Phosphatidylinositol-3-kinase-atypical protein kinase C signaling is required for Wnt attraction and anterior-posterior axon guidance

Abstract

Wnt proteins are conserved axon guidance cues that control growth cone navigation. However, the intracellular signaling mechanisms that mediate growth cone turning in response to Wnts are unknown. We previously showed that Wnt-Frizzled signaling directs spinal cord commissural axons to turn anteriorly after midline crossing through an attractive mechanism. Here we show that atypical protein kinase C (aPKC), is required for Wnt-mediated attraction of commissural axons and proper anterior-posterior (A-P) pathfinding. A PKCzeta pseudosubstrate, a specific blocker of aPKC activity, and expression of a kinase-defective PKCzeta mutant in commissural neurons resulted in A-P randomization in "open-book" explants. Upstream of PKCzeta, heterotrimeric G-proteins and phosphatidylinositol-3-kinases (PI3Ks), are also required for A-P guidance, because pertussis toxin, wortmannin, and expression of a p110gamma kinase-defective construct all resulted in A-P randomization. Overexpression of p110gamma, the catalytic subunit of PI3Kgamma, caused precocious anterior turning of commissural axons before midline crossing in open-book explants and caused dissociated precrossing commissural axons, which are normally insensitive to Wnt attraction, to turn toward Wnt4-expressing cells. Therefore, we propose that atypical PKC signaling is required for Wnt-mediated A-P axon guidance and that PI3K can act as a switch to activate Wnt responsiveness during midline crossing.

Figure 1.

A Ca²⁺-independent PKC pathway mediates A–P guidance of commissural axons. A, Diagram of the open-book assay from E13 rat spinal cord in collagen gel. RP, Roof plate; o/n, overnight. B, Open-book assays for anterior–posterior pathfinding of commissural axons in the presence of PKC inhibitors GF-109203X and Gö-6976. GF-109203X caused A–P randomization, whereas Gö-6976 did not. C, Open-book assays for anterior turning of commissural axons in the absence and presence of myristoylated PKCζ pseudosubstrate (ps). Inhibiting PKCζ caused A–P randomization. D, Open-book assays in the absence and presence of inhibitors of GSK-3β inhibitors (LiCl and SB-216763). Inhibiting GSK-3β caused A–P randomization and stalling. E, Diagram of the postcrossing assay. Explants, including floor plate, are dissected and cultured on a matrix of rat-tail collagen. A COS-7 cell aggregate expressing Wnt4 or vector is placed near the explant and coembedded in collagen. F, G, Postcrossing explant assays for Wnt4-mediated attraction of commissural axons in the presence of PKC inhibitors. The top row is outgrowth in the presence of a control cell aggregate. The bottom row is outgrowth in the presence of a Wnt4-expressing cell aggregate. Arrows mark postcrossing axon outgrowth. H, Postcrossing explant assays for Wnt4-mediated attraction of commissural axons in the presence of GSK-3β inhibitors (LiCl and SB-216763). The top row is outgrowth in the presence of a control cell aggregate. The bottom row is outgrowth in the presence of a Wnt4-expressing cell aggregate. Arrows mark postcrossing axon outgrowth. I, Quantification of open-book explant assay experiments. The graph represents the frequency of the axon turning behavior category as a percentage of all injected sites. n is the total number of explants quantified from three separate experiments. *p < 0.005. J, Quantification of postcrossing assays for Wnt4-mediated attraction in the presence of PKC and GSK3β inhibitors. The graph represents the average total area of outgrowth based on three separate experiments. *p = 0.0347; **p = 0.00527. n is the total number of explants quantified. Scale bars, 100 μm.

Figure 2.

Localization of signaling components necessary for A–P guidance in rat E13 and mouse E11.5 spinal cord. A–A″, Phosphorylated PKCζ in transverse (A, A′) and postcrossing (A″) sections. Arrows indicate immunoreactivity in postcrossing segments. B–B″, Par6 staining in transverse (B, B′) and postcrossing (B″) sections. Arrows indicate immunoreactivity in postcrossing segments. C–C′, Phosphorylated GSK3β (S9) staining in transverse sections. Arrows indicate immunoreactivity in postcrossing segments. D, D″, L1 staining in transverse (D) and postcrossing (D″) sections. Arrows indicate postcrossing staining. E, E″, Tag-1 staining in transverse (E) and postcrossing (E″) sections. Precrossing staining is present (E, E″, arrows), whereas postcrossing segments are negative (E, arrowhead). Inset, Differential interference contrast image indicates presence of axons (black arrow). F, G, Diagrams indicating axon trajectories in transverse (F) and postcrossing (G) sections. The precrossing segment is labeled blue, the crossing segment is labeled red, and the postcrossing segment is labeled green. mE11.5, Mouse E11.5; rE13, rat E13.

Figure 3.

PKCζ is required in the commissural neuron cell autonomously for A–P guidance at the spinal cord midline. A, Diagram of ex utero electroporation. RP, Roof plate. DNA constructs were injected into the central canal of the E13 rat spinal cord, and electroporation targets the dorsal margin of the spinal cord. The spinal cord was then splayed open and cultured in collagen gel. B, Schematic of IRES constructs expressed in commissural neurons. The chick β-actin promoter drives preferential expression in neurons, followed by EGFP, PKCζ-WT, or PKCζ-KD. C, As seen in open-book preparation, ex utero electroporation targets commissural cell bodies along the dorsal margin of the spinal cord. C′, Axons can be visualized approaching and crossing the midline, as well as turning after crossing. C″, Axon projection can be followed all the way into its longitudinal part. D–D″, Open-book assays for commissural axons expressing EGFP or PKCζ constructs. D′, Normal anterior turning of commissural axons expressing wild-type PKCζ. D″, Commissural axons expressing kinase-defective PKCζ showed randomized growth along the A–P axis after midline crossing. Arrowheads mark axons turning after crossing, and brackets and dashed lines mark the borders of the FP. E, Quantification of A–P turning in electroporated open-book explants. The graph shows the percentage of axons turning anteriorly. n is the total number of explants quantified from four separate experiments. *p < 0.00005; **p < 0.0005. F, Similar numbers of commissural axons are observed before midline crossing in explants electroporated with the EGFP, PKCζ wild-type, and PKCζ kinase-defective constructs. Scale bars, 200 μm.

Figure 4.

PI3K signaling is required for appropriate anterior–posterior pathfinding of commissural axons. A, Open-book explants in the absence or presence of inhibitors of Gα_i and Gα_o (PTX) and PI3K (wortmannin). PTX and wortmannin both caused A–P randomization. B, Quantification of open-book explant assay experiments. The graph represents the frequency of the axon turning behavior category as a percentage of all injected sites. n is the total number of explants quantified from three separate experiments. *p < 0.05. C, Expression of p110 family members in E11.5 spinal cord detected by RT-PCR. D, Schematic of EGFP and p110γ-KD IRES EGFP constructs. E, F, Open-book assays. E, Axons expressing EGFP. Arrows indicate anterior directed axons. F, Axons expressing p110γ-KD. Commissural axons wander in all directions after midline crossing. The anterior-turning axons (arrows) turned in wide angles instead of in the normal 90° sharp angles. Arrows indicate anterior-directed axons, and arrowheads indicate posterior-directed axons. G, Quantification of A–P turning in open-book assays with p110γ-KD electroporation. Approximately 65% of axons turned correctly when expressing the kinase-defective construct, as opposed to 96% of EGFP-expressing axons. n indicates the number of explants. H, Schematic of EGFP and p110γ-EGFP fusion constructs. I, J, Open-book assays. I, Navigation of axons expressing EGFP protein. Arrows indicate anterior directed axons. J, Axons expressing p110γ-EGFP. Arrows indicate axons turning anteriorly after midline crossing. Arrowheads indicate axons turning anteriorly before midline crossing. K, Quantification of anterior turning before and after crossing. Approximately 50% of the axons turned anteriorly before midline crossing when p110γ-EGFP fusion protein was expressed. n indicates the number of explants. Scale bars, 100 μm.

Figure 5.

Wnt4 attracts P110γ-EGFP-expressing commissural axons. A, Diagram of the coculture assay of dissociated spinal cord neurons and COS cells expressing guidance cues. Neurons with only one axon shaft of at least 50 μm and their somata at a distance of <200 μm from a COS-7 cell expressing guidance molecules were scored. Directional growth was scored by the direction of growth cone navigation relative to the COS-7 cells expressing guidance molecules. Directional growth refers to a neuron whose axon either invaded or grew on a linear path toward the COS-7 cell secreting the guidance molecules, or to an axon that turned approximately within 30° toward a COS-7 expressing the cues; nondirectional refers to an axon that turned from the initial trajectory at an angle >45° away from the COS-7 cell secreting the guidance molecules or an axon that grew parallel to and then past a COS-7 cell for a distance of >50 μm. RP, Roof plate. B, Left, Commissural axons expressing EGFP grow directionally toward Netrin-1-expressing COS-7 cells. Middle, EGFP-expressing axons growing nondirectionally, indicating that precrossing axons are not yet sensitized to Wnt4-expressing COS-7 cells. However, after P110γ-EGFP overexpression, commissural axons respond to the Wnt4-expressing cells and grow directionally toward them. C, Quantification of the coculture assay. P110γ-EGFP expression results in directional growth to Wnt4. *p < 0.02. Scale bar, 50 μm.

Figure 6.

Diagram of a PI3 kinase–aPKC signaling pathway mediating Wnt attraction in axon guidance. PDK-1, Phosphoinositide-dependent kinase-1.