Different factors promote axonal regeneration of adult rat retinal ganglion cells after lens injury and intravitreal peripheral nerve grafting

We have investigated the differential mediators of the neurotrophic effects of intravitreal peripheral nerve grafting and lens injury on adult rat retinal ganglion cells (RGC). Lens injury and intravitreal peripheral nerve grafting both stimulated RGC neurite growth in vitro and axon regeneration past the optic nerve lesion site in vivo concomitant with activation of retinal glia and invasion of macrophages into the eye. These observations, together with the results of coculture studies using a macrophage-free intact peripheral nerve segment, a macrophage-free intact lens, a macrophage-rich peripheral nerve segment, or a macrophage-rich injured lens in retinal cultures suggest that the stimulation of RGC axon regeneration by lens injury and intravitreal peripheral nerve grafting share a common macrophage-derived component overlain by distinct lens-derived and peripheral nerve-derived neurotrophic factors, respectively. RGC axon regeneration following lens injury and intravitreal peripheral nerve grafting was similar in vivo, correlating with similar retinal glia activation whereas, in vitro, the level of RGC neurite outgrowth was significantly higher following intravitreal peripheral nerve grafting compared with lens injury, concomitant with the presence of increased numbers of activated retinal glia. This suggests that in vivo RGC axon regeneration induced by lens injury and peripheral nerve grafting may be limited, in part, by factors derived from activated retinal glia.     

Global and Regional Damages in Retinal Ganglion Cell Axon Bundles Monitored Non-Invasively by Visible-Light Optical Coherence Tomography Fibergraphy

Retinal ganglion cells (RGCs) exhibit compartmentalized organization, receiving synaptic inputs through their dendrites and transmitting visual information from the retina to the brain through the optic nerve. Little is known about the structure of RGC axon bundles extending from individual RGC somas to the optic nerve head (ONH) and how they respond to disease insults. We recently introduced visible-light optical coherence tomography fibergraphy (vis-OCTF), a technique for directly visualizing and analyzing mouse RGC axon bundles in vivo. In this study, we validated vis-OCTF''s ability to quantify RGC axon bundles with an increased number of RGCs using mice deficient in BCL2-associated X protein (BAX^−/−). Next, we performed optic nerve crush (ONC) injury on wild-type (WT) mice and showed that the changes in RGC axon bundle width and thickness were location-dependent. Our work demonstrates the potential of vis-OCTF to longitudinally quantify and track RGC damage at single axon bundle level in optic neuropathies.SIGNIFICANCE STATEMENT Nearly all clinical and preclinical studies measure the retinal nerve fiber (RNFL) thickness as the sole indicator of retinal ganglion cell (RGC) damage without investigating RGC axon bundles directly. We demonstrated visible-light optical coherence tomography fibergraphy (vis-OCTF) to directly quantify global and regional RGC axon bundle organizations in vivo as a new biomarker for RGC health. We validated in vivo vis-OCTF measures using both confocal microscopy of the immunostained flat-mounted retina and numerical simulations. Vis-OCTF for monitoring RGC axon bundle organization has the potential to bring new insight into RGC damage in optic neuropathies.     

Effect of lens lesion on neurite outgrowth of retinal ganglion cells in vitro

Recent studies have shown that lens lesion promotes axonal regeneration in the optic nerve of adult rats. In the present investigations, dissociated retinal ganglion cells (RGC) from intact postnatal (P) 9-11 rats showed spontaneous neurite outgrowth on laminin-1, in contrast to RGC from intact P14-adult rats. Neurite outgrowth from P9-14 RGC on laminin-1 was promoted by prior lens lesion and also during coculture with lesioned lenses. Neurite outgrowth from adult RGC following prior lens lesion, or in cocultures with lesioned lenses, required the presence of laminin-2. In media conditioned by lesioned lenses, the stimulatory effect on neurite outgrowth was still observed in the presence of K252a (trk receptor blocker) and mAb 228 (which blocks the effects of leukemia inhibitory factor and ciliary neurotrophic factor). Together, these results suggest the existence of a neuritogenic factor(s) associated with the lesioned lens that belongs to neither the neurotrophin nor the gp130 cytokine family.     

Eye drop delivery of pigment epithelium-derived factor-34 promotes retinal ganglion cell neuroprotection and axon regeneration

Axotomised retinal ganglion cells (RGCs) die rapidly by apoptosis and fail to regenerate because of the limited availability of neurotrophic factors and a lack of axogenic stimuli. However, we have recently showed that pigment epithelium-derived factor (PEDF) promotes RGC survival and axon regeneration after optic nerve crush injury. PEDF has multiple fragments of the native peptide that are neuroprotective, anti-angiogenic and anti-inflammatory. Here we investigated the neuroprotective and axogenic properties of a fragment of PEDF, PEDF-34, in retinal neurons in vitro and when delivered by intravitreal injection and eye drops in vivo. We found that PEDF-34 was 43% more neuroprotective and 52% more neuritogenic than PEDF-44 in vitro. Moreover, in vivo, intravitreal delivery of 1.88 nM PEDF-34 was 71% RGC neuroprotective at 21 days after optic nerve crush compared to intact controls, whilst daily eye drops containing 1.88 nM PEDF-34 promoted 87% RGC survival. After topical eye drop delivery, PEDF-34 was detected in the vitreous body within 30 min and attained physiologically relevant concentrations in the retina by 4 h peaking at 1.4 ± 0.05 nM by 14 days. In eye drop- compared to intravitreal-treated PEDF-34 animals, 55% more RGC axons regenerated 250 μm beyond the optic nerve lesion. We conclude that daily topical eye drop application of PEDF-34 is superior to weekly intravitreal injections in promoting RGC survival and axon regeneration through both direct effects on retinal neurons and indirect effects on other retinal cells.     

Erythropoietin is both neuroprotective and neuroregenerative following optic nerve transection

The cytokine hormone erythropoietin (EPO) is neuroprotective in models of brain injury and disease, and protects retinal ganglion cells (RGC) from cell death after axotomy. Here, we assessed EPO's neuroprotective properties in vivo by examining RGC survival and axon regeneration at 4 weeks following intraorbital optic nerve transection in adult rat. EPO was administered as a single intravitreal injection at the time of transection (5, 10, 25, 50 units, PBS control). Intravitreal EPO (5, 10 units) significantly increased RGC somata and axon survival between the eye and transection site. Twenty five units did not improve survival of RGC somata but did increase axon survival between the eye and transection site. In addition, a small proportion of axons penetrated the transection site and regenerated up to 1 mm into the distal nerve. In a second series, intravitreal EPO (25 units) doubled the number of RGC axons regenerating along a length of peripheral nerve grafted onto the retrobulbar optic nerve. Our in vivo evidence of both neuroregeneration and neuroprotection, taken together with the natural occurrence of EPO within the body and its ability to cross the blood-brain barrier, suggests that it offers promise as a therapeutic agent for central nerve repair.     

Lens injury stimulates adult mouse retinal ganglion cell axon regeneration via both macrophage- and lens-derived factors

In the present study the effects of lens injury on retinal ganglion cell axon/neurite re-growth were investigated in adult mice. In vivo, lens injury promoted successful regeneration of retinal ganglion cell axons past the optic nerve lesion site, concomitant with the invasion of macrophages into the eye and the presence of activated retinal astrocytes/Muller cells. In vitro, retinal ganglion cells from lens-lesioned mice grew significantly longer neurites than those from intact mice, which correlated with the presence of enhanced numbers of activated retinal astrocytes/Muller cells. Co-culture of retinal ganglion cells from intact mice with macrophage-rich lesioned lens/vitreous body led to increased neurite lengths compared with co-culture with macrophage-free intact lens/vitreous body, pointing to a neurotrophic effect of macrophages. Furthermore, retinal ganglion cells from mice that had no lens injury but had received intravitreal Zymosan injections to stimulate macrophage invasion into the eye grew significantly longer neurites compared with controls, as did retinal ganglion cells from intact mice co-cultured with macrophage-rich vitreous body from Zymosan-treated mice. The intact lens, but not the intact vitreous body, exerted a neurotrophic effect on retinal ganglion cell neurite outgrowth, suggesting that lens-derived neurotrophic factor(s) conspire with those derived from macrophages in lens injury-stimulated axon regeneration. Together, these results show that lens injury promotes retinal ganglion cell axon regeneration/neurite outgrowth in adult mice, an observation with important implications for axon regeneration studies in transgenic mouse models.     

Basic fibroblast growth factor applied to the optic nerve after injury increases long-term cell survival in the frog retina

The neuroprotective effects of basic fibroblast growth factor (bFGF) on the long-term survival of axotomized retinal ganglion cells (RGCs) were studied in the frog Rana pipiens. Cell loss was quantified in different regions of the ganglion cell layer using Nissl staining and tetramethylrhodamine dextran amine backfilling. All regions of the retina showed a significant decrease (32-66%) in RGC numbers between 4 and 16 weeks after axotomy. Some cells showed morphological and biochemical signs of apoptosis. A single application of bFGF to the optic nerve stump at the time of axotomy protected many of the cells 6 weeks after the injury, but this effect was lost by 12 weeks. A second application of bFGF, 6 weeks after the injury, rescued many RGCs at 12 weeks. In contrast, single or double injections of bFGF into the eyeball had no effect on RGC survival. Axotomized RGCs were significantly enlarged and elongated after axotomy, and these morphological changes were increased by bFGF treatment. In the normal retina and optic nerve, immunocytochemical staining showed bFGF-like immunoreactivity (-LI) in the pigment epithelial layer, in the outer segments of photoreceptors, and in occasional RGCs. Strong bFGF-LI was present in Müller cells and in optic nerve astrocytes and oligodendrocytes. FGF receptor-LI was present in photoreceptors, outer plexiform layer, retinal ganglion cell axons, and Müller cells. FGF receptor-LI was also observed in optic nerve glia.     

FGF-2 modulates expression and distribution of GAP-43 in frog retinal ganglion cells after optic nerve injury

Basic fibroblast growth factor (bFGF or FGF-2) has been implicated as a trophic factor that promotes survival and neurite outgrowth of neurons. We found previously that application of FGF-2 to the proximal stump of the injured axon increases retinal ganglion cell (RGC) survival. We determine here the effect of FGF-2 on expression of the axonal growth-associated phosphoprotein (GAP)-43 in retinal ganglion cells and tectum of Rana pipiens during regeneration of the optic nerve. In control retinas, GAP-43 protein was found in the optic fiber layer and in optic nerve; mRNA levels were low. After axotomy, mRNA levels increased sevenfold and GAP-43 protein was significantly increased. GAP-43 was localized in retinal axons and in a subset of RGC cell bodies and dendrites. This upregulation of GAP-43 was sustained through the period in which retinal axons reconnect with their target in the tectum. FGF-2 application to the injured nerve, but not to the eyeball, increased GAP-43 mRNA in the retina but decreased GAP-43 protein levels and decreased the number of immunopositive cell bodies. In the tectum, no treatment affected GAP-43 mRNA but FGF-2 application to the axotomized optic nerve increased GAP-43 protein in regenerating retinal projections. We conclude that FGF-2 upregulates the synthesis and alters the distribution of the axonal growth-promoting protein GAP-43, suggesting that it may enhance axonal regrowth.     

Induction and axonal localization of epithelial/epidermal fatty acid-binding protein in retinal ganglion cells are associated with axon development and regeneration.

Epithelial/epidermal fatty acid-binding protein (E-FABP) is induced in peripheral neurons during nerve regeneration and is found at high levels in central neurons during neuronal migration and development. Furthermore, E-FABP expression is required for normal neurite outgrowth in PC12 cells treated with nerve growth factor (NGF). The present study examined whether E-FABP plays a role in retinal ganglion cell (RGC) differentiation and axon growth. Rat retinal tissues from embryonic (E) and postnatal (P) development through adulthood were examined using immunocytochemical labeling with E-FABP and growth-associated protein 43 (GAP-43) antibodies. E-FABP colocalized with GAP-43 at E14 through P10. At E14, E-FABP immunoreactivity was confined to the somas of GAP-43-positive cells in the ganglion cell layer, but it was localized to their axons by E15. The axons in the optic nerve were GAP-43-positive and E-FABP-negative on E15, but the two proteins were colocalized by E18. Retinal cultures at E15 confirmed that E-FABP and GAP-43 colocalize in RGCs. Postnatally, labeling was present between P1 and P10 but decreased at older ages and was minimally present or absent in adult animals. Western immunoblotting revealed that at E18, P1, and P10 E-FABP levels were at least fourfold greater than those in the adult. By P15, protein levels were only twofold greater, with adult levels reached by P31. Furthermore, E-FABP could be reinduced during axon regeneration. Dissociated P15 retinal cells cultured in the presence of brain-derived neurotrophic factor, ciliary neurotrophic factor, and basic fibroblast growth factor exhibited sixfold more GAP-43 and E-FABP double-positive RGCs (cell body and axons) than controls. Moreover, all GAP-43-immunoreactive RGCs were also positive for E-FABP. Taken together, these results indicate the following: 1) E-FABP is expressed in RGCs as they reached the ganglion cell layer and 2) E-FABP plays a functional role in the elaboration of RGC axons in both development and regeneration.     

Growth stimulation and chemotropic attraction of rat retinal ganglion cell axons in vitro by co-cultured optic nerves, astrocytes and astrocyte conditioned medium

The effects of explants of optic nerves of different ontogenetic ages (P0-P14, adult), and of cultured astrocytes of various ages on the neurite regeneration of rat retinal ganglion cells (RGC) were assessed in vitro, using a three-dimensional culture system which allows the co-cultivation of various explants. Both co-cultured P0-P12 optic nerves and astrocyte cultures from P2 cerebral cortex stimulated the regeneration of neurites from the retinal explants after 3 days in culture. By contrast, P14 and older explants of the optic nerve, astrocytes from P17 optic nerve and astrocytes that had previously been grown in culture for more than 6 weeks had no effect on RGC neurite outgrowth. Moreover, both the P0–P12 optic nerve explants and the astrocytes from P2 cerebral cortex also seemed to have a chemotropic effect on the regenerating neurites, because the latter were longer on the side facing the co-explantat. The absence of a cellular bridge between retinal and optic nerve explants suggests that the effects are mediated by astroglia-derived diffusible neurite growth promoting factors. Accordingly, astrocyte-conditioned medium from P2 astrocytes also stimulated the outgrowth of neurites from the retinal explants. These findings show that immature astrocytes of a limited ontogenetic period release as yet unknown diffusible neurite growth-promoting factors which stimulate the regeneration of neurites from retinal explants.     

Retinal axon regeneration in peripheral nerve, tectal, and muscle grafts in adult rats.

This study examined whether prior regenerative growth through peripheral nerve (PN) bridging grafts influenced the specificity with which lesioned adult rat retinal ganglion cell (RGC) axons grew into co-grafts of developing target tissue (fetal superior colliculus). Growth into nontarget (muscle) tissue was also examined. Autologous PN was grafted onto the transected optic nerve. After 14 days, the distal ends of the PNs were placed next to, or inserted into, embryonic tectal tissue or into autologous muscle grafts placed in frontal cortex cavities. Host retinal projections were examined 3-8 months later using anterograde and retrograde tracing techniques. In rats in which there was good apposition between PN and tectal tissue, small numbers of RGC axons were observed growing into the tectal grafts (maximum distance of 180 microm). No evidence of specific innervation of appropriate target regions within tectal grafts was detected, even though such regions (identified by acetylcholinesterase histochemistry) were often located close to the PN grafts. In rats with PN/muscle co-grafts, the extent of retinal axon outgrowth was greater (up to 465 microm from the PN tip) and labelled profiles that resembled motor endplates were seen contacting muscle fibres. Previous studies have shown that spontaneously regenerating RGC axons consistently and selectively innervate appropriate target areas in fetal tectal tissue grafted directly into optic tract lesion cavities. Together, the data suggest that exposure to a PN environment may have reduced the extent of adult retinal axon growth into fetal tectal transplants and affected the way regenerating axons responded to specific developmental cues expressed by target cells in the co-grafted tissue.     

Precrushed sciatic nerve grafts enhance the survival and axonal regrowth of retinal ganglion cells in adult rats.

We have examined the influence of normal and precrushed ("conditioned") sciatic nerve grafts on the survival and axonal growth of retinal ganglion cells (RGCs) in adult rats. Normal sciatic nerves (group A) or sciatic nerves which had been crushed 1 week before transplantation (group B, conditioned grafts) were used as grafts. The nerves were removed and sutured to the proximal stump of intraorbitally axotomized optic nerves. Neuronal survival and axon growth were determined by counting the numbers of surviving, DiI-prelabled RGCs, cresyl violet-stained RGCs and the numbers of axons which had grown into the grafts 3 and 6 months after transplantation. Counting of axons was performed by combined use of light and electron microscopy. We observed that the use of conditioned grafts (group B) significantly enhanced RGC survival and axonal regrowth as compared to normal grafts 3 months after transplantation. Six months after grafting, RGC survival (as determined in DiI-stained retinae) and axonal growth were not significantly different in both groups. These results suggest that the functional status of a peripheral nerve used for grafting in the CNS influences neuronal viability and axonal reelongation especially during the first 3 months after grafting. Very long-term RGC survival, however, may be determined by functional reconnection of regenerating RGC axons rather than by the graft itself.     

Developmental changes in the fibre population of the optic nerve follow an avian/mammalian-like pattern in the turtle Mauremys leprosa

The changes in the axon and growth cone numbers in the optic nerve of the freshwater turtle Mauremys leprosa were studied by electron microscopy from the embryonic day 14 (E14) to E80, when the animals normally hatch, and from the first postnatal day (P0) to adulthood (5 years on). At E16, the first axons appeared in the optic nerve and were added slowly until E21. From E21, the fibre number increased rapidly, peaking at E34 (570,000 fibres). Thereafter, the axon number decreased sharply, and from E47 declined steadily until reaching the mature number (about 330,000). These observations indicated that during development of the retina there was an overproduction and later elimination of retinal ganglion cells. Growth cones were first observed in the optic nerve at as early as E16. Their number increased rapidly until E21 and continued to be high through E23 and E26. After E26, the number declined steeply and by E40 the optic nerve was devoid of growth cones. These results indicated that differentiation of the retinal ganglion cells occurred during the first half of the embryonic life. To examine the correlation between the loss of the fibres from the optic nerve and loss of the parent retinal ganglion cells, retinal sections were processed with the TUNEL technique. Apoptotic nuclei were detected in the ganglion cell layer throughout the period of loss of the optic fibres. Our results showed that the time course of the numbers of the fibres in the developing turtle optic nerve was similar to those found in birds and mammals.     

Change in phospholipid species of retinal layer in traumatic optic neuropathy model

Injured optic nerves induce death in almost all retinal ganglion cells (RGC) and cause a loss of axons. To date, we have studied injured RGC axon regeneration by using a traumatic optic nerve injury (TONI) rodent model, and we revealed that axonal regeneration is induced by the graft of an autologous peripheral nerve. The efficient approach to the regeneration of axons thus needs an environmental adjustment of RGC. However, the RGC environment induced by TONI remains unknown. Here, we analyzed female and male C57BL/6 mouse retinal tissue alterations in detail after TONI and focused on the major phospholipid species that are enriched in the whole retina. Reactive astrocyte accumulation, glia scar formation, and demyelination were observed in the injured optic nerve area, while RGC cell death, astrocyte accumulation, and Glial fibrillary acidic protein (GFAP) positive Müller cell increases were detected in the retinal layer. Furthermore, phosphatidylinositol (PI) 18:0/20:4 was localized to three nuclear layer structures: the ganglion cell layer (GCL), the inner nuclear layer (INL), and the outer nuclear layer (ONL) in control retina; however, the localization of 18:0/20:4 PI in TONI was disturbed. Meanwhile, phosphatidylserine (PS) 18:0/22:6 showed that the expression was specifically in the inner plexiform layer (IPL) with similar signal intensity in both cases. Other PS species and phosphatidylethanolamine (PE) were differentially localized in the retinal layer; however, the expressions of PE including docosahexaenoic acid (DHA) were affected by TONI. These results suggest that not only GCL but also other retinal layers were influenced by TONI.     

Optic nerve and vitreal inflammation are both RGC neuroprotective but only the latter is RGC axogenic

Intravitreal inflammation, induced by either lens injury, or intravitreal injection of zymosan (IVZ), protects RGC from apoptosis and stimulates axon regeneration after optic nerve transection. Here, we investigate the differential effects of intra-optic nerve zymosan (ONZ) and IVZ injections on RGC neuroprotection and axogenesis. After both IVZ and ONZ injection, zymosan-induced inflammation promoted a similar 4-/5-fold enhancement in RGC survival, compared to optic nerve transected controls, but only IVZ promoted RGC axon regeneration. IVZ was the most effective in activating retinal astrocyte/Müller cells while regulated intramembraneous proteolysis (RIP) of p75^NTR and inactivation of Rho (key components of the axon growth inhibitory signalling cascade) occurred in both ONZ and IVZ, but only in the latter did RGC axons regenerate. We suggest that neuroprotective factors may be transported to RGC somata by retrograde transport after ONZ and diffuse into the retina after IVZ injection, but an axogenic agent is required to initiate and maintain disinhibited RGC axon regeneration that may be an exclusive property of a Müller cell-derived factor released after IVZ.     

Neuritogenesis of retinal ganglion cells is differentially promoted by target extract

Labeled retinal ganglion cells from neonatal rats extended neurites in dissociated cell culture as a cell type-specific response to ihe influence of a superior collicular extract. The molecule responsible for this neuritogenic effect is soluble and non-dialysable (> 12kDa). Nerve growth factor had a neuritogenic effect both on ganglion cells and other types of retinal cells.     

Estrogen has a neuroprotective effect on axotomized RGCs through ERK signal transduction pathway

The neuroprotective effects of estrogen on neuronal cells in central nervous system have been described previously, however, the mechanisms of neuroprotective effect of estrogen against retinal ganglion cell (RGC) death has not been well identified. To examine the role of endogenous sex steroids produced in ovary, retina samples were prepared from female rats with or without ovariectomy and the density of RGC was calculated. Ovariectomy alone had no effect on the density of fluorogold (FG)-labeled RGC without injury, while the density of surviving RGC after optic nerve axotomy with ovariectomy was significantly decreased compared to that without ovariectomy. To examine the role of exogenous sex steroids, 17beta-estradiol was injected into the vitreous cavity in ovariectomized rats and showed neuroprotective effect on axotomy-induced RGC death while exogenous progesterone showed no effect. Immunoblot and immunohistochemical analysis demonstrated that ERK-c-Fos signal transduction pathway was activated by exogenous 17beta-estradiol in ganglion cell layer. U0126, an ERK inhibitor, inhibited the neuroprotective effect of estrogen on axotomized RGC death. These data suggest that estrogen has neuroprotective effect through activation of ERK-c-Fos signaling pathway on axotomy-induced RGC death. The neuroprotective effect of estrogen may have therapeutic benefits in retinal diseases associated with RGC death such as glaucoma.     

The organization of the fibers in the optic nerve of normal and tectum-less Rana pipiens

We have examined the detailed order of retinal ganglion cell (RGC) axons in the optic nerve and tract of the frog, Ranapipiens. By using horseradish peroxidase (HRP) injections into small regions of theretina, the tectum, and at various points along the visual pathway, it hasbeen possible to follow labelled fibers throughout their course in the nerve and tract. Several surprising features in the order of fibers in the visual pathway were discovered in our investigation. The fascicular pattern of RGC axons in che retina is similar to that described in other vertebrates; however, immediately central to their entry into the optic nerve head, approximately half of the fibers from the nasal or temporal retina cross over to the opposite side of the nerve. Although the axons from the dorsal and ventral regions of the retina generally remain in the dorsal and ventral regions of the nerve, some fiber crossing occurs in those axons as well. The result of this seemingly complex rearrangement is that the optic nerve of Rana pipiens contains mirror symmetric representations of the retinal surface on either side of the dorsal ventral midline of the nerve. The fibers in each of these representation are arranged as semicircles representing the full circumference of the retina. This precise fiber order is preserved in the nerve until immediately periphearal to the optic chiasm, at which point age-related axon from both side of the nerve bundle together. Consequently, when a small pellet of HRP is placed in the chiasmic region of the nerve, an annualus of retinal ganglion cells and a corresponding annulus of RGC terminals in the tectum are la belled. As the age-related bundles of fibers emerge from the chiasm they split to form a medial bundle and a lateral bundle, which grow in the medial and lateral branches of the optic tract, respectively. Although the course followed by RGC axons in the visual pathv/ay is complex, we propose a model in which the organization of fibers in the nerve and tract can arise from a few rules of axon guidance. To determine whether the optic tecta, the primary retinal targets, play a role in the development and organization of the optic nerve and tract, we removed the tectal primordia in Rana embryos and examined the order in the nerve when the animals had reached larval stages. We found that the order in the nerve and tract was well preserved in tectumless frogs. Therefore, we propose that guidance factors independent of the target direct axon growth in the frog visual system.     

Foxn4 is required for retinal ganglion cell distal axon patterning

The regulation of retinal ganglion cell (RGC) axon growth and patterning in vivo is thought to be largely dependent on interactions with visual pathway and target cells. Here we address the hypothesis that amacrine cells, RGCs' presynaptic partners, regulate RGC axon growth or targeting. We asked whether amacrine cells play a role in RGC axon growth in vivo using Foxn4(-/-) mice, which have fewer amacrine cells, but a normal complement of RGCs. We found that Foxn4(-/-) mice have a similar reduction in most subtypes of amacrine cells examined. Remarkably, spontaneous retinal waves were not affected by the reduction of amacrine cells in the Foxn4(-/-) mice. There was, however, a developmental delay in the distribution of RGC projections to the superior colliculus. Furthermore, RGC axons failed to penetrate into the retinorecipient layers in the Foxn4(-/-) mice. Foxn4 is not expressed by RGCs and was not detectable in the superior colliculus itself. These findings suggest that amacrine cells are critical for proper RGC axon growth in vivo, and support the hypothesis that the amacrine cell-RGC interaction may contribute to the regulation of distal projections and axon patterning.