Yimin Zou

Neuropilin-2 Regulates the Development of Select Cranial and Sensory Nerves and Hippocampal Mossy Fiber Projections

Neuropilin-1 and neuropilin-2 bind differentially to different class 3 semaphorins and are thought to provide the ligand-binding moieties in receptor complexes mediating repulsive responses to these semaphorins. Here, we have studied the function of neuropilin-2 through analysis of a neuropilin-2 mutant mouse, which is viable and fertile. Repulsive responses of sympathetic and hippocampal neurons to Sema3F but not to Sema3A are abolished in the mutant. Marked defects are observed in the development of several cranial nerves, in the initial central projections of spinal sensory axons, and in the anterior commissure, habenulo-interpeduncular tract, and the projections of hippocampal mossyfiber axons in the infrapyramidal bundle. Our results show that neuropilin-2 is an essential component of the Sema3F receptor and identify key roles for neuropilin-2 in axon guidance in the PNS and CNS.

Squeezing Axons Out of the Gray Matter: A Role for Slit and Semaphorin Proteins from Midline and Ventral Spinal Cord

Commissural axons cross the nervous system midline and then turn to grow alongside it, neither recrossing nor projecting back into ventral regions. In Drosophila, the midline repellent Slit prevents recrossing: axons cross once because they are initially unresponsive to Slit, becoming responsive only upon crossing. We show that commissural axons in mammals similarly acquire responsiveness to a midline repellent activity upon crossing. Remarkably, they also become responsive to a repellent activity from ventral spinal cord, helping explain why they never reenter that region. Several Slit and Semaphorin proteins, expressed in midline and/or ventral tissues, mimic these repellent activities, and midline guidance defects are observed in mice lacking neuropilin-2, a Semaphorin receptor. Thus, Slit and Semaphorin repellents from midline and nonmidline tissues may help prevent crossing axons from reentering gray matter, squeezing them into surrounding fiber tracts.

Wnt–Ryk signalling mediates medial–lateral retinotectal topographic mapping

Computational modelling has suggested that at least two counteracting forces are required for establishing topographic maps. Ephrin-family proteins are required for both anterior–posterior and medial–lateral topographic mapping, but the opposing forces have not been well characterized. Wnt-family proteins are recently discovered axon guidance cues. We find that Wnt3 is expressed in a medial–lateral decreasing gradient in chick optic tectum and mouse superior colliculus. Retinal ganglion cell (RGC) axons from different dorsal–ventral positions showed graded and biphasic response to Wnt3 in a concentration-dependent manner. Wnt3 repulsion is mediated by Ryk, expressed in a ventral-to-dorsal decreasing gradient, whereas attraction of dorsal axons at lower Wnt3 concentrations is mediated by Frizzled(s). Overexpression of Wnt3 in the lateral tectum repelled the termination zones of dorsal RGC axons in vivo. Expression of a dominant-negative Ryk in dorsal RGC axons caused a medial shift of the termination zones, promoting medially directed interstitial branches and eliminating laterally directed branches. Therefore, a classical morphogen, Wnt3, acting as an axon guidance molecule, plays a role in retinotectal mapping along the medial–lateral axis, counterbalancing the medial-directed EphrinB1–EphB activity.

Ryk-mediated Wnt repulsion regulates posterior-directed growth of corticospinal tract

Guidance cues along the longitudinal axis of the CNS are poorly understood. Wnt proteins attract ascending somatosensory axons to project from the spinal cord to the brain. Here we show that Wnt proteins repel corticospinal tract (CST) axons in the opposite direction. Several Wnt genes were found to be expressed in the mouse spinal cord gray matter, cupping the dorsal funiculus, in an anterior-to-posterior decreasing gradient along the cervical and thoracic cord. Wnts repelled CST axons in collagen gel assays through a conserved high-affinity receptor, Ryk, which is expressed in CST axons. Neonatal spinal cord secretes diffusible repellent(s) in an anterior-posterior graded fashion, with anterior cord being stronger, and the repulsive activity was blocked by antibodies to Ryk (anti-Ryk). Intrathecal injection of anti-Ryk blocked the posterior growth of CST axons. Therefore, Wnt proteins may have a general role in anterior-posterior guidance of multiple classes of axons.

Wnt Signaling in Neural Circuit Assembly

The Wnt family of secreted proteins plays a crucial role in nervous system wiring. Wnts regulate neuronal positioning, polarization, axon and dendrite development, and synaptogenesis. These diverse roles of Wnt proteins are due not only to the large numbers of Wnt ligands and receptors but also to their ability to signal through distinct signaling pathways in different cell types and developmental contexts. Studies on Wnts have shed new light on novel molecular mechanisms that control the development of complex neuronal connections. This review discusses recent advances on how Wnt signaling influences different aspects of neuronal circuit assembly through changes in gene expression and/or cytoskeletal modulation.

Vangl2 Promotes Wnt/Planar Cell Polarity-like Signaling by Antagonizing Dvl1-Mediated Feedback Inhibition in Growth Cone Guidance

Although a growing body of evidence supports that Wnt-Frizzled signaling controls axon guidance from vertebrates to worms, whether and how this is mediated by planar cell polarity (PCP) signaling remain elusive. We show here that the core PCP components are required for Wnt5a-stimulated outgrowth and anterior-posterior guidance of commissural axons. Dishevelled1 can inhibit PCP signaling by increasing hyperphosphorylation of Frizzled3 and preventing its internalization. Vangl2 antagonizes that by reducing Frizzled3 phosphorylation and promotes its internalization. In commissural axon growth cones, Vangl2 is predominantly localized on the plasma membrane and is highly enriched on the tips of the filopodia as well as in patches of membrane where new filopodia emerge. Taken together, we propose that the antagonistic functions of Vangl2 and Dvl1 (over Frizzled3 hyperphosphorylation and endocytosis) allow sharpening of PCP signaling locally on the tips of the filopodia to sense directional cues, Wnts, eventually causing turning of growth cones.

Wnt-Planar Cell Polarity signaling controls the anterior-posterior organization of monoaminergic axons in the brainstem

Monoaminergic neurons [serotonergic (5-HT) and dopaminergic (mdDA)] in the brainstem project axons along the anterior–posterior axis. Despite their important physiological functions and implication in disease, the molecular mechanisms that dictate the formation of these projections along the anterior–posterior axis remain unknown. Here we reveal a novel requirement for Wnt/planar cell polarity signaling in the anterior–posterior organization of the monoaminergic system. We find that 5-HT and mdDA axons express the core planar cell polarity components Frizzled3, Celsr3, and Vangl2. In addition, monoaminergic projections show anterior–posterior guidance defects in Frizzled3, Celsr3, and Vangl2 mutant mice. The only known ligands for planar cell polarity signaling are Wnt proteins. In culture, Wnt5a attracts 5-HT but repels mdDA axons, and Wnt7b attracts mdDA axons. However, mdDA axons from Frizzled3 mutant mice are unresponsive to Wnt5a and Wnt7b. Both Wnts are expressed in gradients along the anterior–posterior axis, consistent with their role as directional cues. Finally, Wnt5a mutants show transient anterior–posterior guidance defects in mdDA projections. Furthermore, we observe during development that the cell bodies of migrating descending 5-HT neurons eventually reorient along the direction of their axons. In Frizzled3 mutants, many 5-HT and mdDA neuron cell bodies are oriented abnormally along the direction of their aberrant axon projections. Overall, our data suggest that Wnt/planar cell polarity signaling may be a global anterior–posterior guidance mechanism that controls axonal and cellular organization beyond the spinal cord.

Repulsive Wnt Signaling Inhibits Axon Regeneration after CNS Injury

Failure of axon regeneration in the mammalian CNS is attributable in part to the presence of various inhibitory molecules, including myelin-associated proteins and proteoglycans enriched in glial scars. Here, we evaluate whether axon guidance molecules also regulate regenerative growth after injury in adulthood. Wnts are a large family of axon guidance molecules that can attract ascending axons and repel descending axons along the length of the developing spinal cord. Their expression (all 19 Wnts) is not detectable in normal adult spinal cord by in situ hybridization. However, three of them are clearly reinduced after spinal cord injury. Wnt1 and Wnt5a, encoding potent repellents of the descending corticospinal tract (CST) axons, were robustly and acutely induced broadly in the spinal cord gray matter after unilateral hemisection. Ryk, the conserved repulsive Wnt receptor, was also induced in the lesion area, and Ryk immunoreactivity was found on the lesioned CST axons. Wnt4, which attracts ascending sensory axons in development, was acutely induced in areas closer to the lesion than Wnt1 and Wnt5a. Injection of function-blocking Ryk antibodies into the dorsal bilateral hemisectioned spinal cord either prevented the retraction of CST axons or promoted their regrowth but clearly enhanced the sprouting of CST collateral branches around and beyond the injury site. Therefore, repulsive Wnt signaling may be a cause of cortical spinal tract axon retraction and inhibits axon sprouting after injury.

Wnt signaling in axon guidance

Recent studies have identified Wnt proteins as conserved axon guidance molecules in vertebrates and invertebrates. Wnt proteins are a large family of diffusible factors that play several important roles, both in embryonic development and in adult function. The signaling mechanisms of Wnt proteins are complex and, because Wnts are newly discovered as axon guidance cues, little is known about how Wnt signaling controls the direction of growth cone navigation - a process that is crucial in development of the nervous system. This review summarizes recent work on the role of Wnts in axon guidance and discusses the possible signaling mechanisms involved in growth cone guidance. Understanding how Wnts regulate axon wiring will not only help us to understand how the nervous system is connected but also provide possible tools for axon regeneration.

Navigating Intermediate Targets: The Nervous System Midline

In a bilaterally symmetric animal, the midline plays a key role in directing axon growth during wiring of the nervous system. Midline cells provide a variety of guidance cues for growing axons, to which different types of axons respond in different ways and at different times. For some axons, the midline is an intermediate target. They first seek it out, but then move on towards their final targets on the opposite side. For others, the midline is a repulsive barrier that keeps them on their own side of the midline. And for many of these axons the midline provides signals that guide them along specific lateral pathways or up and down the longitudinal axis.