Axon outgrowth along segmental nerves in the leech. I. Identification of candidate guidance cells

We studied the development of the major extraganglionic components of the germinal plate in embryos of the glossiphoniid leech Helobdella triserialis to improve our understanding of the mechanism of segmental nerve formation. We examined the outgrowth of groups of axons from ganglionic neurons into the segmental nerves, the migration of peripheral neurons and epidermal specializations to their definitive sites, and the development of circular and longitudinal muscle fibers. We visualized axons, as well as neurons and epidermal specializations, by means of fluorescent cell lineage tracers injected earlier into blastomeres and muscle fibers by means of immunofluorescence. The development of cells in all groups was found to follow a stereotyped pattern. Axons of ganglionic neurons approach some identified peripheral neurons located along the segmental nerve paths but not, in general, epidermal specializations and muscle fibers. Near the somata of a subset of peripheral neurons they approach, axons cease or interrupt their growth. These findings identify a set of candidate guidance cells for axonal outgrowth in the leech, similar to those previously described in the developing nervous system of insects.     

Structure of the intramural nerves of the rat bladder

The bladder of adult female rats receives ～16,000 axons (i.e., is the target of that many ganglion neurons) of which at least half are sensory. In nerves containing between 40 and 1200 axons cross-sectional area is proportional to number of axons; >99% of axons are unmyelinated. A capsule forms a seal around nerves and ends abruptly where nerves, after branching, contain ～10 axons. A single blood vessel is present in many of the large nerves but never in nerves of <600 axons. The number of glial cells was estimated through the number of their nuclei. There is a glial nucleus profile every 76 axonal profiles. Each glial cell is associated with many axons and collectively covers ～1,000 μm of axonal length. In all nerves a few axonal profiles contain large clusters of vesicles independent of microtubules. The axons do not branch; they alter their relative position along the nerve; they vary in size along their length; none has a circular profile. All the axons are fully wrapped by glial cells and never contact each other. The volume of axons is larger than that of glial cells (55%–45%), while the surface of glial cell is twice as extensive as that of axons; there are ～2.27 m² of axolemma and ～4.60 m² of glial cell membrane per gram of nerve. Of the mitochondria of a nerve ～3/4 are in axons and ～1/4 in glial cells.     

Tract formation and axon fasciculation of molecularly distinct peripheral neuron subpopulations during leech embryogenesis.

In leech, the central projections of peripheral sensory neurons segregate into specific axonal tracts, which are distinguished by differential expression of surface antigens recognized by the monoclonal antibodies Lan3-2 and Lan4-2. Lan3-2 recognizes an epitope expressed on axons that segregate into three distinct axon fascicles. In contrast, the Lan4-2-positive axons selectively project into only one of the Lan3-2-positive axon tracts. These observations provide evidence for a hierarchy of guidance cues mediating specific pathway formation in this system. Since the Lan3-2 antibody has been shown to perturb this process and since, as shown here, the Lan3-2 and Lan4-2 antigens are closely molecularly interrelated, these antibodies may help define molecules and epitopes mediating neuronal recognition and axonal guidance.     

EphA4-dependent axon guidance is mediated by the RacGAP alpha2-chimaerin

Neuronal network formation in the developing nervous system is dependent on the accurate navigation of nerve cell axons and dendrites, which is controlled by attractive and repulsive guidance cues. Ephrins and their cognate Eph receptors mediate many repulsive axonal guidance decisions by intercellular interactions resulting in growth cone collapse and axon retraction of the Eph-presenting neuron. We show that the Rac-specific GTPase-activating protein alpha2-chimaerin binds activated EphA4 and mediates EphA4-triggered axonal growth cone collapse. alpha-Chimaerin mutant mice display a phenotype similar to that of EphA4 mutant mice, including aberrant midline axon guidance and defective spinal cord central pattern generator activity. Our results reveal an alpha-chimaerin-dependent signaling pathway downstream of EphA4, which is essential for axon guidance decisions and neuronal circuit formation in vivo.     

Feedback-controlled dynamics of neuronal cells on directional surfaces

The formation of neuronal networks is a complex phenomenon of fundamental importance for understanding the development of the nervous system. The basic process underlying the network formation is axonal growth, a process involving the extension of axons from the cell body and axonal navigation toward target neurons. Axonal growth is guided by the interactions between the tip of the axon (growth cone) and its extracellular environmental cues, which include intercellular interactions, the biochemical landscape around the neuron, and the mechanical and geometrical features of the growth substrate. Here, we present a comprehensive experimental and theoretical analysis of axonal growth for neurons cultured on micropatterned polydimethylsiloxane (PDMS) surfaces. We demonstrate that closed-loop feedback is an essential component of axonal dynamics on these surfaces: the growth cone continuously measures environmental cues and adjusts its motion in response to external geometrical features. We show that this model captures all the characteristics of axonal dynamics on PDMS surfaces for both untreated and chemically modified neurons. We combine experimental data with theoretical analysis to measure key parameters that describe axonal dynamics: diffusion (cell motility) coefficients, speed and angular distributions, and cell-substrate interactions. The experiments performed on neurons treated with Taxol (inhibitor of microtubule dynamics) and Y-27632 (disruptor of actin filaments) indicate that the internal dynamics of microtubules and actin filaments plays a critical role for the proper function of the feedback mechanism. Our results demonstrate that axons follow geometrical patterns through a contact-guidance mechanism, in which high-curvature geometrical features impart high traction forces to the growth cone. These results have important implications for our fundamental understanding of axonal growth as well as for bioengineering novel substrate to guide neuronal growth and promote nerve repair.     

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.     

Ectoderm removal prevents cutaneous nerve formation and perturbs sensory axon growth in the chick hindlimb

Target tissues are thought to provide important cues for growing axons, yet there is little direct evidence that they are essential for axonal pathfinding. Here we examined whether target ectoderm is necessary for the formation of cutaneous nerves, and for the normal growth and guidance of cutaneous axons as they first enter the limb plexus. To do this, we removed a patch of ectoderm from the chick hindlimb at various times during early axon outgrowth. We find there is a critical period when cutaneous nerve formation requires target ectoderm. When the ectoderm is absent during this time, axons progress into the limb more slowly and, although a few sensory axons occasionally diverge a short distance from the plexus, they do not form a discrete nerve that travels to the skin. A few days later, when the nerve pattern is mature, axons normally destined for the 'deprived' cutaneous nerve are not segregated appropriately within the plexus. Some cutaneous axons are instead misdirected along an inappropriate cutaneous nerve, while others have seemingly failed to reach their correct target, or a suitable alternative, and died. These results demonstrate that the target ectoderm is necessary for normal sensory axon growth and guidance in the hindlimb.     

Axonal outgrowth within the abnormal scaffold of brain tracts in a zebrafish mutant

The role of specific axonal tracts for the guidance of growth cones was investigated by examining axonal outgrowth within the abnormal brain tracts of zebrafish cyclops mutants. Normally, the earliest differentiating neurons in the zebrafish brain establish a simple scaffold of axonal tracts. Later-developing axons follow cell-specific pathways within this axonal scaffold. In Cyclops embryos, this scaffold is perturbed due to the deletion of some ventromedial neurons that establish parts of the axonal scaffold and the development of an abnormal crease in the brain. In these mutant embryos, the growth cones projected by the neurons of the nucleus of the posterior commissure (nur PC) are deprived of the two tracts of axons that they sequentially follow to first extend ventrally, then posteriorly. These growth cones respond to the abnormal scaffold in several interesting ways. First, nuc PC growth cones initially always extend ventrally as in wild-type embryos. This suggests that for the first portion of their pathway the axons they normally follow are not required for proper navigation. Second, approximately half of the nuc PC growth cones follow aberrant longitudinal pathways after the first portion of their pathway. This suggests that for the longitudinal portion of the pathway, specific growth cone/axon interactions are important for guiding growth cones. Third, although approximately half of the nuc PC growth cones follow aberrant longitudinal pathways, the rest follow normal pathways despite the absence of the axons that they normally follow. This suggests that cues independent of these axons may be capable of guiding nuc PC growth cones as well. These results suggest that different guidance cues or combinations of cues guide specific growth cones along different portions of their pathway. 1994 John Wiley & Sons, Inc.     

Control of leech swimming activity by the cephalic ganglia

We investigated the role played by the cephalic nervous system in the control of swimming activity in the leech, Hirudo medicinalis, by comparing swimming activity in isolated leech nerve cords that included the head ganglia (supra- and subesophageal ganglia) with swimming activity in nerve cords from which these ganglia were removed. We found that the presence of these cephalic ganglia had an inhibitory influence on the reliability with which stimulation of peripheral (DP) nerves and intracellular stimulation of swim-initiating neurons initiated and maintained swimming activity. In addition, swimming activity recorded from both oscillator and motor neurons in preparations that included head ganglia frequently exhibited irregular bursting patterns consisting of missed, weak, or sustained bursts. Removal of the two head ganglia as well as the first segmental ganglion eliminated this irregular activity pattern. We also identified a pair of rhythmically active interneurons, SRN1, in the subesophageal ganglion that, when depolarized, could reset the swimming rhythm. Thus the cephalic ganglia and first segmental ganglion of the leech nerve cord are capable of exerting a tonic inhibitory influence as well as a modulatory effect on swimming activity in the segmental nerve cord.     

Intrinsic properties guide proximal abducens and oculomotor nerve outgrowth in avian embryos

Proper movement of the vertebrate eye requires the formation of precisely patterned axonal connections linking cranial somatic motoneurons, located at defined positions in the ventral midbrain and hindbrain, with extraocular muscles. The aim of this research was to assess the relative contributions of intrinsic, population-specific properties and extrinsic, outgrowth site-specific cues during the early stages of abducens and oculomotor nerve development in avian embryos. This was accomplished by surgically transposing midbrain and caudal hindbrain segments, which had been pre-labeled by electroporation with an EGFP construct. Graft-derived EGFP+ oculomotor axons entering a hindbrain microenvironment often mimicked an abducens initial pathway and coursed cranially. Similarly, some EGFP+ abducens axons entering a midbrain microenvironment mimicked an oculomotor initial pathway and coursed ventrally. Many but not all of these axons subsequently projected to extraocular muscles that they would not normally innervate. Strikingly, EGFP+ axons also took initial paths atypical for their new location. Upon exiting from a hindbrain position, most EGFP+ oculomotor axons actually coursed ventrally and joined host branchiomotor nerves, whose neurons share molecular features with oculomotor neurons. Similarly, upon exiting from a midbrain position, some EGFP+ abducens axons turned caudally, elongated parallel to the brainstem, and contacted the lateral rectus muscle, their originally correct target. These data reveal an interplay between intrinsic properties that are unique to oculomotor and abducens populations and shared ability to recognize and respond to extrinsic directional cues. The former play a prominent role in initial pathway choices, whereas the latter appear more instructive during subsequent directional choices.     

Cellular expression of a leech netrin suggests roles in the formation of longitudinal nerve tracts and in regional innervation of peripheral targets.

Netrins are secreted, diffusible proteins that direct axonal growth. To study the functions of netrins in the relatively simple and easily accessible nervous system of the leech Hirudo medicinalis, we have cloned a leech netrin and have characterized its expression during embryogenesis. By probing a leech cDNA library at low stringency with chick netrin probes, we have identified a complete cDNA clone that bears significant sequence similarity to netrins of other species. In situ hybridization and dye filling of individual neurons show that this leech netrin is expressed by several identifiable central neurons in every segmental ganglionic primordium during early stages of embryogenesis. Some of these neurons, including the bipolar cells which are thought to be involved in setting up longitudinal tracts, express this gene only transiently during embryogenesis, while others continue to express it in the adult. In addition, leech netrin is expressed by ventral but not dorsal longitudinal muscle cells in each segment before central neurons project their axons to the periphery. These highly specific expression patterns are consistent with the hypothesis that leech netrin plays a role in forming the major interganglionic neuronal tracts and in defining ventral versus dorsal domains of peripheral innervation.     

Schwann cells as regulators of nerve development.

Myelinating and non-myelinating Schwann cells of peripheral nerves are derived from the neural crest via an intermediate cell type, the Schwann cell precursor [K.R. Jessen, A. Brennan, L. Morgan, R. Mirsky, A. Kent, Y. Hashimoto, J. Gavrilovic. The Schwann cell precursor and its fate: a study of cell death and differentiation during gliogenesis in rat embryonic nerves, Neuron 12 (1994) 509-527]. The survival and maturation of Schwann cell precursors is controlled by a neuronally derived signal, beta neuregulin. Other factors, in particular endothelins, regulate the timing of precursor maturation and Schwann cell generation. In turn, signals derived from Schwann cell precursors or Schwann cells regulate neuronal numbers during development, and axonal calibre, distribution of ion channels and neurofilament phosphorylation in myelinated axons. Unlike Schwann cell precursors, Schwann cells in older nerves survive in the absence of axons, indicating that a significant change in survival regulation occurs. This is due primarily to the presence of autocrine growth factor loops in Schwann cells, present from embryo day 18 onwards, that are not functional in Schwann cell precursors. The most important components of the autocrine loop are insulin-like growth factors, platelet derived growth factor-BB and neurotrophin 3, which together with laminin support long-term Schwann cell survival. The paracrine dependence of precursors on axons for survival provides a mechanism for matching precursor cell number to axons in embryonic nerves, while the ability of Schwann cells to survive in the absence of axons is an absolute prerequisite for nerve repair following injury. In addition to providing survival factors to neurones and themselves, and signals that determine axonal architecture, Schwann cells also control the formation of peripheral nerve sheaths. This involves Schwann cell-derived Desert Hedgehog, which directs the transition of mesenchymal cells to form the epithelium-like structure of the perineurium. Schwann cells thus signal not only to themselves but also to the other cellular components within the nerve to act as major regulators of nerve development.     

Axonal Transport in the Garfish Optic Nerve: Comparison with the Olfactory System

Fast and slow axonal transports were studied in the optic nerve of the garfish and compared with previous studies on the olfactory nerve. The composition of fast-transport proteins was very similar in the two nerves. Although the velocity of fast transport was slightly lower in the optic nerve, there was a linear increase in velocity with temperature in both nerves. As in the olfactory nerve, only a single wave of slow-transport protein radioactivity moves along the nerve. The velocity of slow transport also increased linearly with temperature, but the coefficient was less than in the olfactory system. The composition of slow transport in the optic nerve was significantly different from that in the olfactory nerve, a finding reflecting the different cytoskeletal constituents of the two types of axons. The slow wave could be differentiated into several subcomponents, with the order of velocities being a 105-kilodalton protein and actin greater than tubulins and clathrin greater than fodrin much greater than neurofilaments. It can be concluded that the temperature dependence of fast and slow axonal transport in different nerves reflects the influence of temperature on the individual polypeptides constituting the various transport phases. The garfish optic nerve preparation may be advantageous for studies of axonal transport in retinal ganglion cell axons, because its great length avoids the complications of having to study transport in the optic tract or in material accumulating at the tectum.     

Rat optic nerve oligodendrocytes develop in the absence of viable retinal ganglion cell axons.

Retinal ganglion cell axons and axonal electrical activity have been considered essential for migration, proliferation, and survival of oligodendrocyte lineage cells in the optic nerve. To define axonal requirements during oligodendrogenesis, the developmental appearance of oligodendrocyte progenitors and oligodendrocytes were compared between normal and transected optic nerves. In the absence of viable axons, oligodendrocyte precursors migrated along the length of the nerve and subsequently multiplied and differentiated into myelin basic protein-positive oligodendrocytes at similar densities and with similar temporal and spatial patterns as in control nerves. Since transected optic nerves failed to grow radially, the number of oligodendrocyte lineage cells was reduced compared with control nerves. However, the mitotic indices of progenitors and the percentage of oligodendrocytes undergoing programmed cell death were similar in control and transected optic nerves. Oligodendrocytes lacked their normal longitudinal orientation, developed fewer, shorter processes, and failed to form myelin in the transected nerves. These data indicate that normal densities of oligodendrocytes can develop in the absence of viable retinal ganglion axons, and support the possibility that axons assure their own myelination by regulating the number of myelin internodes formed by individual oligodendrocytes.     

Sonic hedgehog is indirectly required for intraretinal axon pathfinding by regulating chemokine expression in the optic stalk

Successful axon pathfinding requires both correct patterning of tissues, which will later harbor axonal tracts, and precise localization of axon guidance cues along these tracts at the time of axon outgrowth. Retinal ganglion cell (RGC) axons grow towards the optic disc in the central retina, where they turn to exit the eye through the optic nerve. Normal patterning of the optic disc and stalk and the expression of guidance cues at this choice point are necessary for the exit of RGC axons out of the eye. Sonic hedgehog (Shh) has been implicated in both patterning of ocular tissue and direct guidance of RGC axons. Here, we examine the precise spatial and temporal requirement for Hedgehog (Hh) signaling for intraretinal axon pathfinding and show that Shh acts to pattern the optic stalk in zebrafish but does not guide RGC axons inside the eye directly. We further reveal an interaction between the Hh and chemokine pathways for axon guidance and show that cxcl12a functions downstream of Shh and depends on Shh for its expression at the optic disc. Together, our results support a model in which Shh acts in RGC axon pathfinding indirectly by regulating axon guidance cues at the optic disc through patterning of the optic stalk.     

Localization of carbonic anhydrase in identified glial cells of the leech central nervous system: application of histochemical and immunocytochemical methods

We localized the enzyme carbonic anhydrase (CA) in frozen sections of the leech (Hirudo medicinalis) central nervous system by two histochemical techniques and the indirect immunofluorescence technique. Hansson's cobalt precipitation method and the use of 1-dimethylamino-naphthalene-5-sulfonamide (DNSA) to build a fluorescent enzyme-substrate complex showed that glial cells are the sites of CA activity in the leech. Neuropil and connective glial cells surrounding the axons had strong CA activity, whereas packet glial cells, which surround neuron cell bodies, and neurons themselves remained unstained. Glial cells reacted markedly with FITC-coupled antibodies against CA isoenzyme II, but experiments with antibodies against CA isoenzyme I showed no reaction.     

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.     

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.     

Modulation of the pattern of axonal projections of a leech motor neuron by ablation or transplantation of its target

The sixth segmental ganglion in the ventral nerve cord of the leech H. medicinalis contains a bilateral pair of rostral penile evertor motor neurons (RPEs) that in the adult innervate the male genitalia. During embryogenesis, the RPEs extend numerous extraganglionic projections. Only two of these innervate the target and are normally retained in the adult, while the others retract. Early, but not late, removal of the male genitalia results in the indefinite retention and continued growth of projections that would normally retract. Any of these projections can innervate targets transplanted to ectopic locations. We conclude that an RPE motor neuron requires a signal, provided by its interaction with the target organ during a critical period, in order to stop extending axons, stabilize those axons that contact the target, and retract those that do not.     

Proximal tissues and patterned neurite outgrowth at the lumbosacral level of the chick embryo: partial and complete deletion of the somite

The development of patterned axon outgrowth and dorsal root ganglion (DRG) formation was examined after partially or totally removing chick somitic mesoderm. Since the dermamyotome is not essential and a full complement of limb muscles developed, alterations in neural patterns could be ascribed to deletion of sclerotome. When somitic tissue was completely removed, axons extended and DRG formed, but in an unsegmented pattern. Therefore the somite does not elicit outgrowth of axons or migration of DRG precursors, it is not a manditory substratum and it is not required for DRG condensation. These results suggest that posterior sclerotome is relatively inhibitory to invasion, an inhibition that is released when sclerotome is absent. When somites were partially deleted, axonal segmentation was not lost proportionally with the amount of sclerotome removed, suggesting that properties that may vary with sclerotome volume (such as diffusible cues) do not play a primary role. Instead, spinal nerves lost segmentation only when ventral sclerotome was deleted, regardless of whether dorsal sclerotome was or was not removed. This strongly suggests that axonal segmentation is imposed by direct interactions between growth cones and extracellular matrices or surfaces sclerotome cells. While DRG tended to be normally segmented when ventral sclerotome was deleted and to lose segmentation when dorsomedial sclerotome was absent, a coordinate loss of DRG segmentation with sclerotome volume could not be ruled out. However it is clear that axonal and DRG segmentation are independent. Observations on a subset of embryos in which the notochord was displaced relative to the spinal cord suggest that the ventromedial sclerotome surrounding the notochord inhibits axon advance. Posterior and ventromedial sclerotome are hypothesized to act as barriers to axon outgrowth due to some feature of their common cartilaginous development. Specific innervation patterns were also examined. When the notochord was displaced toward the control limb, axons on this side made and corrected projection errors, suggesting that the notochord can influence the precision of axonal pathway selection. In contrast, motor axons that entered the limb on all operated sides innervated muscle with their normal precision despite the absence of the somite and axonal segmentation. Therefore, the somite and the process of spinal nerve segmentation are largely irrelevant to the specificity of motoneuron projection.