FGF signaling and target recognition in the developing xenopus visual system

Neuron, Vol. 15, 1017-1028, November, 1995, Copyright © 1995 by Cell Press

FGF Signaling and Target Recognition in the Developing Xenopus Visual System Sarah McFarlane, Lisa McNeill, and Christine E. Holt Department of Biology University of California, San Diego La Jolla, California 92093

Summary We report that the growth cones of Xenopus retinal ganglion cells express fibroblast growth factor receptors (FGFRs) and that bFGF stimulates neurite extension from cultured retinal neurons. Furthermore, bFGF is abundant in the developing optic tract but is reduced in the optic tectum. To test whether FGF signaling plays a role in axonal guidance in vivo, bFGF was exogenously applied to the developing optic pathway in "exposed brain" preparations. FGF-treated retinal axons navigate normally through the optic tract, but the majority veer aberrantly at the tectal border and bypass the target. Our results implicate FGF signaling in target recognition and suggest that diminished levels of bFGF in the tectum cause arriving axons to slow their growth. Introduction To advance through its environment, an axon needs to extend and to steer. These behaviors are mediated by the axon's growth cone, which detects and responds to molecules in the neuroepithelium (see reviews by Dodd and Jessell, 1988; Bixby and Harris, 1991; Goodman and Shatz, 1993). Upon reaching its target, the axon stops advancing and begins to arborize (Harris et al., 1987; Kaethner and Stuermer, 1992). Changes in the expression of molecules in the growth cone could cause this abrupt modification in behavior (Becker et al., 1993; de Curtis and Reichardt, 1993; Liu et al., 1993; Meier et al., 1993). Alternatively, target recognition could require specific molecules present in the target (Baier and Bonhoeffer, 1992; Perez and Halfter, 1993; Yamagata and Sanes, 1995). A number of different classes of molecule have been implicated as target recognition signals, including cell and substrate adhesion molecules and soluble factors (TessierLavigne and Placzek, 1991; Goodman and Shatz, 1993). Growth factors may serve as targeting molecules, in that they are often expressed in the target (Campenot, 1994) and can affect neurite extension, branching, and steering (Campenot, 1977; Gundersen and Barrett, 1979; Zhang et al., 1994), We are particularly interested in the family of fibroblast growth factors (FGFs), whose members are known to promote proliferation, survival, and differentiation of various neuronal types (Sensenbrenner, 1993; Mason, 1994). The possibility that FGF plays a role in steering and target recognition in vivo has not been tested, yet there is evidence to support this idea. For instance, acidic

(aFGF or FGF-1) and basic (bFGF or FGF-2) fibroblast growth factor influence both neurite outgrowth (Walicke et al., 1986; Rydel and Greene, 1987; Hatten et al., 1988; Lipton et al., 1988; Walicke, 1988; Zhou and DiFiglia, 1993) and the expression of extracellul~r matrix molecules in the environment through which axons grow (Drago et al., 1991; Kinoshita et al., 1993; Meiners, et al., 1993; Rettig et al., 1994). In addition, recent evidence implicates FGF signaling in cell migration (Kinoshita et al., 1993; Reichman et al., 1994). To test the idea that FGFs are important for axon navigation, we chose the developing Xenopus visual system for several reasons. The behavior of Xenopus retinal ganglion cell (RGC) axons is well characterized (Chien et al., 1993); the Xenopus embryo is especially amenable to these types of analyses; and FGF signaling is thought to play a role in RGC differentiation (Heuer et al., 1990; Wanaka et al., 1991; Tcheng et al., 1994). In addition, message for FGFs and the FGF receptor (FGFR) is expressed in the developing Xenopus visual system (Friesel and Brown, 1992; I~aacs et al., 1992; Tannahill et al., 1992; Song and Slack, 1994), implicating FGFs in the establishment of the optic pathway. Of the nine known members of the FGF family, bFGF has been particularly well studied in Xenopus because of its involvement in mesoderm induction (Kimelman and Kirschner, 1987; Slack et al., 1987; Amaya et al., 1993). Additionally, bFGF may serve atrophic function for RGCs, since, in other species, it is expressed in differentiating RGCs (de Longh and McAvoy, 1993) and may be involved in the determination of the neural retina (Park and Hollenberg, 1989; Pittack et al., 1991). Furthermore, mammalian retinal culture data indicate that aFGF and bFGF increase RGC survival (Bahr et al., 1989) and the subsequent expression of differentiated markers by RGCs (Guillemot and Cepko, 1992). Finally, in vivo, RGCs bind bFGF and can transport it either in an anterograde (Ferguson et al., 1990) or a retrograde fashion (Sievers et al., 1987). Thus, bFGF is a strong candidate for having a role in RGC axon growth and guidance in vivo. In this study, we investigate the role of bFGF in the formation of the retinotectal projection by determining the distribution of bFGF and FGFR in the developing visual system; the effect of bFGF on RGCs in vitro; and the effect of exogenous bFGF applied to the optic pathway and to RGC somata using an in vivo exposed brain preparation (Chien et al., 1993). Our results suggest a role for bFGF and the FGFR in axonal targeting in the developing Xenopus visual system. Results Basic FGF is Abundant in the Optic Tract but Low in the Tectum The first retinal axons enter the optic chiasm at stage 32 and cross into the contralateral brain at stage 33/34. These

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Figure 1. NormalbFGFand FGFRExpression in the Brain and Localizationof EcotopicbFGF (A) Transversesectionthroughthe diencephaIon and tectum of a stage 39 embryoimmunestained with a rabbit polyclonal antibody againstXenopusbFGF. The neuropil(Np) and cell bodies in the diencephalonare intensely labeled, but only weak labeling is seen in the tectum(Tec).Dottedlinedemarcatesboundary betweenimmunopositiveand immunonegative regions and roughly marks the diencephalictectal border. (B) Whole-mountbrain stainedwith anti-bFGF. All major axon tracts are labeled(TPOC, tract ;" FGFR D Np ~lff-.~of the posterioroptic commissure;TPC, tract of ~ bFGF-Treate( 'J the posteriorcommissure;AOC, anterioroptic commissure). By contrast, immunostainingis reduced in the tectum, especiallyin the midreV Tec OP ;~lf~ ~ gion. (C) Transversesectionthrough the diencepha, Ion of a stage 39 Xenopus embryo immunestainedwith a polyclonalantibodyagainstXenDi ~. ~. opus FGFR. (D and E) FGFR immunostaining(D) and antiNp HRP double-labeledRGC axons (E) from the b~. boxed region in (C) demonstratingthat FGFR c p ~ r ' ~ ..... ~' ' Np staining colocalizeswith the RGC axons. ; ~. i ~• (F) Section throughthe midbrainof an embryo -exposedto 100 ng/mlbFGF for 6 hr and stained with anti-bFGF. Punctate labeling is seen on the exposedside (see arrows),in contrast to the unexposedside. V, ventricle; Di, diencephalon;Pi, pineal;op, optic axons. Bar in (A), 50 pm (for A, C, and F) and 120 p.m (for B). Bar in (D), 20 p_m(for D and E).

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axons then grow dorsalward through the diencephalon to form the optic tract, and they begin to innervate the tectum at stage 37/38 (Holt, 1984). To investigate the distribution of bFGF in the developing visual system, we stained transverse sections of stage 33/34-39 brains and eyes with a polyclonal antibody against Xenopus bFGF (XbFGF). At all stages examined, this antibody labels the neuropil and cell somata in the diencephalon (Figure 1A). In fact, the entire pathway of RGC axons, including the optic tract, optic nerve, and optic nerve head, is immunopositive for bFGF. The neuropil remains immunopositive for bFGF in brains enucleated at stage 30 by removing the contralateral eye, indicating that bFGF is present in the substrate in the absence of retinal fibers. The target of RGC axons, the optic tectum, is only weakly labeled by anti-bFGF, as seen in cross section (Figure 1A) and in a whole-mount brain (Figure 113). Similar immunostaining was observed with a rabbit polyclonal antibody against rat bFGF. In whole mount, it is obvious that other axon tracts in the brain are labeled by anti-bFGF, including the tract of the posterior optic commissure (TPOC), the anterior optic commissure (AOC), and the tract of the posterior commissure (TPC) (Figure 1 B). These results suggest that retinal fibers and axons in other tracts extend in a bFGF-rich environment. In the case of the visual system, the optic fibers then encounter a bFGF-poor region upon entering their target.

RGC Growth Cones Express FGFRs and Bind bFGF Since bFGF is expressed in the optic pathway, we investigated whether RGC axons and growth cones express FGFRs by staining stage 25 eye explant cultures with a

polyclonal antibody that recognizes extracellular and cytoplasmic portions of the Xenopus FGFR (Amaya et al., 1991). Both the axons and growth cones of RGCs are immunopositive for the FGFR (Figure 2A). Immunostaining in the axon and growth cone is punctate, indicative of receptor clustering. Staining is present in the main body of the growth cone, in the lamellopodia, and in some filopodia. To confirm that retinal fibers express the FGFR in vivo, transverse sections of stage 39 embryos were stained with anti-FGFR. Within the diencephalon and tectum, this antibody stains the neuropil, cell profiles, and radially arranged processes (Figure 1C). FGFR is also expressed in the RGC layer, the optic nerve head, and optic nerve (data not shown). Double immunolabeling of HRPfilled optic projections shows that anti-HRP and anti-FGFR staining colocalize (compare Figures 1D and 1E) indicating that retinal axons express FGFRs. To determine whether growth cones bind exogenous bFGF, eye explant cultures were treated with 20 ng/ml bFGF and immunostained with anti-bFGF (Figure 2C). The lamellopodia and filopodia of RGC growth cones are stained in a punctate fashion. Comparable labeling is observed using nonpermeabilized staining conditions, indicating that the staining is extracellular. Specificity of the staining was demonstrated by the fact that the labeling was almost eliminated by preabsorption of the bFGF antibody for 1 hr with 50 ng/ml of XbFGF (data not shown). In dissociated cultures, the intensity of bFGF staining of retinal growth cones correlated with exogenous bFGF being present; cultures treated with bFGF were more intensely labeled than those grown in serum and embryo extract, which in turn were stained more brightly than

FGF Signaling and Axon Targeting 1019

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DFGF Figure 2. RGC Growth Cones in Culture Have FGFRs and Respondto bFGF RGC growth conesfrom 24 hr retinal explant cultures double labeledwith RITC-conjugatedphalloidin(B and D) and anti-FGFR (A) and anti-bFGF (C). The immunostainingpatternfor bFGFand FGFR are very similar. In both cases, punctatelabeling is presentin the lamellopodiaand filopodia of the growth cone and along the axon shaft. The arrow in (A) points to two FGFR clusters in the leading filopodium.Graph (E) showsthe mean length of the longest neurite of RGCs growing in 24 hr dissociatedcultures with and without human bFGF (20 ng/ml). The cultures treated with bFGF have significantlylonger neuritesthan those grownwithout bFGF (*, p < .0001, unpairedtwo-tailedt test), n is the total numberof neurites counted from 4 independentexperiments(total of 8 cultures counted for each condition),and error bars are SEM. Bar in (B), 5 #m. those cultures grown without serum or bFGF (data not shown). These data suggest that at least part of the labeling of RGC axons is due to exogenous bFGF; thus, not only do RGC growth cones have FGFRs, but they also bind bFGF.

Basic FGF Stimulates Neurite Outgrowth from Retinal Cells In Vitro Since RGC growth cones express FGFRs, we investigated whether bFGF can stimulate neurite outgrowth from Xenopus RGCs in culture. Dissociated retinal cultures were grown for 24 hr i n L 1 5 media and 0.1% bovine serum albumin (BSA), either with (+bFGF) or without (-bFGF) 20 ng/ml human bFGF. RGCs were identified morphologically as those cells with large, phase-bright cell bodies (diameter of 15-20 pm) and 1-2 processes; immunostaining of dissociated cultures with an antineurofilament antibody verified that these cells were RGCs (Szaro et al., 1989). Only RGCs whose cell bodies were isolated from other cells were used for neurite measurements. The mean neurite length in bFGF-treated cultures was over twice the length of RGC neurites grown without bFGF (Figure 2E) and, in fact, was greater than that measured for serum-treated cultures (data not shown). These results indicate that bFGF stimulates extension of neurites from RGCs. Exogenously Applied bFGF Disrupts Targeting of RGC Axons In Vivo Our data indicate that RGCs express FGFRs, bFGF stimulates neurite extension, and bFGF localizes discretely

within the visual system with strong expression in the optic pathway but not the tectum. These results led us to investigate what happens to retinal axon growth and navigation when the specific pattern of bFGF expression is disrupted by exogenously applying bFGF to the entire developing optic pathway. Using an exposed brain preparation (Chien et al., 1993), a recombinant form of XbFGF (100 ng/ml) was applied to the brain of stage 33/34 embryos. At this stage, the first RGC axons have crossed the optic chiasm and entered the base of the optic tract in the contralateral brain. At stage 40, we filled the RGC fibers with HRP to visualize the optic projection. Representative optic projections of control, epidermal growth factor-treated, and XbFGF-treated embryos are shown in Figure 3. In control embryos, neither the exposure nor the BSA carrier had any effect on the optic projections, which were very similar to those of unexposed controls (data not shown). The control optic projection takes a stereotypical route, making a wide 45 ° posterior turn in the middiencephalon; then running in a dorsal-caudal direction; and finally entering the tectum and arborizing (seen in Figures 3A and 3B). Figures 3D-3F show the optic projections of embryos that developed in the presence of XbFGF. The XbFGF-treated axons pathfind normally through the diencephalon. However, when they reach the diencephalon-midbrain border, most XbFGF-treated RGC axons fail to enter the tectum and instead veer ventrally or dorsally around it (Figure 3E and 3F). Treated fibers did not appear to branch, but since single retinal axons grew in either direction, the optic projections were often seen to bifurcate at the tectal entry point. This pheno-

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Figure 3. Exogenous Basic FGF Causes RGC Axons to Bypass the Tectum Control retinal projection (A and B) and retinal projections in brains exposed to 250 ng/ml EGF (C) and 100 ng/ml XbFGF (D-F). Panels (B) and (E) are higher magnification Nomarski images of (A) and (D), respectively. Control optic projections enter the tectum (Tec), the anterior and posterior borders of which are marked with arrowheads. EGF-treated optic projections are normal (C). In contrast, the majority of bFGF-treated axons fail to enter and grow past the tectum (D-F). Axons head either dorsally or ventrally around the tectal border, often resulting in a split optic projection. Aberrant behavior at the tectal border can be severe, as seen in (F) where the RGC fibers make an abrupt change m direction and fail to enter the target. Ot, optic tract; Di, diencephalon; Tel, telencephalon; Hb, hindbrain. Bar in (C), 100 pm (for A, C, and D) and 50 #m (for B, E, and F).

type, in which R G C fibers fail to r e c o g n i z e the t e c t u m a s their target, o c c u r r e d in 8 2 % of c a s e s (37/45). To d e t e r m i n e w h e t h e r the e x o g e n o u s b F G F e n t e r s the n e u r o e p i t h e l i u m , w e s t a i n e d t r a n s v e r s e s e c t i o n s of b r a i n s e x p o s e d for 6 hr to b F G F with a n t i - b F G F (see Figure 1F).

E x o g e n o u s b F G F labeling is punctate, p e n e t r a t e s as far as the v e n t r i c u l a r surface, a n d is f o u n d within the dienc e p h a l o n , f o r e b r a i n , midbrain, a n d h i n d b r a i n on the exp o s e d side of the brain. Similar staining is s e e n for brains e x p o s e d to b F G F for 20 hr (data not shown)• T h e s e d a t a

FGF Signalingand AxonTargeting 1021

indicate that exogenous bFGF is present in the optic tecturn, where it is expressed normally at low levels.

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Trajectories of Mistargeted Axons Retinal axons growing through a brain treated with bFGF exhibit a range of mistargeting phenotypes. First, the majority of axons fail to enter the target, and the optic projection bifurcates around the tectum (Figure 4A). Second, some fibers do enter the tectum but either stall, grow in abnormal directions, or grow out of the target (see arrows and arrowheads in Figure 4A). Fibers that stall remain in a ventral location, stopping approximately 50-100 ~m distant from the area of the tectum they would normally innervate. Other fibers make abrupt turns and grow either dorsally or ventrally, and commonly exit the tectum. Third, axons grow beyond the target along either the rostral or ventral border of the tectum. Axons that head dorsally along the rostral border often continue across the dorsal midline into the contralateral brain (58%, 26/45 brains). This results in a striking phenotype with retinal axons forming a commissural tract across the dorsal midline of the diencephalon (Figure 4B). Finally, axons turn orthogonally and grow deep in the brain. Normally, RGC axons grow close to the pia in the optic tract and never invade the ventricular zone (Silver and Rutishauser, 1984; Bovolenta and Mason, 1987; Easter and Taylor, 1989; Holt, 1989). However, some bFGF-treated optic fibers grow deep in the brain neuroepithelium, sometimes even reaching the ventricular surface (Figure 4C). Axons headed deep in 9/9 transversely sectioned bFGF-treated brains, in contrast to 0/4 control brains. Thus, mistargeted axons showed a range of aberrant trajectories, with growth around the tectal border being the predominant behavior.

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Dose-Dependence of the bFGF-Induced Mistargeting Phenotype The frequency of the mistargeting phenotype was determined for different doses of bFGF (Figure 5A). The strongest effect of bFGF is seen at 50-100 ng/ml, though optic projections treated with 10 ng/ml frequently bifurcated around the tectum (see Figure 4A). Similar results were seen with a human recombinant form of bFG F at a concentration of 100 ng/ml (data not shown). Even at doses as low as 1 ng/ml of XbFGF, many of the axons turn aberrantly near the diencephalic-tectal border and grow either dorsally or ventrally. Other behaviors, such as axons crossing the midline, require higher doses of bFGF; at 50 ng/ml, the majority of treated optic projections cross the midline. Because exogenous bFGF has to cross 2-10 ~m of neuroepithelium to reach the RGC growth cones, the absolute concentration of bFGF at the axon tip is unknown. However, the dose is within the range we and others have found has effects in culture experiments.

Figure 4. Three Different Aspects of Aberrant Targeting Camera lucida representations of the optic projections of control and bFGF-treated brains from a lateral view (A), viewed dorsally (B), and in transverse section (C). (A) Trajectories of optic fibers at the border between the diencephalon and the tectum. The control axons grow into the tectal mass before arborizing. The majority of XbFGF-treated fibers grow either dorsally (*) or ventrally (A) around the rectum. Some fibers enter the rectum but either stall in ventral regions of the tectum (arrowheads), or exit the tectum ventrally (arrows). Dotted lines show the approximate borders of the rectum.

(B) The XbFGF-treatedopticprojectioncrossesthe dorsalmidlineand enters the contralateralbrain. (C)Thecontroloptic projectionis restrictedto the pialsurface,whereas fibers treated with XbFGF can grow deep in the brain, sometimes reaching the ventricularsurface. Vent, ventricle;Pi, pineal;ot, optictract; Tec, tectum;Tel, telencephaIon; D, dorsal;V, ventral; L, lateral;A, anterior; P, posterior.

Quantitation of Mistargeted Projections To quantitate the bFGF-induced phenotype, the width of control and bFGF-treated optic projections was measured (Figure 5B). Figure 5C shows the mean width plotted as a function of the distance along the optic projection. In

control brains, the optic projection widens gradually within the diencephalon and widens further upon entering the tectum as retinal fibers branch and arborize. The graph reveals two aspects of the bFGF phenotype. First, optic

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To test the specificity of the bFGF effect, other growth factors were used in the exposed brain preparation. Nerve growth factor (NGF) and epidermal growth factor (EGF) are found in Xenopus (Carriero et al., 1991; Lee et al., 1993) and are expressed, along with their respective receptors, in the eyes of other species (Anchan et al., 1991; Zanellato et al., 1993). In bath experiments, NGF (n = 4) and EGF (n = 10), at concentrations of up to 250 ng/ml, had no effect on the optic projection, in terms of either pathfinding or target recognition. A brain treated with EGF (250 ng/ml) is shown in Figure 3C. Activin, known to be a powerful mesodermal inducer in Xenopus (Thomsen et al., 1990), similarly had no effect (5 nM; Andreas Walz, personal communication). A h u m a n recombinant form of aFGF caused axons to bypass the tectum, but with a somewhat different phenotype from that induced by bFGF. Unlike the phenotype observed with bFGF treatment, fibers rarely grew ventrally; instead, the RGC axons grew dorsally along the rostral border of the tectum, often entering the tectum at a more dorsal location than normal. High concentrations (500 ng/ml) of aFGF were needed to generate this phenotype reliably. Thus, the tectal bypass effect is not generally caused by growth factors, but appears to be specific to FGF and particularly sensitive to bFGF.

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Figure 5. Quantitation of the Mistargeting Phenotype (A) The percentage of optic projections that bypass the tectum at different concentrations of Xenopus bFGF. Mistargeted projections were those where the majority of axons grew around the target, with few or no axons entering the tectum. (B) Projection width was used to quantitate the bypass phenotype as described in the Experimental Procedures. Method of analysis for a control brain and a brain treated with 10 ng/ml bFGF is shown. A standard reference line (1 bru) was drawn between the optic chiasm and the midbrain-hindbrain isthmus (Chien et al., 1993). Concentric circles were placed at 0.1 intervals and centered at the optic chiasm, and the widest projection width was measured for bins centered on each concentric circle (e.g., bin 0.1 comprises the region between 0.05 and 0.15 bru). Boxes demarcate the outermost parts of the projection for each bin. (C) Mean optic projection widths (brus have been converted to microns, with 1 bru = 620 pro, Chien et al., 1993). Control (n = 32), 1 ng/ml (n = 11), 10 ng/ml (n = 19), and 100 ng/ml (n = 29). Basic FGF-treated projections are significantly narrower in the diencephalon (tested at 186 pro) and significantly wider at the tectal border as compared with control projections (* *, p < .005, * * * p < .0001, unpaired two-tailed t test). Errors bars are SEM and, if not visible, are smaller than the symbols.

projections treated with 100 ng/ml of bFGF are significantly narrower than control projections within the diencephalon (as seen at 186 p~m). Second, as the optic projection approaches the tectum (370 ~tm), it becomes significantly wider in bFGF-treated brains (1, 10, and 100 ng/ml) than in control brains, as a result of treated optic projections splitting around and bypassing the tectum.

To elucidate further how e x o g e n o u s bFGF affects optic fibers, we determined how late bFGF could be applied to the optic projection and still cause axons to miss the target. If bFGF is applied at stage 35136, when the first axons have reached the middiencephalon, m a n y retinal fibers miss the target (95% of cases, n = 19), as seen in Figure 6A. However, the optic projections of brains treated after the first fibers have arrived at the target (stage 37•38) show a mixture of normal axons and axons exhibiting a mistargeting phenotype (Figure 6B). Most optic projections have axons that grow into the appropriate area of the tectum (91%, n = 22), yet many axons are misdirected: over 5 0 % of the optic projections have axons that grow around the tectal border, or enter and then exit the target. In addition, in many optic projections (82%, n = 22), fibers turned and headed either dorsally or ventrally, a behavior that is observed rarely in control projections (13%, n = 22). These results indicate that the onset of action of bFGF must be fairly rapid (within 2 - 3 hr), since bFGF is effective even though the majority of axons are approaching the tectal entry point at stage 37/38. They also suggest that retinal fibers do not use each other to recognize the tecturn, since s o m e optic fibers are misdirected despite the existence of retinal axons in the target that have appropriately innervated the tectum.

Somatic Application of bFGF Does Not Cause Mistargeting Exogenous bFGF could influence either the RGC growth cones or the environment through which they grow. If bFGF causes mistargeting by acting directly on RGC axons, then exposing RGC s o m a t a to e x o g e n o u s bFGF

FGF Signaling and Axon Targeting 1023

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B stag

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Figure 6. Application of bFGF to Axons, but not Somata, Elicits Mistargeting Response within Hours Camera lucida representations of the brain and optic projection of stage 40 embryos exposed to 100 ng/ml bFGF either at stage 35/36 when the first RGC axons are making the turn in the middiencephalon (A) or at stage 37/38 when RGC axons first begin to enter the tectum (B). Mistargeted axons turn and grow dorsally or ventrally around the tectal border, or exit the tectum upon entering (see arrowheads). Panel (C) shows an optic projection of a stage 40 embryo whose eye was exposed to bFGF at stage 33/34. Basic FGF acting on the somata does not cause mistargeting Arrows mark the approximate borders of the tectum, and open circles mark the presumptive diencephalicmidbrain border.

known to stimulate proliferation, increased mitosis in the tectum could produce an immature tectum that the retinal fibers might not be able to recognize. We compared the number of proliferating cells in bFGF-treated and control tecta. EGF-treated brains were used as a positive control since EGF, also a mitogen, has no effect on target recognition (see Figure 3C). Both bFGF and EGF cause a small increase in the number of BrdU-labeled cells in the midbrain region including the tectum (Table 1). However, the approximate 2-fold increase corresponds to less than 1% of the total number of cells in the midbrain at this stage ( - 20,000 cells). In addition, there is no evidence of ectopic hotspots of proliferation anywhere in the treated brains. Because EGF causes a similar increase in proliferation, but has no effect on the optic projection, it is unlikely that bFGF causes axons to avoid the tectum by stimulating proliferation. To investigate further whether the identity of the tectum is altered with bFGF treatment, we used immunocytochemistry and Western analysis to investigate possible changes in levels of the homeobox protein engrailed, which is expressed in a posterior to anterior gradient in the Xenopus tectum (Hemmati-Brivanlou et al., 1991). Engrailed protein levels in bFGF-treated brains were 108% - 8% of control levels (mean ± SEM, 5 independent experiments), indicating that bFGF does not alter engrailed expression. In addition, we examined whether tenascin expression in the tectum is altered by bFGFtreatment. Tenascin is an extracellular matrix molecule expressed in the chick tectum that inhibits RGC neurite outgrowth in culture (Perez and Halfter, 1993; Taylor et al., 1993) and whose levels are upregulated in astrocytes by bFGF (Meiners et al., 1993). We used an antibody against Xenopus tenascin to immunostain control and bFGF-treated whole-mount brains and found that bFGF does not obviously affect the intensity or pattern of tenascin labeling (data not shown). Thus, at least two tectal molecules continue to be expressed at normal levels, indicating that bFGF does not change the gross identity of the target.

Discussion might similarly affect the optic projections. RGCs take up bFGF and anterogradely transport it to their nerve terminals (Ferguson et al., 1990). In addition, ciliary neurotrophic factor affects synaptic transmission regardless of whether it is applied to the soma or to the nerve terminal (Stoop and Poo, 1995). However, we found that exposing the RGC somata to bFGF by removing the lens at stage 33/34 had no effect on the optic projections, which in 90% of cases (28/31) resembled those in control (Figure 6C). These results indicate that exogenous bFGF exerts its action not on the cell body but on the growth cone, either directly or through changes in the neuroepithelial substrate.

Basic FGF Does Not Change the Gross Identity of the Target N e x t , w e a s k e d w h e t h e r e x o g e n o u s b F G F alters t h e p r o p erties of t h e n e u r o e p i t h e l i a l

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Three main results implicate bFGF in guiding retinal axons. First, endogenous bFGF shows a regionalized distriTable 1. Basic FGF and EGF Cause a Small Increase in Cell Proliferation in the Tectum Condition

Control

bFGF

EGF

Number of BrdUlabeled cells per embryo

42 _+ 6

88.6 _+ 11 (*)

124 __. 19 (**)

The number of BrdU-labeled cells was counted in midbrain sections of control, bFGF-, and EGF-treated embryos. A total of 4 sections was counted for each embryo, and data show the mean number of cells counted for each embryo. Control, n = 4 embryos; XbFGF, n = 5 embryos; EGF, n = 4 embryos. EGF and XbFGF caused small but significant increases (*, p < .05; * *, p < .01 ; Dunnett multiple comparisons test) in the number of proliferating cells as compared with control. Errors are SEM.

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bution in the visual pathway with high levels in the optic tract and low levels in the optic tectum. Second, the growth cones of retinal axons express FGFRs, and cultured retinal neurons respond to bFGF by increased neurite outgrowth. Third, ectopic bFGF causes retinal axons to veer in abnormal directions at the diencephalic-midbrain boundary and, consequently, to bypass the optic tectum. Together, these results suggest that bFGF normally acts to stimulate the extension of axons through the optic tract and that a reduction of this growth factor in the tectum may play a role in target recognition. In the exposed brain preparation used here, both the retinal axons and their cellular substrate are simultaneously exposed to the bathing medium, making it difficult to determine which population of cells bFGF exerts its action on. Thus, the axon mistargeting phenotype described here could be mediated indirectly by bFGFinduced changes in the tectum or directly by action on the retinal axons. A number of pieces of evidence favor the idea that axon mistargeting is a result of bFGF acting directly on retinal axons. First, RGC growth cones express the FGFR, and in culture bFGF stimulates neurite extension. Second, the bFGF effect occurs rapidly, within a couple of hours, as applying bFGF when retinal fibers are reaching the tectum still causes mistargeting of some axons. Third, our data indicate that bFGF does not alter the gross molecular identity of the tectum in that some tectal molecules, such as tenascin and engrailed, are expressed normally. In this respect, we also show that it is unlikely that ectopic bFGF drives the tectal primordium into an immature and thus unrecognizable state, as EGF has a similar effect to bFGF in terms of cell proliferation but not target recognition. Nonetheless, the possibility remains that bFGF could act on FGFRs expressed in the tectum and alter the expression of as yet unidentified target molecules, perhaps upregulating the expression of an inhibitory molecule (Baler and Bonhoeffer, 1992; Perez and Halfter, 1993; Nose et al., 1994). As retinal axons navigate through the diencephalon, they must receive signals that tell them to grow and signals that tell them to steer. Our data suggest that bFGF acts as a growth stimulatory molecule. The presence of bFGF in the optic tract and, indeed, in other developing axon tracts (TPOC, TPC, and AOC) raises the possibility that bFGF acts to stimulate the extension of RGC axons in vivo. We show that RGC axons and growth cones express FGFRs and that in vitro bFGF stimulates a 2-fold increase in the length of retinal neurites, both of which lend support to this idea. Furthermore, suramin, a reagent known to block the action of bFGF, inhibits the extension of axons in the optic tract (McFarlane et al., 1993, Soc. Neurosci., abstract). Previously, aFGF has been shown to stimulate axon outgrowth from RGCs (Lipton et al., 1988). If, indeed, the role of endogenous bFGF is to stimulate axon growth within the optic tract, rather than to guide, then application of exogenous bFGF would not be expected to cause misrouting in this area. Our finding that axon trajectories are normal in the optic tract when bFGF is added is consistent with this. Previous timelapse imaging studies of Dil-filled growth

cones have shown that retinal axons abruptly change their behavior when leaving the optic tract and entering the tectum (Harris et al., 1987). They advance through the optic tract rapidly (50-80 p~m/hr) with expanded and complex growth cones. On nearing and entering the target, they slow down, lose their expanded tips, and begin to arborize by elaborating back branches. These two distinct modes of behavior are likely to be regulated by molecular cues in the pathway and target. The regionalized distribution of bFGF in the optic pathway, bFGF-rich in the tract and bFGF-poor in the tectum, raises the possibility that the transition between high and low levels of growth factor at the tectal border might trigger retinal axons to slow their growth and switch from a rapidly extending to an arborizing mode of growth. The idea that an axon recognizes its target by registering a concentration difference of growth factor between its growth cone and the target is also suggested by studies in transgenic mice that show that sympathetic axons made to overexpress NGF reach their NGFexpressing targets but fail to innervate them appropriately (Hoyle et al., 1993). If the role of bFGF is to stimulate extension of retinal axons, one might expect that bath application of bFGF, which introduces bFGF to both the optic tract and the tectum, would smooth out regionalized differences in bFGF and cause axons to grow indiscriminately through the tecturn. instead, our results show that retinal axons turn and avoid entering the tectum in the presence of exogenous bFGF. This result is clearly puzzling. One possible explanation is that the reduced levels of bFGF encountered normally at the tectum act not only to slow axons down, but also to modify their responsiveness to certain target molecules. This change may be required for the retinal axons to invade the tectum, a region which expresses several inhibitory molecules (Baier and Bonhoeffer, 1992; Perez and Halfter, 1993; Luo et al., 1995). In bFGF-treated brains, RGC axons would not receive the necessary signal at the target border and thus would remain unresponsive to the target and grow preferentially in areas that are permissive to axon extension. Such regions would include areas where axons of other tracts are actively extending, such as the TPC and TPOC. In fact, following surgical ablation of the target, RGC axons follow the TPC and TPOC that are present along the rostral and ventral borders of the tectum, respectively (Taylor, 1990). Recently, it has been postulated that the FGFR acts as a downstream signaling molecule for cell adhesion molecules (CAMs; Williams et al., 1994). Experiments suggest that CAMs promote retinal axon outgrowth in vivo and in vitro (Neugebauer et al., 1988; R. Riehl, personal communication). In addition, overexpression of Fas II, a Drosophila CAM homolog, in motoneuron growth cones causes the axons to mistarget in a similar fashion to retinal fibers treated with bFGF (Lin and Goodman, 1994). These authors hypothesize that increased fasciculation between axons prevents them from reading cues in their environment and finding their target. Growth factors can increase neurite fasciculation (Ure et al., 1992); therefore, an alternative mechanism for the retinal axon mistargeting is that exogenous bFGF mimics CAM signaling and increases

FGF Signaling and Axon Targeting 1025

fasciculation. Interestingly, within the diencephalon, bFGFtreated optic projections are significantly narrower than controls, suggesting that the tracts are more tightly fasciculated. There are at least three different types of FGFR (Johnson and Williams, 1993), including a family of membrane receptor tyrosine kinases (RTKs), heparan sulfate proteoglycans (HSPGs), and a cysteine rich FGFR (Burrus et al., 1992). The activated FGFR is currently thought to exist as a ternary complex of the high affinity RTK, a low affinity HSPG, and FGF. Thus, the mere presence of bFGF does not always guarantee a function, since biological activity depends on its ability to bind specific cell surface or extracellular matrix HS (Neufeld et al., 1987; Klagsbrun, 1990; Givol and Yayon, 1992; Nurcombe et al., 1993). This raises the possibility that heparin, a member of the HS family, might mimic bath application of bFGF by releasing bFGF bound to HSPG and redistributing it within the brain (Flaumenhaft et al., 1990; Givol and Yayon, 1992). Indeed, heparin also causes mistargeting of retinal axons (McFarlane et al., 1993, Soc. Neurosci., abstract). The heparin and bFGF results support the idea that bFGF normally interacts with specific HSPGs to provide localized signals for growth of RGC axons and that disruption of this pattern causes axons to miss their target. The FGFR family of RTKs consists of at least four receptor types, and different FGFs can activate the same type of receptor (Givol and Yayon, 1992; Johnson and Williams, 1993). This promiscuity may be important, since a number of different FGFs have been identified in Xenopus, including bFGF (Kimelman and Kirschner, 1987; Slack et al., 1987), FGF-3 (Tannahill et al., 1992), and XeFGF, an embryonically expressed FGF homologous to FGF-4 and FGF-6 (Isaacs et al., 1992). FGF-3 and XeFGF are coexpressed with FGFR-2 at the back of the tectum (Friesel and Brown, 1992; Isaacs et al., 1992; Tannahill et al., 1992); therefore, it is possible that exogenous bFGF mimics the action of an endogenous FGF other than bFGF. Soluble growth factors probably act as specific growth stimulatory molecules along the pathways of many axons. Recently, a number of soluble molecules secreted by target cells and deposited in the extracellular matrix have been shown to act as chemoattractant molecules (see review in Tessier-Lavigne, 1994). At least one growth factor, brain-derived neurotrophic factor, is expressed in the Xenopus tectum (Cohen-Cory and Fraser, 1994), but at a later stage when RGC axons have already reached the tectum. In fact, in the developing Xenopus visual system, experimental evidence does not support the existence of a target-derived attractant factor (Harris et al., 1985; Taylor, 1990). Certainly, since bFGF levels are low in the tectum, bFGF does not appear to serve this function. We suggest alternatively, that the absence of a specific growth factor in the target that was present in the pathway may signal to axons that they have reached their destination.

Experimental Procedures Animals

Eggs were obtained from adult Xenopus laevis stimulated to breed

by treatment with human chorionic gonadotropin. Embryos were raised in 10% Holtfreter's solution (Holtfreter, 1943) at 14°C-25°C and staged according to the Nieuwkoop and Faber staging tables (Nieuwkoop and Faber, 1967). Retinal Cell Cultures

Eye primordia were dissected from stage 25 embryos and cultured as described previously (Harris et al., 1985; Harris and Messersmith, 1992). Dissociated cells or entire eyes were plated onto polyornithine/ laminin-coated coverslips in 35 mm petri dishes containing 2 ml of culture media. Culture media consisted of 60% L15 (CORE cell culture facility, UCSD) supplemented with 10% fetal bovine serum (Gemini Products), 1% tungibact (CORE cell culture facility), and 1% embryo extract (Harris et al., 1985). Some cultures were grown in the absence of serum and embryo extract, and in either the presence or absence of 20 ng/ml human recombinant bFGF (GIBCO) and 0.1% BSA (fraction V, Fisher Scientific). Dissociated and explant cultures were fixed for 45 rain in 2% paraformaldehyde. Neurites were drawn using camera lucida and measured using a flexible ruler. Bathing Media and Growth Factors

The exposed brain preparation was performed largely as described previously (Chien et al., 1993). Briefly, embryos were anesthetized in modified Barth's saline (Gurdon, 1977) supplemented with 0.4 rng/ml tricaine (ethyl 3-aminobenzoate methanesulfonie acid, Aldrich), 1% Fungibact, 50 mg/ml gentamicin sulfate (Gemini Products), and 10 mg/rnl phenol red. The embryos were pinned in a Sylgard dish (K. R. Anderson Co.), and the skin and eye over the left brain were removed. This procedure exposes the entire anterior brain on one side, reaching as far caudal as the posterior rectum. Surgery was performed on all embryos before they were randomly divided to develop in either experimental or control solutions for another 18-24 hr until stage 40. Control bath solution consisted of modified Barth's saline (pH 7.4), 0.1 mg/ml tricaine, and 0.1% BSA. To make the experimental bath solutions, different growth factors were added to this control solution: recombinant XbFGF (0.05-5 nM, 1-100 ng/ml, kindly provided by both J. Slack and D. Kimelman); human bFGF (100 ng/ml); EGF (100-500 ng/ml, murine natural, GIBCO); 7S NGF (250 ng/ml, murine natural, GIBCO); and aFGF (100-500 ng/ml, human recombinant, GIBCO). Visualization of the Optic Projection

To visualize the optic projection, RGC axons were labeled using horseradish peroxidase (HRP, type VI; Sigma) as described previously (Cornel and Holt, 1992). The lens of the right eye was surgically removed and HRP, dissolved in 1% lysolecithin, was placed in the eye cavity. After allowing time for anterograde labeling of RGCs (25 min), embryos were fixed at room temperature in 1% gluteraldehyde in 0.1 M sodium phosphate buffer (pH 7.4). After fixation, brains were washed in phosphate-buffered saline, reacted with diaminobenzidine (Sigma), dehydrated through a graded series of alcohols, and cleared in 2:1 benzyl benzoate:benzyl alcohol. They were mounted in Permount (Fisher Scientific) under a coverstip supported by two plastic reinforcement rings (Avery). The outlines of brains and optic projections were drawn using a camera lucida attachment on a Leitz microscope. Quantitation of Optic Projection Width

Width measurements were made along the full contralateral extent of optic projections in control and bFGF-treated brains. Camera lueida representationsof mounted brains were scanned (ScanJet IIc, HewlettPackard) to provide digital images. Samples were used only if they were mounted without significant rolling and had well-filled optic projections. Analysis was performed on a Macintosh Quadra 700 computer using the public domain NIH Image program (National Institutes of Health). Macros were used to normalize brain size by rotating and scaling them to a line drawn between the anterior optic chiasm and the midbrain-hindbrain isthmus and matching this line to a standard reference line (Chien et al., 1993). The optic ehiasm and the isthmus were chosen as easily identified and reliable morphological markers in the Xenopus brain. The reference line was used to define an artificial unit, a brain reference unit (bru); one bru is -620 pm in an unfixed brain (Chien et al., 1993). This unit line was divided into 0.1 intervals through which concentric circles were drawn (see Figure 5B). The projection width was measured as the widest part of the tract within

Neuron 1026

bins centered around each concentric circle (for example, 0.5-0.15, 0.15-0.25, etc.). The lateral boundaries of the projection were defined by the presence of >3 axons.

Immunofiuorescence Fixed embryos were washed in 0.1 M sodium phosphate buffer (pH 7.4). Some were sunk in 30% sucrose, embedded in optimal cutting temperature (Baxter), and quick frozen at -20°C; and 12 I~m sections were cut on a Slee cryostat and collected on gelatin-coated slides. Others had their brains dissected out and processed as whole mounts. Standard immunostaining procedures were used for cryostat sections, whole-mount brains, and cultures (Cornel and Holt, 1992). Samples were incubated overnight at 4°C in the primary antibody diluted in PBT (phosphate buffered saline, 0.2% BSA, with or without 0.2% Triton X-100; Sigma)with 5o/0 goat serum (Gemini Products). Samples were incubated with a secondary fluorescent antibody for 1 hr at room temperature. After washing, samples were mounted in glycerol with an antibleaching agent, p-phenylenediamine (1 mg/ml in 9 parts glycerol, 1 part 1 M Tris-HCI; pH 8.5; Sigma). Cultures were frequently counterstained with RITC-conjugated phalloidin (Sigma) added to the secondary incubation at a dilution of 1:100, to help visualize growth cones. Samples were photographed either with a camera attachment on a Zeiss Axioskop or using a cooled CCD camera (Spectrasource) attached to a Nikon optiphot-2 microscope. Photographs taken with the CCD camera were captured using a Gateway 2000 PC and processed using NIH Image and Adobe Photoshop software.

Antibodies We used a rabbit polyclonal anti-Xenopus bFGF antibody (provided by D. Kimelman) that recognizes a protein of 19 kDa (corresponding to bFGF) on a Western blot (data not shown). The specificity of the antibody was demonstrated by successful elimination of the 19 kDa band by preabsorption of the antibody with bFGF for 2 hr at room temperature. For immunostaining and Western blots, the serum antibody was diluted 1:500 with PBT and 5% goat serum and then preabsorbed with a homogenate of - 30 embryos (stages 30-40) in a volume of 1 ml for 2 hr at room temperature. This procedure eliminated background staining on the Western. For FGFR labeling, we used an affinity-purified rabbit polyclonal antibody against the Xenopus FGFR (T. Musci; Amaya et al., 1993) at a concentration of 1:400. This antibody was generated against a glutathione transferase partial FGFR fusion protein that includes most of the external portion of the FGFR, the transmembrane domain, and a portion of the intracellular domain including the tyrosine kinase domains (Amaya et al., 1991). Occasionally, eyes were filled with HRP before fixation. Double labeling with an antibody against HBP (Sigma, dilution 1:500) and either anti-bFGF or anti-FGFR allowed visualization of the optic projection relative to bFGF and FGFR staining. A rabbit polyclonal antibody against Xenopustenascin (kindly provided by J. Riou)was used at a dilution of 1:250. Fluorescein isothiocyanate-conjugated or rhodamine isothiocyanateconjugated goat antirabbit secondary antibodies (Jackson Laboratories) were used at a dilution of 1:500. A monoclonal antibody against the engrailed protein (4D9, provided by N. Patel) was used for immunocytochemistry and Western analysis at a dilution of 1:500. For analysis of engrailed levels, Western blots were scanned to make digital images, and the intensity of the bands was determined using NIH Image gel analysis macros.

BrdU Labeling Brains were exposed to control, bFGF (100 ng/ml) or EGF (200 ng/ ml) solutions at stage 33•34. At stage 39, the gut of each embryo was injected with 5 mg/ml BrdU diluted in water and phenol red (to visualize injections). Three hours after injection, embryos were fixed in 4% paraformaldehyde for 4 hr at room temperature and processed for paraffin sectioning. Sections (12 ~m)were im munostained as described above. An anti-BrdU monoclonal antibody (Sigma) was used at a dilution of 1:10. The secondary was a rhodamine isothiocyanate-conjugated goat antimouse antibody used at a dilution of 1:500. Photos of serial sections were taken and BrdU-positive ceils were counted in every second section starting from the midbrain- hindbrain isthmus (back of tectum) and moving in an anterior direction. For each embryo a total of 4 sections were analyzed.

Acknowledgments We would like to thank B. Harris, C.-B. Chien, S. Retaux, R. Wilson, and R. Riehl for comments and advice. Elsa Cornel provided excellent technical assistance. David Kimelman very generously provided us with the Xenopus-specific bFGF antibody and with Xenopus recombinant bFGF. We would like to thank Tom Musci for his gift of a Xenopusspecific FGFR antibody, J. Riou for the Xenopus tenascin antibody, J. Slack for Xenopus recombinant bFGF, and A. Baird for the rat bFGF antibody. S. McFarlane is funded by a postdoctoral fellowship from MRC of Canada, and the work was supported by NIH grant NS23780 and a Pew Scholars Award. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC Section 1734 solely to indicate this fact. Received May 25, 1995; revised July 19, 1995.

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