PTPμ signaling via PKCδ is instructive for retinal ganglion cell guidance

www.elsevier.com/locate/ymcne Mol. Cell. Neurosci. 25 (2004) 558 – 571

PTPM signaling via PKCD is instructive for retinal ganglion $ cell guidance Sonya E. Ensslen and Susann M. Brady-Kalnay * Department of Neurosciences, School of Medicine, Case Western Reserve University, Cleveland, OH 44106-4960, USA Department of Molecular Biology and Microbiology, School of Medicine, Case Western Reserve University, Cleveland, OH 44106-4960, USA Received 16 October 2003; revised 24 November 2003; accepted 3 December 2003

The receptor protein tyrosine phosphatase (RPTP) PTPM mediates distinct cellular responses in nasal and temporal retinal ganglion cell (RGC) axons. PTPM is permissive for nasal RGC neurite outgrowth and inhibitory to temporal RGCs. In addition, PTPM causes preferential temporal growth cone collapse. Previous studies demonstrated that PTPM associates with the scaffolding protein RACK1 and the protein kinase C-D (PKCD) isoform in chick retina and that PKCD activity is required for PTPM-mediated RGC outgrowth. Using in vitro stripe and collapse assays, we find that PKCD activity is required for both inhibitory and permissive responses of RGCs to PTPM, with higher levels of PKCD activation associated with temporal growth cone collapse and repulsion. A potential mechanism for differential PKCD activation is due to the gradient of PTPM expression in the retina. PTPM is expressed in a high temporal, low nasal step gradient in the retina. In support of this, overexpression of exogenous PTPM in nasal neurites results in a phenotypic switch from permissive to repulsive in response to PTPM. Together, these results suggest that the differential expression of PTPM within the retina is instructive for RGC guidance and that the magnitude of PKCD activation in response to PTPM signaling results in the distinct cellular behaviors of nasal and temporal RGCs. D 2004 Elsevier Inc. All rights reserved.

Introduction Axons are guided to their targets by cues in the extracellular environment that bind receptors on the surface of growth cones (Goodman, 1996; Skaper et al., 2001; Tessier-Lavigne and Goodman, 1996). Members of the receptor protein tyrosine phosphatase (RPTP) family have been demonstrated to function as target recognition or ‘‘stop’’ signals in vivo (for a review, see Johnson and Van Vactor, 2003). Loss of function mutations of RPTPs in Drosophila result in bypass neuron guidance phenotypes suggesting that RPTPs instruct neurons to ‘‘stop’’ and innervate their

$ Supplementary data associated with this article can be found at doi: 10.1016/S1044-7431(03)00377-4. * Corresponding author. Department of Molecular Biology and Microbiology, School of Medicine, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, OH 44106-4960. Fax: +1-216-368-3055. E-mail address: [email protected] (S.M. Brady-Kalnay). Available online on ScienceDirect (www.sciencedirect.com.)

1044-7431/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.mcn.2003.12.003

targets (Clandinin et al., 2001; Garrity et al., 1999; Maurel-Zaffran et al., 2001; Sun et al., 2001). The RPTP, PTPA, has been shown to regulate retinal ganglion cell (RGC) neurite outgrowth in vitro (Burden-Gulley and Brady-Kalnay, 1999; Burden-Gulley et al., 2002) and axon growth within the vertebrate retina (Ensslen et al., 2003). PTPA is a tyrosine phosphatase (Brady-Kalnay and Tonks, 1993) that mediates cell – cell adhesion (Brady-Kalnay et al., 1993). In the developing chick visual system, PTPA promotes neurite outgrowth from a subset of RGCs (Burden-Gulley and Brady-Kalnay, 1999), associates with the cadherin – catenin complex (Brady-Kalnay et al., 1995, 1998), and regulates N-cadherinmediated neurite outgrowth (Burden-Gulley and Brady-Kalnay, 1999). The only known ligand of PTPA is itself, that is, it is a homophilic binding protein (Brady-Kalnay and Tonks, 1994; Brady-Kalnay et al., 1993). The precise signaling pathway triggered in response to PTPA homophilic binding is not known. However, PTPA-interacting proteins have been identified, including the receptor for activated C kinase 1 (RACK1) (Mourton et al., 2001). RACK1 was originally identified as a protein that binds to PKC h, y, and q isoforms (Ron et al., 1994) but has subsequently been shown to be a scaffolding protein for other signaling molecules (reviewed in Schechtman and Mochly-Rosen, 2001). RACK1 binding to activated PKCs results in their translocation to the plasma membrane, regulating PKC substrate availability (reviewed in Jaken and Parker, 2000). PTPA associates with RACK1 and protein kinase C-y (PKCy) in retinal cells, and PKCy activity is required for retinal ganglion cell (RGC) neurite outgrowth on a PTPA substrate (Rosdahl et al., 2002). Embryonic day 8 of chick development corresponds to the peak period of retinotectal pathfinding (Thanos and Mey, 2001). At this time, PTPA is expressed in a high-temporal, low-nasal gradient in the retina (Burden-Gulley et al., 2002). There is also a gradient of PTPA expression in the target of the RGCs, the tectum, corresponding to high anterior low posterior expression (BurdenGulley et al., 2002). PTPA elicits distinct behavioral responses in nasal and temporal RGC axons in vitro (Burden-Gulley et al., 2002). For example, nasal neurites grow on a purified PTPA substrate, whereas temporal neurites avoid PTPA (Burden-Gulley et al., 2002). Furthermore, temporal growth cones collapse in response to PTPA whereas nasal growth cones do not (Burden-

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Gulley et al., 2002). In vivo, PTPA may act as an instructive ‘‘stop’’ cue for temporal RGC axons to innervate the anterior tectum. Both the in vivo function of PTPA in axon guidance and the molecular mechanisms responsible for producing these distinct behaviors in nasal and temporal RGCs in response to PTPA are unknown. In this manuscript, we demonstrate that the magnitude of PKCy activation regulates both nasal outgrowth and temporal repulsion in response to PTPA. In addition, our results demonstrate that the gradient of PTPA expression in the retina accounts for the differences in PKCy activation in nasal and temporal retina. In fact, a phenotypic switch from growth permission to repulsion is observed in nasal neurites following PTPA overexpression. We demonstrate that the ability of overexpressed PTPA to induce nasal growth cone collapse requires PKCy activation. This study emphasizes that the magnitude of similar signals, in this case PTPA and PKCy, can yield highly distinct cellular responses that are important for axon guidance.

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marked bundling or fasciculation of nasal neurites on the laminin lanes (Fig. 1c). Temporal RGC neurites have previously been described to be highly fasciculated in the stripe assay (Walter et al., 1987b). When nasal neurites are presented with two growth permissive substrates, they do not tend to fasciculate (Walter et al., 1987b). However, when only one growth permissive substrate remains, the neurites do fasciculate (Drescher et al., 1995), as in Fig. 1c. This is supported by previous studies that demonstrated that RGC tend to be highly fasciculated on a laminin substrate (Lemmon et al., 1992). Inhibition of PKCy activity with the antagonist peptide did not alter the behavior of temporal neurites (Figs. 1d and e) due to the lack of a growth permissive substrate (see below). Addition of an inhibitor peptide to another novel PKC isoform, PKCq, or a scrambled PKCq peptide did not alter the crossing behavior of nasal or temporal neurites (Fig. 1e). Therefore, PKCy antagonist peptide and the chemical inhibitor Rottlerin (Rosdahl et al., 2002) both block nasal neurite outgrowth on a PTPA substrate (Fig. 1f).

Results PKCd isoform-specific antagonist peptides block nasal neurite crossing on PTPl The Bonhoeffer stripe assay (Vielmetter et al., 1990; Walter et al., 1987a,b) examines the ability of neurons to grow on two substrates presented in alternating lanes. As previously described (Burden-Gulley et al., 2002), nasal neurites readily cross between PTPA and a growth permissive molecule, such as laminin (Fig. 1a), whereas temporal neurites avoid the PTPA substrate lanes (Fig. 1b) and grow preferentially on the laminin lanes. When the stripe assay is quantified for the average degree of avoidance (for details, see Experimental methods), nasal neurites show little preference for either substrate (mean degree of avoidance = 0.3) whereas temporal axons avoid the PTPA lanes (mean degree of avoidance = 2.6, P < 0.0001; Fig. 1e). It is clear from this preferential temporal repulsion that PTPAdependent signaling in the temporal retina must be distinct from the PTPA-dependent signals generated in the nasal retina. PKCy activity has been shown to be required for neurite outgrowth on a PTPA substrate using the PKCy-specific chemical inhibitor Rottlerin (Rosdahl et al., 2002). To examine the role of PKCs in mediating distinct temporal and nasal responses to PTPA, we perturbed PKC activity with isoform-specific PKC antagonist peptides. The PKC family is divided into three groups: classical, novel, and atypical (Newton, 2001). When activated, PKCs are translocated to the plasma membrane by scaffolding proteins known as RACKs (Mochly-Rosen et al., 1991). This permits phosphorylation of physiological substrates (Newton, 2001). The isoform-specific peptide antagonists prevent association between PKCs and their appropriate RACKs to block PKC function (Chen et al., 2001). The peptide modulators of PKC are conjugated to a peptide derived from the Drosophila antennapedia transcription factor to mediate direct protein transduction into cells (Chen et al., 2001). To verify that activity of the novel PKC isoform, PKCy, is required for nasal neurite outgrowth on PTPA and that the antagonist peptides functioned in our system, we added the isoform-specific PKC antagonist peptides to explants growing on alternating lanes of laminin and PTPA. As predicted, inhibition of PKCy signaling prevented nasal neurite crossing onto the PTPA lanes (Figs. 1c and e). In addition to reducing nasal neurite outgrowth on PTPA, blocking PKCy activity also resulted in a

Signaling via PKCd regulates temporal growth cone avoidance of PTPl When mixed substrate lanes of PTPA and laminin are placed on alternating lanes next to laminin only lanes, nasal neurites readily grow between the laminin and the mixed PTPA/laminin lanes (mean degree of avoidance = 0.9; Figs. 2a and e), while temporal neurites avoid the PTPA/laminin mixed lanes (mean degree of avoidance = 2.9; Figs. 2b and e). This demonstrates that the temporal repulsive PTPA signal is dominant over the permissive laminin signal (Fig. 2f). As a test for the efficacy of the mixed substrate assay, we blocked the known PTPA-dependent nasal signal, PKCy (Fig. 1), and examined whether nasal neurites were able to cross onto the mixed PTPA/laminin lanes. PKCy inhibition has no effect on laminin-dependent neurite outgrowth (Rosdahl et al., 2002). The laminin cue in the mixed lanes should be growth permissive to nasal neurites, although the signal required to respond to the growth permissive PTPA component is blocked (Fig. 2f). As predicted, in the presence of the PKCy antagonist, nasal neurites are able to cross over the PTPA/laminin lanes (Figs. 2c and e). In addition, inhibition of PKCy activity induced temporal neurite crossing over the PTPA/laminin lanes (mean degree of avoidance = 0.8 versus 2.9 in the controls, P < 0.0001; Figs. 2d and e). This indicates that PKCy activity is required for the repulsive PTPA signal (Fig. 2f). In the single substrate stripe assays shown in Fig. 1d, temporal neurites were unable to cross over the PTPA lanes because no permissive growth cue remained in the PTPA lanes following PKCy inhibition. Inhibition of the novel PKC isoform, PKCq, had no effect on nasal or temporal crossing (Fig. 2e), suggesting that PKCy is the specific PKC isoform required for PTPA-mediated repulsion. Another standard assay to test growth inhibitory cues is the growth cone collapse assay (Cox et al., 1990; Raper and Kapfhammer, 1990). The collapse assay tests the ability of an exogenous protein to induce a response in a growth cone’s morphology even in the presence of highly growth permissive substrates, such as laminin (Cox et al., 1990; Raper and Kapfhammer, 1990; Stepanek et al., 2001). It was developed as a means of identifying proteins that could function as inhibitory guidance molecules in vivo (Cox et al., 1990; Raper and Kapfhammer, 1990). Changes in growth cone morphology can also be viewed in time-lapse movies of growth cones in culture. Following stimulation with media

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Fig. 1. PKCy activity is required for nasal neurites to grow on PTPA substrate lanes. E8 retinal explants were grown on alternating stripes of PTPA and laminin in the presence of either a control antennapedia peptide (a and b) or a PKCy antagonist peptide (c and d). Phase contrast images show neurite outgrowth, while the fluorescent images (right side of the panel) illustrate the alternating substrate lanes of PTPA and laminin/Texas Red BSA. Inhibition of PKCy activity with the antagonist peptide reduced nasal crossing onto PTPA lanes (c). Inhibition of PKCy activity with the antagonist peptide did not alter the behavior of temporal neurites due to the lack of a growth permissive substrate (d). The average degree of avoidance (mean F SEM) was plotted for each condition (e). Treatment of nasal neurites with the PKCy antagonist prevented nasal crossing over PTPA lanes and was statistically different from nasal neurite behavior following control peptide, PKCq antagonist, or PKCq scrambled peptide addition (Fisher’s PLSD, *P < 0.0002). None of the peptide antagonists altered the behavior of temporal neurites. The behavior of nasal and temporal neurites in the stripe assay is schematically shown in (f). Nasal neurites are able to cross between PTPA and laminin lanes because PTPA is permissive. Temporal neurites avoid PTPA lanes. Blocking PKCy activity reduces nasal neurite crossing of PTPA lanes but does not change the behavior of temporal neurites to PTPA. The results suggest PKCy activity is required for the PTPA permissive signal. Scale bar: 100 Am.

alone, both nasal (Supplemental movie 1) and temporal (Supplemental movie 2) growth cones display prominent lamellipodial and filopodial activity and forward growth. Nasal growth cones respond to a PTPA stimulus as they do to the control stimulus, with prominent lamellipodial veil movement and forward growth (Supplemental movie 3). Stimulation of temporal growth cones with PTPA, however, induces growth cone collapse, characterized by the loss of all lamellipodial veils and neurite retraction (Supplemental movie 4). These movies are in agreement with our previously published static images (Burden-Gulley et al., 2002), demonstrating that PTPA is only inhibitory to temporal RGC growth cones. Furthermore, these movies confirm that the PTPA-collapsing signal is dominant over laminin, as the growth cones are growing on a laminin substrate.

To examine whether PKCy activity is also required for PTPAmediated growth cone collapse, collapse assays were performed in the presence of the isoform-specific peptide antagonists of PKCy and PKCq. Retinal explants treated with the control antennapedia only peptide then stimulated with exogenous PTPA display preferential collapse of temporal growth cones over nasal growth cones similar to untreated neurons (Fig. 3, 47.5% for temporal versus 19.7% for nasal). Treatment with the PKCy-specific peptide antagonist (conjugated to antennapedia) before PTPA stimulation prevented temporal growth cone collapse in response to PTPA (Fig. 3, 15.4% versus 47.5% for control). Inhibition of PKCy with a PKCy-specific peptide antagonist conjugated to another protein transduction sequence, TAT (Chen et al., 2001), also prevented temporal growth cone collapse (data not shown). Treatment with

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Fig. 2. PKCy activity is required for temporal avoidance of PTPA. E8 retinal explants were grown on alternating stripes of laminin and a PTPA/laminin (PTPA/ LN) mixture. Nasal neurites treated with the control peptide readily crossed between the substrate lanes (a and e) because both PTPA and laminin are permissive to nasal neurons. Temporal neurites grow exclusively on the laminin only lanes (b and e), demonstrating that the PTPA inhibitory signal was dominant over the laminin permissive signal. Treatment with the PKCy antagonist results in extensive nasal neurite crossing over PTPA/LN lanes (c), suggesting that nasal neurites were able to grow on the laminin component of the PTPA/LN mixture when the PKCy signal necessary for growth on PTPA was blocked. Treatment with the PKCy antagonist likewise resulted in extensive crossing of temporal neurites (d), indicating that the growth inhibitory cue previously present in the PTPA lanes was abolished and that temporal neurites could now respond to the growth permissive laminin cue in the mixed substrate lanes. The average degree of avoidance (mean F SEM) was plotted for each condition (e). The tendency of temporal neurites to avoid the PTPA substrate lanes was significantly decreased following the inhibition of PKCy activity with the PKCy antagonist peptide (Fisher’s PLSD, *P < 0.0001). The behavior of nasal and temporal neurites in the mixed stripe assay is schematically shown in f. Blocking the PTPA-dependent PKCy signal induces nasal and temporal neurites to cross over the PTPA/LN lanes using the laminin permissive cue. These results suggest PKCy activity is required for the PTPA inhibitory signal. Scale bar: 100 Am.

the PKCq antagonist peptide did not change temporal growth cone collapse in response to PTPA (Fig. 3, 48.3% versus 47.5% for controls). Therefore, PKCy activity is required for temporal growth cone collapse in response to PTPA. The magnitude of PKCd activation is greater in temporal than in nasal RGCs Surprisingly, PKCy activity is downstream of both PTPAmediated growth permissive (Fig. 1c) and repulsive (Figs. 2d and 3) events. PTPA is expressed in a high-temporal, low-nasal gradient

in the E8 retina (Burden-Gulley et al., 2002). Because PTPA is differentially expressed in the retina, the magnitude of PTPAdependent signals may be differentially activated in temporal and nasal growth cones. To examine whether PKCy activation is different in nasal versus temporal growth cones in response to exogenous PTPA stimulation, growth cones were immunolabeled with antibodies to PKCy and to an activated form of PKCy, which is phosphorylated on threonine 505 (Newton, 2001). Following PTPA stimulation, temporal growth cones preferentially collapse (Fig. 3). The morphology of PTPA-collapsed temporal growth cones is small, often club shaped, and devoid of any lamellipodia

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the average intensity of the immunolabel normalized to the surface area of the growth cone for each stimulation condition. The average fluorescence intensity per unit area of the growth cone verified that phospho-PKCy labeling is fivefold greater in PTPAstimulated temporal growth cones than in control-stimulated temporal growth cones (Fig. 4b; P < 0.0001). Stimulation of growth cones with a control stimulus results in no difference in either PKCy or phospho-PKCy immunolabeling in nasal and temporal growth cones (Figs. 4a and b). To ensure that PKCy is not activated in all growth cones with a collapsed morphology, we infected retinal explants with a constitutively active mutant form of RhoA (CA-RhoA). This mutant induces a collapsed growth cone morphology in nasal and temporal neurons

Fig. 3. PKCy activity is required for temporal growth cone collapse. E8 retinal explants were grown on a laminin substrate for 17 h, treated with either a control antennapedia peptide (white bars), a peptide antagonist to PKCy (black bars), or PKCq (grey bars) for 3 h before stimulation with PTPA or control for 10 min. The percentage of collapsed growth cones (mean F SEM) is shown for each condition and analyzed with Fisher’s PLSD at a 95% confidence level. Temporal growth cones collapse in response to PTPA stimulation (47.5%) in the presence of the control peptide, whereas nasal growth cones do not. Collapse of temporal growth cones was abolished following treatment with PKCy antagonist (15.4%, *P < 0.0001) but was not affected by treatment with the PKCq antagonist (48.3%). Nasal growth cone behavior to PTPA was not altered in the presence of any of the peptide antagonists.

or filopodia (see Supplemental movie 4 for an example). Nasal growth cones, however, tend to have broad lamellipodia and prominent filopodia (see Supplemental movie 3). PTPA stimulation of temporal growth cones growing on laminin results in a marked increase in phospho-PKCy labeling compared to nasal PTPAstimulated growth cones (Fig. 4a). To compare the differences in immunolabeling of PKCy and phospho-PKCy in nasal and temporal growth cones, we determined

Fig. 4. PKCy is preferentially activated in temporal growth cones in response to PTPA stimulation. E8 retinal explants were grown on laminincoated glass dishes for 20 h, then stimulated with either control or PTPA for 10 min before fixation and immunolabeling with PKCy and phosphoPKCy antibodies (a). In addition, retinal explants were infected with a constitutively active mutant form of RhoA (CA-RhoA) to induce collapsed growth cone morphology. Phosphorylated PKCy, which represents activated PKCy, is higher in PTPA-collapsed temporal growth cones than in any other condition. CA-RhoA-induced collapse of growth cones does not increase PKCy activation indicating that the PKCy increased activity is a specific response to a PTPA signal. The fluorescence intensity for each immunolabel was normalized to the unit area of the growth cone and plotted (mean F SEM) in b. An average of 15 growth cones were evaluated for each condition. Phospho-PKCy intensity is approximately fivefold greater in PTPA-stimulated temporal than in control-stimulated and CA-RhoA-infected temporal growth cones (Fisher’s PLSD, P < 0.0001). This demonstrates that PKCy is preferentially activated in temporal growth cones following PTPA stimulation. Dashed lines indicate the boundaries of the growth cone as seen in phase contrast images. Scale bar: 60 Am.

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characterized by the lack of lamellipodia and filopodia (Fig. 4a). Very little phospho-PKCy is observed in CA-RhoA-infected growth cones (Figs. 4a and b) despite the presence of PKCy in these growth cones (Figs. 4a and b grey bars). Therefore, PKCy is not activated in all growth cones with a collapsed morphology. These data suggest an explanation for the difference in nasal and temporal RGC growth cone behavior in response to PTPA; high PKCy activity correlates with PTPA-mediated growth cone collapse and repulsion, while low levels of PKCy activity correlate with PTPA-mediated promotion of neurite outgrowth.

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assay by increasing PKCy activity using a PKCy-specific agonist peptide (Chen and Mochly-Rosen, 2001). When the PKCy agonist peptide is added to retinal explants growing on alternating lanes of laminin and PTPA only, nasal neurites avoid the PTPA substrate lanes (mean degree of avoidance = 2.6 versus 0.3 for control; Figs. 5c and e). PKCy agonist-treated temporal neurites continue to avoid the PTPA lanes (Figs. 5d and e). These data demonstrate that high levels of PKCy activity induce repulsion to PTPA substrate lanes (Fig. 5f). Increasing expression of PTPl in the retina induces nasal growth cone collapse and avoidance

PKCD agonist treatment of retinal explants induces nasal neurite repulsion to PTPm The hypothesis that high levels of PKCy activation lead to growth cone collapse in response to PTPA was tested in the stripe

PKCy activity is higher in temporal than in nasal growth cones following PTPA stimulation (Figs. 4a and b). The high-temporal, low-nasal gradient of PTPA expression in the retina suggests that more PKCy may be activated in temporal retina because of the

Fig. 5. High levels of PKCy activity in nasal retina induce nasal neurite avoidance of PTPA in the stripe assay. E8 retinal explants were grown on alternating stripes of PTPA and laminin and treated with the PKCy agonist peptide (c and d) or with the antennapedia alone (a and b). Treatment with the PKCy agonist induces nasal neurite avoidance of PTPA lanes (c). Temporal neurite behavior is unchanged (d). The degree of avoidance for each of the treatments was compared and plotted (mean F SEM) for the various conditions (e). PKCy agonist-treated nasal neurite avoidance is significantly different from control peptide-treated nasal neurites (Fisher’s PLSD, *P < 0.0001). These data suggest that sustained high levels of PKCy activity induce repulsion in response to PTPA (f). Scale bar: 100 Am.

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higher PTPA expression in temporal retina. To address this possibility, PTPA expression was manipulated in E8 retinal explants growing on alternating stripes of laminin and mixed PTPA/laminin lanes by infecting explants with a herpes simplex virus (HSV) encoding wild-type PTPA (WT-PTPA) or GFP alone. WT-PTPA overexpressing nasal neurites avoid the PTPA lanes (mean degree of avoidance = 2.4 compared to 0.6 for nasal GFPinfected controls, P < 0.0001; Figs. 6a, c, and e). Wild-type and GFP infection does not alter the behavior of temporal neurites to PTPA (mean degree of avoidance = 3.0 for both conditions, Figs. 6b, d, and e). These data demonstrate that high levels of PTPA

expression in nasal RGC axons are sufficient to switch nasal axon behavior from growth permissive to repulsive in response to PTPA (Fig. 6f). In the growth cone collapse assay, collapse of WT-PTPAinfected nasal growth cones (Fig. 7) was twofold greater than collapse of GFP-infected nasal growth cones (37% versus 16.8% for GFP-infected controls). Infection of retinal explants with WTPTPA did not influence the percent of collapsed temporal growth cones in response to PTPA (Fig. 7, 43.1% for both). This demonstrates that increasing PTPA expression in nasal retina induces nasal growth cone collapse and repulsion in response to PTPA.

Fig. 6. Increased PTPA expression in nasal retina induces nasal avoidance of PTPA lanes. E8 retinal explants were grown on alternating stripes of PTPA and PTPA/laminin and infected with either GFP (a and b) or wild-type PTPA (c and d). Infection of explants with wild-type PTPA results in a reduction of nasal crossing (c) but does not alter the behavior of temporal neurites (d). GFP-infected nasal neurites readily cross between the substrate lanes (a), while temporal neurites do not cross over PTPA lanes (b), similar to uninfected explants (Figs. 1a and b). The tendency of nasal and temporal neurites to avoid PTPA was determined. The average degree of avoidance was plotted for each condition (mean F SEM; e). Following infection with wild-type PTPA, nasal neurite avoidance of PTPA substrate lanes is significantly increased compared to GFP-infected nasal neurites (Fisher’s PLSD, *P < 0.0001). Temporal neurite behavior infected with wild-type PTPA is not different from GFP-infected temporal neurites. This demonstrates that increasing PTPA expression in nasal neurons induces repulsion in response to PTPA (f). Scale bar: 100 Am.

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Fig. 7. Increased PTPA expression in retinal explants induces nasal growth cone collapse in response to PTPA. E8 retinal explants grown on a laminin substrate were infected with either GFP (grey bars) or wild-type PTPA (black bars) for 20 h before stimulation with either a control stimulus or PTPA. The percentage of collapsed growth cones (mean F SEM) is shown for each condition. Infection with wild-type PTPA followed by stimulation with PTPA results in a statistically significant increase in nasal growth cone collapse compared to GFP-infected nasal growth cones (37% versus 16%, Fisher’s PLSD, *P < 0.0001). Collapse of temporal growth cones infected with wild-type PTPA is unaffected compared to GFP-infected controls (43.1% for both). Infection with wild-type PTPA does not alter the collapse of either nasal or temporal growth cones in response to the control stimulus compared to GFP-infected explants.

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PTPA (Fig. 9b). Inhibiting PKCy activity prevents temporal growth cone repulsion in response to PTPA (Fig. 2), while artificially increasing PKCy activity in nasal neurites induces repulsion to PTPA (Figs. 5 and 9b). A lower level of PKCy activity is required for nasal neurite outgrowth on a PTPA substrate (Fig. 9b; Rosdahl et al., 2002). Our immunocytochemistry data demonstrate that PTPA-stimulated nasal growth cones have less activated PKCy than PTPA-stimulated temporal growth cones (Figs. 4a and b). We propose that a low level of PKCy activity is sufficient to mediate nasal neurite outgrowth on PTPA (Fig. 9c). Increasing the magnitude of the PKCy signal by overexpressing PTPA (Figs. 6 and 7) or by using PKCy agonist peptides induces nasal growth cone collapse and repulsion in response to PTPA (Figs. 5 and 9). Thus, regulation of the magnitude of PKCy signaling is key to regulating PTPAmediated permissive and inhibitory guidance events. Previously, PKCs have been shown to mediate both growth permissive (Bixby and Jhabvala, 1990; Kabir et al., 2001; Kolkova et al., 2000) and inhibitory responses in neurons (Mikule et al., 2003; Powell et al., 2001; Theodore et al., 1995; Xiang et al., 2002). For nasal neurite outgrowth on PTPA, sustained low levels of PKC activity could regulate PKC substrates important for axon outgrowth, including GAP-43 and MARCKS (Jaken and Parker, 2000). Higher levels of PKC activation could result in the phosphorylation of unique PKC substrates or a greater phosphorylation of the same substrates. Interestingly, PKC activation has

PKCd activity is required for the nasal collapse induced by increased PTPl expression To determine whether PKCy is directly activated downstream of high levels of PTPA-binding to mediate growth cone collapse, WT-PTPA overexpressing retinal explants were treated with either the PKCy or PKCq antagonist peptide, then subjected to the collapse assay (Fig. 8). Inhibition of PKCy activity in WT-PTPA overexpressing explants results in a significant reduction in nasal (23.7% versus 48.1% for PKCq antagonist, P < 0.0001) and temporal (24.5% versus 57.7% for PKCq antagonist, P < 0.0001) growth cone collapse in response to PTPA stimulation (Fig. 8). This demonstrates that increasing PTPA expression in nasal neurons induces collapse via a PKCydependent mechanism.

Discussion PTPA is a homophilic adhesion molecule that is a permissive substrate for nasal RGCs (Fig. 1a; Burden-Gulley et al., 2002) and an inhibitory substrate for temporal RGCs (Fig. 1b; Burden-Gulley et al., 2002). Our model (Fig. 9c) proposes that PTPA – PTPA homophilic binding activates PKCy, likely via RACK1 (see below), and that the magnitude of the PKCy signal generated determines whether the neurite exhibits a growth permissive or inhibitory response to PTPA. High PTPA expression in temporal RGC neurites corresponds to repulsion and growth cone collapse in response to PTPA (Figs. 1 – 3, 6, and 9a). High PTPA expression in the growth cone correlates with high PKCy activation (Figs. 4 and 9c), which results in temporal neurite repulsion in response to

Fig. 8. PKCy activity is required for nasal growth cone collapse induced by wild-type PTPA overexpression. E8 retinal explants infected with either GFP or wild-type PTPA were treated with either PKCq (grey bars) or PKCy (black bars) antagonist peptides before stimulation with PTPA or a control. The percentage of collapsed growth cones (mean F SEM) is shown for each condition and analyzed with Fisher’s PLSD at a 95% confidence level. Treatment of the wild-type PTPA-infected explants with the PKCy antagonist peptide reduced the collapse of both nasal (from 48.1% in PKCq antagonists treated to 23.7%) and temporal (from 57.7% to 24.5%) growth cones in response to PTPA (*P < 0.0001). PKCq antagonist treated, GFP-infected temporal growth cones collapse preferentially in response to PTPA (48.8%) compared to GFP-infected nasal growth cones (22.0%) similar to uninfected neurons. Addition of the PKCy antagonist peptide to GFP-infected explants reduced the collapse of temporal growth cones to PTPA (26.0%, *P < 0.0001). These results demonstrate that inhibition of PKCy activity is required for the elevated collapse of nasal growth cones following increased PTPA expression.

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Fig. 9. Model of PTPA growth permissive and repulsive signaling events in nasal and temporal retina. In a and b, the stripe assays represent single PTPA (grey) stripes versus laminin (white) stripes, except for the last set of stripe assays that represents mixed PTPA + laminin (grey) versus laminin (white) stripe. The level of PTPA expression and PKCy activity is represented by the grey gradient in the retina (i.e., high PTPA and PKCy activity are represented with dark grey shading). PTPA is more highly expressed in temporal (T) than in nasal (N) retina (a). PTPA causes the preferential repulsion of temporal growth cones but is permissive to nasal growth cones. Overexpression of PTPA (indicated by darker grey shading in entire retina) in nasal retina can induce nasal neurite repulsion to PTPA (a). One potential mechanism for PTPA-mediated repulsion is by regulating the magnitude of the PKCy signal activated in response to PTPA signaling (b). Low levels of activated PKCy in nasal retina (light grey shading) are permissive for crossing onto PTPA, while high levels of PKCy activity in temporal retina (dark grey shading) are repulsive (b). Increasing PKCy activity with a PKCy agonist (dark grey shading throughout retina) induces nasal neurite repulsion in response to PTPA. The PKCy antagonist (no shading) blocks both the permissive and repulsive signals in response to PTPA (b). Lower levels of PTPA expression results in a low level of PKCy activation required for permissive nasal neurite outgrowth (c). Higher PTPA expression levels yield a greater magnitude of PKCy activation, leading to temporal growth cone collapse and repulsion (c). Therefore, our model suggests that the magnitude of the PKCy signal generated in response to PTPA-PTPA homophilic binding regulates whether the neurite response is permissive or repulsive (c).

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been shown to be required for Rho kinase (ROCK) activity (Barandier et al., 2003). ROCK has been implicated in mediating growth cone collapse in response to other guidance cues (Arimura et al., 2000; Wahl et al., 2000) and therefore may be activated by high levels of PKCy activity in response to PTPA stimulation in temporal growth cones. Recent data suggest a role for PKCs in regulating RPTP-mediated processes (Stetak et al., 2001; Tracy et al., 1995), including PKCy interaction with (Stetak et al., 2001) and phosphorylation of (Tracy et al., 1995) PTPa. It is interesting to speculate therefore that PKCs may be a common signal activated downstream of RPTPs. In addition to signaling via PKCy, our model of PTPAmediated outgrowth and repulsion includes a role for the scaffolding protein RACK1. The translocation inhibitor peptides specifically block the association of PKCs with their appropriate RACK (Johnson et al., 1996; Schechtman and Mochly-Rosen, 2001; Yedovitzky et al., 1997), suggesting that targeting of PKCy to the plasma membrane by RACK1 is important for PTPA-mediated growth events. In addition to binding PKCy and PTPA (Mourton et al., 2001; Rosdahl et al., 2002), RACK1 also interacts with a number of signaling molecules including the src tyrosine kinase, phospholipase Cg, RasGAP, dynamin-1, h subunit of integrin receptors, and the cyclic AMP phosphodiesterase isoform 4D5 (reviewed in Schechtman and Mochly-Rosen, 2001). RACK1 contains seven WD40 repeats (Ron et al., 1994), making it possible that some of these proteins may bind to RACK1 at the same time as PTPA. Interestingly, RasGAP binding to EphB2 receptors is required for ephrin-B1-induced neurite retraction (Tong et al., 2003). Regulation of cAMP levels by cAMP phosphodiesterases (Conti et al., 2003), such as the RACK1interacting protein cAMP phosphodiesterase isoform 4D5, is also a likely mechanism for regulating growth cone guidance (see below). Therefore, additional RACK1-associated proteins may also contribute to regulating PTPA-mediated permissive and repulsive growth events. Besides PKCs, the Rho family of GTPases (reviewed in Giniger, 2002), calcium (Nishiyama et al., 2003; Zheng, 2000), cyclic nucleotides (Song et al., 1997, 1998), and MAPK kinases (Dimitropoulou and Bixby, 2000; Perron and Bixby, 1999; Tong et al., 2003) have all been shown to mediate repulsive and attractive behaviors in response to guidance cues (for a review, see Skaper et al., 2001), including those generated by RPTPs (Drosopoulos et al., 1999; Stepanek et al., 2001). In the case of cyclic nucleotides, alteration of the relative level of either cAMP or cGMP within the growth cone influences whether growth cones are repulsed or attracted by the same guidance cues (Song et al., 1997, 1998). Therefore, our PKCy data are similar to what was observed with cyclic nucleotide signaling in response to guidance cues; namely, regulation of the relative level of PKCy activation influences the behavioral responses of growth cones to be either growth permissive or repulsive. Temporal neurites are virtually all repulsed by PTPA in the stripe assay (see Figs. 1b and e); whereas in the collapse assay, only 45 – 50% of temporal growth cones collapse in response to PTPA. The discrepancy in the magnitude of the response generated between these two assays is likely twofold. First, in the stripe assay, neurites tend to fasciculate along preexisting axons (Walter et al., 1987b), thus giving the impression that all axons are affected, whereas in reality they may not be. It is likely that later growing neurites grow along preexisting neurites regardless of whether they are or are not inhibited by PTPA. Second, the growth

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cone collapse assays are calculated using static images of stimulated growth cones. Using static images to assess the extent of collapse, which is a highly dynamic process (see Supplemental movies), may result in an underestimation of the number of collapsed growth cones. Together, these two factors likely account for the apparent discrepancy in the magnitude of behavior observed in these two assays. In the retinotectal system, RGCs project to the tectum in a topographic manner, thereby maintaining positional relationships from the retina to the tectum (Thanos and Mey, 2001). For example, temporal RGC axons innervate the anterior tectum and nasal axons innervate the posterior tectum. Along the route to the tectum, chick RGC axons bundle together in the optic fissure, nerve, and tract and cross the midline at the optic chiasm. Between the retina and tectum, RGC axons are guided by a host of cues expressed near growing RGC axons and by RGC axons themselves. In the tectum, other cues direct the axons to stop and innervate their appropriate targets (Thanos and Mey, 2001). While in the optic nerve and chiasm, RGC axons fasciculate and maintain close association with one another. PTPA is expressed in a high-temporal, low-nasal gradient in the retina and this pattern is maintained in the optic nerve and chiasm (Burden-Gulley et al., 2002). The data presented here suggest that PTPA does not appear to act as a negative regulator of axon fasciculation. For example, in the stripe assays, alteration of PKCy signaling (Figs. 1, 2, and 5) or increasing PTPA expression (Fig. 6) does not interfere with the ability of RGC neurites to grow along one another in the laminin lanes. We propose that in the presence of other adhesive cues that mediate axon bundling (Rathjen et al., 1987; Rutishasuer et al., 1988) PTPA is not acting as a repulsive cue in the optic nerve. Our data suggest that PTPA functions as guidance cue, perhaps by instructing temporal RGC axons to ‘‘stop’’ and innervate their targets in the anterior tectum. This is supported by the fact that PTPA is able to mediate both permissive and inhibitory guidance events. The PTPA signal is dominant, as it is repulsive even in the presence of the highly growth permissive cue, laminin. Second, all CAMs do not mediate RGC guidance. For example, RGC neurites grow equally well on alternating stripes of laminin next to Ncadherin or L1 (Lemmon et al., 1992). Laminin is a potent neurite outgrowth-promoting molecule but does not mediate either neurite attraction or repulsion (McKenna and Raper, 1988; Sun et al., 2000). Third, studies of FGF and ephrin function demonstrate that although these cues are inhibitory in vitro (Mann et al., 2002; Webber et al., 2003), they act as target innervation cues in vivo (McFarlane et al., 1995, 1996; Mann et al., 2002). Incorrect target arborization patterns are observed following the disruption of Eph – ephrin interaction (Mann et al., 2002), and target bypass phenotypes are observed following the disruption of FGF signaling (McFarlane et al., 1995, 1996). Fourth, loss of function mutations in RPTPs often produce bypass and branching defects, suggesting that other RPTPs instruct neurons to ‘‘stop’’ and innervate their correct targets (reviewed in Johnson and Van Vactor, 2003). Finally, the gradient of PTPA expression within the retina and tectum (hightemporal retina and high-anterior tectum; Burden-Gulley et al., 2002) suggests that high levels of PTPA-PTPA homophilic binding may instruct temporal axons to ‘‘stop’’ and innervate the anterior tectum. Although Eph receptors and their ephrin ligands have been shown to regulate retinotectal pathfinding, perturbation studies of

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ephrins do not completely account for topographic map formation within the visual system (Feldheim et al., 2000). Therefore, other guidance cues, including PTPA, may be required for retinotectal projection formation. Future in ovo perturbation studies of PTPA expression and signaling in the developing visual system will illuminate whether PTPA does in fact act as an instructive guidance cue for temporal RGC axons in vivo.

Experimental methods Materials PKCy antibodies (SC-937; Santa Cruz Biotechnology, Santa Cruz, CA) and antibodies to an activated form of PKCy, phospho-PKCy threonine 505 (9374; Cell Signaling Technology, Beverly, MA), were used for immunocytochemistry. Phosphorylation of PKCy on threonine 505 by PDK-1 is necessary for activation and autophosphorylation of this enzyme (Newton, 2001). Antennapedia-conjugated peptide regulators of PKCy and PKCq, as well as antennapedia carrier peptides alone, were obtained from Dr. D. Mochly-Rosen (Stanford University, Stanford, CA). The peptide antagonists function by binding to the RACK-binding sites of PKCy and q isoforms, thus functioning as competitive inhibitors of RACK binding (Chen et al., 2001). The PKCy agonist peptide functions to destabilize the inactive conformation of PKCy (Chen et al., 2001), thereby increasing the association with PKCy and its RACK. The PKCy antagonist peptide corresponds to amino acids 8 – 17 of PKCy, while the PKCy agonist peptide corresponds to amino acids 74 – 81 of PKCy (Chen et al., 2001). The PKCq antagonist peptide corresponds to amino acids 14 – 21 of PKCq (Chen and MochlyRosen, 2001). All of these peptides were conjugated to Antennapedia sequence as described (Chen et al., 2001). Herpes simplex viruses (HSV) encoding wild-type PTPA and GFP were prepared as previously described (Ensslen et al., 2003). The Q63L constitutively active RhoA (CA-RhoA) mutant has been described (Wong et al., 2001). The pBPSTRI-CA-RhoA plasmid was digested with BamHI and NotI and ligated into pIRES-HSVGFP-MCS (Ensslen et al., 2003) to generate Q63LRho/pIRESHSV-GFP-MCS. Production of HSV encoding this mutant was performed as described (Ensslen et al., 2003). PTPA was purified from rat brain as previously described (Burden-Gulley and BradyKalnay, 1999). The PTPA-Fc chimera was described previously (Rosdahl et al., 2003). Culturing of chick retinal explants Chick embryonic day 8 (stage 32) embryos were used for all experiments and staged according to Hamburger and Hamilton (1951). Retinal explants were prepared as described (Drazba and Lemmon, 1990; Halfter et al., 1983). Briefly, neural retinas were dissected, flattened on concanavalin-coated nitrocellulose filters, and cut into 350-Am wide explants perpendicular to the optic fissure. Explants were placed retinal ganglion side down on substrate-coated dishes and grown in 10% fetal bovine serum (Atlas, Fort Collins, CO), 2% chick serum (Sigma, St. Louis, MO), 2 mM L-glutamine (Invitrogen, Carlsbad, CA), and 2 units/ ml penicillin, 2 Ag/ml streptomycin, 5 ng/ml amphotericin in RPMI-1640 (Invitrogen) for 20 h for collapse assays and immunocytochemistry, and 48 h for stripe assays.

Bonhoeffer stripe assay The substrate lane assay used was a slightly modified version of the Bonhoeffer method (Vielmetter et al., 1990), performed as previously described (Burden-Gulley et al., 2002). Briefly, tissue culture dishes were coated with nitrocellulose (Lagenaur and Lemmon, 1987) and dried before applying the silicon lane matrix to the dish surface. Laminin (40 Ag/ml) (Sigma) containing a small amount of Texas-Red conjugated bovine serum albumin (BSA) (Molecular Probes, Eugene, OR) was injected into the channels of the matrix, incubated for 7 min, aspirated then replaced with a fresh aliquot of the same substrate for several cycles. All remaining binding sites within the lanes were blocked with BSA (fraction V; Sigma) and rinsed with calcium – magnesium-free phosphate buffer (CMF). The matrix was removed and PTPA was spread across the lane area and incubated for 30 min. For mixed substrate lanes, 40 Ag/ml laminin with Texas-Red BSA (for visualization) was used as the first substrate as described above. For the second substrate in the mixed substrate experiments, 20 Ag/ml of laminin was combined with PTPA before spreading over the lane area. The entire dish was blocked with BSA, then rinsed with RPMI. Explants were cultured for 48 h before imaging. For viral perturbations, 1 Al of virus was added at the time of explanting. For antennapedia peptide transduction experiments, 250 nM peptide was added at time of explanting and fresh peptide was supplemented 24 h later. Representative images from a minimum of three separate experiments are shown. Quantitation of the stripe assays was performed using a rating scale as previously described (Walter et al., 1987b). Neurites that show no preference for either substrate are rated 0, and neurites that grow exclusively on one substrate are rated 3. A rating of 2 indicates that most of the neurites grow on the laminin lanes with an occasional neurite crossing over PTPA lanes, while a rating of 1 is given when there is a significant amount of neurite crossing but a tendency to fasciculate on laminin. Data from a minimum of three experiments were combined (with an average sample size of six explants per condition) to determine the average degree of avoidance for each condition then analyzed with Fisher’s PLSD (Statview 4.51; Abacus Concepts, Inc., Calabasas, CA) at a 95% confidence level. Growth cone collapse assay Growth cone collapse was performed as previously described (Burden-Gulley et al., 2002). Petri dishes were coated with 40 Ag/ ml laminin, blocked with BSA, and rinsed with RPMI. Retinal explants were cultured on laminin-coated dishes for 20 h before stimulation with either purified PTPA (9 Ag/ml) dialyzed in RPMI or RPMI alone for 10 min. Alternatively, 2.5 Ag/ml PTPA-Fc chimera also induces preferential temporal growth cone collapse. After 10 min, explants were fixed with 4% paraformaldehyde and 0.01% glutaraldehyde in PEM buffer (80 mM Pipes, 5 mM EGTA, 1 mM MgCl2, 3% sucrose) before viewing on a Nikon TE200 (Tokyo, Japan) inverted microscope. Both nasal and temporal growth cones were scored for collapse. Growth cone collapse (Cox et al., 1990; Raper and Kapfhammer, 1990) was defined as a complete loss of broad lamellipodial veils and filopodia, as described previously (Burden-Gulley et al., 2002). In addition, partially collapsed growth cones that had a bullet-like shape and were devoid of almost all lamellipodia were also scored as collapsed. Data from a minimum of three separate experiments were combined and analyzed using Fisher’s PLSD at a 95% confidence level. A minimum of 250 growth cones were scored per treatment.

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For viral perturbations, 1 Al of virus was added to cultures at the time of explanting, and for antennapedia peptide transduction, 250 nM peptide was added 3 h before stimulation. Movies were made by acquiring sequential images (40 magnification) of E8 nasal and temporal growth cones stimulated with either RPMI control or PTPA. Images were collected at 20 s intervals for 10 min on a Nikon inverted TE-200 microscope with a Spot-RT digital camera. The images were converted into AVI movies (3  1/30 s per frame) using Metamorph software (Universal Imaging Corp., West Chester, PA). Immunocytochemistry of retinal growth cones Glass bottom tissue culture dishes were coated with 0.1 mg/ml poly-L-lysine for 12 h, rinsed, coated with nitrocellulose then 40 Ag/ ml of laminin. Dishes were blocked with BSA and rinsed with RPMI. Retinal explants were placed on laminin-coated slides and cultured for 20 h. For constitutively active RhoA mutant experiments, retinal explants were infected with 1 Al of virus at time of plating. Purified proteins were added to the culture media for 10 min as described previously (Burden-Gulley et al., 2002). Explants were fixed with 4% paraformaldehyde and 0.01% glutaraldehyde in PEM buffer for 1 h. Explants were rinsed in phosphate-buffered saline (PBS), blocked and permeabilized with 20% goat serum, 1% BSA, and 1% saponin in PBS, and incubated overnight at 4jC in primary antibody diluted in block buffer. Explants were rinsed in TNT buffer (0.1 M Tris, 0.15 M NaCl, 0.05% Tween 20), incubated in fluorophore-conjugated secondary antibodies for 90 min at room temperature. After extensive rinsing with TNT, explants were coverslipped with Slowfade Light antifade reagent (Molecular Probes) and photographed with a Spot RT digital camera and image acquisition software (Diagnostic Instruments, Inc., Sterling Heights, MI) on a Nikon TE200 inverted microscope. All images of immunolabeled growth cones were acquired with the same settings. Analysis of immunolabel intensity was performed on a MetaMorph image analysis program (Universal Imaging). The growth cones were outlined based on phase contrast images and the intensity of the immunolabel was measured after subtracting background fluorescence intensities. The average fluorescent intensity over unit area of the growth cone was determined and population averages for each immunolabel were calculated. An average of 15 growth cones were evaluated for each condition. The data were analyzed with Fisher’s PLSD (Statview 4.51) at a 95% confidence level.

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