Pioneer growth cone behavior at a differentiating limb segment boundary in the grasshopper embryo

The first neurons to extend axons through embryonic grasshopper limbs are a pair of sibling pioneer neurons. After migrating proximally along the limb axis, the pioneer growth cones normally make an abrupt ventral turn. In some cases (less than 20%) this turn is directly toward the proximo-ventrally located Cx1 guidepost neurons. However, in the majority of cases (greater than 80%) the pioneer growth cones make a more acute ventral turn along a single circumferential line which lies distal to the Cx1 neurons. Growth cones from other afferent neurons orient along the same line. Growth cones can extend along this line around more than half of the circumference of the limb and can grow in either direction along it. The circumferential line appears to be the prospective trochanter-coxa segment boundary. Afferent axons on the segment boundary leave it and contact the proximo-ventrally located Cx1 neurons. The site at which pioneer growth cones leave the boundary is variable and appears to be the point from which filopodial contact with Cx1 cells is first established. In addition to the trochanter-coxa segment boundary, the pioneer growth cones and axons also respond to the tibia-femur and femur-trochanter segment boundaries. The role of segment boundaries as barriers to growth cone movement and the effect of such barriers on the timing and placement of differentiation of pioneer neurons are discussed.     

Pioneer neuron pathfinding from normal and ectopic locations in vivo after removal of the basal lamina

The contribution of the basal lamina to Ti1 pioneer axon guidance in grasshopper limb buds was investigated by allowing growth cones to migrate in 30%-31% stage limbs from which the basal lamina had been removed by enzymatic treatment. When the Ti1 axons extended from their normal location, the pathways established in the absence of basal lamina were normal. This indicates that the basal lamina is not required for initial proximal axon outgrowth, recognition of limb segment boundaries, or selective interaction with neuronal somata. Removal of the basal lamina from slightly older (32% stage) embryos resulted in displacement of the Ti1 somata to ectopic locations in approximately 50% of the limbs. Pathfinding from ectopic locations was aberrant in 45% of the cases observed. This demonstrates that if orienting information is present in the basal lamina-free epithelium at this stage, it is not the predominant factor in determining growth cone orientation from ectopic locations.     

The restricted spatial and temporal expression of a nervous-system-specific antigen involved in axon outgrowth during development of the grasshopper

To identify molecules important for pathfinding by growing axons, monoclonal antibodies (mAb) have been generated against embryonic grasshopper tissue. One mAb, 2B2, shows labeling exclusively in the nervous system. It recognizes a surface epitope on neuronal growth cones, filopodia and axons in the central nervous system (CNS). Initially, the antigen is expressed on all processes of the CNS; after 70% of embryonic development, localization of the 2B2 mAb is restricted to a small subset of axon tracts within the ganglia. Immunoprecipitation from embryonic membrane extracts with the 2B2 mAb reveals a unique band of 160 x 10(3) Mr. Functional studies with the 2B2 mAb demonstrate that the antigen is important in growth cone-axon interactions during process outgrowth. Growth cones that extend along axonal substrata are either blocked in growth or grow along an aberrant pathway when embryos are cultured in the presence of the 2B2 mAb. However, pioneer neurons that extend processes on non-neuronal substrata grow normally.     

Pathfinding by pioneer neurons in isolated, opened and mesoderm-free limb buds of embryonic grasshoppers

The Ti1 afferent neurons are the first neurons to undergo axonogenesis in limb buds of embryonic grasshoppers. Their growth cones pioneer a stereotyped pathway through the limb which becomes the route of one of the major leg nerve trunks. The growth cones appear to be oriented by several kinds of guidance cues, including guidepost neurons, a developing limb segment boundary, and an additional proximally orienting cue(s). In the experiments reported here, we have investigated the possible nature and source of proximally orienting and segment boundary cues by surgical manipulations of the limb. Before the onset of pioneer axonogenesis, limbs were isolated from the body, opened longitudinally and pinned out flat, or stripped of mesoderm. Pioneer axon routes in cultured, surgically manipulated limb buds were compared to routes in cultured control limbs. The results indicate that proximal extension of pioneer growth cones along the limb axis does not require (during the period of growth) tissue extrinsic to the limb, contact guidance by the limb contour, an axial electrical field, a diffusion gradient generated by a localized source, mesodermal cells, or guidepost neurons; adequate guidance information for proximal growth apparently can be provided by the limb epidermal epithelium (including the basal lamina) and/or by internal polarity of the pioneer neurons. Adequate guidance information for the segment boundary portion of the pioneer route apparently can be provided by the limb epithelium.     

Migrating mesoderm establish a uniform distribution of laminin in the developing grasshopper embryo

The basal lamina is composed of molecules which physically interact to form a network that serves as a migrational scaffold for many cell types. In the developing peripheral nervous system of the grasshopper, neuronal growth cones are intimately associated with the basal lamina as they migrate. Laminin is a major component of the basal lamina and is a potent promoter of neurite outgrowth in vitro. However, it is unclear what the source of laminin is or how the distribution of laminin within the basal lamina is established. To address this question, grasshopper laminin subunit genes were cloned. As expected, laminin was found within the basal lamina throughout the embryo, in particular in the limb bud, where its expression is coincident with the outgrowth and guidance of the Tibial (Til) pioneer neurons. Surprisingly, the synthesis of beta and gamma chains of laminin was restricted to migratory mesodermal cells, while in other nonmigratory tissues, such as epithelium and presumptive muscle, beta and gamma chains of laminin were not detected. In spite of this, laminin immunoreactivity in the basal lamina appears uniform and is available as a substrate for axonal outgrowth.     

Pioneer neurones use basal lamina as a substratum for outgrowth in the embryonic grasshopper limb

During axonogenesis, contacts made by the growth cone with its substratum are important in guiding the direction of neurone outgrowth. This study examines the contacts made by the growth cones of pioneer neurones in the embryonic grasshopper limb. Individual pioneer neurones at different stages of development were injected with horseradish peroxidase and the contacts made by the filopodia at the tip of their growth cones were examined by electron microscopy. Filopodia made few contacts with mesodermal cells, some contacts with ectodermal cells and very frequent contacts with basal lamina underlying the ectoderm. Components of the basal lamina may therefore play a role in guiding pioneer axon outgrowth.     

Nitric oxide and cGMP influence axonogenesis of antennal pioneer neurons

The grasshopper embryo has been used as a convenient system with which to investigate mechanisms of axonal navigation and pathway formation at the level of individual nerve cells. Here, we focus on the developing antenna of the grasshopper embryo (Schistocerca gregaria) where two siblings of pioneer neurons establish the first two axonal pathways to the CNS. Using immunocytochemistry we detected nitric oxide (NO)-induced synthesis of cGMP in the pioneer neurons of the embryonic antenna. A potential source of NO are NADPH-diaphorase-stained epithelial cells close to the basal lamina. To investigate the role of the NO/cGMP signaling system during pathfinding, we examined the pattern of outgrowing pioneer neurons in embryo culture. Pharmacological inhibition of soluble guanylyl cyclase (sGC) and of NO synthase (NOS) resulted in an abnormal pattern of pathway formation in the antenna. Axonogenesis of both pairs of pioneers was inhibited when specific NOS or sGC inhibitors were added to the culture medium; the observed effects include the loss axon emergence as well as retardation of outgrowth, such that growth cones do not reach the CNS. The addition of membrane-permeant cGMP or a direct activator of the sGC enzyme to the culture medium completely rescued the phenotype resulting from the block of NO/cGMP signaling. These results indicate that NO/cGMP signaling is involved in axonal elongation of pioneer neurons in the antenna of the grasshopper.     

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.     

Fasciclin IV: sequence, expression, and function during growth cone guidance in the grasshopper embryo.

Monoclonal antibody 6F8 was used to characterize and clone fasciclin IV, a new axonal glycoprotein in the grasshopper, and to study its function during growth cone guidance. Fasciclin IV is dynamically expressed on a subset of axon pathways in the developing CNS and on circumferential bands of epithelial cells in developing limb buds. One of these bands corresponds to the location where the growth cones of the Ti1 pioneer neurons make a characteristic turn while extending toward the CNS. Embryos cultured in the 6F8 antibody or Fab exhibit aberrant formation of this axon pathway. cDNA sequence analysis suggests that fasciclin IV has a signal sequence; long extracellular, transmembrane, and short cytoplasmic domains; and shows no homology with any protein in the available data bases. Thus, fasciclin IV appears to be a novel integral membrane protein that functions in growth cone guidance.     

Discrete roles for secreted and transmembrane semaphorins in neuronal growth cone guidance in vivo

From the initial stages of axon outgrowth to the formation of a functioning synapse, neuronal growth cones continuously integrate and respond to multiple guidance cues. To investigate the role of semaphorins in the establishment of appropriate axon trajectories, we have characterized a novel secreted semaphorin in grasshopper, gSema 2a. Sema 2a is expressed in a gradient in the developing limb bud epithelium during Ti pioneer axon outgrowth. We demonstrate that Sema 2a acts as chemorepulsive guidance molecule critical for axon fasciculation and for determining both the initial direction and subsequent pathfinding events of the Ti axon projection. Interestingly, simultaneous perturbation of both secreted Sema 2a and transmembrane Sema I results in a broader range and increased incidence of abnormal Ti pioneer axon phenotypes, indicating that different semaphorin family members can provide functionally distinct guidance information to the same growth cone in vivo.     

Spatial and temporal variation in the structure of the basal lamina in embryonic grasshopper limbs during pioneer neurone outgrowth

The pioneer neurones of the embryonic grasshopper limb use the basal lamina underlying the limb ectoderm as a substratum over which to grow from the periphery to the CNS (Anderson & Tucker, 1988). In this paper we use transmission electron microscopy to describe the structure of this substratum before, during, and after the time of axon navigation. The organization of the basal lamina varies considerably in different regions and at different times of development of the embryonic limbs, and is unlike that of the fully developed limb at the time of hatching. We suggest that this spatial and temporal variation could play a role in regulating the direction of outgrowth of pioneer neurones.     

Mechanisms of growth cone guidance and motility in the developing grasshopper embryo

During neuronal pathfinding in vivo, growth cones must reorient their direction of migration in response to extracellular guidance cues. The developing grasshopper limb bud has proved to be a model system in which to examine mechanisms of growth cone guidance and motility in vivo. In this review we examine the contributions of adhesion and multiple guidance cues (semaphorins 1 and 2) in directing a growth cone steering event. Recent observations have suggested that the tibial pioneer growth cones are not directed via mechanisms of differential adhesivity. We present a model of growth cone steering that suggests a combination of adhesive and guidance receptors are important for a correct steering event and that guidance molecules may be important regulators of adhesive interactions with the actin cytoskeleton.     

Accumulation of actin in subsets of pioneer growth cone filopodia in response to neural and epithelial guidance cues in situ

Directed outgrowth of neural processes must involve transmission of signals from the tips of filopodia to the central region of the growth cone. Here, we report on the distribution and dynamics of one possible element in this process, actin, in live growth cones which are reorienting in response to in situ guidance cues. In grasshopper embryonic limbs, pioneer growth cones respond to at least three types of guidance cues: a limb axis cue, intermediate target cells, and a circumferential band of epithelial cells. With time-lapse imaging of intracellularly injected rhodamine-phalloidin and rhodamine-actin, we monitored the distribution of actin during growth cone responses to these cues. In distal limb regions, accumulation of actin in filopodia and growth cone branches accompanies continued growth, while reduction of actin accompanies withdrawal. Where growth cones are reorienting to intermediate target cells, or along the circumferential epithelial band, actin selectively accumulates in the proximal regions of those filopodia that have contacted target cells or are extending along the band. Actin accumulations can be retrogradely transported along filopodia, and can extend into the central region of the growth cone. These results suggest that regulation and translocation of actin may be a significant element in growth cone steering.     

GPI-anchored human placental alkaline phosphatase has a nonpolarized distribution on the cell surface of mouse cerebellar granule neurons in vitro

The glycosyl phosphatidylinositol (GPI) lipid anchor, which directs GPI-anchored proteins to the apical cell surface in certain polarized epithelial cell types, has been proposed to act as an axonal protein targeting signal in neurons. However, as several GPI-anchored proteins have been found on both the axonal and somatodendritic cell-surface domains of a variety of neuronal cell types, the role of the GPI anchor in protein localization to the axon remains unclear. To begin to address the role of the GPI anchor in neuronal protein localization, we used a replication-incompetent retroviral vector to express a model GPI-anchored protein, human placental alkaline phosphatase (hPLAP), in early postnatal mouse cerebellar granule neurons developing in vitro. Purified granule neurons were cultured in large mitotically active cellular reaggregates to allow retroviral infection of undifferentiated, proliferating granule neuron precursors. To more easily visualize hPLAP localization during the sequence of differentiation of single postmitotic granule neurons, reaggregates were dissociated following infection, plated as high-density monolayers, and maintained for 1-9 days under serum-free culture conditions. As we previously demonstrated for uninfected granule neurons developing in monolayer culture, hPLAP-expressing granule neurons likewise developed in vitro through a series of discrete temporal stages highly similar to those observed in situ. hPLAP-expressing granule neurons first extended either a single neurite or two axonal processes, and subsequently attained a mature, well-polarized morphology consisting of multiple short dendrites and one or two axons that extended up to 3 mm across the culture substratum. hPLAP was expressed uniformly on the entire cell surface at each stage of granule neuron differentiation. Thus, it appears that the GPI anchor is not sufficient to confer axonal localization to an exogenous GPI-anchored protein expressed in a well-polarized primary neuronal cell type in vitro; other signals, such as those present in the extracellular domain of these proteins, may be necessary for the polarized targeting or retention of axon-specific GPI-anchored proteins.     

Expression and release of phosphatidylinositol anchored cell surface molecules by a cell line derived from sensory neurons.

Early postnatal mouse dorsal root ganglion neurons were found to express several glycosylphosphatidylinositol-anchored (GPI) molecules from the immunoglobulin superfamily (neural cell adhesion molecule 120 kD isoform, F3, Thy1) whose expression is developmentally regulated. A hybrid cell line (ND26), made by fusing postmitotic rat dorsal root ganglion (DRG) neurons with the mouse neuroblastoma N18Tg2, could be induced to differentiate by manipulating the composition of the culture medium and expressed similar GPI molecules to DRG neurons. We used this model system to investigate the metabolism of GPI-anchored molecules. We found that neural cell adhesion molecule 120 Kd isoform expression decreased upon differentiation, whereas the level of F3 and Thy1 increased, suggesting a role in neurite outgrowth processes. The ratio of molecules cleavable by exogenous phosphatidylinositol phospholipase C (PI-PLC) was similar for all the GPI-anchored molecules, which could mean that cell-specific modifications of the basic anchoring structure determine the level of potentially releasable molecules. Measurements of spontaneous release indicated that this reflected the overall level of expression of these molecules by the ND26 cell line. Finally, we observed an effect of dibutyryl cAMP on the level of expression of F3 and Thy1 but not of N-CAM. However, we could not detect any significant effect of nerve growth factor (NGF) either on the level of expression or on the amount of spontaneously released molecules.     

Phospholipase-C sensitive GPI-anchored proteins of goat sperm: possible role in sperm protection

The role of glycosylphosphatidylinositol (GPI)-anchored sperm proteins in reproduction has been investigated. SDS-polyacrylamide gels (PAGE) analysis of goat sperm (Capra indica) indicated that several GPI-anchored proteins were released by phosphatidylinositol-specific phospholipase-C (PI-PLC) treatment. The distribution of this category of PI-PLC-sensitive GPI-anchored proteins on the surface of sperm was examined by indirect immunofluorescence. The fluorescence microscopic study clearly demonstrated that the PI-PLC-sensitive GPI-anchored proteins are confined predominantly to the head region of goat sperm. Further experiments were conducted on intact and PI-PLC treated sperm in order to decipher the function of GPI proteins. Co-incubation of sperm with peritoneal macrophages led to the enhanced phagocytosis of PI-PLC treated sperm by macrophages compared with the untreated intact sperm. Transmission electron micrographs of the macrophages acquired from the phagocytosis assay are provided to corroborate the same. From the results obtained it is inferred that one or more of the PI-PLC-sensitive GPI-anchored proteins on the sperm surface could act as protection factor(s) that shield the sperm from macrophages.     

Listeria monocytogenes phosphatidylinositol (PI)-specific phospholipase C has low activity on glycosyl-PI-anchored proteins.

The ability of the phosphatidylinositol-specific phospholipase C (PI-PLC) from Listeria monocytogenes to hydrolyze glycosyl phosphatidylinositol (GPI)-anchored membrane proteins was compared with the ability of the PI-PLC from Bacillus thuringiensis to hydrolyze such proteins. The L. monocytogenes enzyme produced no detectable release of acetylcholinesterase from bovine, sheep, and human erythrocytes. The cleavage of the GPI anchors of alkaline phosphatase from rat and rabbit kidney slices was less than 10% of the cleavage seen with the PI-PLC from B. thuringiensis. Activity for release of Fc gamma receptor IIIB (CD16) on human granulocytes was also low. Variations in pH and salt concentration had little effect on the release of GPI-anchored proteins. Our data show that L. monocytogenes PI-PLC has low activity on GPI-anchored proteins.     

Phosphatidylinositol-specific phospholipase C of Bacillus anthracis down-modulates the immune response

Phosphatidylinositol-specific phospholipases (PI-PLCs) are virulence factors produced by many pathogenic bacteria, including Bacillus anthracis and Listeria monocytogenes. Bacillus PI-PLC differs from Listeria PI-PLC in that it has strong activity for cleaving GPI-anchored proteins. Treatment of murine DCs with Bacillus, but not Listeria, PI-PLC inhibited dendritic cell (DC) activation by TLR ligands. Infection of mice with Listeria expressing B. anthracis PI-PLC resulted in a reduced Ag-specific CD4 T cell response. These data indicate that B. anthracis PI-PLC down-modulates DC function and T cell responses, possibly by cleaving GPI-anchored proteins important for TLR-mediated DC activation.     

Outgrowth by fin motor axons in wildtype and a finless mutant of the Japanese medaka fish

The outgrowth of motor axons to the developing pectoral fin of the Japanese medaka fish (Oryzias latipes) was investigated both in wildtype embryos and in the pectoral finless (pl) mutants in which adults are missing pectoral fins. Late in embryogenesis the pectoral fin is a simple limb which contains two antagonist muscles which are innervated by presumptive motor neurons from the first four spinal segments (S1-4). The pectoral fin develops from a fin bud located in S1 and S2 centered on the border between S1 and S2 and, as with other limbs, one of the earliest signs of differentiation is the apical ectodermal ridge (AER). By the time the AER is well formed the growth cones of the presumptive motor neurons have reached the base of the fin bud and formed a plexus by extending toward the fin bud upon emergence from the spinal cord. This is especially evident on the ventral surface of the metamerically arranged axial muscles. For example, growth cones from S2 extend in a diagonal direction (both anterior and lateral) towards the fin bud. One hypothesis which can account for the pattern of motor outgrowth is that growth cones are attracted to the base of the fin bud, perhaps via a long distance cue. This hypothesis was tested by examining outgrowth of segmental nerves in pl embryos in which the fin buds arrest early in development following the initial appearance of the AER. In pl, nerves from S1-4 converged to form a plexus at the base of the abnormal fin bud, but the pattern of outgrowth varied from wildtype in a way consistent with a diminished capacity of the fin bud to attract segmental nerves to it.