Dual function of polysialic acid during zebrafish central nervous system development.

Polysialic acid (PSA), a carbohydrate epitope attached to the neural cell adhesion molecule, serves as a modulator of axonal interactions during vertebrate nervous system development. We have used PSA-specific antibodies and whole-mount immunocytochemistry to describe the spatiotemporal expression pattern of PSA during zebrafish central nervous system development. PSA is transiently expressed on all cell bodies and, except for the posterior commissure, it is not found on axons. Floorplate cells in the spinal cord and hindbrain strongly express PSA throughout development. Enzymatic removal of PSA leads to a defasciculated growth pattern of the posterior commissure and also affects distinct subsets of commissural axons in the hindbrain, which fail to cross the midline. Whereas the disordered growth pattern of hindbrain commissures produced by PSA-removal could be mimicked by injections of soluble PSA, the growth of axons in the posterior commissure was unaffected by such treatment. These results suggest that there are distinct mechanisms for PSA action during axon growth and pathfinding in the developing zebrafish CNS.     

The restricted spatial and temporal expression of a nervous-system-specific antigen involved in axon outgrowth during development of the grasshopper

To identify molecules important for pathfinding by growing axons, monoclonal antibodies (mAb) have been generated against embryonic grasshopper tissue. One mAb, 2B2, shows labeling exclusively in the nervous system. It recognizes a surface epitope on neuronal growth cones, filopodia and axons in the central nervous system (CNS). Initially, the antigen is expressed on all processes of the CNS; after 70% of embryonic development, localization of the 2B2 mAb is restricted to a small subset of axon tracts within the ganglia. Immunoprecipitation from embryonic membrane extracts with the 2B2 mAb reveals a unique band of 160 x 10(3) Mr. Functional studies with the 2B2 mAb demonstrate that the antigen is important in growth cone-axon interactions during process outgrowth. Growth cones that extend along axonal substrata are either blocked in growth or grow along an aberrant pathway when embryos are cultured in the presence of the 2B2 mAb. However, pioneer neurons that extend processes on non-neuronal substrata grow normally.     

Robo2 is required for establishment of a precise glomerular map in the zebrafish olfactory system

Olfactory sensory neurons (OSNs) expressing a given odorant receptor project their axons to specific glomeruli, creating a topographic odor map in the olfactory bulb (OB). The mechanisms underlying axonal pathfinding of OSNs to their precise targets are not fully understood. Here, we demonstrate that Robo2/Slit signaling functions to guide nascent olfactory axons to the OB primordium in zebrafish. robo2 is transiently expressed in the olfactory placode during the initial phase of olfactory axon pathfinding. In the robo2 mutant, astray (ast), early growing olfactory axons misroute ventromedially or posteriorly, and often penetrate into the diencephalon without reaching the OB primordium. Four zebrafish Slit homologs are expressed in regions adjacent to the olfactory axon trajectory, consistent with their role as repulsive ligands for Robo2. Masking of endogenous Slit gradients by ubiquitous misexpression of Slit2 in transgenic fish causes posterior pathfinding errors that resemble the ast phenotype. We also found that the spatial arrangement of glomeruli in OB is perturbed in ast adults, suggesting an essential role for the initial olfactory axon scaffold in determining a topographic glomerular map. These data provide functional evidence for Robo2/Slit signaling in the establishment of olfactory neural circuitry in zebrafish.     

The transmembrane protein Golden goal regulates R8 photoreceptor axon-axon and axon-target interactions

During Drosophila visual system development, photoreceptor (R) axons choose their correct paths and targets in a step-wise fashion. R axons with different identities make specific pathfinding decisions at different stages during development. We show here that the transmembrane protein Golden goal (Gogo), which is dynamically expressed in all R neurons and localizes predominantly to growth cones, is required in two distinct steps of R8 photoreceptor axon pathfinding: Gogo regulates axon-axon interactions and axon-target interactions in R8 photoreceptor axons. gogo loss-of-function and gain-of-function phenotypes suggest that Gogo mediates repulsive axon-axon interaction between R8 axons to maintain their proper spacing, and it promotes axon-target recognition at the temporary layer to enable R8 axons to enter their correct target columns in the medulla. From detailed structure-function experiments, we propose that Gogo functions as a receptor that binds an unidentified ligand through its conserved extracellular domain.     

Directional guidance of nerve growth cones

The intricate connections of the nervous system are established, in part, by elongating axonal fibers that are directed by complex guidance systems to home in on their specific targets. The growth cone, the major motile apparatus at the tip of axons, explores its surroundings and steers the axon along a defined path to its appropriate target. Significant progress has been made in identifying the guidance molecules and receptors that regulate growth cone pathfinding, the signaling cascades underlying distinct growth cone behaviors, and the cytoskeletal components that give rise to the directional motility of the growth cone. Recent studies have also shed light on the sophisticated mechanisms and new players utilized by the growth cone during pathfinding. It is clear that axon pathfinding requires a growth cone to sample and integrate various signals both in space and in time, and subsequently to coordinate the dynamics of its membrane, cytoskeleton and adhesion to generate specific responses.     

The growth cones of identified motoneurons in embryonic zebrafish select appropriate pathways in the absence of specific cellular interactions

Developing motoneurons in zebrafish embryos follow a stereotyped sequence of axonal outgrowth and accurately project their axons to cell-specific target muscles. During axonal pathfinding, an identified motoneuron pioneers the peripheral motor pathway. Growth cones of later motoneurons interact with the pioneer via contact, coupling, and axonal fasciculation. In spite of these interactions, ablation of the pioneer motoneuron does not affect the ability of other identified motoneurons to select the pathways that lead to appropriate target muscles. We conclude that interactions between these cells during pathfinding are not required for accurate pathway selection.     

Wnt Signaling in the Vertebrate Central Nervous System: From Axon Guidance to Synaptic Function

Regulation of cell signaling by Wnt proteins is critical for the formation of neuronal circuits. Wnts modulate axon pathfinding, dendritic development, and synaptic assembly. Through different receptors, Wnts activate diverse signaling pathways that lead to local changes on the cytoskeleton or global cellular changes involving nuclear function. Recently, a link between neuronal activity, essential for the formation and refinement of neuronal connections, and Wnt signaling has been uncovered. Indeed, neuronal activity regulates the release of Wnt and the localization of their receptors. Wnts mediate synaptic structural changes induced by neuronal activity or experience. New emerging evidence suggests that dysfunction in Wnt signaling contributes to neurological disorders. In this article, the attention is focused on the function of Wnt signaling in the formation of neuronal circuits in the vertebrate central nervous system.The formation of neuronal connections requires the navigation of axons to their appropriate synaptic targets, the formation of terminal branches, and the assembly of functional synapses. These processes greatly depend on the proper dialogue between axons and their environment as they navigate to their target, and between axons and their postsynaptic dendrites during synapse assembly. A combination of secreted molecules and transmembrane proteins modulates these processes. Studies over the last 10 years have revealed an essential role for Wnt signaling in axon pathfinding, dendritic development, and synapse assembly in both central and peripheral nervous systems. Wnts also modulate basal synaptic transmission and the structural and functional plasticity of synapses in the central nervous system. Studies of Wnts in the nervous system have significantly contributed to our current understanding of the molecular mechanisms that control neuronal circuit assembly. These studies have also shed light into fundamental aspects of cell signaling such as novel mechanisms of protein secretion (Korkut et al. 2009) and receptor dynamics (Sahores et al. 2010). Here I review the mechanisms by which Wnts modulate axon guidance and synapse formation in the vertebrate central nervous system. I also discuss the increasing evidence in support for a role of Wnts in basal synaptic transmission, synaptic plasticity, and neurological disorders.     

Pathfinding, target recognition, and synapse formation of single regenerating fibers in the adult grasshopper Schistocerca gregaria

After lesion of the peripheral tympanal nerve of the adult locust (Schistocerca gregaria), sensory axons regenerate into their original target areas. We examined the individual behavior of single regenerating auditory afferents during pathway and target selection by intracellularly recording and labeling them at different times postlesion. During axotomy, spontaneous activity is not increased in either the distal or proximal part of the cells. Stimulus response properties of lesioned cells with or without regenerating axons are not influenced. Surprisingly, only 55% of sensory neurons regenerate through the lesion site and often give rise to more than one axonal fiber. Within the central nervous system, 70% of regenerated axons consistently follow an incorrect pathway to reach the correct target region. Often, one of two processes formed by a cell chooses the correct pathway, and the other the incorrect one. In the target region, regenerated axons reconstitute somatotopically ordered projections and form synapses that resemble those of intact fibers in number and structure. The regeneration process does not induce a detectable expression of antigens that are known to be expressed during neural development in these neurons. Our study clearly demonstrates that precise synaptic regeneration is possible in adult animals within a completely differentiated central nervous system, although pathfinding and formation of arborizations are disturbed in a particular and probably system-related manner. The results strongly suggest that accurate pathfinding is unlikely to be a decisive factor in target area recognition and synaptogenesis.     

Trying to understand axonal regeneration in the CNS of fish.

In contrast to the situation in mammals and birds, neurons in the central nervous system (CNS) of fish--such as the retinal ganglion cells--are capable of regenerating their axons and restoring vision. Special properties of the glial cells and the neurons of the fish visual pathway appear to contribute to the success of axonal regeneration. The fish oligodendrocytes lack the axon growth inhibiting molecules that interfere with axonal extension in mammals. Instead, fish optic nerve oligodendrocytes support--at least in vitro--axonal elongation of fish as well as that of rat retinal axons. Moreover, the fish retinal ganglion cells re-express upon injury a set of growth-associated cell surface molecules and equip the regenerating axons throughout their path and up into their target, the tectum opticum with these molecules. This may indicate that the injured fish ganglion cells reactivate the cellular machinery necessary for axonal regrowth and pathfinding. Furthermore, the target itself provides positional marker molecules even in adult fish. These marker molecules are required to guide the regenerating axons back to their retinotopic home territory within the tectum.     

Functional development of the olfactory system in zebrafish

The olfactory system has become a popular model to study the function of neuronal circuits and the molecular and cellular mechanisms underlying the development of neurons and their connections. An excellent model to combine studies of function and development is the zebrafish because it not only permits sophisticated molecular and genetic analyses of development, but also functional measurements of neuronal activity patterns in the intact brain. This article reviews insights into the functional development of the olfactory system that have been obtained in zebrafish. The focus is on the specification of olfactory sensory neurons (OSNs), the mechanisms controlling odorant receptor expression and OSN identity, the pathfinding of OSN axons towards target glomeruli in the olfactory bulb (OB), the development of glomeruli and functional topographic maps in the OB, and the development of inhibitory interneurons in the OB.     

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.The complex guidance decisions at the midline have made it a particularly fascinating model for investigating the mechanisms and logic of axon pathfinding. Much of this work has focused on the ventral midline of the vertebrate spinal cord and its Drosophila analog, the ventral nerve cord. This work has sought to explain why some axons cross the midline, whereas others do not; why these axons respond differently to midline cues before and after crossing; and how the midline directs axon traffic along the lateral pathways. Striking similarities, as well as intriguing differences, have been documented in the way these guidance decisions are regulated in vertebrates and in Drosophila. Here, we briefly introduce the two systems, then review our current understanding of each of the key guidance decisions, and finally discuss some of the general principles and open questions that have emerged from these studies.     

Autonomous and nonautonomous functions for Hox/Pbx in branchiomotor neuron development

The vertebrate branchiomotor neurons are organized in a pattern that corresponds with the segments, or rhombomeres, of the developing hindbrain and have identities and behaviors associated with their position along the anterior/posterior axis. These neurons undergo characteristic migrations in the hindbrain and project from stereotyped exit points. We show that lazarus/pbx4, which encodes an essential Hox DNA-binding partner in zebrafish, is required for facial (VIIth cranial nerve) motor neuron migration and for axon pathfinding of trigeminal (Vth cranial nerve) motor axons. We show that lzr/pbx4 is required for Hox paralog group 1 and 2 function, suggesting that Pbx interacts with these proteins. Consistent with this, lzr/pbx4 interacts genetically with hoxb1a to control facial motor neuron migration. Using genetic mosaic analysis, we show that lzr/pbx4 and hoxb1a are primarily required cell-autonomously within the facial motor neurons; however, analysis of a subtle non-cell-autonomous effect indicates that facial motor neuron migration is promoted by interactions amongst the migrating neurons. At the same time, lzr/pbx4 is required non-cell-autonomously to control the pathfinding of trigeminal motor axons. Thus, Pbx/Hox can function both cell-autonomously and non-cell-autonomously to direct different aspects of hindbrain motor neuron behavior.     

Cellular Strategies of Axonal Pathfinding

Axons follow highly stereotyped and reproducible trajectories to their targets. In this review we address the properties of the first pioneer neurons to grow in the developing nervous system and what has been learned over the past several decades about the extracellular and cell surface substrata on which axons grow. We then discuss the types of guidance cues and their receptors that influence axon extension, what determines where cues are expressed, and how axons respond to the cues they encounter in their environment.This article provides an overview of how growth cones respond to the cellular substrata and molecular cues they encounter as they extend through the developing nervous system. It elaborates on the primer by Kolodkin and Tessier-Lavigne (2010) and touches on many of the topics covered in greater detail in the articles that follow. The first sections describe how axons extend in a directed manner, the substrata on which they grow, interactions between pioneer and follower axons, and growth cone behaviors in emerging tracts and at decision points. The subsequent sections discuss examples of specific cues, their distributions, how their distributions are determined, and how growth cones integrate multiple cues during pathfinding.     

Mutations in the stumpy gene reveal intermediate targets for zebrafish motor axons

Primary motoneurons, the earliest developing spinal motoneurons in zebrafish, have highly stereotyped axon projections. Although much is known about the development of these neurons, the molecular cues guiding their axons have not been identified. In a screen designed to reveal mutations affecting motor axons, we isolated two mutations in the stumpy gene that dramatically affect pathfinding by the primary motoneuron, CaP. In stumpy mutants, CaP axons extend along the common pathway, a region shared by other primary motor axons, but stall at an intermediate target, the horizontal myoseptum, and fail to extend along their axon-specific pathway during the first day of development. Later, most CaP axons progress a short distance beyond the horizontal myoseptum, but tend to stall at another intermediate target. Mosaic analysis revealed that stumpy function is needed both autonomously in CaP and non-autonomously in other cells. stumpy function is also required for axons of other primary and secondary motoneurons to progress properly past intermediate targets and to branch. These results reveal a series of intermediate targets involved in motor axon guidance and suggest that stumpy function is required for motor axons to progress from proximally located intermediate targets to distally located ones.     

Perturbations of MicroRNA Function in Mouse Dicer Mutants Produce Retinal Defects and Lead to Aberrant Axon Pathfinding at the Optic Chiasm

Background

During development axons encounter a variety of choice points where they have to make appropriate pathfinding decisions. The optic chiasm is a major decision point for retinal ganglion cell (RGC) axons en route to their target in order to ensure the correct wiring of the visual system. MicroRNAs (miRNAs) belong to the class of small non-coding RNA molecules and have been identified as important regulators of a variety of processes during embryonic development. However, their involvement in axon guidance decisions is less clear.

Methodology/Principal Findings

We report here that the early loss of Dicer, an essential protein for the maturation of miRNAs, in all cells of the forming retina and optic chiasm leads to severe phenotypes of RGC axon pathfinding at the midline. Using a conditional deletion approach in mice, we find in homozygous Dicer mutants a marked increase of ipsilateral projections, RGC axons extending outside the optic chiasm, the formation of a secondary optic tract and a substantial number of RGC axons projecting aberrantly into the contralateral eye. In addition, the mutant mice display a microphthalmia phenotype.

Conclusions

Our work demonstrates an important role of Dicer controlling the extension of RGC axons to the brain proper. It indicates that miRNAs are essential regulatory elements for mechanisms that ensure correct axon guidance decisions at the midline and thus have a central function in the establishment of circuitry during the development of the nervous system.     

A GFP-based genetic screen reveals mutations that disrupt the architecture of the zebrafish retinotectal projection

The retinotectal projection is a premier model system for the investigation of molecular mechanisms that underlie axon pathfinding and map formation. Other important features, such as the laminar targeting of retinal axons, the control of axon fasciculation and the intrinsic organization of the tectal neuropil, have been less accessible to investigation. In order to visualize these processes in vivo, we generated a transgenic zebrafish line expressing membrane-targeted GFP under control of the brn3c promoter/enhancer. The GFP reporter labels a distinct subset of retinal ganglion cells (RGCs), which project mainly into one of the four retinorecipient layers of the tectum and into a small subset of the extratectal arborization fields. In this transgenic line, we carried out an ENU-mutagenesis screen by scoring live zebrafish larvae for anatomical phenotypes. Thirteen recessive mutations in 12 genes were discovered. In one mutant, ddl, the majority of RGCs fail to differentiate. Three of the mutations, vrt, late and tard, delay the orderly ingrowth of retinal axons into the tectum. Two alleles of drg disrupt the layer-specific targeting of retinal axons. Three genes, fuzz, beyo and brek, are required for confinement of the tectal neuropil. Fasciculation within the optic tract and adhesion within the tectal neuropil are regulated by vrt, coma, bluk, clew and blin. The mutated genes are predicted to encode molecules essential for building the intricate neural architecture of the visual system.