ATYPICAL PKC MEDIATES WNT SIGNALING IN GROWTH CONE GUIDANCE

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No. 60/822,581, filed August 16, 2006, which is incorporated herein by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with United States government support awarded by NINDS, under grant number RO1-NS47484 and a National Institutes of Health Individual Predoctoral NRSA, under grant number 1F31-NS049753. The United States has certain rights in this invention.

INTRODUCTION

Wnt polypeptides are secreted cysteine-rich glycosylated polypeptides that play a role in the development of a wide range of organisms. Wnts are encoded by a large gene family, whose members have been found in round worms, insects, cartilaginous fish and vertebrates. The Wnt family of polypeptides binds to an extracellular domain of a family of cell surface proteins called Frizzled receptors.

The Wnt proteins play a number of roles in early development, including embryonic patterning and cell fate determination (Logan and Nusse, Annu Rev Cell Dev Biol 20, 781- 810, 2004, which is incorporated herein by reference). They are also essential to the processes that occur later in nervous system development, cell migration, axon guidance, dendrite morphogenesis and synapse formation (Ciani and Salinas, Nat Rev Neurosci 6, 351- 362, 2005, which is incorporated herein by reference) (Fradkin et al., J Neurosci 25, 10376- 10378, 2005, which is incorporated herein by reference). Their diverse functions indicate that this family of signaling proteins plays significant roles in nervous system development. However, relatively little is known about how these functions are mediated, and whether these functions can be modulated. A need exists in the art to identify agents that may be used to modulate Wnt-frizzled mediated functions.

SUMMARY OF THE INVENTION

In one aspect, methods of assaying an agent for the ability to modulate the frizzled3 mediated signal transduction pathway are provided in which a cultured neuronal cell is

contacted with the agent under conditions that allow the agent to enter the cell. The axonal morphology of the cell is monitored. The axonal morphology of the neuronal cell contacted with the agent is then compared to the axonal morphology of a control cell. A difference in axonal morphology is indicative of the ability of the agent to modulate the frizzled3 mediated signal transduction pathway.

In another aspect, methods of introducing a polynucleotide into a spinal cord explant are provided. The neural tube of a spinal cord explant is contacted with the polynucleotide and the neural tube is electroporated to deliver the polynucleotide into the cells of the explant.

In a further aspect, an assay system for assaying an agent for the ability to modulate a frizzled3 mediated signal transduction pathway is provided. The assay system includes a cultured neuronal cell.

In yet another aspect, methods of stimulating axonal turning of commissural axons are provided. A polynucleotide encoding a polypeptide capable of stimulating the frizzled3 mediated signal transduction pathway is introduced into a neuronal cell having a commissural axon. Expression of the polypeptide stimulates axonal turning.

BRIEF DESCRIPTION OF DRAWINGS

Fig. 1. A Ca^-independent PKC pathway mediates anterior-posterior (A-P) guidance of commissural axons. (A) "Open-book" assays for anterior turning of commissural axons in the presence of PKC and phospholipase C inhibitors. (B) Quantification of "open-book" explant assays. (C) Post-crossing assays for Wnt4-mediated attraction of commissural axons in the presence of PKC and phospholipase C inhibitors. (D) Quantification of post-crossing assays for Wnt4-mediated attraction in the presence of PKC and phospholipase C inhibitors. Scale bar: 100 μm.

Fig. 2. Atypical PKC, PKCζ, mediates A-P guidance of commissural axons. (A) "Open-book" assays testing the effects of PKC pseudosubstrates and inhibitor, rottlerin. (B) Quantification of "open-book" assays with PKC pseudosubstrates and rottlerin. (C) Post- crossing assays for Wnt4-mediated attraction in the presence of PKC pseudosubstrates and rottlerin. (D) Quantification of post-crossing assays for Wnt4-meidated attraction in the presence of PKC pseudosubstrates and rottlerin. Scale bar: 100 μm. Fig. 3. GPCR signaling, PI3 kinase and GSK-3β are required for A-P guidance of commissural axons. (A) "Open-book" explants in the presence of inhibitors of Gαi and Gαo

(Purtussis toxin), PI3 kinase (wortmannin) and GSK-3β inhibitors (LiCl and SB-216763).

(B) Quantification of "open-book" assays. (C) Post-crossing assays in the presence of inhibitors of God and Gαo (Purtussis toxin), PB kinase (wortmannin) and GSK-3β inhibitors (LiCl and SB-216763). (D) Quantification of post-crossing assays. Scale bar: 100 μm.

Fig. 4. Localization of signaling components in El 1.5 mouse spinal cord. (A) (C) (E) (G) Overview of protein distribution using antibodies against PI3Kγ, PKCζ, GSK3β and an axonal marker Ll. (B) (D) (F) (H) Higher magnification showing distribution of PI3Kγ, PKCζ, GSK3β and an axonal marker Ll. Scale bars: 100 μm.

Fig. 5. PKCζ is required in the commissural neuron cell-autonomously for A-P guidance at the spinal cord midline. (A) Ex utero electroporation of El 3 rat spinal cord to deliver dominant-negative PKCζ construct in dorsal spinal cord neurons. (B) Commissural axons expressing GFP control showed anterior turning after midline crossing. (C) Commissural axons expressing dominant-negative PKCζ construct and GFP through an IRES sequence showed randomization of turning after midline crossing. (D) Quantification of A-P turning. Scale bar: 100 μm. Fig. 6. Diagram of a novel Wnt signaling pathway mediating axon guidance.

Fig. 7. PI3Kγ is required for A-P guidance of commissural axons. (A) Schematic diagram of the constructs used to electroporate spinal cord explants in B and B'. (B) Photograph of commissural axons after electroporation with the EGFP construct. (B') Photograph of commissural axons after electroporation with the pl lOγ-KD-EGFP construct. (C) Graph comparing the percent of axons turning correctly in B and B'. (D) Schematic diagram of the constructs used to electroporate spinal cord explants in E and E'. (E) Photograph of commissural axons after electroporation with the EGFP construct. (E') Photograph of commissural axons after electroporation with the pi lOγ- WT-EGFP construct. (F) Graph comparing the percent of axons turning before and after midline crossing in E and E'.

DETAILED DESCRIPTION

Wnts and frizzleds play important roles in nervous system wiring but the signaling pathways have not been unveiled. Although several studies suggest that Wnts guide axons in development, the intracellular signaling mechanisms mediating Wnts involvement in axon guidance are unknown. Axon growth cone turning requires coordinated intracellular events involving changes in actin dynamics, microtubule cyctoskeleton organization and membrane trafficking. These processes are highly regulated in pathfinding axonal growth cones. Three

major Wnt signaling pathways have been described in mediating various processes, the canonical/β-catenin, planar cell polarity and calcium/Protein Kinase C pathways. To uncover the downstream signaling components mediating anterior turning, the known Wnt signaling pathways were tested initially. The role of the PKC pathway in Wnt-mediated anterior turning of commissural axons was tested. Surprisingly, a novel Wnt signaling pathway was identified, involving PB kinase γ and an atypical PKCζ (calcium-independent), which mediates attraction in growth cone guidance.

The Examples below describe the elucidation of a downstream signaling pathway for Wnt-mediated axon attraction via the frizzled3 receptor involving PKCζ. As shown in Figure 6, the novel signaling pathway involves GPCR->PI3K->PKCζ->GSK3β. Because the G- protein family is very large, which Ga protein is required in Frizzled-3 mediated Wnt repulsion has not been pinpointed.

A fundamental mechanism of complex neuronal network wiring is the usage of intermediate targets or guideposts, where axons change responsiveness to guidance cues. With limited numbers of intermediate targets, highly complex axon networks can be built by switching on and off responsiveness to cues present at or around the intermediate targets. The mechanisms of response changes at the intermediate target have been an active area of research and several models have been proposed, including hierarchical order among guidance systems, local translational control or receptor sorting, particularly at the midline intermediate target. These mechanisms are likely complementary to each other. PI3 kinase γ may act as a potential switch mechanism, because it is only present in the post-crossing commissural axons as demonstrated in the Examples. Moreover, the activated form of its downstream target, PKCζ, and Par6, a component of the PKCζ/Par6 complex, are also enriched in the post-crossing axon segment. Methods are provided for assaying an agent for the ability to modulate the Wnt- frizzled3 mediated signal transduction pathway. Modulation of the frizzled3 mediated signal transduction pathway includes, but is not limited to, activation or inactivation of any of the signaling mediators, alterations in the cellular distribution or localization of any of the signaling mediators, and alterations in expression of any of the signaling mediators. The signaling mediators include, but are not limited to, PKCζ, PI3Kγ, GSK-3β, PDKl, and Par6. For example, the agent may be capable of phosphorylating or modulating the phosphorylation of PKCζ, thus inducing activation of the frizzled3 signal transduction pathway.

Alternatively, the agent may result in dephosphorylation of PKCζ after Wnt4-frizzled3 binding and activation of the signal transduction pathway, thereby blocking the frizzled3 mediated signal transduction pathway. One of skill in the art appreciates that the agent could affect the activation state of any of the downstream signal mediators in the frizzled3 signal transduction pathway and result in a difference in axonal morphology relative to that of control cells not contacted with the agent. In addition, agents may affect the distribution of a member of the signal transduction pathway. As demonstrated in the Examples, PKCζ, PDKγ, and Par6 are enriched in post-crossing commissural axons. Agents capable of altering the distribution or localization of any of these mediators may alter the frizzled3 signal transduction pathway as well.

The methods involve contacting a cultured neuronal cell with the agent. In the context of the invention, contacting may be in vitro, ex vivo or in vivo. The agent may be added in solution or provided on a solid or semi-solid support. The cell is contacted with the agent under conditions that allow the agent to enter the cell. One of skill in the art will appreciate that various mechanisms exist for allowing agents to enter cells. Suitably, the agents may be capable of crossing the membrane of a cultured neuronal cell. Agents may be membrane permeable or may be made membrane permeable by any suitable means. For example, the agent may be N-myristoylated, such that it can cross the cellular membrane. Agents may also be transported across the neuronal cell membrane, e.g. by a membrane transport protein. Additionally, a small molecule or nucleic acid may be electroporated into a cell.

The agent may be a small molecule, a polypeptide, a polynucleotide or a library of small molecules, polypeptides or polynucleotides. Small molecules include, but are not limited to, pharmaceuticals (i.e. antibiotics), pharmaceutical candidates, nucleosides, nucleotides and peptides. Agents do not include the native polypeptides and proteins that bind to frizzled3, such as the Wnts, or antibodies, such as anti-frizzled3 antibodies. In the Examples, agents known to inhibit activation of various members of the frizzled3 signal transduction pathway were used. One of skill in the art will appreciate that other agents are known that may inhibit members of the frizzled3 signal transduction pathway. Cultured neuronal cells for use in the methods may be sensory or motor neurons. The cultured neuronal cells are suitably mammalian neurons. The cultured neuronal cells may be harvested from a tissue (i.e. primary neuronal cells), encompassed within a tissue (e.g. a spinal cord explant), or derived from stem cells or from an immortalized neuronal cell line.

Suitable tissue from which neuronal cells may be obtained includes, but is not limited to, the brain, spinal cord and cerebellum. In the Examples, spinal cord tissue was used. Primary neuronal cells and tissues may be harvested from an embryonic animal, such as an El 1 -El 3 mouse embryo. Suitably, the neuronal cells are from a spinal cord explant. Neuronal cells may be obtained by stimulating differentiation of embryonic stem cells using methods known to those of skill in the art. One of skill in the art will appreciate that a number of neuronal cells lines exist which may be stimulated to develop axons in vitro.

In one embodiment, the neuronal cell is damaged. A neuron may be damaged by any injury or disease resulting in the loss of axonal connections including, but not limited to, traumatic injury, neurologic disease, degenerative disease, hypoxia, ischemia, anoxia and stroke.

One of skill in the art will appreciate that axonal morphology of the neuronal cell in response to contact with the agent can be monitored in a variety of ways including, but not limited to, microscopically and electrochemically. A difference in morphology of the axon includes, but is not limited to, enhanced or reduced growth, a change in the direction of growth, a change in the width or thickness of the axon, and a change in the topography of growth (i.e. straight, wavy, curly, sinusoidal). The growth may be enhanced in the anterior, posterior, medial, or lateral direction in a tissue. Enhanced or stimulated growth is indicative of activation of the frizzled3 mediated signal transduction pathway. Differences in axonal morphology may also include stalling, or lack of growth as compared to control cells, changes in shape, size or number of axons, changes in axonal organization such as increased/decreased fasciculation (bundling), or randomization. Differences in axonal morphology are measured by comparison to axonal morphology in control cells.

Control cells are cells similar to neuronal cells contacted with the agent that are either left untreated or contacted with a control agent that does not affect axonal growth cone guidance or axonal morphology.

Methods of modulating axon growth cone guidance are also provided. Modulating axonal growth includes, but is not limited to, attracting, stimulating, repressing, inhibiting, or repulsing axonal growth. The growth may be limited to the axon or may include growth of the neuron as a whole and includes, but is not limited to, extension/retraction of an axon, redirection/randomization of an axon's growth, an increase/decrease in volume of an axon, an increase/decrease in length of an axon relative to the cell body or a change in the organization of the axons (e.g., randomization or bundling). The axonal growth can be modulated in an

anterior-posterior orientation or in a medial-lateral orientation relative to a specific spatial address, for example a region of a spinal cord.

The methods of the invention may be conducted in the absence of addition of a Wnt protein or polypeptide. Alternatively, a Wnt protein or polypeptide may be added to bind and stimulate signaling through the frizzled3 receptor. When the method is conducted in the absence of Wnt or another ligand of frizzled3, agents capable of stimulating the frizzled3 signal transduction pathway in the absence of receptor-ligand interaction may be identified, i.e. agonists of the pathway. Alternatively, if a Wnt, such as Wnt4, or another ligand of frizzled3 is added to stimulate the frizzled3 signal transduction pathway, the method may be used to screen for agents capable of blocking or down-regulating the pathway, i.e. antagonists of the pathway.

Also provided herein is a method of introducing a polynucleotide into a spinal cord explant by contacting the explant with the polynucleotide and electroporating the neural tube to deliver the polynucleotide into at least one cell of the spinal cord explant. Another method of contacting the neural tube of the spinal cord explant with a polynucleotide is by injecting the polynucleotide into the neural tube. In the Examples, 5 μl of the polynucleotide was injected into the neural tube at a concentration of 1 μg/μl prior to electroporation. Electroporation comprises passing current across the dorsal neural tube of the spinal cord explant. In the Examples, an electroporator with 5mm gold plated electrodes was used to pass square wave current across the dorsal neural tube. In the Examples the cells were electroporated at 25 volts for 100ms pulses, repeated five times at one second intervals. One of skill in the art appreciates that different current and times of exposure to the current may be chosen based on considerations of efficiency and minimization of cellular damage. After electroporation, the spinal cord explant may be cultured in a collagen matrix for a sufficient time to allow gene expression and cellular recovery. In the Examples, the explants were cultured for 48 hours after electroporation. One skilled in the art will appreciate that the resultant spinal cord explants could be used in a variety of ways including, but not limited to, assays of gene expression, signal transduction, axonal growth and morphology.

Methods of stimulating axonal turning of commissural axons are also provided. A polynucleotide may be introduced into a neuronal cell having a commissural axon. The polynucleotide may be introduced into a spinal cord explant as described above and in the Examples. Those of skill in the art will appreciate that other methods may be used to introduce a polynucleotide into a neuronal cell. The polynucleotide encodes a polypeptide

capable of stimulating the frizzled3 mediated signal transduction pathway. Polynucleotides suitable for use in the method include those polynucleotides encoding constitutively activated members of the frizzled3 signal transduction pathway, polynucleotides encoding polypeptides capable of stimulating members of the frizzled3 signal transduction pathway and polynucleotides that will lead to overexpression and/or redistribution of members of the frizzled3 signal transduction pathway. In the Examples, pl lOγ was overexpressed causing redistribution of PI3Kγ and stimulation of the frizzled3 signal transduction pathway. One of skill in the art will appreciate that other polynucleotides may be used to stimulate the frizzled3 mediated signal transduction pathway and thus stimulate axonal turning. All references cited herein are hereby incorporated by reference in their entireties.

The following examples are meant to be illustrative only and are not intended as a limitation on the concepts and principles of the invention.

EXAMPLES Reagents Pharmacological inhibitors and PKC pseudosubstrates were purchased from various vendors: GF- 109203 (Alexis Biochemicals, Lausen, Switzerland, Catalog # 270-019-M001), Go-6967 (Calbiochem, San Diego, CA, Catalog # 365250), U-73122 (Cayman Chemical, Ann Arbor, MI, Catalog # 70740), neomycin sulfate (Biomol, Plymouth Meeting, PA, Catalog # El 180), rottlerin (Biomol, Plymouth Meeting, PA, Catalog # El-270), myristoylated PKC pseudosubstrates (Biomol, Plymouth Meeting, PA, Catalog # for ζ: P-219; α: P-205), Pertussis Toxin (Sigma, St. Louis, MO, Catalog # P-2980), wortmannin (Biomol, Plymouth Meeting, PA, Catalog # ST-415), lithium chloride (LiCl) (Sigma, St. Louis, MO, Catalog # L4408), SB-216763 (Sigma, St. Louis, MO, Catalog # S3442).

The following antibodies were purchased from vendors as indicated below: PI3K pi lOγ (rabbit polyclonal (H- 199), Santa Cruz Biotechnology, Santa Cruz, CA, Catalog # sc- 7177), PKCζ (rabbit polyclonal, Santa Cruz Biotechnology, Santa Cruz, CA, Catalog # sc- 216), phosporylated PKCζ (Thr 410, rabbit polyclonal, Santa Cruz Biotechnology, Santa Cruz, CA, Catalog # sc- 12894), PAR6 (rabbit polyclonal, Santa Cruz Biotechnology, Santa Cruz, CA, Catalog # sc- 14405), GSK3β (Chemicon, Temecula, CA, Catalog # AB8687), EGFP (rabbit polyclonal, Molecular Probes, Eugene, OR, Catalog # Al 11122) and β-tubulin E7 (Developmental Biology Hybridoma Bank). TAG-I antibody was generated from

hybridoma cells obtained from Developmental Studies Hybridoma Bank (cell line #4D7/Tag- 1). Ll antibodies were a kind gift from Dr. Rathjen.

Immunohistochemistry

El 1.5 mouse embryos were first fixed in 4% paraformaldehyde (PFA), embedded in OCT compound and sectioned into 10 μm sections. Slides were washed in phosphate buffered saline (PBS), then incubated in blocking solution which contained 1% or 5% donkey serum, 0.1% Triton X-IOO in PBS for 2-3 hours at room temperature. Antibodies were diluted in the blocking solution and the slides were incubated in primary antibody for 48 hours at 4C°. The concentrations of primary antibodies used were as follows: PBKγ 1:50, PKCζ 1 :1000, pPKCζ 1:50, PAR6 1:50, GSK3β 1 :1000, Tag-1 1 :50, Ll- 1 :10,000. The slides were washed 3 times for 10 minutes in PBS then incubated for 2 hours in 1 :500 secondary antibody, washed again and mounted using Fluoromount G. Pictures were taken using a Zeiss Axioplan and Openlab software.

Pharmacological treatment of explants "Open-book" or post-crossing explants were prepared as described (Lyuksyutova et al., Science 302, 1984-1988, 2003, which is incorporated herein by reference) (Zou et al., Cell 102, 363-375, 2000, which is incorporated herein by reference). After approximately 8 hours of culture, explants were treated with pharmacological or peptide inhibitor or a control treatment. Following another 10-12 hours of incubation, explants were fixed with warm 4% PFA. For "open-book" preparations axons were visualized using ionotophoretic injection of lipophilic DiI at 1/3-1/4 from the dorsal margin. After allowing the DiI to diffuse along the axon length, the explants were visualized and recorded. For quantification, as each DiI injection labels a cohort of axons, injection sites were scored based on axonal behavior as previously described (Zou et al., 2000) (Lyuksyutova et al., 2003). If all axons turned anteriorly in one injection, that injection site was scored "anterior (correct) turn"; if many axons stalled after midline crossing, the injection site was classified as "stalling"; if a significant number of axons projected posteriorly, the site was scored as "random turn (AZP)". If both "stalling" and "random turn (A/P)" phenotypes were observed, the injection site was classified as "random turn (A/P)". The frequency of each category was presented as percentage of all inj ected sites .

Three sets of experiments for each experimental condition were performed. The total number of samples tested in each condition were as follows: (1) control (17 explants) and

neomycin treated (27 explants); (2) control (13 explants) and Wortmannin (11 explants); (3) control (13 explants) and U-73122 (21 explants); (4) control (19 explants) and Go-6976 (25 explants); (5) control (15 explants) and GF-102903 (23 explants); (6) control (29 explants) and LiCl (28 explants); (7) control (9 explants) and Pertussis Toxin (20 explants); (8) control (14 explants) and SB-216763 only (15 explants); (9) Wn4t only (23 explants) and Wnt4 plus SB-216763 (17 explants).

For post-crossing preparations, explants were fixed with warm 4% PFA after 16-hour culture and immunostained with b-tubulin E7 antibody and visualized through a DAB reaction with a secondary antibody linked to horseradish peroxidase (HRP). Area of total axon outgrowth was measured for each explant and averaged over the experiment for control and treatment groups. For quantification, the explants were photographed at the same magnification using Zeiss AxioPlan with OpenLab software. The images were then imported into NIH Image J. Each image was turned into a black and white composite using the Threshold function. Each experimental set was quantified using the same Threshold parameters. The total area of black pixels was measured using the Analyze Particles function and then the particles showing axonal outgrowth were erased using the Eraser tool. The total area of black particles was measured again and the difference was recorded as total area of axonal outgrowth. The measurements for each explant in a set were averaged and then the ratios of axonal outgrowth of experimental conditions compared to control condition were calculated.

Three sets of experiments for each condition were performed and the resulting ratios or area of outgrowth were averaged. Standard deviations were calculated based on these sets of experiments. The total number of explants tested at each condition are as follows: vector only (23), vector only with neomycin (24), Wnt4 (29) and Wnt4 with neomycin conditions (25); vector only (21), vector only with Wortmannin (16), Wnt4 (27) and Wnt4 with Wortmannin (23); vector only (26), vector only with U-73122 (20), Wnt4 (17) and Wnt4 with U-73122 (22); vector only (26), vector only with Go-6976 (23), Wnt4 (17) and Wnt4 with Go-6976 (16); vector only (26), vector only with GF-102903 (16), Wnt4 (17) and Wnt4 with GF-102903 (16); vector only (42), vector only with Rottlerin (45), Wnt4 (34)and Wnt4 with Rottlerin (45); vector only (19), vector only with PKCα pseudosubstrate (20), Wnt4 (39) and Wnt4 with PKCα pseudosubstrate (39).

Calcium-independent Protein Kinase C signaling is required for Wnt4 attraction

To determine whether the PKC family plays a role in anterior-posterior (A-P) guidance, inhibitors that block all PKCs (GF-109203X) and the conventional PKC (Go-6976) were used in El 3 rat "open-book" spinal cord explants as previously described (Lyuksyutova et al., Science 302, 1984-1988, 2003, which is incorporated herein by reference). PKC inhibitors were added to the medium six hours after initiation of culture when the dorsal populations of commissural axons normally reach the contralateral side of the spinal cord and turn anteriorly. Spinal cord explant cultures were then continued for a total of 20 hours. Commissural axons were visualized by DiI injection into the dorsal spinal cord after fixation. The general PKC inhibitor, GF-109293X, caused consistent randomized turning along the A- P axis of commissural axons after midline crossing, whereas, commissural axons turned normally after midline crossing in the presence of a conventional PKC inhibitor, Go-6976 (Figure IA). As activation of conventional PKCs requires calcium and diacylglycerol, to further determine whether conventional PKCs are involved in A-P guidance of commissural axons, phospholipase C inhibitors were tested. Consistent with the previous results, PLC inhibitors, U -73122 and neomycin, had little effect on A-P guidance of post-crossing commissural axons (Figure IA).

To further confirm that the pan-PKC inhibitor (GF-109203X) blocks Wnt-mediated guidance, we tested whether it can perturb Wnt4-induced growth stimulation of post-crossing commissural axons (Lyuksyutova et al., Science 302, 1984-1988, 2003). Using post-crossing commissural axon explants, GF-109203X blocked Wnt4 stimulation, whereas Go-6976, U- 73122 and neomycin did not (Figure 1C). Therefore, the calcium/PKC pathway is not required for Wnt4-mediated anterior-directed turning; rather, a calcium-independent PKC pathway is involved. Interestingly, Wnt 4 causes an increase in fasciculation of post-crossing commissural axons and GF-109203X not only caused a decrease in outgrowth but also de- fasciculation. The post-crossing axons were thin and displayed a wavy morphology. In contrast, Go-6976, U-73122 and neomycin did not cause such axon morphology (Figure 1C).

Atypical PKC, PKCζ, mediates A-P guidance of commissural axons

To identify the PKC pathway involved in A-P axon guidance of commissural neurons, specific inhibitors for each subclass of PKC were added to the "open-book" explant cultures: pseudosubstrates for PKCα (conventional PKC) and PKCζ (all atypical PKCs, including ζ, ι, and λ), namely P-205 and P-219, respectively, and a pharmacological inhibitor for PKCδ

(novel PKC), rottlerin. These pseudosubstrates are N-terminus myristoylated to allow cell membrane permeability. As shown in figure 2A, the PKCζ pseudosubstrate caused A-P randomization and stalling of post-crossing commissural axons, whereas the PKCα pseudosubstrate and rottlerin had no significant effect. To test whether PKCζ mediates Wnt4 stimulated outgrowth, these inhibitors were included in post-crossing explant assays. PKCζ pseudosubstrate partially blocked Wnt4-stimulated outgrowth, while the PKCα pseudosubstrate did not significantly block Wnt4 attraction (Figure 2C). The PKCζ inhibitor also caused de-fasciculation and axon morphology change (sinusoidal axons), which suggests axonal retraction (Baas and Ahmad, Trends Cell Biol 11, 244-249, 2001, which is incorporated herein by reference). The PKCα pseudosubstrate and the PKCδ inhibitor, rottlerin, showed growth promoting and growth inhibition effects, respectively, on post- crossing commissural axons with or without Wnt4, therefore, conventional and novel PKCs may have a general effect on axon growth but not related to the Wnt4-stimulated effects (Figure 2D). Therefore, an atypical PKC subfamily, PKCζ, is an essential component of the Wnt4-mediated anterior turning of post-crossing commissural axons.

Upstream regulators of PKCζ, GPCR signaling and PI3 kinase γ, are required for A-P guidance of commissural axons

To further characterize this pathway, upstream regulators of atypical PKCs were targeted. As Frizzleds belong to the GPCR superfamily, pertussis toxin, a Ga inhibitor, was tested to determine whether it blocked A-P guidance of commissural axons. Pertussis toxin caused A-P randomization of post-crossing commissural axons at 800 ng/ml (6.8 nM) (Figure 3A). PKCζ can be activated by PB kinase signaling (Cantley, Science 296, 1655-1657, 2002, which is incorporated herein by reference), and among the PI3 kinase family, PI3 kinase γ has been shown to be activated by G-protein signaling (Rickert et al., Trends Cell Biol 10, 466-473, 2000, which is incorporated herein by reference), therefore phosphoinositol signaling may be a mediator of Frizzled signaling leading to PKCζ activation. Inhibitors were used to test whether PI3 kinase is required for Wnt-mediated A-P guidance of commissural axons. Using the open book and post-crossing assay, Wortmannin, a PI3 kinase inhibitor, caused A-P randomization in the open book assay (Figure 3A). In addition, both wortmannin and pertussis toxin blocked Wnt4-mediated attraction of post-crossing

commissural axons, suggesting that G protein signaling and PB kinase γ are required for mediating Wnt attraction in A-P axon guidance (Figure 3C).

A downstream target of PKCζ, GSK3β, is required for A-P guidance of commissural axons Looking downstream of PKCζ, inhibitors were used to test whether GSK3β was involved in Wnt mediated A-P guidance of commissural axons, because previous studies showed that the PKCζ/PAR6 complex is required for cell polarity and GSK3β is downstream of this complex (Etienne-Manneville and Hall, Curr Opin Cell Biol 15, 67-72, 2003b, which is incorporated herein by reference) (Etienne-Manneville and Hall, Nature 421, 753-756, 2003a, which is incorporated herein by reference). GSK3β inhibitors, lithium chloride and SB-216763, were tested in the "open-book" and post-crossing assays. Both inhibitors caused A-P randomization of commissural axons after midline crossing (Figure 3A). In addition, both inhibitors of GSK3β also blocked Wnt4 stimulation of outgrowth of post-crossing commissural axons (Figure 3C). Taken together, a novel Wnt signaling pathway, including the following components; trimeric G proteins, PI3 kinase γ, PKCζ, and GSK3β, were identified as mediating signaling in directional control of commissural axons along the A-P axis.

PI3 kinase γ and phosphorylated PKCζ are specifically enriched in post-crossing commissural axons In order to ask whether this signaling pathway involving PKCζ operates in commissural axons during anterior-posterior pathfinding in vivo, the protein distribution of PI3 kinase γ, PKCζ and GSK3β in embryonic spinal cord was examined. GSK3β was broadly expressed throughout the mantle zone of the spinal cord including the ventral funiculus areas where post-crossing commissural axons pathfind during this stage of development (Figure 4 G-H). PKCζ was also broadly expressed in the mantle zone but the phosporylated form (activated form) was relatively concentrated in the ventral funiculus (Figure 4E-F). A key component of the PKCζ complex is Par6 and Par6 was present both in pre- and post-crossing commissural axons but particularly enriched in the post-crossing segment. Interestingly, we found that PI3 kinase γ, a key component of this pathway is enriched specifically in the post-crossing commissural axons in transverse spinal cord sections in El 1.5 embryos (Figure 4C-D). The staining is present only in the ventral

funiculus, where post-crossing commissural axons turn and grow anteriorly. To confirm that the staining represents the protein localization in axons, irnrnunostaining of sections of post- crossing commissural axon explants were completed. Prior to crossing, no PB kinase γ protein was detected and only after midline crossing, the axons stained positively with the PB kinase γ (pi 10) antibodies (Figure 4M). Similarly, phosphorylated PKCζ was found enriched in the post-crossing segment (Figure 4N). Therefore, the components of the pathways described above are present in the right place at the right time. The specific localization of PB kinase γ and phosphorylated PKCζ suggests that they may be part of the switch mechanism that allows commissural axons to only respond to Wnts after midline crossing.

PKCζ functions cell autonomously in commissural axons during A-P pathfinding

Electroporation procedure. El 3 rat embryos were eviscerated and the notochord was removed. Using a pulled glass needle, 5 μl control or experimental DNA was injected into the neural tube at a concentration of lμg/μl. Using 5mm gold plated electrodes (Therma Apparatus, BTX electroporater # ECM 830, Catalog #45-0115), square wave current was passed across the dorsal neural tube. Electroporation conditions: 5 pulses, 25 V, 100 msec pulse, 1 -second interval. After electroporation, the spinal cord was dissected and "open- book" explants were cultured in a 3D collagen matrix for 48 hours. After culture, explants were fixed with 37°C 4% PFA. After fixation, explants were removed from collagen matrix and blocked with 5% normal donkey serum (NDS), 1% bovine serum albumin (BSA), 1% Triton X-100 overnight. This was followed by overnight incubation with primary antibody, α-GFP (1 :7500). Explants were washed for 4-6 hours with multiple IX PBS washes and incubated with secondary antibody Cy2 conjugated α-rabbit IgG overnight. After multiple washes, explants were mounted between two cover slips for microscopic analysis. Quantification was summarized from a total of 8 experiments, involving 87 explants for EGFP controls and 97 explants for PKCζ dominant-negative construct.

DNA constructs: A dominant-negative construct of PKCζ was provided by Alex Toker (Romanelli et al., MoI Cell Biol 19, 2921-2928, 1999, which is incorporated herein by reference). A point mutation at a conserved lysine residue to tryptophan in the ATP -binding domain inactivates the kinase activity of PKCζ and can partially inhibit signaling activated by both EGF and a constitutively active mutant of PBK. This mutant construct was cloned into pCIG2 vector followed by IRES GFP via the EcoRl site.

To further address whether this PKCζ pathway functions cell autonomously during the anterior turning of commissural axons within the spinal cord tissue, the electroporation procedure described above was used to introduce a dominant-negative construct of PKCζ in dorsal spinal cord commissural axons and the behavior of commissural axons at the midline was examined. The dominant-negative PKCζ was driven by a chick actin promoter enhanced by CMV-IE (pCIG2) with an IRES-EGFP so that the behavior of commissural axons that are expressing the dominant-negative PKCζ, and EGFP could be observed (Figure 5A). The dominant-negative PKCζ construct caused significant A-P randomization after midline crossing compared to controls where only GFP was expressed (Figure 5C). Therefore, this PKCζ pathway functions in the commissural neuron growth cones to mediate Wnt-Frizzled signaling.

PI3Kγ is required for A-P guidance of commissural axons and may act as an on-switch of Wnt responsiveness during midline crossing

The catalytic subunit if PI3Kγ is pl lOγ. To determine if pl lOγ functions cell- autonomously for proper A-P guidance of commissural axons, a construct containing kinase- defective human pl lOγ containing a lysine-to-arginine substitution in the ATP binding domain (pi 10-KD) linked to an EGFP reporter was electroporated into a spinal cord explant as described above. (See Figure 7A) This construct has previously been shown to exhibit 0.1% of the activity of wild-type PI3Kγ . After introduction of this construct into commissural axons (as shown in Figure 7B and Figure 7B'), a significant increase in A-P randomization after midline crossing was observed. Only 61% of axons projected correctly in the spinal explant electroporated with the pi 10-KD as compared to 86% in EGFP control electroporated explants (p<0.005) (Figure 7C). As with the PKC ζ mutant constructs, axons expressing pl lOγ-KD-EGFP reached the midline in comparable numbers to EGFP expressing axons, indicating that expression of this construct did not disrupt Netrin-1 signaling (not shown).

Based on the finding that pl lOγ protein is only present in the post-crossing segment of commissural axons, we hypothesized that it may serve as a switch to turn on Wnt responsiveness upon midline crossing, and that introducing ectopic pl lOγ protein before crossing may be sufficient to cause premature Wnt responsiveness. To test this, the catalytic subunit of PI3Kγ was fused to EGFP as shown in Figure 7D and used to electroporate commissural axons in an open-book preparation (Figure 7E and E'). It has been shown

previously that the catalytic domain alone can act as a constitutively active form of PBKγ. As depicted in Figure 7E and Figure 7F many commissural axons which usually only turn anteriorly after midline crossing now turned anteriorly prior to midline crossing (arrowheads in E' as compared to arrows in both E and E'). These data suggest that the ectopic pl lOγ allowed the axons to respond to Wnts before crossing. Approximately 50% of the axons expressing pl lOγ turned anteriorly before midline crossing, compared to 10% of axons expressing EGFP alone in the "open-book" culture (Figure 7F). Notably, the A-P choice of commissural axons of both the pre-crossing and post-crossing longitudinal tracts expressing pl lOγ remains correct (not shown). In the electroporation experiments, axons that did not turn at all but projected toward the dorsal margin of the contralateral spinal cord were sometimes observed. These projections have been described previously, and we did not observe any differences in the frequency or behavior of these axons between control and experimental conditions.

PI3Kγ knockout mouse commissural axons do not turn anteriorly after midline crossing In wild-type mice, commissural axons cross the midline of the spinal cord and turn anteriorly during normal development. The inventors have demonstrated that commissural axon guidance is dependent on PBKγ by demonstrating that in PBKγ knockout mice the commissural axons grow randomly after midline crossing (i.e. some axons turn anteriorly while others turn posteriorly. This data suggests that PBKγ is required for normal anterior- posterior guidance of commissural axons along the spinal cord during normal development.

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