Fibroblast Growth Factor Receptor Function Is Required for the Orderly Projection of Ganglion Cell Axons in the Developing Mammalian Retina

MCN

Molecular and Cellular Neuroscience 8, 120–128 (1996) Article No. 0051

Fibroblast Growth Factor Receptor Function Is Required for the Orderly Projection of Ganglion Cell Axons in the Developing Mammalian Retina Perry A. Brittis, Jerry Silver,* Frank S. Walsh, and Patrick Doherty1 Department of Experimental Pathology UMDS, Guy’s Hospital, London Bridge, London SE1 9RT, United Kingdom; and *Department of Neurosciences, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106

During the early stages of development various cell adhesion molecules (CAMs) and fibroblast growth factor receptors (FGFR) are expressed throughout the retinal neuroepithelium. The ability of retinal ganglion cells to project their axons to the optic fissure depends, in part, on cell–cell interactions mediated by cell adhesion molecules. In the present study we show that the ability of the firstborn rat retinal ganglion cells to extend axons in vitro can be stimulated by NCAM and L1, but not N-cadherin. Both CAM responses can be fully inhibited by antibodies that block neuronal fibroblast growth factor receptor function and by agents that block defined steps in the FGFR signal transduction cascade. When added to living E13.5 rat retinal whole-mount preparations the same agents induced errors in the orderly establishment of young axon patterns in the retinal periphery and caused axons in the retinal center to defasciculate. These results suggest that the activation of the fibroblast growth factor receptor signal cascade not only promotes survival and proliferation of various cell types but can also mediate intraretinal axon guidance.

INTRODUCTION The orderly projection of retinal ganglion cell (RGC) axons toward the optic fissure is an exquisite example of the precise and stereotyped nature of initial axonal growth in the developing mammalian central nervous system. In the rat, the first axons to exit the retina at around E13.0 belong to the ganglion cells that differentiate in close proximity to the optic fissure and these ‘‘pioneer’’ axons grow in close association with the pial 1 To whom correspondence should be addressed. Fax: 0171 403 8883. http://gramercy.ios.com:80/,pab9/.

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endfeet of the surrounding neuroepithelial cells (e.g., see Rager and von Oeynhausen, 1979; Krayanek and Goldberg, 1981; Silver and Sapiro, 1981). The remainder of the RGCs differentiate over a 3- to 4-day period in a synchronous progression from the center to the retinal periphery (Goldberg et al., 1972; Silver and Sidman, 1980; Halfter et al., 1985) and the orderly exit of axons belonging to these cells may depend upon positive guidance cues present on the endfeet of the neuroepithelial cells and/or the axons that have developed immediately ahead of them (e.g., see Rager and von Oeynhausen, 1979; Silver and Sapiro, 1981; Silver and Rutishauser, 1984; Halfter et al., 1987; Williams et al., 1991; Halfter, 1996). These cues may also act in concert with repulsive cues that prevent the RGC axons from projecting into inappropriate territories (Snow et al., 1991; Brittis et al., 1992; Brittis and Silver, 1995). A number of observations suggest that cell adhesion molecules (CAMs) are good candidates for the positive cues that promote and/or guide axons out of the developing retina. Evidence that CAMs can stimulate axonal growth has been obtained by culturing these neurons on monolayers of control or transfected fibroblasts that express physiological levels of N-cadherin (Matsunaga et al., 1988) or neural cell adhesion molecule (NCAM) (Doherty et al., 1990a, 1991). Furthermore, studies with explants of chick retina have shown that antibodies to NCAM, N-cadherin, and L1, but not antibodies to integrins, inhibit RGC axonal growth on Muller cells (Drazba and Lemmon, 1990). CAMs can also stimulate rodent RGC axon growth in culture (Hankin and Lagenauer, 1994) and antibodies to NCAM and L1 can alter axonal growth and guidance in the developing rat retina (Brittis et al., 1995). 1044-7431/96 $18.00 Copyright r 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.

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We have recently shown that the ability of rat cerebellar granule cells to extend neurites in response to NCAM, N-cadherin, and L1 can be inhibited by antibodies that block the function of neuronal fibroblast growth factor receptors (FGFRs) and by agents that block the FGFR signal transduction cascade (Williams et al., 1994a). This and other evidence has led us to propose that all three CAMs promote axonal growth by directly or indirectly activating neuronal FGFRs (see Saffell et al., 1994; Williams et al., 1994a; Doherty et al., 1995; Hall et al., 1996a). A role for FGFs and, by implication, FGFRs on RGCs in the developing retina has been suggested by a number of groups. For example, there is evidence that FGFs can regulate the differentiation of retinal ganglion progenitor cells (Park and Hollenberg, 1989; Pittack et al., 1991; Guillemot and Cepko, 1992). Furthermore, during the early stages of development, the FGFR is expressed throughout the retinal neuroepithelium (Cirillo et al., 1990; Heuer et al., 1990; Wanaka et al., 1991) and immunoreactivity for acidic and basic FGF is found associated with the first rat RGCs that develop at around E13.0–14.0 (de Iongh and McAvoy, 1993). FGFs are also known to promote the survival and morphological differentiation of a wide range of neuronal cell types (e.g., see Hatten et al., 1988; Walicke, 1988; Morrison et al., 1986; Hughes et al., 1993; Williams et al., 1994b). In the present study we have addressed three questions. By culturing E13.5 RGCs on monolayers of control and transfected fibroblasts we have determined if NCAM, N-cadherin, and L1 can stimulate growth of their axons. We then determined if CAM-stimulated axonal growth can be inhibited by agents that block neuronal FGFR function. Finally, we have determined whether agents that block FGFR function can perturb the establishment of the orderly axonal projection pattern in the developing retina.

RESULTS AND DISCUSSION NCAM and L1, but Not N-cadherin, Stimulate Axonal Growth from E13.5 RGCs Dissociated rat RGCs from E13.5 retina were cultured on top of monolayers of parental 3T3 cells or transfected 3T3 cells which express physiological levels of NCAM, N-cadherin, or L1 (for details of monolayer cells see Doherty et al., 1990a, 1991; Williams et al., 1992). After 13 h the cocultures were fixed and the length of the primary axons on the RGCs were determined. There was a robust neurite outgrowth response over the parental 3T3 cell monolayers, but axons were almost twice as long on monolayers expressing NCAM or L1

(Fig. 1). In contrast, axonal growth over N-cadherinexpressing cells was only slightly higher than the control basal response. Previous studies have reported that N-cadherin stimulates neurite outgrowth from RGCs (Matsunaga et al., 1988; Doherty et al., 1990b, 1991). However, these studies used populations of developmentally older chick RGCs and one study noted an increased responsiveness to N-cadherin with increasing developmental age (Doherty et al., 1991). In a similar vein, neurons isolated from the rat hippocampus at E17.0 do not respond to N-cadherin, whereas those isolated at PND4 do (Doherty et al., 1992). Thus it would appear that NCAM and L1, but not N-cadherin, can stimulate axonal growth from the first cohort of differentiating RGCs isolated from the developing rat retina.

Axonal Growth Stimulated by NCAM and L1 Is Inhibited by Agents That Block FGFR Function Neurite outgrowth stimulated by basic FGF can be inhibited by antibodies that directly perturb FGFR function, or by a number of pharmacological agents that block identified steps in the downstream signal transduction cascade leading to the axonal growth response (Williams et al., 1994b, 1995a). All of the pharmacological agents that block FGF-stimulated neurite outgrowth also inhibit neurite outgrowth stimulated by NCAM, N-cadherin, and L1 (Williams et al., 1994a, 1995a) and there is some evidence that following homophilic binding, CAMs can either directly or indirectly activate neuronal FGFRs (Williams et al., 1994a; Doherty et al., 1995). All of this work has been done with postnatal cerebellar granule cells, which, on average, extend a neurite of only 60 µm over a 16- to 20-h period of culture on CAM-expressing cells. It was therefore important to determine if CAM-stimulated neurite outgrowth from long projection neurons also requires FGFR function. Fibroblast growth factor receptors contain a stretch of 20 amino acids called the cell adhesion molecule homology domain (CHD) which shows identity with amino acid sequences found in L1, NCAM, and N-cadherin (Williams, 1994a). When an antisera against the FGFR-CHD was applied to cultured-dissociated E13.5 rat retinal ganglion cells, immunoreactivity was predominantly concentrated on the cell bodies and growth cones (Fig. 1A). In the present study we found that basal RGC neurite outgrowth over 3T3 fibroblasts is not inhibited by antibodies that block FGFR function, or by three pharmacological perturbations that block independent steps in the signal transduction cascade that lie downstream from activation of the FGFR (Table 1). These agents include RHC-80267 (50 µM) which is a diacylglycerol

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FIG. 1. (A) An E13.5 retinal ganglion cell grown in culture over a laminin substrate stained with antibodies to the FGFR-CHD. FGFR expression was predominantly localized to the cell body and growth cone (arrow). (B) Growth of E13.5 retinal ganglion cell axons over parental and transfected 3T3 cells. The FGFR-CHD antibody, and various pharmacological reagents that specifically block the FGFR signal cascade, inhibit the axon’s response to L1 and NCAM. The results show the mean axon length (mean 6 SEM) pooled from three independent experiments. Individual values in each experiment were obtained from 100 to 150 neurons. All of the above agents are nontoxic (Williams et al., 1994a,b,c) and, thus, had no effect on basal axon outgrowth. *, The NCAD response was statistically significant but not substantial at this early age. BPB is 4-bromophenadyl bromide.

lipase inhibitor, a combination of v-conotoxin (0.25 µM) and diltiazem (10 µM) which antagonize N- and L-type calcium channels, and KN-62 (10 µM) which blocks the activity of the calcium/calmodulin-dependent kinase in neurons. We can therefore conclude that in this coculture system FGFR function is not required for the

TABLE 1 The Effects of a Variety of Agents on Axonal Growth over 3T3 Monolayers Agent

Axonal growth (µm)

Control Anti-FGFR antibody (1:200) RHC-80267 (50 µM) KN-62 (10 µM) BPB (50 µM) v´ -Conotoxin/diltiazem (0.25/10 µM)

74.4 6 1.8 65.7 6 7.9 74.5 6 3.5 74.1 6 1.1 77.7 6 3.4 67.2 6 1.8

Note. E13.5 RGCs were grown over confluent monolayers of parental 3T3 fibroblasts in control media or media supplemented with a variety of agents as indicated. After 13 h the cultures were fixed and immunostained for GAP43, and the length of RGC axons was determined. The results show the mean axon length (6SEM) pooled from three independent experiments. Measurements were made on 100–150 neurons in each experiment. BPB is 4-bromophenacyl bromide.

survival of the RGCs, nor does it contribute to the basal axonal growth response. By implication, it would appear that the cultures do not contain functional levels of endogenous FGFs. These experiments also clearly show that the antibodies to the FGFR and the various pharmacological reagents have no nonspecific effects on axonal growth. In contrast to their lack of effects on neurite outgrowth over parental 3T3 cells, which is likely to be attributable, in part, to integrin receptors on the neurons (Reichardt and Tomaselli, 1991; Doherty et al., 1990b; Williams et al., 1994c), the FGFR antibody and the three pharmacological perturbations completely inhibited the NCAM and L1 component of the axonal growth response (Fig. 1B). We have shown that these agents all inhibit neurite outgrowth over a purified L1–Fc chimeric protein (Williams et al., 1995b), consistent with a neuronal site of action in the present study. Activation of the FGFR leads to the production of arachidonic acid via the diacylglycerol lipase pathway and this is inhibited by RHC-80267 (Williams et al., 1995b). Arachidonic acid stimulates neurite outgrowth and this requires calcium influx into neurons and activation of the calcium/ calmodulin-dependent kinase. This response can be inhibited by a combination of v-conotoxin and diltiazem, or by KN-62 (Williams et al., 1994b, 1995a). As an

FGF Receptor Function in Developing Retina

additional control we found that 4-bromophenacyl bromide, which can inhibit neurite outgrowth stimulated by agents that activate phospholipase A2 (Williams et al., 1994b), had no effect on basal neurite outgrowth (Table 1) or the CAM responses (Fig. 1B). The fact that the CAM responses are inhibited by antibodies that bind to the neuronal cell body and growth cone (Fig. 1A) and specifically inhibit FGFR function, together with three agents that block independent steps in the FGFR signal transduction cascade, suggests that the CAMs are again operating via activation of this second messenger pathway in retinal ganglion cells.

Axonal Projection Patterns in the Developing Retina Are Perturbed by Agents That Block FGFR Function Within the retina at E13.5, ganglion cells with axons appear just dorsal to the optic fissure. Thereafter, axons emerge from ganglion cell bodies located progressively more peripherally and these axons grow in a very precise manner to the optic fissure (e.g., see Fig. 2A). In an effort to test whether perturbation of the FGFR and its signal cascade could also affect axonal growth within a native environment, E13.5 retinas were dissected, and whole-mounted preparations were prepared. Groups of retinas were allowed to develop in control media or media supplemented with the anti-FGFR antibody, RHC-

123 80267 (50 µM), KN-62 (10 µM), or 4-bromophenacyl bromide (50 µM). After 7 or 13 h the preparations were fixed and the ganglion cell axons were labeled with either the neuron-specific TUJ1 monoclonal antibody or the membrane label DiI. In control preparations axons consistently grew in the vitreal margin, but only in a restricted path leading to the optic fissure (Fig. 2A). In the retinal periphery, the region of newly recruited neurons, there were no ectopically positioned axons. In contrast, in preparations grown in the presence of the FGFR antibody, RHC-80267, or KN-62, newly generated axons in the retinal periphery displayed gross guidance abnormalities (Fig. 2B; Table 2). After 7 h these ectopically located axons extended in the wrong direction toward the retinal periphery until they eventually made smooth turns and grew more laterally (Figs. 3B and 3C). After 13 h of treatment, the axon pattern became more disrupted as axons continued to grow in the wrong direction but were less likely to turn away from the periphery (Fig. 2B). When filled with DiI, the majority of ectopic axons at both time periods contained large, flattened growth cones at their tips (Fig. 3C, inset). In the central portion of the retina, large DiI-labeled growth cones were also found on top of other axons (Fig. 3D). In the area of the optic fissure, the thin bundle of fasciculating axons found in control preparations was greatly expanded (Fig. 2B). Thus, many of the axons failed to

FIG. 2. Axon guidance in TUJ1-labeled retinal wholemounts. (A) E13.5 whole-mounted retina grown in control media (anti-human IgG, 1:200) for 13 h. Notice the highly orchestrated pattern of retinal ganglion cell differentiation and the tightly knit fascicles of axons at the optic fissure (bottom of retina). (B) At E13.5, 13 h of FGFR-CHD antibody treatment (1:200) caused newly added axons in the periphery to grow ectopically (top arrow), away from the optic fissure. As the axons defasiculated in the center of the retina (asterisk), many of them could no longer exit the retina (bottom arrow). Scale bar represents 25 µm.

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TABLE 2 Quantification of Severely Misrouted Axons in the Retinal Periphery Agent Control Anti-FGFR antibody (1:100) BPB (50 µM) RHC-80267 (50 µM) KN-62 (10 µM)

1

2

3

4

5

0

2

1

0

1

12

8

16

10

7

1

0

2

2

1

13

15

11

9

12

9

16

12

14

11

Note. E13.5 whole-mounted retinas were grown for 7 h in control media or in the presence of the above agents as indicated. Axons that had clearly grown away from the optic fissure into the retinal periphery (the checkered box region and see Figs. 3B and 3C for examples) were counted in a total of five preparations for individual experiments. Similar results were obtained in at least three independent experiments for each age. BPB is 4-bromophenacyl bromide.

FIG. 3. E13.5 intraretinal axon perturbation. (A) The corresponding magnified viewing area of the retinal neuroepithelium. In the retinal periphery, retinal whole mounts were incubated for 7 h (B) in the presence of anti-FGFR-CHD antibodies (1:200) or (C) in the diacylglycerol lipase inhibitor RHC-80267 (50 µM) and immunostained with MAb TUJ1. Both treatments caused identical deficits in axonal guidance. Axons were no longer directed toward the optic fissure but rather grew toward the retinal periphery (arrows in B and C). Notice how some of the perturbed axons curved laterally as they entered into more peripheral territories (arrows in B). Misguided axons in this region had large DiI-labeled growth cones (inset in C). In the retinal periphery, the anti-FGFR-CHD treatment most likely prevented newly differentiated ganglion cell axons from making stable L1–L1 contacts with the axons ahead of them. (D) DiI-labeled growth cones (arrows) in the retinal center after anti-FGFR-CHD perturbation. Notice how these growth cones in the center of the retina became large and flattened but, after 7 h of perturbation, did not change their direction of growth. Scale bar in (D) represents 20 µm; in (B and C) 10 µm.

FGF Receptor Function in Developing Retina

properly exit the fissure and began to grow up the sides of the neuroepithelium. These data demonstrate that axonal guidance within the retina can be perturbed by antibodies that directly block FGFR function or by two pharmacological agents that block defined steps in the FGFR signal transduction cascade. Other agents that block the signal transduction cascade, such as calcium channel antagonists, were also tested in this paradigm but had clear toxic effects toward the preparation. The fact that the diacylglycerol lipase and calcium/calmodulin kinase inhibitors induced the same effect as the antibodies supports the argument that the antibodies are working in a specific, rather than a nonspecific, manner such as generally perturbing recognition events as a consequence of steric hindrance. It is also of interest that the phospholipase A2 inhibitor had no effect, suggesting that this signaling pathway does not contribute to intraretinal axonal guidance. Furthermore it is clear that the effects of the antibodies and drugs were primarily on guidance of the axons, rather than the growth of the axons, and this argues against these agents having nonspecific effects on axonal growth. Indeed, in the retinal periphery, misrouted axons grew for long distances until they were shunted away from the repulsive environment (for example, see Fig. 3B). This ectopic guidance phenomenon was very similar to the results obtained from retinal whole-mount time-lapse experiments in which anti-L1 antiserum was added to the growth medium (Brittis et al., 1995). Interestingly in the present study, after 13 h the growth cones no longer made direct turns as they grew toward the periphery. A repulsive environment which recedes in a uniform fashion across the retinal neuroepithelium has been described (Snow et al., 1991; Brittis et al., 1992; Brittis and Silver, 1995). It is possible that the receding wave of repulsion, responsible for crudely directing the growth cones toward the center, was no longer present or active in this area after 13 h and that the misrouted axons abandoned CAM pathways and grew over other non-FGFR-dependent permissive substrates such as laminin present within the neuroepithelium (Halfter, 1996). Indeed, recent studies have suggested that retinal ganglion cells show a slight preference for growth on cell adhesion molecules such as L1 (Hankin and Lagenauer, 1994) relative to matrix molecules such as laminin (Burden-Gulley et al., 1995). In accordance with this hypothesis, previous time-lapse studies of L1 antibody-perturbed growth cones in retinal whole mounts revealed a time period in which the growth cones became large, stalled for a few hours, became streamlined, made sharp course changes, and then preceded to cross the neuroepithelium at twice

125 the original growth rate (Brittis et al., 1995). In this context, the morphology of a growth cone can be quite distinct depending on the nature of the substrate (Lemmon et al., 1992; Burden-Gulley, 1995). In the present study, the perturbed growth cones which were located both in the center and at the periphery of the retina were much larger than control growth cones (Fig. 3D), a phenomenon also observed in previous whole-mount CAM perturbations (Brittis et al., 1995). However, the percentage of affected growth cones in both the retinal center and the periphery could not be calculated without time-lapse videomicroscopy. Last, it is important to note that the agents that perturbed the FGFR pathway in this present study had no effect on the ability of the RGCs to extend axons over 3T3 monolayers (Fig. 1) and FGFR function is not required for integrin-dependent neurite outgrowth over laminin (Williams et al., 1994a,b,c). In primary neurons, activation of an FGFR–phospholipase C gamma cascade leads to the production of DAG, its conversion to AA, and a subsequent increase in calcium influx into neurons. This pathway is both necessary and sufficient to account for the stimulation of neurite outgrowth by FGF and CAMs (Williams et al., 1994c, 1995b; Hall et al., 1996b; reviewed in Hall et al., 1996a). The demonstration that three agents that block independent steps in this pathway (the FGF receptor antibody, the DAG lipase inhibitor, and a CAM kinase inhibitor) all cause the same misrouting phenotype in the developing retina provides the first evidence that this signaling cascade is important for axonal guidance in living tissue. The fact that all three agents cause the same phenotype also argues that they are acting in a common specific manner, namely to block the above cascade. More specifically, the data suggest that the signaling through the FGFR is not only responsible for allowing retinal cells to survive and proliferate (Pittak et al., 1991; Guillemot and Cepko, 1992; Hicks and Courtois, 1992), but that it can also mediate CAM-stimulated RGC axonal growth in vitro, and the appropriate guidance of axons in the developing retina. A role for the FGF/FGFR signaling cascade in the next step of RGC development, namely axon growth in the optic tract and target recognition, has recently been demonstrated by McFarlane et al. (1995). These workers showed that the tectal target contains diminished levels of FGF compared to the optic pathway, and that the addition of FGF to ‘‘exposed brain’’ preparations resulted in the majority of axons growing around and past the target. Although the results from the present study suggest a role for the FGFR in axon guidance, they do not address the nature of the ligand that activates the

126 receptor during this period of development. Growth stimulated by FGFs and CAMs requires neuronal FGFR function. However, there is other evidence that it is the CAMs rather than FGF that play a direct role in allowing the orderly exit of RGC axons from the retina. Cell–cell interactions can modulate the expression of L1 (Kobayashi et al., 1992; Martini et al., 1994; Brittis and Silver, 1995a). For example, in the retinal periphery, L1 is concentrated at points of early neurite–neurite contact, and the application of L1 antisera to retinal whole mounts has led to ectopic growth cone guidance. Thus, it has been suggested that the concentration of L1 on the youngest ganglion cells at neurite–neurite contact points may be critical for initially guiding RGC axons and, thereby, may help focus the axon toward the optic fissure (Brittis and Silver, 1995; Brittis et al., 1995). It is therefore plausible to speculate that concentration of L1 at points of neurite–neurite contact may lead to greater efficiency of FGFR–CAM interaction. If as is the case in vitro, L1-stimulated growth in vivo requires FGFR function; this step might be the one that is perturbed by the anti-FGFR antibodies and the pharmacological agents. This in turn might increase the growth cone’s probability to interact with inappropriate substrate molecules present within the neuroepithelium. As a consequence the axon would grow in a misrouted fashion. The exact manner in which the homophilic binding of CAMs might lead to activation of the FGFR is not known; however, there is good evidence that such an activation can take place (Williams et al., 1994a; Doherty et al., 1995) and it has been postulated that CAMs can directly interact with FGFRs in cis, in the neuronal membrane (Hall et al., 1996a). Taken as a whole, this study suggests that the effects of the FGF receptor on growth cone behavior may also be important during CAM-dependent growth, guidance, or remodeling within target structures in many other areas of the nervous system during various developmental stages.

EXPERIMENTAL METHODS Cell Culture Embryonic Day 13.5 retinas were dissected away from the sclera, cornea, lens, and pigment epithelium, subjected to gentle trituration and incubated at 37°C for 10 min in calcium/magnesium-free Dulbecco’s modified Eagle’s medium (DMEM) containing 1% trypsin and 0.1% DNAse. After adding an equal volume of DMEM containing 10% fetal calf serum (FCS), the cells were pelleted and resuspended in DMEM/SATO medium (Doherty et al., 1990a). RGC cocultures with 3T3 cells

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were established by seeding 3000 retinal cells onto confluent monolayers of parental and transfected CAMexpressing clones of 3T3 cells (140-kDa isoform of NCAM, N-cadherin, and L1; 100,000 cells per individual chamber of an eight-chamber LabTek slide) established over a 24-h period (for details see Doherty et al., 1990a, 1991; Williams et al., 1992). Retinal cultures were maintained for 13 h in DMEM/SATO supplemented with 2% FCS and various reagents as indicated.

Immunocytochemistry The FGFR antiserum used in the present study was raised against a peptide that corresponds to an extracellular region of the FGFR1 dubbed the CAM homology domain (amino acids 151–170; see Williams et al., 1994a for details). In Western blots, this antibody binds to the FGFR and shows no cross-reactivity with purified NCAM or L1 (Williams et al., 1994a). The FGFR antiserum does not inhibit integrin-dependent neurite outgrowth, and whole serum preabsorbed against the immunogen does not inhibit neurite outgrowth stimulated by NCAM, N-cadherin, or L1 (Williams et al., 1994a). Because 3T3 cells also express the FGFR, in some experiments the retinal cells were cultured on laminin– polylysine-coated tissue culture slides as previously described (Williams et al., 1994a), fixed in 4% paraformaldehyde, incubated in FGFR antiserum (1:400 dilution) at 4°C overnight, washed and incubated in biotin-conjugated goat anti-rabbit secondary, washed and incubated in streptavidin Texas red, and viewed with the appropriate filters (both reagents at 1:500; Amersham). To visualize the neurons on top of the fibroblast monolayer, the RGC cultures were fixed with paraformaldehyde, methanol permeabilized, and incubated in anti-GAP-43 antiserum followed by immunostaining as above (for details see Williams et al., 1994a). Because of the early stage of retinal differentiation (E13.5), the GAP-43-positive neurons were predominantly of the RGC class. Only those neurons which morphologically resembled retinal ganglion cells as seen in vivo were counted (i.e., one GAP-43-positive axon with a bona fide growth cone). The length of each axon was determined using a Sight System Image Manager (Sight Systems, Newbury, England).

Whole Mount Culture Embryonic Day 13.5 retinas were dissected away from the sclera, cornea, lens, and pigment epithelium and mounted vitreous side up as described previously (Brittis et al., 1992, 1995; Brittis and Silver 1995). Retinas were

FGF Receptor Function in Developing Retina

grown under control conditions (in media alone, in media with nonspecific IgG antibodies, or in 4-bromophenacyl bromide) or in the presence of the FGFR antibody (1:200), fixed with paraformaldehyde, permeabilized with Triton, and immunostained as above with either MAb TUJ1 (1:500) or anti-GAP-43 antiserum (1:2000) (not shown). Under the culture conditions, retinas remained healthy. When all the groups were visualized in cross section, the radial nature of the nonneuronal cells remained intact. The exception to the above was in the presence of calcium channel antagonists which were found to be toxic to the nonneuronal cells. Note added in proof. A recent manuscript by McFarlane et al. (1996, Neuron 17, 245–254) reports that 44% of retinal ganglion cells that express a kinase-deleted ‘‘dominant negative’’ form of the FGF receptor fail to send an axon out of the developing Xenopus retina. An almost identical result (37% of axons remaining in the retina) was obtained with a kinase-deleted receptor that does not bind FGF and, as a consequence, is not a dominant negative receptor for FGF. These data underscore the importance of FGF receptor function for the orderly exit of axons from the developing retina, and are more in keeping with CAMs, rather than FGFs, being the activating ligands within the retina.

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