on the effect of activity on axon growth have been more controversial. Neuronal activity may increase the rate of axon arborization in vitro and in vivo by stabilizing growing branches (Cantallops et al., 2000; Cohen-Cory, 1999; Rashid and Cambray-

Figure 33.3 Effects of electrical activity on axon growth and survival in response to BDNF. A, Photograph of silicon chip growth substrate and diagram of burst-stimulation protocol. B, DiI-labeled RGCs cultured on silicon chips extended rudimentary axons in response to BDNF (top). Axon growth was enhanced if the neurons were stimulated with electrical activity (bottom). C, Mean longest axon/RGC ± SEM measured after 16 hours in the presence of BDNF, with stimulation, SQ, or TTX, as marked and as described in the text. D, DiI-labeled RGCs (left panels) were plated in media containing BDNF and stimulated by electrical activity as marked. RGC survival was assessed 48 hours later by labeling with calceinAM (right panels). DiI-labeled RGCs stained with calcein are alive (arrows); those lacking calcein are dead (arrowheads). E, RGC survival in response to BDNF was enhanced 10-fold by electrical stimulation. Scale bar = 50 μm (B) or 200 μm (D). (Reprinted from Goldberg, J. L., Espinosa, J. S., Xu, Y., Davidson, N., Kovacs, G. T., Barres, B. A. [2002]. Retinal ganglion cells do not extend axons by default: Promotion by neurotrophic signaling and electrical activity. Neuron 33(5):689–702. Copyright 2002, with permission from Elsevier.)

Deakin, 1992). On the other hand, growth cones may collapse acutely in response to electrical stimulation, but then desensitize and recommence axon growth (Fields et al., 1990). Therefore, after a brief accommodation at the growth cone, the longer-term effects of electrical stimulation appear to promote axon growth.

This trophic dependence contrasts with PNS neurons, which survive and regenerate their axons in response to trophic peptides in the absence of cAMP elevation or electrical activity, raising the hypothesis that the trophic signaling of axon growth may differ between CNS and PNS neurons. Could this difference contribute to their different abilities to regenerate in vivo? Previous studies have suggested that neither trophic factor delivery alone nor electrical stimulation alone induces CNS axonal regeneration. If axon growth normally depends on activity in vivo, and if damaged cells are less active or elevate cAMP less effectively in response to activity, a CNS neuron’s ability to regenerate its axon could be impaired. Therefore, it may be crucial to provide trophic peptides as well as signals such as cAMP elevation to ensure an optimal axon growth response. Although cAMP elevation has been implicated in overcoming inhibitory signals at the growth cone (Ming et al., 2001), it is interesting to speculate whether some of the effect of cAMP in promoting regeneration in vivo is actually attributable to improving the neuron’s response to trophic or other positive peptide signals (Hu et al., 2006; Qui et al., 2002).

Intrinsic control of axon growth

Intrinsic Growth Ability of Central Nervous System Neurons Is Developmentally Regulated Are the rate and extent of axon growth purely dependent on extracellular signals and substrates, or do they also depend on the intrinsic state of the neuron? This is a critical if largely unanswered question in research on axon growth and regeneration. Embryonic CNS neurons can regenerate their axons quite readily, but they lose their capacity to regenerate with age. For example, in the spinal cord, axons lose the ability to regenerate between P4 and P20 (Kalil and Reh, 1982; Reh and Kalil, 1982; Saunders et al., 1992). This developmental loss of regenerative ability has generally been attributed to the maturation of CNS glial cells, both astrocytes and oligodendrocytes, and to the production of CNS myelin, all of which strongly inhibit regenerating axons after injury. In experiments in which the inhibitory environment is removed or molecularly blocked, however, only a few percent of axons regenerate, and functional recovery typically proceeds remarkably slowly. For example, RGCs take 2 months to regenerate through a peripheral nerve graft (Aguayo et al., 1987; Bray et al., 1987). These experiments indicate that an inhibitory environment is likely only part of the explanation.

Are the neurons themselves partly responsible? Axons from P2 or older hamster retinas have lost the ability to reinnervate even embryonic tectal explants (Chen et al., 1995). Similar explant preparations have shown a developmental loss of regenerative ability in cerebellar Purkinje cells and brainstem neurons (Blackmore and Letourneau, 2006; Dusart et al., 1997). This suggests that changes in a CNS neuron’s intrinsic ability to grow could also explain this developmental loss of regenerative ability. The inability of postnatal neurons to reextend their axons might also be explained by the development of glial cells, however, which are largely generated postnatally. Therefore, the ability to separate neurons from CNS glia remains critical to determining whether CNS neurons actually change in their intrinsic axon growth ability during development.

When cultured in strongly trophic environments in the complete absence of CNS glia and at clonal density, embryonic RGCs extend axons up to 10 times faster than postnatal RGCs (figure 33.4) (Goldberg et al., 2002b). The evidence for this decreased growth ability being intrinsically maintained is twofold. First, embryonic RGCs grow at a faster rate than postnatal RGCs in a variety of environments that should facilitate growth, including in media containing neurotrophic factors, in media conditioned by cells from the embryonic visual pathway, and after transplantation into developing pathways in vivo. In all cases, embryonic RGCs extended their axons at rates substantially higher than did the postnatal RGCs, suggesting that any extrinsic growthpromoting environment is dependent on an intrinsically set maximal growth rate. Second, RGCs purified from either embryonic or postnatal ages and cultured away from all of the other cell types with which they normally interact retained their faster or slower growth phenotypes, respectively. Therefore, the difference in the abilities of embryonic and postnatal RGCs to elongate axons is not dependent on continued signaling by neighboring cell types but is intrinsically maintained.

Do these neurons lose their axon growth ability as the result of intrinsic aging? Embryonic RGCs aged up to 10 days in purified cultures, to the age they would decrease their axon growth ability in vivo, continue to elongate their axons rapidly, suggesting that the change in axon growth ability is signaled by an extrinsic cue (Goldberg et al., 2002b). The decrease in axon growth ability occurs sharply at birth during the period of target innervation, but E20 RGCs cocultured with superior collicular slices retain their rapid axon growth ability, suggesting that target contact is not responsible for this change. Indeed, retinal maturation, and specifically a membrane-associated signal from retinal amacrine cells, was shown to be sufficient to induce the developmental decrease in RGC axon growth ability. Thus, a membrane-associated cue from these presynaptic amacrine cells signals embryonic

Figure 33.4 Difference in axon growth ability of E20 and P8 RGCs. A–D, RGCs purified by immunopanning were cultured at clonal density (<5/mm2) on poly-D-lysine (PDL) and laminin in growth medium containing BDNF, ciliary neurotrophic factor (CNTF), insulin, and forskolin (GM). A, RGC axons immunolabeled for the axonal protein-tubulin III after 18 hours. B, Percentage of RGCs at each age elaborating at least one axon more than 20 μm after 3 days. C, Average length of each neuron’s longest axon at days 1, 2, and 3. E20 and P8 axon lengths did not differ statistically at day 1. P8 axon lengths at days 2 and 3 were longer than on the day before (Dunnett’s P < 0.05). D, Time course of change

RGCs to decrease their axon growth ability, but once this signal is sent, the loss is permanent: removal of the amacrine cells does not allow the RGCs to speed up again.

Does Dendrite Growth Ability Replace Axon Growth

Ability? Why would a presynaptic cell type signal these CNS neurons to decrease their intrinsic axon growth ability? Remarkably, at about the same time that neonatal RGCs lose their ability to rapidly elongate axons, they gain the ability to rapidly generate dendrites (Goldberg et al., 2002b). This increase in dendritic growth ability is not an RGCautonomous result of intrinsic aging, because RGCs do not

in intrinsic axon growth ability of RGCs purified simultaneously from rats of different ages and cultured in target-conditioned growth medium. Means ± SEM. E, RGC axon length after 3 days at clonal density in minimal medium conditioned by superior collicular (SC), retinal (Ret), optic nerve (ON), or sciatic nerve (SN) cells. Scale bars = 50 μm. (Reprinted from Goldberg, J. L., Klassen, M. P., Hua, Y., Barres, B. A. [2002]. Amacrine-signaled loss of intrinsic axon growth ability by retinal ganglion cells. Science 296: 1860–1864. Copyright 2002, with permission from Science Publishing.)

increase their dendrite growth ability with time in purified cultures, but rather is signaled by a retinal cue (Goldberg et al., 2002a). Therefore, retinal cues trigger neonatal RGCs to irreversibly switch from an axonal to a dendritic growth mode. Whether the increase in dendritic growth ability and the decrease in axon growth ability are the result of the same signal is not yet known, but an interesting implication of these data is that the control of axon versus dendrite growth may be largely intrinsic, rather than determined by separate extracellular cues (figure 33.5). Axons and dendrites appear to respond to many, or possibly all, of the same growth and guidance signals. It is

Figure 33.5 Is axon versus dendrite growth a function of environment, a function of the intrinsic growth state of the neuron, or both? A, In the extrinsic control hypothesis, axon-specific and dendrite-specific growth signals are presented to the neuron at different developmental windows or locations. B, In the intrinsic control hypothesis, growth signals are available in general, and the neuron’s intrinsic growth state, either axonal or dendritic, determines which processes are preferentially responsive to extrinsic trophic signals. The change in intrinsic state could come from an intrinsic, preprogrammed clock or aging mechanism or could be elicited by an extrinsic signal; for example, amacrine cells are able to signal RGCs to decrease their intrinsic axon growth ability (Goldberg et al., 2002b). Note, however, that the amacrine signal does not have to be present in an ongoing fashion: Once it signals RGCs to decrease their axon growth ability, they retain this new phenotype intrinsically. (Reprinted from Goldberg, J. L. [2004]. Intrinsic neuronal regulation of axon and dendrite growth. Current Opinion in Neurobiology 14(5):551–557. Copyright 2004, with permission from Elsevier.)

unknown whether some of the signals found previously to increase dendrite outgrowth, such as bone morphogenic protein-7 (Higgins et al., 1997), actually modulate the growth mode of the neuron from axonal to dendritic, rather than stimulating dendritic growth cones preferentially. Many of the previous studies that have shown that presynaptic cell types stimulate dendritic growth may reflect a switch in the neurons from axonal to dendritic growth modes, after which the neurons extend dendrites in response to growth signals already present. Thus, the ability of neurotrophic factors to stimulate axon and dendrite growth may strongly depend on whether a neuron is in an axonal or dendritic growth state, and raises the question of the identity of the extracellular signal that induces a dendritic growth mode (Goldberg et al., 2002b).

What Molecular Changes Underlie the Developmental Loss in Rapid Axon Growth Ability? In response to this amacrine cell-associated cue, RGCs could gradually increase the expression of genes that limit axon growth or decrease the expression of genes necessary for faster axonal elongation, or both. For instance, in the developing chick, RGCs lose axon growth responsiveness to laminin either by downregulating specific integrins (laminin receptors) (Cohen et al., 1989; de Curtis and Reichardt, 1993; de Curtis et al., 1991) or by downregulating the activation of such integrins (Ivins et al., 2000), although they continue to respond to laminin-2 (merosin) (Cohen and Johnson, 1991). At least for mammalian RGCs, such changes are not responsible for the observed decrease in growth ability, as postnatal RGCs continue to be responsive to laminin-1, as well as a variety of other substrates, and laminin-2 is no more effective than laminin-1 in promoting postnatal RGC axon growth (Goldberg et al., 2002a). Similarly, a difference in trophic receptor levels or responsiveness to trophic signals could explain these differences. For example, depolarization rapidly elevates Trk-B receptors on the surface of CNS neurons (Du et al., 2000; Meyer-Franke et al., 1998). Exogenously elevating Trk-B levels, however, fails to increase P8 axon growth rates to embryonic levels (Goldberg et al., 2002a). The antiapoptotic protein Bcl-2 was proposed as an intrinsic genetic switch that decreases axon growth rate by RGCs (Chen et al., 1997), but Bcl-2 overexpression by purified RGCs in culture neither promotes axon growth nor enhances axon growth in response to neurotrophic signaling in vitro or in vivo (Goldberg et al., 2002a), a result consistent with other findings (Goldberg and Barres, 2000; Chierzi et al., 1999; Greenlund et al., 1995; Lodovichi et al., 2001; Michaelidis et al., 1996).

What molecular mechanisms could regulate the axonal or dendritic growth mode of a neuron? If properly regulated, almost any molecule in the cell could be crucial to determining axon versus dendrite growth (Goldberg, 2003). It is attractive to first consider transcription factors that could activate the expression of whole programs of axon or dendrite growth and could also maintain an intrinsic phenotype in the absence of ongoing extrinsic signaling. For example, the POU domain transcription factor Brn-3b is essential for RGC development. In Brn-3b−/− mice, RGCs fail to extend axons properly, and 80% die before birth; in addition, Brn3b−/− RGCs in culture extend only rudimentary axons and instead elaborate abnormal, dendrite-like processes (Erkman et al., 1996, 2000; Gan et al., 1996, 1999; Wang et al., 2000). RGCs also express the related family members Brn-3a and Brn-3c: about 50% of RGCs express both Brn-3b and Brn3c. The Brn-3b−/− Brn-3c−/− double knockout shows an almost total loss of axon outgrowth from explanted retinas, suggesting that both Brn-3b and Brn-3c contribute to controlling RGC axon growth (Wang et al., 2002). In these experiments,