The timing of initial neuropeptide expression by an identified insect neuron does not depend on interactions with its normal peripheral target

To study the developmental regulation of a neuropeptide phenotype, we have analyzed the biochemical and morphological differentiation of two identifiable neurons in embryos of the moth, Manduca sexta. The central cell, CF, and the peripheral cell, L1, are both neuroendocrine neurons that express neuropeptides related to the molluscan tetrapeptide FMRFamide. Both neurons project axons to the transvers nerve in each thoracic segment. Within the CF and L1 cells, neuropeptide-like immunoreactivity was localized to secretory granules that had cell specific morphologies and sizes. The onset of neuropeptide expression in the two cell types displayed a similar pattern: immunoreactivity was first detected in distal processes and soon after within cells bodies. However, the onsets occurred at different times: for the CF cell, neuropeptides were first seen at 60%-63% of embryonic development, after the neuron had extended a long axon into the periphery, while L1 neuropeptide expression began at ～42%, as it first extended its growth cone. These times were related in that they corresponded to the arrival times of the respective growth cones at a similar position in the developing peripheral nerve. Withinthis region of the nerve, the growth cones of both cell typesexhibited a transient and cell-specific interaction with an identified mesodermal cell, called the Syncytium. Like the L1 and B neurons (Carr and Taghert, 1988b), the CF growth cones typically grew past this cell, yet remained attached to it by lamellipodial and filopodial processes of the axon. Ultrastructurally, the interaction involved filopodial adhesion to and insertion within the Syncytial cell. Two other nonneuroendocrine cell types grew axons past this same region, but showed no such tendencies. To test the hypothesis that the morphological and biochemical differentiation of these cells was somehow linked, central ganglia were isolated (as individuals or connected as ganglionic chains) in tissue culture, prior to the time when CF growth cones entered the periphery and prior to the development of CF neuropeptide expression. In the majority of cases, CF neurons nevertheless displayed their neuropeptide phenotype at a normal and cell-specific stage. We conclude that the initiation of neuropeptide expression is highly correlated with schedules of morphological differentiation in these neurons, but that, in the case of the CF neuron, it is not regulated by interactions of the growth cone with peripheral structures.     

Modulation of growth cone morphology by substrate-bound adhesion molecules.

The growth cone, a terminal structure on developing and regenerating axons, is specialized for motility and guidance functions. In vivo the growth cone responds to environmental cues to guide the axon to its appropriate target. These cues are thought to be responsible for position-specific morphological changes in the growth cone, but the molecules that control growth cone behavior are poorly characterized. We used scanning electron microscopy to analyze the morphology of retinal ganglion cell growth cones in vitro on different adhesion molecules that axons normally encounter in vivo. L1/8D9, N-cadherin, and laminin each induced distinctive morphological characteristics in growth cones. Growth cones elaborated lamellipodial structures in response to the cell adhesion molecules L1/8D9 and N-cadherin, whereas laminin supported filopodial growth cones with small veils. On L1/8D9, the growth cones were larger and produced more filopodia. Filopodial associations between adjacent growth cones and neurites were frequent on L1/8D9 but were uncommon on laminin or N-cadherin. These results demonstrate that different adhesion molecules have profoundly different effects on growth cone morphology. This is consistent with previous reports suggesting that changes in growth cone morphology in vivo occur in response to changes in substrate composition.     

Axon initiation by ciliary neurons in culture

A nerve culture system for the study of axon initiation is described. A population of individual chick embryo ciliary neurons, free from contact with other cells and attached to a polyornithinecoated culture dish, is exposed to heart cell-conditioned medium (HCM). Within 30 min after the addition of HCM the majority of neurons have formed growth cones, and by 90 min more than 80% of the neurons bear at least one axon longer than 15 μm. Before the addition of HCM, ciliary neurons generate membrane ruffles and extend filopodia around the entire periphery of the rounded cell body. Axon initiation, following addition of HCM, consists of two distinctive changes in the cell surface: (1) organization of the randomly distributed surface movements into localized highly active growth cones, which then form axons; and (2) the cessation of surface movements elsewhere on the cell periphery. Heart cell-conditioned medium may induce these changes by increasing the adhesion between parts of the nerve cell surface and the substratum.     

Non-Muscle Myosin II Regulates Neuronal Actin Dynamics by Interacting with Guanine Nucleotide Exchange Factors

Background

Non-muscle myosin II (NM II) regulates a wide range of cellular functions, including neuronal differentiation, which requires precise spatio-temporal activation of Rho GTPases. The molecular mechanism underlying the NM II-mediated activation of Rho GTPases is poorly understood. The present study explored the possibility that NM II regulates neuronal differentiation, particularly morphological changes in growth cones and the distal axon, through guanine nucleotide exchange factors (GEFs) of the Dbl family.

Principal Findings

NM II colocalized with GEFs, such as βPIX, kalirin and intersectin, in growth cones. Inactivation of NM II by blebbistatin (BBS) led to the increased formation of short and thick filopodial actin structures at the periphery of growth cones. In line with these observations, FRET analysis revealed enhanced Cdc42 activity in BBS-treated growth cones. BBS treatment also induced aberrant targeting of various GEFs to the distal axon where GEFs were seldom observed under physiological conditions. As a result, numerous protrusions and branches were generated on the shaft of the distal axon. The disruption of the NM II–GEF interactions by overexpression of the DH domains of βPIX or Tiam1, or by βPIX depletion with specific siRNAs inhibited growth cone formation and induced slender axons concomitant with multiple branches in cultured hippocampal neurons. Finally, stimulation with nerve growth factor induced transient dissociation of the NM II–GEF complex, which was closely correlated with the kinetics of Cdc42 and Rac1 activation.

Conclusion

Our results suggest that NM II maintains proper morphology of neuronal growth cones and the distal axon by regulating actin dynamics through the GEF–Rho GTPase signaling pathway.     

Neuron differentiation and axon growth in the developing wing of Drosophila melanogaster

Sensory neurons in the wing of Drosophila originate locally from epithelial cells and send their axons toward the base of the wing in two major bundles, the L1 and L3 nerves. We have estimated the birth times of a number of identified wing sensory neurons using an X-irradiation technique and have followed the appearance of their somata and axons by means of an immunohistochemical stain. These cells become immunoreactive and begin axon growth in a sequence which mirrors the sequence of their birth times. The earliest ones are born before pupariation and begin axonogenesis within 1 to 2 hr after the onset of metamorphosis; the last are born and differentiate some 12 to 14 hr later. The L1 and L3 nerves are formed in sections, with specific neurons pioneering defined stretches of the pathways during the period between 0 and 4 hr after pupariation (AP), and finally joining together around 12 hr AP. By 16 hr AP the adult complement of neurons is present and the adult peripheral nerve pattern has been established. Pathway establishment appears to be specified by multiple cues. In places where neurons differentiate in close proximity to one another, random filopodial exploration followed by axon growth to a neighboring neuron soma might be the major factor leading to pathway construction. In other locations, filopodial contact between neighboring somata does not appear to occur, and axon pathways joining neural neighbors by the most direct route are not established. We propose that in these cases additional factors, including veins which are already present at the time of axonogenesis, influence the growth of axons through non-neural tissues.     

Differential expression of neuropeptide gene mRNA within the LUQ cells of Aplysia californica.

Two neuropeptide precursor cDNAs (LUQ-1 and L5-67) have been recently isolated from the Left Upper Quadrant (LUQ) neurons of the abdominal ganglion of Aplysia californica (Shyamala, Fisher, and Scheller, 1986; Wickham and DesGroseillers, 1991). Using in situ hybridization techniques as well as dot blot and polymerase chain reaction (PCR) assays, we have studied the expression of these genes in the central nervous system (CNS) of Aplysia californica. The LUQ-1 gene was found to be expressed in neuron L5 in the abdominal ganglion, whereas the expression of the L5-67 gene was observed in the other four LUQ cells (L2-4 and L6). When in situ hybridization was performed on paraffin sections of the abdominal ganglion, clusters of smaller cells located in the left hemiganglion, were also found to express either the LUQ-1 or the L5-67 gene, never both. In many sections, the mRNAs coding for the two neuropeptides were found not only in cell bodies but also in the axon of individual LUQ neurons and even as far as the pericardial nerve. The presence of neuropeptide mRNA in axons, pericardial nerve, and kidney has been confirmed by polymerase chain reaction. A specific, although diffuse hybridization in the left upper quadrant also suggests that mRNA is present in the neuritic field. Taken together these results indicate that neuron L5 is the only giant neuron expressing the LUQ-1 gene and might therefore have a physiological function different from the other four LUQ cells. Neuropeptide mRNAs were also found in the axon and/or the neuritic field of giant neurons and could play important roles related to cell signalling in axons and nerve termini.     

Spatial regulation of axonal glycoprotein expression on subsets of embryonic spinal neurons

The identification of surface proteins restricted to subsets of embryonic axons and growth cones may provide information on the mechanisms underlying axon fasciculation and pathway selection in the vertebrate nervous system. We describe here the characterization of a 135 kd cell surface glycoprotein, TAG-1, that is expressed transiently on subsets of embryonic spinal cord axons and growth cones. TAG-1 is immunochemically distinct from the cell adhesion molecules N-CAM and L1 (NILE) and is expressed on commissural and motor neurons over the period of initial axon extension. Moreover, TAG-1 and L1 appear to be segregated on different segments of the same embryonic spinal axons. These observations provide evidence that axonal guidance and pathway selection in vertebrates may be regulated in part by the transient and selective expression of distinct surface glycoproteins on subsets of developing neurons.     

Embryogenesis of peripheral nerve pathways in grasshopper legs. I. The initial nerve pathway to the CNS

The founding of the first nerve path of the grasshopper metathoracic leg was examined at the level of identified neurons, using intracellular dye fills, immunohistochemistry, Nomarski optics, and scanning and transmission electron microscopy. The embryonic nerve is established by the axonal trajectory of a pair of afferent pioneer neurons, the tibial 1 (Ti1) cells. Following a period of profuse filopodial sprouting, the Ti1 axonal growth cones, possessing 75- to 100-microns-long filopodia, navigate a stereotyped path across the limb bud epithelium to the base of the appendage and into the CNS. The Ti1 axons grow from cell to cell along a chain of preaxonogenesis neurons spaced at intervals along the pathway, forming dye-passing junctions with them. The contacted neurons subsequently undergo axonogenesis and follow the pioneer axons into the CNS. Later arising neurons project their axons onto the cell bodies of the chain, thereby establishing the principal branch points of the nerve. Among the later arising afferents are the sensory neurons of the femoral chordotonal and subgenual organs. The morphology of the adult nerve appears to be determined by the stereotyped positioning of neurons in the differentiating limb bud and by the resultant axonal trajectories established during the first 10% of peripheral neurogenesis.     

Differential expression of neuropeptide gene mRNA within the LUQ cells of Aplysia californica

Two neuropeptide precursor cDNAs (LUQ-1 and L5-67) have been recently isolated from the Left Upper Quadrant (LUQ) neurons of the abdominal ganglion of Aplysia californica (Shyamala, Fisher, and Scheller, 1986; Wickham and DesGroseillers, 1991). Using in situ hybridization techniques as well as dot blot and polymerase chain reaction (PCR) assays, we have studied the expression of these genes in the central nervous system (CNS) of Aplysia californica. The LUQ-1 gene was found to be expressed in neuron L5 in the abdominal ganglion, whereas the expression of the L5-67 gene was observed in the other four LUQ cells (L2-4 and L6). When in situ hybridization was performed on paraffin sections of the abdominal ganglion, clusters of smaller cells located in the left hemiganglion, were also found to express either the LUQ-1 on the L5-67 gene, never both. In many sections, the mRNAs coding for the two neuropeptides were found not only in cell bodies but also in the axon of individual LUQ neurons and even as far as the pericardial nerve. The presence of neuropeptide mRNA in axons, pericardial nerve, and kidney has been confirmed by polymerase chain reaction. A specific, although diffuse hybridization in the left upper quadrant also suggests that mRNA is present in the neuritic field. Taken together these results indicate that neuron L5 is the only giant neuron expressing the LUQ-1 gene and might therefore have a physiological function different from the other four LUQ cells. Neuropeptide mRNAs were also found in the axon and/or the neuritic field of giant neurons and could play important roles related to cell signalling in axons and nerve termini.     

Reciprocal interactions between olfactory receptor axons and olfactory nerve glia cultured from the developing moth Manduca sexta

In olfactory systems, neuron-glia interactions have been implicated in the growth and guidance of olfactory receptor axons. In the moth Manduca sexta, developing olfactory receptor axons encounter several types of glia as they grow into the brain. Antennal nerve glia are born in the periphery and enwrap bundles of olfactory receptor axons in the antennal nerve. Although their peripheral origin and relationship with axon bundles suggest that they share features with mammalian olfactory ensheathing cells, the developmental roles of antennal nerve glia remain elusive. When cocultured with antennal nerve glial cells, olfactory receptor growth cones readily advance along glial processes without displaying prolonged changes in morphology. In turn, olfactory receptor axons induce antennal nerve glial cells to form multicellular arrays through proliferation and process extension. In contrast to antennal nerve glia, centrally derived glial cells from the axon sorting zone and antennal lobe never form arrays in vitro, and growth-cone glial-cell encounters with these cells halt axon elongation and cause permanent elaborations in growth cone morphology. We propose that antennal nerve glia play roles similar to olfactory ensheathing cells in supporting axon elongation, yet differ in their capacity to influence axon guidance, sorting, and targeting, roles that could be played by central olfactory glia in Manduca.     

Dynamic behavior of the ends of growing parallel fibers in early postnatal rat cerebellum

The molecular layer of the cerebellum contains parallel fibers, the axons of granule neurons. We have examined the morphology and behavior of parallel fiber growth cones in the early postnatal rat cerebellum using the fluorescent tracer DiI. Parallel fiber growth cones distributed into three categories based on size and shape: short torpedo-like, long torpedo-like, and lamellopodial in form. The torpedo-like growth cones were modified by the addition of lamellopodia and/or filopodia, and the lamellopodial growth cones were often decorated with a filopodium. These three different growth cone morphologies were found throughout the growing region of the molecular layer. The nascent axons elaborated by premigratory granule neurons differed from the longer axons of more developed neurons in that they often had forked growth cones and extensive lamellopodial decoration along the axon shaft. Growth cones in living slices closely resembled those observed in the fixed preparations. The living growth cones exhibited frequent lamellopodial rearrangement and a side-to-side head-waving movement. The axon proximal to the growth cone was also dynamic. The axons curved and undulated, and mobile swellings formed along the axon shaft. These observations show that the growth cones of parallel fibers are similar to growth cones described for axons in other developing systems in terms of size, morphological characteristics, and dynamic behavior. © 1998 John Wiley & Sons, Inc. J Neurobiol 36: 91–104, 1998     

Common mechanisms underlying growth cone guidance and axon branching

During development, growth cones direct growing axons into appropriate targets. However, in some cortical pathways target innervation occurs through the development of collateral branches that extend interstitially from the axon shaft. How do such branches form? Direct observations of living cortical brain slices revealed that growth cones of callosal axons pause for many hours beneath their cortical targets prior to the development of interstitial branches. High resolution imaging of dissociated living cortical neurons for many hours revealed that the growth cone demarcates sites of future axon branching by lengthy pausing behaviors and enlargement of the growth cone. After a new growth cone forms and resumes forward advance, filopodial and lamellipodial remnants of the large paused growth cone are left behind on the axon shaft from which interstitial branches later emerge. To investigate how the cytoskeleton reorganizes at axon branch points, we fluorescently labeled microtubules in living cortical neurons and imaged the behaviors of microtubules during new growth from the axon shaft and the growth cone. In both regions microtubules reorganize into a more plastic form by splaying apart and fragmenting. These shorter microtubules then invade newly developing branches with anterograde and retrograde movements. Although axon branching of dissociated cortical neurons occurs in the absence of targets, application of a target-derived growth factor, FGF-2, greatly enhances branching. Taken together, these results demonstrate that growth cone pausing is closely related to axon branching and suggest that common mechanisms underlie directed axon growth from the terminal growth cone and the axon shaft.     

Regulation of SC1/DM-GRASP during the migration of motor neurons in the chick embryo brain stem

The hindbrain of the chick embryo contains three classes of motor neurons: somatic, visceral, and branchial motor. During development, somata of neurons in the last two classes undergo a laterally directed migration within the neuroepithelium; somata translocate towards the nerve exit points, through which motor axons are beginning to extend into the periphery. All classes of motor neuron are immunopositive for the SC1/DM-GRASP cell surface glycoprotein. We have examined the relationship between patterns of motor neuron migration, axon outgrowth, and expression of the SC1/DM-GRASP mRNA and protein, using anterograde or retrograde axonal tracing, immunohistochemistry, and in situ hybridization. We find that as motor neurons migrate laterally, SC1/DM-GRASP is down-regulated, both on neuronal somata and axonal surfaces. Within individual motor nuclei, these lateral, more mature neurons are found to possess longer axons than the young, medial cells of the population. Labelling of sensory or motor axons growing into the second branchial arch also shows that motor axons reach the muscle plate first, and that SC1/DM-GRASP is expressed on the muscle at the time growth cones arrive. 1994 John Wiley & Sons, Inc.     

Peanut agglutinin and chondroitin-6-sulfate are molecular markers for tissues that act as barriers to axon advance in the avian embryo

Axon outgrowth between the spinal cord and the hindlimb of the chick embryo is constrained by three tissues that border axon pathways. Growth cones turn to avoid the posterior sclerotome, perinotochordal mesenchyme, and pelvic girdle precursor during normal development and after experimental manipulation. We wanted to know if these functionally similar barriers to axon advance also share a common molecular composition. Since the posterior sclerotome differentially binds peanut agglutinin (PNA) and since PNA binding is also typical of prechondrogenic differentiation, we examined the pattern of expression of PNA binding sites and cartilage proteoglycan epitopes in relation to axon outgrowth. We found that all three barrier tissues preferentially express both PNA binding sites and chondroitin-6-sulfate (C-6-S) immunoreactivity at the time when growth cones avoid these tissues. Moreover, both epitopes are expressed in the roof plate of the spinal cord and in the early limb bud, two additional putative barriers to axon advance. In contrast, neither epitope is detected in peripheral axon pathways. In the somites, this dichotomous pattern of expression clearly preceded the invasion of the anterior sclerotome by either motor growth cones or neural crest cells. However, in the limb, barrier markers disappeared from presumptive axon pathways in concert with the invasion of axons. Since this coordinate pattern suggested that the absence of barrier markers in these axon pathways requires an interaction with growth cones, we analyzed the pattern of barrier marker expression following unilateral neural tube deletions. We found that PNA-negative axon pathways developed normally even in the virtual absence of axon outgrowth. We conclude that the absence of staining with carbohydrate-specific barrier markers is an independent characteristic of the cells that comprise axon pathways. These results identify two molecular markers that characterize known functional barriers to axon advance and suggest that barrier tissues may impose patterns on peripheral nerve outgrowth by virtue of their distinct molecular composition.     

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.     

Identification of the major proteins that promote neuronal process outgrowth on Schwann cells in vitro

Schwann cells have a unique role in regulating the growth of axons during regeneration and presumably during development. Here we show that Schwann cells are the best substrate yet identified for promoting process growth in vitro by peripheral motor neurons. To determine the molecular interactions responsible for Schwann cell regulation of axon growth, we have examined the effects of specific antibodies on process growth in vitro, and have identified three glycoproteins that play major roles. These are the Ca2+-independent cell adhesion molecule (CAM), L1/Ng-CAM; the Ca2+-dependent CAM, N-cadherin; and members of the integrin extracellular matrix receptor superfamily. Two other CAMs present on neurons and/or Schwann cells-N-CAM and myelin-associated glycoprotein-do not appear to be important in regulating process growth. Our results imply that neuronal growth cones use integrin-class extracellular matrix receptors and at least two CAMs--N-cadherin and L1/Ng-CAM-for growth on Schwann cells in vitro and establish each of these glycoproteins as a strong candidate for regulating axon growth and guidance in vivo.     

Growth cones isolated from identified Aplysia neurons in vitro: biochemical and morphological characterization

The right upper quadrant (RUQ) cells (R3-R13) of Aplysia regenerating in dissociated cell culture form unusually large growth cones. The movement of these growth cones was observed by time-lapse phase microscopy and their ultrastructure was examined by transmission electron microscopy. Their behavior and ultrastructure have features that are typical of growth cones in vitro. Additionally, they contain neurosecretory granules similar to those found in these cells in vivo. Because RUQ growth cones are large, they can be isolated by manual dissection. RUQ cells were grown in the presence of [35S]methionine and the labeled proteins transported to the growth cones were analyzed by SDS-PAGE. These proteins were compared to those in RUQ cell bodies, RUQ neurites, and to those in the neurites and cell bodies of other identified neurons grown in vitro. Most proteins synthesized by RUQ cells in vitro are transported to their growth cones, including several glycoproteins and the precursor to the R3-R14 neuropeptide. Neuropeptides are also synthesized by a number of other Aplysia neurons growing in vitro. We examined R2, LPL1, R15, and left upper quadrant neurons and found that their precursor peptides, like those of R3-R14, are readily recognized as major cell-specific radiolabeled bands on SDS gels. The presence in regenerating growth cones of neuropeptides, neurosecretory granules, and glycoproteins known to be rapidly transported toward synapses in vivo supports the emerging view that the growth cone in vitro contains not only a motility apparatus but also a macromolecular assembly capable of forming an active synapse immediately upon or shortly after contacting targets.     

Mesenchyme of embryonic reproductive ducts directs process outgrowth of Retzius neurons in the medicinal leech.

In the two segments of the medicinal leech (Hirudo medicinalis) that contain the male (segment 5) and the female (segment 6) reproductive ducts, the paired Retzius (Rz) neurons are distinguished by several unique properties. For example, the muscles and glands of the body wall are the primary peripheral targets of Rz neurons in standard segments [Rz(X)], whereas the muscles and glands of the reproductive ducts are the primary peripheral targets of Rz neurons in the two reproductive segments [Rz(5,6)]. In this paper, we show that organogenesis and differentiation, which generate an epithelial tube surrounded by mesenchymal cells, occur in the embryonic reproductive ducts at approximately the time when Rz processes first contact these structures. The growth cones leading one branch of the posterior axon of Rz(5,6) contact the duct mesenchymal cells. Following initiation of this contact, these posterior growth cones enlarge and send out numerous filopodia. Secondarily, growth cones leading the anterior axon of each Rz(5,6) also modify their shapes and trajectories. When embryonic reproductive ducts were transplanted into posterior (nonreproductive) segments, the branch of the posterior Rz axon near the ectopic reproductive tissue produced enlarged growth cones and extended several secondary branches into the mesenchyme of the ectopic tissue. This result suggests that the reproductive mesenchyme is attractive to, and can modify the growth of, all Rz neurons. The behavior of Rz(5,6) growth cones suggests that the reproductive mesenchyme cells provide guidance cues that control the location in which Rz axons elaborate their peripheral arborization and form synapses, and that the mesenchyme may also stimulate the production of a densely branched arbor.