Zebrafish topped is required for ventral motor axon guidance

Zebrafish primary motor axons extend along stereotyped pathways innervating distinct regions of the developing myotome. During development, these axons make stereotyped projections to ventral and dorsal myotome regions. Caudal primary motoneurons, CaPs, pioneer axon outgrowth along ventral myotomes; whereas, middle primary motoneurons, MiPs, extend axons along dorsal myotomes. Although the development and axon outgrowth of these motoneurons has been characterized, cues that determine whether axons will grow dorsally or ventrally have not been identified. The topped mutant was previously isolated in a genetic screen designed to uncover mutations that disrupt primary motor axon guidance. CaP axons in topped mutants fail to enter the ventral myotome at the proper time, stalling at the nascent horizontal myoseptum, which demarcates dorsal from ventral axial muscle. Later developing secondary motor nerves are also delayed in entering the ventral myotome whereas all other axons examined, including dorsally projecting MiP motor axons, are unaffected in topped mutants. Genetic mosaic analysis indicates that Topped function is non-cell autonomous for motoneurons, and when wild-type cells are transplanted into topped mutant embryos, ventromedial fast muscle are the only cell type able to rescue the CaP axon defect. These data suggest that Topped functions in the ventromedial fast muscle and is essential for motor axon outgrowth into the ventral myotome.     

Collagen XIXa1 is crucial for motor axon navigation at intermediate targets

During development, motor axons navigate from the spinal cord to their muscle targets in the periphery using stereotyped pathways. These pathways are broken down into shorter segments by intermediate targets where axon growth cones are believed to coordinate guidance cues. In zebrafish stumpy mutants, embryonic development proceeds normally; however, as trunk motor axons stall at their intermediate targets, suggesting that Stumpy is needed specifically for motor axon growth cones to proceed past intermediate targets. Fine mapping and positional cloning revealed that stumpy was the zebrafish homolog of the atypical FACIT collagen collagenXIXa1 (colXIX). colXIX expression was observed in a temporal and spatial pattern, consistent with a role in motor axon guidance at intermediate targets. Knocking down zebrafish ColXIX phenocopied the stumpy phenotype and this morpholino phenotype could be rescued by adding back either mouse or zebrafish colXIX RNA. The stumpy phenotype was also partially rescued in mutants by first knocking down zebrafish ColXIX and adding back colXIX RNA, suggesting that the mutation is acting as a dominant negative. Together, these results demonstrate a novel function for a FACIT collagen in guiding vertebrate motor axons through intermediate targets.     

Sema3a1 guides spinal motor axons in a cell- and stage-specific manner in zebrafish

In order for axons to reach their proper targets, both spatiotemporal regulation of guidance molecules and stepwise control of growth cone sensitivity to guidance molecules is required. Here, we show that, in zebrafish, Sema3a1, a secreted class 3 semaphorin, plays an essential role in guiding the caudal primary (CaP) motor axon that pioneers the initial region of the motor pathway. The expression pattern of Sema3a1 suggests that it delimits the pioneer CaP axons to the initial, common pathway via a repulsive action, but then CaP axons become insensitive to Sema3a1 beyond the common pathway. Indeed, nrp1a, which probably encodes a component of the Sema3a1 receptor, is specifically expressed by CaP during the early part of its outgrowth but not during later stages when extending into sema3a1-expressing muscle cells. To examine this hypothesis directly, expression of sema3a1 and/or nrp1a was manipulated in several ways. First, antisense knockdown of Sema3a1 induced CaP axons to branch excessively, stall and/or follow aberrant pathways. Furthermore, dynamic analysis showed they extended more lateral filopodia and often failed to pause at the horizontal myoseptal choice point. Second, antisense knockdown of Nrp1a and double knockdown of Nrp1a/Sema3a1 induced similar outgrowth defects in CaP. Third, CaP axons were inhibited by focally misexpressed sema3a1 along the initial common pathway but not along their pathway beyond the common pathway. Thus, as predicted, Sema3a1 is repulsive to CaP axons in the common region of the pathway, but not beyond the common pathway. Fourth, induced ubiquitous overexpression of sema3a1 caused the CaP axons but not the other primary motor axons to follow aberrant pathways. These results suggest that the repulsive response to Sema3a1 of the primary motor axons along the common pathway is both cell-type specific and dynamically regulated, perhaps via regulation of nrp1a.     

Motoneurons are essential for vascular pathfinding

The neural and vascular systems share common guidance cues that have direct and independent signaling effects on nerves and endothelial cells. Here, we show that zebrafish Netrin 1a directs Dcc-mediated axon guidance of motoneurons and that this neural guidance function is essential for lymphangiogenesis. Specifically, Netrin 1a secreted by the muscle pioneers at the horizontal myoseptum (HMS) is required for the sprouting of dcc-expressing rostral primary motoneuron (RoP) axons and neighboring axons along the HMS, adjacent to the future trajectory of the parachordal chain (PAC). These axons are required for the formation of the PAC and, subsequently, the thoracic duct. The failure to form the PAC in netrin 1a or dcc morphants is phenocopied by laser ablation of motoneurons and is rescued both by cellular transplants and overexpression of dcc mRNA. These results provide a definitive example of the requirement of axons in endothelial guidance leading to the parallel patterning of nerves and vessels in vivo.     

Position fine-tuning of caudal primary motoneurons in the zebrafish spinal cord

In zebrafish embryos, each myotome is typically innervated by three primary motoneurons (PMNs): the caudal primary (CaP), middle primary (MiP) and rostral primary (RoP). PMN axons first exit the spinal cord through a single exit point located at the midpoint of the overlying somite, which is formed beneath the CaP cell body and is pioneered by the CaP axon. However, the placement of CaP cell bodies with respect to corresponding somites is poorly understood. Here, we determined the early events in CaP cell positioning using neuropilin 1a (nrp1a):gfp transgenic embryos in which CaPs were specifically labeled with GFP. CaP cell bodies first exhibit an irregular pattern in presence of newly formed corresponding somites and then migrate to achieve their proper positions by axonogenesis stages. CaPs are generated in excess compared with the number of somites, and two CaPs often overlap at the same position through this process. Next, we showed that CaP cell bodies remain in the initial irregular positions after knockdown of Neuropilin1a, a component of the class III semaphorin receptor. Irregular CaP position frequently results in aberrant double exit points of motor axons, and secondary motor axons form aberrant exit points following CaP axons. Its expression pattern suggests that sema3ab regulates the CaP position. Indeed, irregular CaP positions and exit points are induced by Sema3ab knockdown, whose ectopic expression can alter the position of CaP cell bodies. Results suggest that Semaphorin-Neuropilin signaling plays an important role in position fine-tuning of CaP cell bodies to ensure proper exit points of motor axons.     

Pathfinding by zebrafish motoneurons in the absence of normal pioneer axons.

Individually identified primary motoneurons of the zebrafish embryo pioneer cell-specific peripheral motor nerves. Later, the growth cones of secondary motoneurons extend along pathways pioneered by primary motor axons. To learn whether primary motor axons are required for pathway navigation by secondary motoneurons, we ablated primary motoneurons and examined subsequent pathfinding by the growth cones of secondary motoneurons. We found that ablation of the primary motoneuron that pioneers the ventral nerve delayed ventral nerve formation, but a normal-appearing nerve eventually formed. Therefore, the secondary motoneurons that extend axons in the ventral nerve were able to pioneer that pathway in the absence of the pathway-specific primary motoneuron. In contrast, in the absence of the primary motoneuron that normally pioneers the dorsal nerve, secondary motoneurons did not pioneer a nerve in the normal location, instead they formed dorsal nerves in an atypical position. This difference in the ability of these two groups of motoneurons to pioneer their normal pathways suggests that the guidance rules followed by their growth cones may be very different. Furthermore, the observation that the atypical dorsal nerves formed in a consistent incorrect location suggests that the growth cones of the secondary motoneurons that extend dorsally make hierarchical pathway choices.     

Netrin signaling breaks the equivalence between two identified zebrafish motoneurons revealing a new role of intermediate targets

Background

We previously showed that equivalence between two identified zebrafish motoneurons is broken by interactions with identified muscle fibers that act as an intermediate target for the axons of these motoneurons. Here we investigate the molecular basis of the signaling interaction between the intermediate target and the motoneurons.

Principal Findings

We provide evidence that Netrin 1a is an intermediate target-derived signal that causes two equivalent motoneurons to adopt distinct fates. We show that although these two motoneurons express the same Netrin receptors, their axons respond differently to Netrin 1a encountered at the intermediate target. Furthermore, we demonstrate that when Netrin 1a is knocked down, more distal intermediate targets that express other Netrins can also function to break equivalence between these motoneurons.

Significance

Our results suggest a new role for intermediate targets in breaking neuronal equivalence. The data we present reveal that signals encountered during axon pathfinding can cause equivalent neurons to adopt distinct fates. Such signals may be key in diversifying a neuronal population and leading to correct circuit formation.     

sidestep encodes a target-derived attractant essential for motor axon guidance in Drosophila

At specific choice points in the periphery, subsets of motor axons defasciculate from other axons in the motor nerves and steer into their muscle target regions. Using a large-scale genetic screen in Drosophila, we identified the sidestep (side) gene as essential for motor axons to leave the motor nerves and enter their muscle targets. side encodes a target-derived transmembrane protein (Side) that is a novel member of the immunoglobulin superfamily (IgSF). Side is expressed on embryonic muscles during the period when motor axons leave their nerves and extend onto these muscles. In side mutant embryos, motor axons fail to extend onto muscles and instead continue to extend along their motor nerves. Ectopic expression of Side results in extensive and prolonged motor axon contact with inappropriate tissues expressing Side.     

The divergent TGF-beta ligand Dawdle utilizes an activin pathway to influence axon guidance in Drosophila

Axon guidance is regulated by intrinsic factors and extrinsic cues provided by other neurons, glia and target muscles. Dawdle (Daw), a divergent TGF-beta superfamily ligand expressed in glia and mesoderm, is required for embryonic motoneuron pathfinding in Drosophila. In daw mutants, ISNb and SNa axons fail to extend completely and are unable to innervate their targets. We find that Daw initiates an activin signaling pathway via the receptors Punt and Baboon (Babo) and the signal-transducer Smad2. Furthermore, mutations in these signaling components display similar axon guidance defects. Cell-autonomous disruption of receptor signaling suggests that Babo is required in motoneurons rather than in muscles or glia. Ectopic ligand expression can rescue the daw phenotype, but has no deleterious effects. Our results indicate that Daw functions in a permissive manner to modulate or enable the growth cone response to other restricted guidance cues, and support a novel role for activin signaling in axon guidance.     

Pathfinding by sensory axons in Drosophila: substrates and choice points in early lch5 axon outgrowth

We have examined the pattern of axon growth from the lateral chordotonal (lch5) neurons in the body wall of the Drosophila embryo and identified cellular substrates and choice points involved in early axon pathfinding by these sensory neurons. At the first choice point (TP1), the lch5 growth cones contact the most distal cells of the spiracular branch (SB) of the trachea. The SB provides a substrate along which the axons extend internally to the level of the intersegmental nerve (ISN). In the absence of the SB, the lch5 axons often stall near TP1 or follow aberrant routes towards the CNS. At the second choice point (TP2), the lch5 growth cones make their first contact with other axons and turn ventrally toward the CNS, fasciculating specifically with the motor axons of the ISN.     

Local translation and directional steering in axons

The assembly of functional neural circuits in the developing brain requires neurons to extend axons to the correct targets. This in turn requires the navigating tips of axons to respond appropriately to guidance cues present along the axonal pathway, despite being cellular 'outposts' far from the soma. Work over the past few years has demonstrated a critical role for local translation within the axon in this process in vitro, making axon guidance another process that requires spatially localized translation, among others such as synaptic plasticity, cell migration, and cell polarity. This article reviews recent findings in local axonal translation and discusses how new protein synthesis may function in growth cone guidance, with a comparative view toward models of local translation in other systems.     

Genes necessary for directed axonal elongation or fasciculation in C. elegans.

The outgrowth of single axons through different cellular environments requires distinct sets of genes in the nematode C. elegans. Three genes are required for the pioneering circumferential outgrowth of identified motor neuron axons between the lateral hypodermal cell membrane and the basal lamina. Three other genes are required for the longitudinal outgrowth of these axons along preexisting axon bundles as well as for the fasciculation of axons within these neuron bundles. Five additional genes are required for circumferential outgrowth, longitudinal outgrowth, and fasciculation; mutations in three of these genes disrupt axon ultrastructure, suggesting that they function in axon formation rather than in axon guidance.     

Slit-mediated repulsion is a key regulator of motor axon pathfinding in the hindbrain

The floor plate is known to be a source of repellent signals for cranial motor axons, preventing them from crossing the midline of the hindbrain. However, it is unknown which molecules mediate this effect in vivo. We show that Slit and Robo proteins are candidate motor axon guidance molecules, as Robo proteins are expressed by cranial motoneurons, and Slit proteins are expressed by the tissues that delimit motor axon trajectories, i.e. the floor plate and the rhombic lip. We present in vitro evidence showing that Slit1 and Slit2 proteins are selective inhibitors and repellents for dorsally projecting, but not for ventrally projecting, cranial motor axons. Analysis of mice deficient in Slit and Robo function shows that cranial motor axons aberrantly enter the midline, while ectopic expression of Slit1 in chick embryos leads to specific motor axon projection errors. Expression of dominant-negative Robo receptors within cranial motoneurons in chick embryos strikingly perturbs their projections, causing some motor axons to enter the midline, and preventing dorsally projecting motor axons from exiting the hindbrain. These data suggest that Slit proteins play a key role in guiding dorsally projecting cranial motoneurons and in facilitating their neural tube exit.     

The branchial arches and HGF are growth-promoting and chemoattractant for cranial motor axons

During development, cranial motor neurons extend their axons along distinct pathways into the periphery. For example, branchiomotor axons extend dorsally to leave the hindbrain via large dorsal exit points. They then grow in association with sensory ganglia, to their targets, the muscles of the branchial arches. We have investigated the possibility that pathway tissues might secrete diffusible chemorepellents or chemoattractants that guide cranial motor axons, using co-cultures in collagen gels. We found that explants of dorsal neural tube or hindbrain roof plate chemorepelled cranial motor axons, while explants of cranial sensory ganglia were weakly chemoattractive. Explants of branchial arch mesenchyme were strongly growth-promoting and chemoattractive for cranial motor axons. Enhanced and oriented axon outgrowth was also elicited by beads loaded with Hepatocyte Growth Factor (HGF); antibodies to this protein largely blocked the outgrowth and orientation effects of the branchial arch on motor axons. HGF was expressed in the branchial arches, whilst Met, which encodes an HGF receptor, was expressed by subpopulations of cranial motor neurons. Mice with targetted disruptions of HGF or Met showed defects in the navigation of hypoglossal motor axons into the branchial region. Branchial arch tissue may thus act as a target-derived factor that guides motor axons during development. This influence is likely to be mediated partly by Hepatocyte Growth Factor, although a component of branchial arch-mediated growth promotion and chemoattraction was not blocked by anti-HGF antibodies.     

Analysis of zebrafish sidetracked mutants reveals a novel role for Plexin A3 in intraspinal motor axon guidance

One of the earliest guidance decisions for spinal cord motoneurons occurs when pools of motoneurons orient their growth cones towards a common, segmental exit point. In contrast to later events, remarkably little is known about the molecular mechanisms underlying intraspinal motor axon guidance. In zebrafish sidetracked (set) mutants, motor axons exit from the spinal cord at ectopic positions. By single-cell labeling and time-lapse analysis we show that motoneurons with cell bodies adjacent to the segmental exit point properly exit from the spinal cord, whereas those farther away display pathfinding errors. Misguided growth cones either orient away from the endogenous exit point, extend towards the endogenous exit point but bypass it or exit at non-segmental, ectopic locations. Furthermore, we show that sidetracked acts cell autonomously in motoneurons. Positional cloning reveals that sidetracked encodes Plexin A3, a semaphorin guidance receptor for repulsive guidance. Finally, we show that sidetracked (plexin A3) plays an additional role in motor axonal morphogenesis. Together, our data genetically identify the first guidance receptor required for intraspinal migration of pioneering motor axons and implicate the well-described semaphorin/plexin signaling pathway in this poorly understood process. We propose that axonal repulsion via Plexin A3 is a major driving force for intraspinal motor growth cone guidance.     

Paraxial mesoderm specifies zebrafish primary motoneuron subtype identity

We provide the first analysis of how a segmentally reiterated pattern of neurons is specified along the anteroposterior axis of the vertebrate spinal cord by investigating how zebrafish primary motoneurons are patterned. Two identified primary motoneuron subtypes, MiP and CaP, occupy distinct locations within the ventral neural tube relative to overlying somites, express different genes and innervate different muscle territories. In all vertebrates examined so far, paraxial mesoderm-derived signals specify distinct motoneuron subpopulations in specific anteroposterior regions of the spinal cord. We show that signals from paraxial mesoderm also control the much finer-grained segmental patterning of zebrafish primary motoneurons. We examined primary motoneuron specification in several zebrafish mutants that have distinct effects on paraxial mesoderm development. Our findings suggest that in the absence of signals from paraxial mesoderm, primary motoneurons have a hybrid identity with respect to gene expression, and that under these conditions the CaP axon trajectory may be dominant.