Axonal Growth Patterns Research Articles

Enhanced axon outgrowth of spinal motor neurons in co-culturing with dorsal root ganglions antagonizes the growth inhibitory environment

IntroductionForming a bridge made of functional axons to span the lesion is essential to reconstruct the motor circuitry following spinal cord injury (SCI). Dorsal root ganglion (DRG) axons are robust in axon growth and have been proved to facilitate the growth of cortical neurons in a process of axon-facilitated axon regeneration. However, whether DRG transplantation affects the axon outgrowth of spinal motor neurons (SMNs) that play crucial roles in motor circuitry remains unclear. MethodsWe investigated the axonal growth patterns of co-cultured DRGs and SMN aggregates (SMNAs) taking advantage of a well-designed 3D-printed in vitro system. Chondroitin sulphate proteoglycans (CSPG) induced inhibitory matrix was introduced to imitate the inhibitory environment following SCI. Axonal lengths of DRG, SMNA or DRG & SMNA cultured on the permissive or CSPG induced inhibitory matrix were measured and compared. ResultsOur results indicated that under the guidance of full axonal connection generated from two opposing populations of DRGs, SMNA axons were growth-enhanced and elongated along the DRG axon bridge to distances that they could not otherwise reach. Quantitatively, the co-culture increased the SMNA axonal length by 32.1 %. Moreover, the CSPG matrix reduced the axonal length of DRGs and SMNAs by 46.2 % and 17.7 %, respectively. This inhibitory effect was antagonized by the co-culture of DRGs and SMNAs. Especially for SMNAs, they extended the axons across the CSPG-coating matrix, reached the lengths close to those of SMNAs cultured on the permissive matrix alone. ConclusionsThis study deepens our understanding of axon-facilitated reconstruction of the motor circuitry. Moreover, the results support SCI treatment utilizing the enhanced outgrowth of axons to restore functional connectivity in SCI patients.

Physical understanding of axonal growth patterns on grooved substrates: groove ridge crossing versus longitudinal alignment

Surface topographies such as micrometric edges and grooves have been widely used to improve neuron outgrowth. However, finding the mechanism of neuron–surface interactions on grooved substrates remains a challenge. In this work, PC12 cells and chick forebrain neurons (CFNs) were cultured on grooved and smooth polyacrylonitrile substrates. It was found that CFNs showed a tendency of growing across groove ridges; while PC12 cells were only observed to grow in the longitudinal direction of grooves. To further investigate these observations, a 3D physical model of axonal outgrowth was developed. In this model, axon shafts are simulated as elastic 3D beams, accounting for the axon outgrowth as well as the focal contacts between axons and substrates. Moreover, the bending direction of axon tips during groove ridge crossing is governed by the energy minimization principle. Our physical model predicts that axonal groove ridge crossing is contributed by the bending compliance of axons, caused by lower Young’s modulus and smaller diameters. This work will aid the understanding of the mechanisms involved in axonal alignment and elongation of neurons guided by grooved substrates, and the obtained insights can be used to enhance the design of instructive scaffolds for nerve tissue engineering and regeneration applications.

A rapid and accurate method to quantify neurite outgrowth from cell and tissue cultures: Two image analytic approaches using adaptive thresholds or machine learning

BackgroundAssessments of axonal outgrowth and dendritic development are essential readouts in many in vitro models in the field of neuroscience. Available analysis software is based on the assessment of fixed immunolabelled tissue samples, making it impossible to follow the dynamic development of neurite outgrowth. Thus, automated algorithms that efficiently analyse brightfield images, such as those obtained during time-lapse microscopy, are needed. New methodWe developed and validated algorithms to quantitatively assess neurite outgrowth from living and unstained spinal cord slice cultures (SCSCs) and dorsal root ganglion cultures (DRGCs) based on an adaptive thresholding approach called NeuriteSegmantation. We used a machine learning approach to evaluate dendritic development from dissociate neuron cultures. ResultsNeuriteSegmentation successfully recognized axons in brightfield images of SCSCs and DRGCs. The temporal pattern of axonal growth was successfully assessed. In dissociate neuron cultures the total number of cells and their outgrowth of dendrites were successfully assessed using machine learning. Comparison with existing methodsThe methods were positively correlated and were more time-saving than manual counts, having performing times varying from 0.5−2 min. In addition, NeuriteSegmentation was compared to NeuriteJ®, that uses global thresholding, being more reliable in recognizing axons in areas of intense background. ConclusionThe developed image analysis methods were more time-saving and user-independent than established approaches. Moreover, by using adaptive thresholding, we could assess images with large variations in background intensity. These tools may prove valuable in the quantitative analysis of axonal and dendritic outgrowth from numerous in vitro models used in neuroscience.

Long-Distance Axonal Growth and Protracted Functional Maturation of Neurons Derived from Human Induced Pluripotent Stem Cells After Intracerebral Transplantation.

The capacity for induced pluripotent stem (iPS) cells to be differentiated into a wide range of neural cell types makes them an attractive donor source for autologous neural transplantation therapies aimed at brain repair. Translation to the in vivo setting has been difficult, however, with mixed results in a wide variety of preclinical models of brain injury and limited information on the basic in vivo properties of neural grafts generated from human iPS cells. Here we have generated a human iPS cell line constitutively expressing green fluorescent protein as a basis to identify and characterize grafts resulting from transplantation of neural progenitors into the adult rat brain. The results show that the grafts contain a mix of neural cell types, at various stages of differentiation, including neurons that establish extensive patterns of axonal growth and progressively develop functional properties over the course of 1 year after implantation. These findings form an important basis for the design and interpretation of preclinical studies using human stem cells for functional circuit re‐construction in animal models of brain injury. Stem Cells Translational Medicine 2017;6:1547–1556

Mechanosensing is critical for axon growth in the developing brain.

During nervous system development, neurons extend axons along well-defined pathways. The current understanding of axon pathfinding is based mainly on chemical signalling. However, growing neurons interact not only chemically but also mechanically with their environment. Here we identify mechanical signals as important regulators of axon pathfinding. In vitro, substrate stiffness determined growth patterns of Xenopus retinal ganglion cell (RGC) axons. In vivo atomic force microscopy revealed striking stiffness gradient patterns in the embryonic brain. RGC axons grew towards the tissue’s softer side, which was reproduced in vitro in the absence of chemical gradients. To test the importance of mechanical signals for axon growth in vivo, we altered brain stiffness, blocked mechanotransduction pharmacologically, and knocked down the mechanosensitive ion channel Piezo1. All treatments resulted in aberrant axonal growth and pathfinding errors, suggesting that local tissue stiffness–read out by mechanosensitive ion channels–is critically involved in instructing neuronal growth in vivo.

Self-organization of "fibro-axonal" composite tissue around unmodified metallic micro-electrodes can form a functioning interface with a peripheral nerve: A new direction for creating long-term neural interfaces.

A long-term peripheral neural interface is an area of intense research. The use of electrode interfaces is limited by the biological response to the electrode material. We created an electrode construct to harbor the rat sciatic nerve with interposition of autogenous adipose tissue between the nerve and the electrode. The construct was implanted for 10 weeks. Immunohistochemistry showed a unique laminar pattern of axonal growth layered between fibro-collagenous tissue, forming a physical interface with the tungsten micro-electrode. Action potentials transmitted across the intrerface showed mean conduction velocities varying between 6.99 ± 2.46 and 20.14 ± 4 m/s. We have demonstrated the feasibility of a novel peripheral nerve interface through modulation of normal biologic phenomena. It has potential applications as a chronic implantable neural interface.

The olfactory system as a model to study axonal growth patterns and morphology in vivo.

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of the olfactory epithelium continuously give rise to new sensory neurons that extend their axons into the olfactory bulb, where they face the challenge to integrate into existing circuitry. Because of this particular feature, the olfactory system represents a unique opportunity to monitor axonal wiring and guidance, and to investigate synapse formation. Here we describe a procedure for in vivo labeling of sensory neurons and subsequent visualization of axons in the olfactory system of larvae of the amphibian Xenopus laevis. To stain sensory neurons in the olfactory organ we adopt the electroporation technique. In vivo electroporation is an established technique for delivering fluorophore-coupled dextrans or other macromolecules into living cells. Stained sensory neurons and their axonal processes can then be monitored in the living animal either using confocal laser-scanning or multiphoton microscopy. By reducing the number of labeled cells to few or single cells per animal, single axons can be tracked into the olfactory bulb and their morphological changes can be monitored over weeks by conducting series of in vivo time lapse imaging experiments. While the described protocol exemplifies the labeling and monitoring of olfactory sensory neurons, it can also be adopted to other cell types within the olfactory and other systems.

Sea urchin neural development and the metazoan paradigm of neurogenesis.

Summary:Urchin embryos continue to prove useful as a means of studying embryonic signaling and gene regulatory networks, which together control early development. Recent progress in understanding the molecular mechanisms underlying the patterning of ectoderm has renewed interest in urchin neurogenesis. We have employed an emerging model of neurogenesis that appears to be broadly shared by metazoans as a framework for this review. We use the model to provide context and summarize what is known about neurogenesis in urchin embryos. We review morphological features of the differentiation phase of neurogenesis and summarize current understanding of neural specification and regulation of proneural networks. Delta-Notch signaling is a common feature of metazoan neurogenesis that produces committed progenitors and it appears to be a critical phase of neurogenesis in urchin embryos. Descriptions of the differentiation phase of neurogenesis indicate a stereotypic sequence of neural differentiation and patterns of axonal growth. Features of neural differentiation are consistent with localized signals guiding growth cones with trophic, adhesive, and tropic cues. Urchins are a facile, postgenomic model with the potential of revealing many shared and derived features of deuterostome neurogenesis.

Cyclic AMP promotes axon regeneration, lesion repair and neuronal survival in lampreys after spinal cord injury

Axon regeneration after spinal cord injury in mammals is inadequate to restore function, illustrating the need to design better strategies for improving outcomes. Increasing the levels of the second messenger cyclic adenosine monophosphate (cAMP) after spinal cord injury enhances axon regeneration across a wide variety of species, making it an excellent candidate molecule that has therapeutic potential. However, several important aspects of the cellular and molecular mechanisms by which cAMP enhances axon regeneration are still unclear, such as how cAMP affects axon growth patterns, the molecular components within growing axon tips, the lesion scar, and neuronal survival. To address these points, we took advantage of the large, identified reticulospinal (RS) neurons in lamprey, a vertebrate that exhibits robust axon regeneration after a complete spinal cord transection. Application of a cAMP analog, db-cAMP, at the time of spinal cord transection increased the number of axons that regenerated across the lesion site. Db-cAMP also promoted axons to regenerate in straighter paths, prevented abnormal axonal growth patterns, increased the levels of synaptotagmin within axon tips, and increased the number of axotomized neurons that survived after spinal cord injury, thereby increasing the pool of neurons available for regeneration. There was also a transient increase in the number of microglia/macrophages and improved repair of the lesion site. Taken together, these data reveal several new features of the cellular and molecular mechanisms underlying cAMP-mediated enhancement of axon regeneration, further emphasizing the positive roles for this conserved pathway.

Short- and Long-Term Effects of LRRK2 on Axon and Dendrite Growth

Mutations in leucine-rich repeat kinase 2 (LRRK2) underlie an autosomal-dominant form of Parkinson's disease (PD) that is clinically indistinguishable from idiopathic PD. The function of LRRK2 is not well understood, but it has become widely accepted that LRRK2 levels or its kinase activity, which is increased by the most commonly observed mutation (G2019S), regulate neurite growth. However, growth has not been measured; it is not known whether mean differences in length correspond to altered rates of growth or retraction, whether axons or dendrites are impacted differentially or whether effects observed are transient or sustained. To address these questions, we compared several developmental milestones in neurons cultured from mice expressing bacterial artificial chromosome transgenes encoding mouse wildtype-LRRK2 or mutant LRRK2-G2019S, Lrrk2 knockout mice and non-transgenic mice. Over the course of three weeks of development on laminin, the data show a sustained, negative effect of LRRK2-G2019S on dendritic growth and arborization, but counter to expectation, dendrites from Lrrk2 knockout mice do not elaborate more rapidly. In contrast, young neurons cultured on a slower growth substrate, poly-L-lysine, show significantly reduced axonal and dendritic motility in Lrrk2 transgenic neurons and significantly increased motility in Lrrk2 knockout neurons with no significant changes in length. Our findings support that LRRK2 can regulate patterns of axonal and dendritic growth, but they also show that effects vary depending on growth substrate and stage of development. Such predictable changes in motility can be exploited in LRRK2 bioassays and guide exploration of LRRK2 function in vivo.

Protein Tyrosine Phosphatase Receptor Type O Inhibits Trigeminal Axon Growth and Branching by Repressing TrkB and Ret Signaling

Axonal branches of the trigeminal ganglion (TG) display characteristic growth and arborization patterns during development. Subsets of TG neurons express different receptors for growth factors, but these are unlikely to explain the unique patterns of axonal arborizations. Intrinsic modulators may restrict or enhance cellular responses to specific ligands and thereby contribute to the development of axon growth patterns. Protein tyrosine phosphatase receptor type O (PTPRO), which is required for Eph receptor-dependent retinotectal development in chick and for development of subsets of trunk sensory neurons in mouse, may be such an intrinsic modulator of TG neuron development. PTPRO is expressed mainly in TrkB-expressing (TrkB(+)) and Ret(+) mechanoreceptors within the TG during embryogenesis. In PTPRO mutant mice, subsets of TG neurons grow longer and more elaborate axonal branches. Cultured PTPRO(-/-) TG neurons display enhanced axonal outgrowth and branching in response to BDNF and GDNF compared with control neurons, indicating that PTPRO negatively controls the activity of BDNF/TrkB and GDNF/Ret signaling. Mouse PTPRO fails to regulate Eph signaling in retinocollicular development and in hindlimb motor axon guidance, suggesting that chick and mouse PTPRO have different substrate specificities. PTPRO has evolved to fine tune growth factor signaling in a cell-type-specific manner and to thereby increase the diversity of signaling output of a limited number of receptor tyrosine kinases to control the branch morphology of developing sensory neurons. The regulation of Eph receptor-mediated developmental processes by protein tyrosine phosphatases has diverged between chick and mouse.

Quantification of dendritic and axonal growth after injury to the auditory system of the adult cricket Gryllus bimaculatus

Dendrite and axon growth and branching during development are regulated by a complex set of intracellular and external signals. However, the cues that maintain or influence adult neuronal morphology are less well understood. Injury and deafferentation tend to have negative effects on adult nervous systems. An interesting example of injury-induced compensatory growth is seen in the cricket, Gryllus bimaculatus. After unilateral loss of an ear in the adult cricket, auditory neurons within the central nervous system (CNS) sprout to compensate for the injury. Specifically, after being deafferented, ascending neurons (AN-1 and AN-2) send dendrites across the midline of the prothoracic ganglion where they receive input from auditory afferents that project through the contralateral auditory nerve (N5). Deafferentation also triggers contralateral N5 axonal growth. In this study, we quantified AN dendritic and N5 axonal growth at 30 h, as well as at 3, 5, 7, 14, and 20 days after deafferentation in adult crickets. Significant differences in the rates of dendritic growth between males and females were noted. In females, dendritic growth rates were non-linear; a rapid burst of dendritic extension in the first few days was followed by a plateau reached at 3 days after deafferentation. In males, however, dendritic growth rates were linear, with dendrites growing steadily over time and reaching lengths, on average, twice as long as in females. On the other hand, rates of N5 axonal growth showed no significant sexual dimorphism and were linear. Within each animal, the growth rates of dendrites and axons were not correlated, indicating that independent factors likely influence dendritic and axonal growth in response to injury in this system. Our findings provide a basis for future study of the cellular features that allow differing dendrite and axon growth patterns as well as sexually dimorphic dendritic growth in response to deafferentation.

Activation of glial FGFRs is essential in glial migration, proliferation, and survival and in glia-neuron signaling during olfactory system development.

Development of the adult olfactory system of the moth Manduca sexta depends on reciprocal interactions between olfactory receptor neuron (ORN) axons growing in from the periphery and centrally-derived glial cells. Early-arriving ORN axons induce a subset of glial cells to proliferate and migrate to form an axon-sorting zone, in which later-arriving ORN axons will change their axonal neighbors and change their direction of outgrowth in order to travel with like axons to their target areas in the olfactory (antennal) lobe. These newly fasciculated axon bundles will terminate in protoglomeruli, the formation of which induces other glial cells to migrate to surround them. Glial cells do not migrate unless ORN axons are present, axons fail to fasciculate and target correctly without sufficient glial cells, and protoglomeruli are not maintained without a glial surround. We have shown previously that Epidermal Growth Factor receptors and the IgCAMs Neuroglian and Fasciclin II play a role in the ORN responses to glial cells. In the present work, we present evidence for the importance of glial Fibroblast Growth Factor receptors in glial migration, proliferation, and survival in this developing pathway. We also report changes in growth patterns of ORN axons and of the dendrites of olfactory (antennal lobe) neurons following blockade of glial FGFR activation that suggest that glial FGFR activation is important in reciprocal communication between neurons and glial cells.

Chlorpyrifos-Oxon Disrupts Zebrafish Axonal Growth and Motor Behavior

Axonal morphology is a critical determinant of neuronal connectivity, and perturbation of the rate or extent of axonal growth during development has been linked to neurobehavioral deficits in animal models and humans. We previously demonstrated that the organophosphorus pesticide (OP) chlorpyrifos (CPF) inhibits axonal growth in cultured neurons. In this study, we used a zebrafish model to determine whether CPF, its oxon metabolite (CPFO), or the excreted metabolite trichloro-2-pyridinol (TCPy) alter spatiotemporal patterns of axonal growth in vivo. Static waterborne exposure to CPFO, but not CPF or TCPy, at concentrations ≥ 0.03 μM from 24- to 72-h post fertilization significantly inhibited acetylcholinesterase, and high-performance liquid chromatography detected significantly more TCPy in zebrafish exposed to 0.1 μM CPFO versus 1.0 μM CPF. These data suggest that zebrafish lack the metabolic enzymes to activate CPF during these early developmental stages. Consistent with this, CPFO, but not CPF, significantly inhibited axonal growth of sensory neurons, primary motoneurons, and secondary motoneurons at concentrations ≥ 0.1 μM. Secondary motoneurons were the most sensitive to axonal growth inhibition by CPFO, which was observed at concentrations that did not cause mortality, gross developmental defects, or aberrant somatic muscle differentiation. CPFO effects on axonal growth correlated with adverse effects on touch-induced swimming behavior, suggesting the functional relevance of these structural changes. These data suggest that altered patterns of neuronal connectivity contribute to the developmental neurotoxicity of CPF and demonstrate the relevance of zebrafish as a model for studying OP developmental neurotoxicity.

Prenatal stress alters presynaptic marker proteins in the hippocampus of rat pups

Exposure to stress during critical periods of an organism's maturation can result in permanent behavioral changes and induced hyper-responsive to aversive stimuli as adult. Hippocampus is a plastic and vulnerable brain structure that is susceptible to damage during aging and repeated stress. The present study examines the effect of maternal restraint stress on the level of GAP-43, pGAP-43 and synaptophysin in the hippocampus of rat pups. Prenatal stress (PS) causes a significant increase of GAP-43 and pGAP-43 ( p ≤ 0.001) in the pup's hippocampus during postnatal days 7 and 14, but not at later ages. Up-regulation of GAP-43 and pGAP-43 may alter the pattern of axonal growth and synapses’ formation in the pup's brain since the first two postnatal weeks are correlated with peak period of synaptogenesis in the rat brain. We also examined the level of synaptophysin, a synaptic vesicle membrane protein, in the pup's brain. Our finding revealed that, PS causes a significant decrease of synaptophysin in the pup's hippocampus as compared to control ( p ≤ 0.001). These changes are due to the direct effects of maternal stress hormone since repeated injection with corticosterone (CORT, 40 mg/kg) to pregnant rat during gestation days (GDs) 14–21 also gave the same results. Abnormal axonal sprouting and reorganization together with the alterations in synaptic vesicle membrane protein during the critical period of synaptogenesis may lead to a defect in synapse formation and axonal pruning in the hippocampus. These changes may be associated with stress-induced impairment of hippocampal function that occurs in later life of the offspring.

Distinct Modes of Neuritic Growth in Purkinje Neurons at Different Developmental Stages: Axonal Morphogenesis and Cellular Regulatory Mechanisms

BackgroundDuring development, neurons modify their axon growth mode switching from an elongating phase, in which the main axon stem reaches the target territory through growth cone-driven extension, to an arborising phase, when the terminal arbour is formed to establish synaptic connections. To investigate the relative contribution of cell-autonomous factors and environmental signals in the control of these distinct axon growth patterns, we examined the neuritogenesis of Purkinje neurons in cerebellar cultures prepared at elongating (embryonic day 17) or arborising (postnatal day zero) stages of Purkinje axon maturation.Methodology/Principal FindingsWhen placed in vitro, Purkinje cells of both ages undergo an initial phase of neurite elongation followed by the development of terminal ramifications. Nevertheless, elongation of the main axon stem prevails in embryonic Purkinje axons, and many of these neurons are totally unable to form terminal branches. On the contrary, all postnatal neurites switch to arbour growth within a few days in culture and spread extensive terminal trees. Regardless of their elongating or arborising pattern, defined growth features (e.g. growth rate and tree extension) of embryonic Purkinje axons remain distinct from those of postnatal neurites. Thus, Purkinje neurons of different ages are endowed with intrinsic stage-specific competence for neuritic growth. Such competence, however, can be modified by environmental cues. Indeed, while exposure to the postnatal environment stimulates the growth of embryonic axons without modifying their phenotype, contact-mediated signals derived from granule cells specifically induce arborising growth and modulate the dynamics of neuritic elongation.Conclusions/SignificanceCultured Purkinje cells recapitulate an intrinsically coded neuritogenic program, involving initial navigation of the axon towards the target field and subsequent expansion of the terminal arborisation. The execution of this program is regulated by environmental signals that modify the growth competence of Purkinje cells, so to adapt their endogenous properties to the different phases of neuritic morphogenesis.

Conservation of arthropod midline netrin accumulation revealed with a cross‐reactive antibody provides evidence for midline cell homology

Although many similarities in arthropod CNS development exist, differences in axonogenesis and the formation of midline cells, which regulate axon growth, have been observed. For example, axon growth patterns in the ventral nerve cord of Artemia franciscana differ from that of Drosophila melanogaster. Despite such differences, conserved molecular marker expression at the midline of several arthropod species indicates that midline cells may be homologous in distantly related arthropods. However, data from additional species are needed to test this hypothesis. In this investigation, nerve cord formation and the putative homology of midline cells were examined in distantly related arthropods, including: long- and short-germ insects (D. melanogaster, Aedes aeygypti, and Tribolium castaneum), branchiopod crustaceans (A. franciscana and Triops longicauditus), and malacostracan crustaceans (Porcellio laevis and Parhyale hawaiensis). These comparative analyses were aided by a cross-reactive antibody generated against the Netrin (Net) protein, a midline cell marker and regulator of axonogenesis. The mechanism of nerve cord formation observed in Artemia is found in Triops, another branchiopod, but is not found in the other arthropods examined. Despite divergent mechanisms of midline cell formation and nerve cord development, Net accumulation is detected in a well-conserved subset of midline cells in branchiopod crustaceans, malacostracan crustaceans, and insects. Notably, the Net accumulation pattern is also conserved at the midline of the amphipod P. hawaiensis, which undergoes split germ-band development. Conserved Net accumulation patterns indicate that arthropod midline cells are homologous, and that Nets function to regulate commissure formation during CNS development of Tetraconata.

Getting into shape: Optimal ligand gradients for axonal guidance

During neural development, neurons from downstream, presynaptic regions of the nervous system (such as the retina) send spatially patterned axonal projections to upstream, target regions (the tectum or superior colliculus). A servomechanism model has been proposed to explain the pattern and time-course of axonal growth between these two regions [Honda, H., 1998. Topographic mapping in the retinotectal projection by means of complementary ligand and receptor gradients: a computer simulation study. J. Theor. Biol., 192, 235-246]. Here, we show that a modification of this model incorporating a different criterion for axonal decision-making, called the local optimum rule, is guaranteed to converge to a topographic map under a wide range of conditions encountered during neural development. A theoretical investigation of these conditions leads to new hypotheses regarding map formation.

Divergent axon growth patterns among arthropods coincide with changes in netrin expression patterns

Divergent axon growth patterns among arthropods coincide with changes in netrin expression patterns

Cell interactions that affect axonogenesis in the leech Theromyzon rude.

The leech nervous system comprises a relatively simple network of longitudinal (connective) and transverse (segmental) nerves. We have followed the normal pattern of axon development in the glossiphoniid leech Theromyzon rude by immunostaining embryonic preparations with antibody to acetylated alpha-tubulin. The dependence of the normal pattern of axon growth on cells in the mesodermal (M) and ectodermal (N, O, P and Q) lineages was examined by selectively ablating subsets of these lineages in developing embryos. We found that ablating mesoderm severely disrupted overall axonogenesis, while various ectodermal ablations induced a range of more specific phenotypes. In particular, formation of the posterior segmental nerve (PP) was abnormal in embryos deficient in primary neuroectoderm (N lineage). More specific ablations demonstrated that a subset of N-derived cells were required for establishing the PP nerve root. Previous studies have shown that the PP nerve root is normally pioneered by an O lineage-derived neuron (PD). Our results suggest that the role of the N lineage-derived cells is to induce the migration of neuron P(D) to its normal position in the posterior compartment of the hemiganglion.