Elimination of a brain tract increases errors in pathfinding by follower growth cones in the zebrafish embryo

The early zebrafish brain contains a simple axon scaffold of longitudinal tracts connected by commissures. Neurons in the nucleus of the posterior commissure (nuc PC) project growth cones along a specific route in this axonal scaffold, raising the possibility that specific axons in the early scaffold guide nuc PC growth cones. We tested this possibility by analyzing the behavior of nuc PC growth cones in embryos in which a portion of the scaffold, normally traversed by nuc PC growth cones, was surgically prevented from forming. Under these conditions nuc PC growth cones extended along both normal and aberrant pathways. This suggests that specific axons do provide guidance cues, since their removal leads to errors. However, these cues are not obligatory, since some growth cones still followed normal pathways.     

A specific brain tract guides follower growth cones in two regions of the zebrafish brain.

Neurons of the nucleus of the posterior commissure (nuc PC), an identifiable cluster of neurons in the embryonic zebrafish brain, project growth cones ventrally along the posterior commissure to the anterior tegmentum where the PC intersects two longitudinal tracts, the tract of the postoptic commissure (TPOC) and the medial longitudinal fasciculus (MLF). Once at the intersection, nuc PC growth cones turn posteriorly onto the TPOC in the dorsal tegmentum and follow it to the hindbrain. Previously we showed that in the absence of the TPOC, nuc PC growth cones often extended along aberrant pathways suggesting that fasciculation, that is, contact with TPOC axons is an important factor in guiding growth cones along their normal pathway. However, a significant number of nuc PC growth cones also followed their normal pathway suggesting that cues associated with the dorsolateral tegmentum, independent of the TPOC, can also guide nuc PC growth cones. We have now confirmed using electron microscopy that nuc PC growth cones fasciculate with axons in the TPOC. In the absence of the TPOC, the nuc PC growth cones that extend along their normal pathway do so in contact with dorsolateral neuroepithelial cells. This suggests that cues associated with these cells can also guide the nuc PC growth cones. Furthermore, in the absence of the TPOC axons, these growth cones now inappropriately turn onto axons that normally intersect the TPOC near the border of the midbrain and hindbrain, that is, at a second intersection of tracts. This suggests that fasciculation with TPOC axons may also guide nuc PC growth cones in this second region of the brain.     

A specific brain tract guides follower growth cones in two regions of the zebrafish brain

Neurons of the nucleus of the posterior commissure (nuc PC), an identifiable cluster of neurons in the embryonic zebrafish brain, project growth cones ventrally along the posterior commissure to the anterior tegmentum where the PC intersects two longitudinal tracts, the tract of the postoptic commissure (TPOC) and the medial longitudinal fasciculus (MLF). Once at the intersection, nuc PC growth cones turn posteriorly onto the TPOC in the dorsal tegmentum and follow it to the hindbrain. Previously we showed that in the absence of the TPOC, nuc PC growth cones often extended along aberrant path ways suggesting that fasciculation, that is, contact with TPOC axons is an important factor in guiding growth cones along their normal pathway. However, a significant number of nuc PC growth cones also followed their normal pathway suggesting that cues associated with the dorsolateral tegmentum, independent of the TPOC, can also guide nuc PC growth cones. We have now confirmed using electron microscopy that nuc PC growth cones fasciculate with axons in the TPOC. In the absence of the TPOC, the nuc PC growth cones that extend along their normal pathway do so in contact with dorsolateral neuroepithelial cells. This suggests that cues associated with these cells can also guide the nuc PC growth cones. Furthermore, in the absence of the TPOC axons, these growth cones now inappropriately turn onto axons that normally intersect the TPOC near the border of the midbrain and hindbrain, that is, at a second intersection of tracts. This suggests that fasciculation with TPOC axons may also guide nuc PC growth cones in this second region of the brain. © 1992 John Wiley & Sons, Inc.     

Growth cones utilize both widespread and local directional cues in the zebrafish brain

The distribution of cues that provide directional information for specific growth cones in the zebrafish brain was functionally assayed by transplanting epiphysial neurons to ectopic locations in the embryonic brain followed by determining the pathways taken by the donor axons. Epiphysial axons normally first extend ventrally from their position in the dorsal diencephalon and then turn and extend anteriorly in the ventral diencephalon. When transplanted to ectopic sites at other axial levels of the brain, where in principle the axons could extend in any direction, epiphysial axons consistently extended ventrally. Furthermore, following initial ventral extension ectopic epiphysial axons turned randomly in the anterior and posterior directions. These results suggest that the cues for ventral extension are widely distributed along the rostrocaudal axis of the zebrafish brain, but the cues for subsequent anterior extension are restricted to the site where the epiphysial axons normally turn longitudinally.     

Are pioneer axons guided by regulatory gene expression domains in the zebrafish forebrain? High-resolution analysis of the patterning of the zebrafish brain during axon tract formation

Although the principles of axon growth are well understood in vitro the mechanisms guiding axons in vivo are less clear. It has been postulated that growing axons in the vertebrate brain follow borders of neuroepithelial cells expressing specific regulatory genes. In the present study we reexamined this hypothesis by analysing the earliest growing axons in the forebrain of embryonic zebrafish. Confocal laser scanning microscopy was used to determine the spatiotemporal relationship between growing axons and the expression pattern of eight regulatory genes in zebrafish brain. Pioneer axons project either longitudinally or dorsoventrally to establish a scaffold of axon tracts during this developmental period. Each of the regulatory genes was expressed in stereotypical domains and the borders of some were oriented along dorsoventral and longitudinal planes. However, none of these borders clearly defined the trajectories of pioneer axons. In two cases axons coursed in proximity to the borders of shh and pax6, but only for a relatively short portion of their pathway. Only later growing axons were closely apposed to the borders of some gene expression domains. These results suggest that pioneer axons in the embryonic forebrain do not follow continuous pathways defined by the borders of regulatory gene expression domains.     

Migration of zebrafish spinal motor nerves into the periphery requires multiple myotome-derived cues

In vertebrate embryos, spinal motor neurons project through segmentally reiterated nerves into the somites. Here, we report that zebrafish secondary motor neurons, which are similar to motor neurons in birds and mammals, depend on myotomal cues to navigate into the periphery. We show that the absence of myotomal adaxial cells in you-too/gli2 embryos severely impairs secondary motor axonal pathfinding, including their ability to project into the somites. Moreover, in diwanka mutant embryos, in which adaxial cells are present but fail to produce cues essential for primary motor growth cones to pioneer into the somites, secondary motor axons display similar pathfinding defects. The similarities between the axonal defects in you-too/gli2 and diwanka mutant embryos strongly suggest that pathfinding of secondary motor axons depends on myotome-derived cues, and that the diwanka gene is a likely candidate to produce or encode such a cue. Our experiments also demonstrate that diwanka plays a central role in the migration of primary and secondary motor neurons, suggesting that both neural populations share mechanisms underlying axonal pathfinding. In summary, we provide compelling evidence that myotomal cells produce multiple signals to initiate and control the migration of spinal nerve axons into the somites.     

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.     

Growth cone guidance by floor plate cells in the spinal cord of zebrafish embryos.

The spinal cord of early zebrafish embryos contains a small number of neuronal classes whose growth cones all follow stereotyped, cell-specific pathways to their targets. Two classes of spinal neurons make cell-specific turns at or near the ventral midline of the spinal cord, which is occupied by a single row of midline floor plate cells. We tested whether these cells guide the growth cones by examining embryos missing the midline floor plate cells due either to laser ablation of the cells or to a mutation (cyc-1). In these embryos the growth cones followed both normal and aberrant pathways once near the ventral midline. This suggests that normally the midline floor plate cells do provide guidance cues, but that these cues are not obligatory.     

Morphology of pioneer and follower growth cones in the developing cerebral cortex

In the developing nervous systems of both invertebrates and vertebrates, neurons must develop precise sets of axonal connections. One strategy used by both orders of animals is to generate a special class of neurons whose axons "pioneer" the first pathways between these cells and their targets. In the developing mammalian telencephalon, the subplate neurons (which are among the first neurons to be generated in development) extend axons to long-distance subcortical targets before the neurons of the deep cortical layers 5 and 6 have been generated. The axons of layer 5 and 6 neurons later follow a similar pathway to form permanent subcortical projections to the thalamus and tectum, and thereafter the vast majority of subplate neurons die. These results have generated the hypothesis that subplate axons may actually be required for the axons of layer 5 and 6 neurons to innervate their appropriate subcortical targets. The complexity of growth cones has previously been correlated with axonal decision making: differences in growth cone morphologies have been noted in comparisons of leading versus following axons (LoPresti, Macagno, and Levinthal, 1973; Nordlander, 1987; Yaginuma, Homma, Kunzi, and Oppenheim, 1991), and at choice points along axon pathways (Raper, Bastiani, and Goodman, 1983; Tosney and Landmesser, 1985; Caudy and Bentley, 1986a,b; Bovolenta and Mason, 1987; Holt, 1989; Bovolenta and Dodd, 1990; Yaginuma et al., 1991). Thus, as a first step toward addressing the question of whether the axons of deep-layer neurons simply follow subplate axons to their targets, we have studied the morphology of cortical growth cones at various points along the corticothalamic pathway and at different stages of development. We examined the brains of fetal ferrets and cats at ages ranging from embryonic days (E) 24 to E50, using the fluorescent lipophilic tracer 1,1-dioctadecyl-3,3,3',3'-tetramethyl indocarbocyanine perchlorate (DiI) to reveal the axons and growth cones of cortical neurons. Growth cones were drawn, and quantitative measurements of their complexity were made by counting filopodia and calculating their surface area. No morphological differences were found among growth cones at different points along the corticothalamic pathway at a given age. However, growth cones belonging to early-generated cells (likely to be subplate neurons) are significantly larger and more complex than are the growth cones of later-generated cortical neurons. This evidence is consistent with the suggestion that subplate growth cones actively pioneer the corticothalamic pathway, and that the axons of layer 5 and 6 neurons follow it.     

A Dual Role for the Zebrafish unplugged Gene in Motor Axon Pathfinding and Pharyngeal Development

On their way toward their synaptic targets, motor growth cones encounter multiple choice points, where they are confronted with trajectory choices. We have previously shown that the zebrafish unplugged gene acts as a somite-derived cue controlling pathway choice of primary motor axons. Here, we demonstrate that this trajectory choice is not exclusively controlled by a single unplugged-dependent process, but depends on the coordinated function of additional cues. We also show that secondary motor neurons, most similar to those in birds and mammals, depend on the unplugged gene to navigate a choice point, suggesting that primary and secondary motor neurons share common mechanisms controlling axonal path selection. Moreover, we show that the unplugged gene plays an additional role guiding secondary motor axons through a single segmental nerve. Finally, we report that unplugged larvae display a striking pharyngeal arch defect, consistent with a dual function of the unplugged gene in axonal guidance and cell motility.     

Growth cone guidance in the zebrafish central nervous system.

The accessibility and simplicity of the zebrafish embryo have allowed researchers to make a detailed characterization of pathfinding by identifiable growth cones. The growth cones follow precise cell-specific pathways to their targets. Analyses of pathfinding in mutant and experimentally manipulated wild type embryos have shown that growth cones accomplish this by interacting with specific cellular cues in their environment, many of which are likely to be redundant.     

Growth pattern of pioneering chick spinal cord axons

The early growth pattern of axons in the embryonic chick spinal cord was studied by electron microscopy. Serial perisagittal thin sections were obtained from the lateral margins of spinal cords of stage 17 (S17) and S19 embryos. A simple stereotypic pattern of axonal growth was found. Axons originated from a dispersed population of presumptive interneurons located along the lateral spinal cord margin. They first grew ventrally in a nonfasciculative pattern and later turned at right angles and grew in a fasciculative manner longitudinally in the ventrolateral fasciculus. Growth along the circumferential pathway was analyzed in detail by reconstructing individual axons and growth cones from the S17 specimen. Most circumferential axons, regardless of their site of origin, grew in a parallel orientation, and each of their growth cones projected ventrally. This pattern suggested that circumferential growth cones were guided at many, if not all, points along their path. Study of the region in front of these seven growth cones, however, revealed no apparent structural basis for their guidance. Alternative guidance mechanisms are discussed. In conjunction with previous studies (e.g., Windle and Baxter, 1936; Lyser, 1966), these findings suggest that the circumferential-nonfasciculative and the longitudinal-fasciculative patterns of axonal growth are the two fundamental patterns followed by most early forming axons in the brain stem and spinal cord of all higher vertebrates.     

Unique Responses of Differentiating Neuronal Growth Cones to Inhibitory Cues Presented by Oligodendrocytes

During central nervous system development, neurons differentiate distinct axonal and dendritic processes whose outgrowth is influenced by environmental cues. Given the known intrinsic differences between axons and dendrites and that little is known about the response of dendrites to inhibitory cues, we tested the hypothesis that outgrowth of differentiating axons and dendrites of hippocampal neurons is differentially influenced by inhibitory environmental cues. A sensitive growth cone behavior assay was used to assess responses of differentiating axonal and dendritic growth cones to oligodendrocytes and oligodendrocyte- derived, myelin-associated glycoprotein (MAG). We report that >90% of axonal growth cones collapsed after contact with oligodendrocytes. None of the encounters between differentiating, MAP-2 positive dendritic growth cones and oligodendrocytes resulted in growth cone collapse. The insensitivity of differentiating dendritic growth cones appears to be acquired since they develop from minor processes whose growth cones are inhibited (nearly 70% collapse) by contact with oligodendrocytes. Recombinant MAG(rMAG)-coated beads caused collapse of 72% of axonal growth cones but only 29% of differentiating dendritic growth cones. Unlike their response to contact with oligodendrocytes, few growth cones of minor processes were inhibited by rMAG-coated beads (20% collapsed). These results reveal the capability of differentiating growth cones of the same neuron to partition the complex molecular terrain they navigate by generating unique responses to particular inhibitory environmental cues.     

Pathway selection by ectopic motoneurons in embryonic zebrafish.

Primary motoneurons in embryonic zebrafish innervate cell-specific muscles. During pathfinding, motoneuronal growth cones encounter three distinct regions: a common pathway, a choice point, and separate cell-specific pathways. To learn whether the order in which these regions are encountered influences pathway choice, we transplanted individual motoneurons to the choice point region. These cells selected their appropriate cell-specific pathways. Thus, the sequence in which pathway regions are encountered may not be important for accurate path-finding, and the cell-specific pathways may be delineated by distinct cues that individual growth cones recognize. Moreover, these cues are unlikely to be general ones, since primary sensory neurons transplanted to the same location do not extend growth cones along the motoneuronal pathways.     

Hierarchical guidance cues in the developing nervous system of C. elegans

During embryogenesis, the basic axon scaffold of the nervous system is formed by special axons that pioneer pathways between groups of cells. To find their way, the pioneer growth cones detect specific cues in their extracellular environment. One of these guidance cues is netrin. Observations and experimental manipulations in vertebrates and nematodes have shown that netrin is a bifunctional guidance cue that can simultaneously attract and repel axons. During the formation of this basic axon scaffold in Caenorhabditis elegans, the netrin UNC-6 is expressed by neuroglia and pioneer neurons, providing hierarchical guidance cues throughout the animal. Each cue has a characteristic role depending on the cell type, its position and the developmental stage. These roles include activities as global, decussation and labeled-pathway cues. This hierarchical model of UNC-6 netrin-mediated guidance suggests a method by which guidance cues can direct formation of basic axon scaffolds in developing nervous systems.     

Genetic screens for genes controlling motor nerve-muscle development and interactions

Motor growth cones navigate long and complex trajectories to connect with their muscle targets. Experimental studies have shown that this guidance process critically depends on extrinsic cues. In the zebrafish embryo, a subset of mesodermal cells, the adaxial cells, delineates the prospective path of pioneering motor growth cones. Genetic ablation of adaxial cells causes profound pathfinding defects, suggesting the existence of adaxial cell derived guidance factors. Intriguingly, adaxial cells are themselves migratory, and as growth cones approach they migrate away from the prospective axonal path to the lateral surface of the myotome, where they develop into slow-twitching muscle fibers. Genetic screens in embryos stained with an antibody cocktail identified mutants with specific defects in differentiation and migration of adaxial cells/slow muscle fibers, as well as mutants with specific defects in axonal pathfinding, including exit from the spinal cord and pathway selection. Together, the genes underlying these mutant phenotypes define pathways essential for nerve and muscle development and interactions between these two cell types.     

Patched regulation of axon guidance is by specifying neural identity in the<Emphasis Type="Italic"> Drosophila</Emphasis> nerve cord

Within an axon bundle, one or two are pioneering axons and the rest are follower axons. Pioneering axons are projected first and the follower axons are projected later but follow a pioneering axon(s) pathway. It is not clear whether the pioneering axons have a guidance role for follower axons. In this paper, we have investigated the role of Patched (Ptc) in regulating the guidance of medial tract, one of the longitudinal tracts in the nerve cord. In patched mutants the medial longitudinal tract fails to fasciculate on its own side along the nerve cord, instead it abnormally crosses the midline and fasciculates with the contralateral tract. Interestingly, the medial tracts cross the midline ignoring the axon-repellant Slit on the midline and Roundabout on growth cones. The medial tract is pioneered by neurons pCC and vMP2. Our results show that guidance defects of this tract are due to loss and mis-specification of vMP2, which results in the projection from pCC to either stall or project outward near the location of vMP2. Thus, both pioneering neurons are necessary for the proper guidance of pioneering and follower axons. We also show that the loss of Ptc activity in the neuroectoderm prior to the formation of S1 and S2 neuroblasts causes the majority of axon guidance defects. These results provide insight into how mis-specification and loss of neurons can non-autonomously contribute to defects in axon pathfinding.     

A dual role for the zebrafish unplugged gene in motor axon pathfinding and pharyngeal development.

On their way toward their synaptic targets, motor growth cones encounter multiple choice points, where they are confronted with trajectory choices. We have previously shown that the zebrafish unplugged gene acts as a somite-derived cue controlling pathway choice of primary motor axons. Here, we demonstrate that this trajectory choice is not exclusively controlled by a single unplugged-dependent process, but depends on the coordinated function of additional cues. We also show that secondary motor neurons, most similar to those in birds and mammals, depend on the unplugged gene to navigate a choice point, suggesting that primary and secondary motor neurons share common mechanisms controlling axonal path selection. Moreover, we show that the unplugged gene plays an additional role guiding secondary motor axons through a single segmental nerve. Finally, we report that unplugged larvae display a striking pharyngeal arch defect, consistent with a dual function of the unplugged gene in axonal guidance and cell motility.     

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.