Pathway selection by ectopic motoneurons in embryonic zebrafish.

Primary motoneurons in embryonic zebrafish innervate cell-specific muscles. During pathfinding, motoneuronal growth cones encounter three distinct regions: a common pathway, a choice point, and separate cell-specific pathways. To learn whether the order in which these regions are encountered influences pathway choice, we transplanted individual motoneurons to the choice point region. These cells selected their appropriate cell-specific pathways. Thus, the sequence in which pathway regions are encountered may not be important for accurate path-finding, and the cell-specific pathways may be delineated by distinct cues that individual growth cones recognize. Moreover, these cues are unlikely to be general ones, since primary sensory neurons transplanted to the same location do not extend growth cones along the motoneuronal pathways.     

HuC–eGFP mosaic labelling of neurons in zebrafish enables in vivo live cell imaging of growth cones

The field of axon guidance is taking advantage of the powerful genetic and imaging tools that are now available to visualise growth behaviour in living cells, both in vivo and in real time. We have developed a method to visualise individual neurons within the living zebrafish embryo which provides exceptional cellular resolution of growth cones and their filopodia. We generated a DNA construct in which the HuC promoter drives expression of eGFP. Injection of the plasmid into single cell fertilised zebrafish egg resulted in mosaic expression of eGFP in neurons throughout the developing embryo. By manipulating the concentration of injected plasmid, it was possible to optimise the numbers of neurons that expressed the construct so that individual growth cones could be easily visualised. We then used time-lapse high magnification widefield epifluorescence microscopy to visualise the growth cones as they were exploring their environment. Growth cones both near the surface of the embryo as well as deep within the developing brain of embryos at 20?h post fertilisation were clearly imaged. With time-lapse sequence imaging with intervals between frames as frequent as 1?s there was minimal loss of fluorescence intensity and the dynamic nature of the growth cones became evident. This method therefore provides high magnification, high resolution time-lapse imaging of living neurons in vivo and by use of widefield epifluorescence rather than confocal it is a relatively inexpensive microscopy method.     

Distinct calcium signaling within neuronal growth cones and filopodia

Previous findings indicate that spatial restriction of intracellular calcium levels within growth cones can regulate growth cone behavior at many levels, ranging from filopodial disposition to neurite extension. By combining techniques for focal stimulation of growth cones with those for measurement of filopodia and for capturing low intensity calcium signals, we demonstrate that filopodia on individual growth cones can respond to imposed stimuli independently from one another. Moreover, filopodia and their parent growth cones appear to represent functionally and morphologically distinct domains of calcium regulation, possessing distinct calcium sources and sinks. Both are sensitive to calcium influx; however, application of the calcium ionophore A23187 to cells in calcium-free medium demonstrated the presence of potential intracellular calcium pools in the growth cone proper, but not in isolated filopodia. Thapsigargin significantly reduced the rise in growth cone calcium levels associated with excitatory neurotransmitters, further implicating release from calcium pools as one component of growth cone calcium regulation. The relative contributions of these pools were examined in response to excitatory neurotransmitters by quantitative calcium measurements made in both growth cones and isolated filopodia. Striking differences were observed; filopodia were sensitive to a low concentration of dopamine and serotonin, while growth cones displayed an amplified rise at a higher concentration. The spatial distribution of organelles that could serve as morphological correlates to such calcium amplification was examined using confocal microscopy. While the majority of organelles were located in the central core of the growth cone proper, peripheral organelles were detected at the base of a subset of filopodia. The distinctive distribution of calcium regulation within motile growth cones suggests one mechanism by which growth cones may regulate their complex behavior. © 1996 John Wiley & Sons, Inc.     

GAP-43 phosphorylation is dynamically regulated in individual growth cones

In vivo, kinase C phosphorylation of the growth-associated protein GAP-43 is spatially and temproally associated with the proximity of growing axons to their targets. Here we have used dissociated dorsal root ganglia (DRG)s and an antibody specific for the phosphorylated form of GAP-43 to demonstrate that neurite regeneration in culture also begins in the absence of detectable levels of phosphorylated GAP-43. Since the β isoform of kinase C was found to be enriched in growth cones before stably phosphorylated GAP-43 was detected, it may normally be inactive during initial neurite outgrowth; however, premature phosphorylation of GAP-43 could be stimulated in newly dissociated DRGs by plating them on cultures in which phosphorylation had already been initiated; media conditioned by such cultures caused no response suggesting an effect of either cell-cell or cell-substrate contact. Increased GAP-43 phosphorylation correlated with a reduced extent of neurite outgrowth but not with the rate at which individual growth cones translocated so that motile growth cones contained very low levels of phosphorylated GAP-43, whereas stationary growth cones showed much more immunoreactivity. Downregulation of kinase C by phorbol ester prevented increased GAP-43 phosphorylation and led to growth cone collapse. Finally, phosphorylated GAP-43 was found to be differently distributed within growth cones. Increased immunoreactivity was frequently observed in the neck of the growth cone and was heterogeneously distributed in lamellae and filopodia. These results, which demonstrate the dynamic regulation of GAP-43 phosphorylation in individual growth cones, are discussed with reference to the association between changes in growth cone shape and the ability to translocate and change direction. © 1992 John Wiley & Sons, Inc.     

Specific pathway selection by the early projections of individual peripheral sensory neurons in the embryonic medicinal leech

In leech, the central annulus of each midbody segment possesses seven pairs of sensilla, which are mixed clusters of primary peripheral sensory neurons that extend their axons into the CNS where they segregate into distinct fascicles. Pathway selection by individual afferent growth cones of sensillar neurons was examined by double labeling using intracellular dye-filling with anitobody labeling in early Hirudo medicinalis embryos. The monoclonal antibody Lan3–2 was used because sensillar neuronal tracts are specifically labeled by this antibody. Examining 68 individually filled neurons we found that sensillar neuron growth cones bifurcate within the CNS, that they project long filopodia capable to sampling the local environment, and that all of them appeared to choose a single particular CNS fascicle without apparent retraction or realignment of growth cones. Furthermore, each side of the bifurcating afferent growth cones always chose the same fascicle, implying a specific choice of a distinct labeled pathway. By dye-filling individual central neurons (P-cells), we show that there are centrally projecting axons present at the time sensillar afferents enter the ganglionic primordia and select a particular fascicle, and we confirm that at least the dorsal peripheral nerve is likely to be pioneered by central neurons, not by the peripheral afferent. In the sensillum studied here, we sound examples of sensory neurons extending axons into one of all the avilable fascicles. Thus, an individual embryonic sensillum possesses a heterogeneous population of afferents with respect to the central fascicle chosen. This is consistent with the idea that segregation into distinct axon fascicles may be based upon functional differences between individual afferent neurons. Our findings argue strongly in favor of specific pathway selection by afferents in this system and are consistent with previous suggestions that there exists a hierarchy of cues, including surface glycoconjugates that mediate navigation of the sensillar growth cones and the fasciculation of their axons. 1994 John Wiley & Sons, Inc.     

The cadherin Flamingo mediates level-dependent interactions that guide photoreceptor target choice in Drosophila

Quantitative differences in cadherin activity have been proposed to play important roles in patterning connections between pre- and postsynaptic neurons. However, no examples of such a function have yet been described, and the mechanisms that would allow such differences to direct growth cones to specific synaptic targets are unknown. In the Drosophila visual system, photoreceptors are genetically programmed to make a complex, stereotypic set of synaptic connections. Here we show that the atypical cadherin Flamingo functions as a short-range, homophilic signal, passing between specific R cell growth cones to influence their choice of postsynaptic partners. We find that individual growth cones are sensitive to differences in Flamingo activity through opposing interactions between neighboring cells and require these interactions to be balanced in order to extend along the appropriate trajectory.     

Growth cone guidance in the zebrafish central nervous system.

The accessibility and simplicity of the zebrafish embryo have allowed researchers to make a detailed characterization of pathfinding by identifiable growth cones. The growth cones follow precise cell-specific pathways to their targets. Analyses of pathfinding in mutant and experimentally manipulated wild type embryos have shown that growth cones accomplish this by interacting with specific cellular cues in their environment, many of which are likely to be redundant.     

Growth pattern of pioneering chick spinal cord axons

The early growth pattern of axons in the embryonic chick spinal cord was studied by electron microscopy. Serial perisagittal thin sections were obtained from the lateral margins of spinal cords of stage 17 (S17) and S19 embryos. A simple stereotypic pattern of axonal growth was found. Axons originated from a dispersed population of presumptive interneurons located along the lateral spinal cord margin. They first grew ventrally in a nonfasciculative pattern and later turned at right angles and grew in a fasciculative manner longitudinally in the ventrolateral fasciculus. Growth along the circumferential pathway was analyzed in detail by reconstructing individual axons and growth cones from the S17 specimen. Most circumferential axons, regardless of their site of origin, grew in a parallel orientation, and each of their growth cones projected ventrally. This pattern suggested that circumferential growth cones were guided at many, if not all, points along their path. Study of the region in front of these seven growth cones, however, revealed no apparent structural basis for their guidance. Alternative guidance mechanisms are discussed. In conjunction with previous studies (e.g., Windle and Baxter, 1936; Lyser, 1966), these findings suggest that the circumferential-nonfasciculative and the longitudinal-fasciculative patterns of axonal growth are the two fundamental patterns followed by most early forming axons in the brain stem and spinal cord of all higher vertebrates.     

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.     

RNAi of the receptor tyrosine phosphatase HmLAR2 in a single cell of an intact leech embryo leads to growth-cone collapse

Receptor protein tyrosine phosphatases (RPTPs) are important for growth-cone migration [1-5], but their specific roles have yet to be defined. Previously, we showed that the growth cones of the Comb cell, an embryonic cell in the leech, express high levels of an RPTP called HmLAR2 [6,7]. Here, we report the use of RNA interference (RNAi) to block expression of HmLAR2 in individual Comb cells in the developing embryo. HmLAR2 mRNA levels were reduced in the soma, processes and growth cones of Comb cells injected with double-stranded RNA (dsRNA) for HmLAR2, but no decrease was detected when control dsRNAs were injected. Consistent with this observation, the level of phosphotyrosine increased significantly in the growth cones of Comb cells injected with HmLAR2 dsRNA. Within 24 hours, the growth cones of treated cells showed a distinct collapsed phenotype, with sharp reductions in lamellipodial surface area and in numbers of filopodia. These experiments indicate a key role for LAR-like RPTPs in maintaining the integrity of the growth cone.     

Measurements of growth cone adhesion to culture surfaces by micromanipulation

Neurons were grown on plastic surfaces that were untreated, or treated with polylysine, laminin, or L1 and their growth cones were detached from their culture surface by applying known forces with calibrated glass needles. This detachment force was taken as a measure of the force of adhesion of the growth cone. We find that on all surfaces, lamellipodial growth cones require significantly greater detachment force than filopodial growth cones, but this differences is, in general, due to the greater area of lamellipodial growth cones compared to filopodial growth cones. That is, the stress (force/unit area) required for detachment was similar for growth cones of lamellipodial and filopodial morphology on all surfaces, with the exception of lamellipodial growth cones on L1-treated surfaces, which had a significantly lower stress of detachment than on other surfaces. Surprisingly, the forces required for detachment (760-3,340 mudynes) were three to 15 times greater than the typical resting axonal tension, the force exerted by advancing growth cones, or the forces of retraction previously measured by essentially the same method. Nor did we observe significant differences in detachment force among growth cones of similar morphology on different culture surfaces, with the exception of lamellipodial growth cones on L1-treated surfaces. These data argue against the differential adhesion mechanism for growth cone guidance preferences in culture. Our micromanipulations revealed that the most mechanically resistant regions of growth cone attachment were confined to quite small regions typically located at the ends of filopodia and lamellipodia. Detached growth cones remained connected to the substratum at these regions by highly elastic retraction fibers. The closeness of contact of growth cones to the substratum as revealed by interference reflection microscopy (IRM) did not correlate with our mechanical measurements of adhesion, suggesting that IRM cannot be used as a reliable estimator of growth cone adhesion.     

Fine structure of growth cones in the upper dorsal horn of the adult primate spinal cord in the course of reactive synapto-neogenesis

Summary Following transganglionic degenerative atrophy of primary afferent terminals induced by a crush-injury of the sciatic nerve, a regenerative process takes places in the upper dorsal horn of the lumbar spinal cord in the primate Macacus rhesus. Axonal growth cones are characterized by cisterns of axoplasmic reticulum; filopodia emanating from growth cones are electron-optically translucent sheet-like expansions, often containing growth-cone vesicles. Axoplasmic reticulum appears also in preterminal portions of regenerating axons. Dendritic growth cones contain a fine, filamentous matrix; electron-dense membrane specializations can be seen in well-defined areas of their surfaces. Immature synapses are formed between filopodia of axonal growth cones and dendritic growth cones. Electron-microscopic structures of this unique CNS regeneration are similar to those seen in the course of embryonic development of the spinal cord.     

Guidance of pioneer growth cones: Filopodial contacts and coupling revealed with an antibody to Lucifer Yellow

We are interested in the factors that guide individual neuronal growth cones during embryonic development. We have developed an antibody to the fluorescent dye Lucifer Yellow. We use the antibody here to examine the specific filopodial contacts and dye coupling by the first growth cones in the grasshopper embryo that navigate in an axonless environment. We have studied the distribution and apparent selective adhesion of the filopodia from these pioneering growth cones in the central nervous system and periphery. Our results suggest that selective filopodial adhesion to specific “landmark” cells may play an important role in the guidance of pioneer growth cones.     

Morphology of pioneer and follower growth cones in the developing cerebral cortex

In the developing nervous systems of both invertebrates and vertebrates, neurons must develop precise sets of axonal connections. One strategy used by both orders of animals is to generate a special class of neurons whose axons "pioneer" the first pathways between these cells and their targets. In the developing mammalian telencephalon, the subplate neurons (which are among the first neurons to be generated in development) extend axons to long-distance subcortical targets before the neurons of the deep cortical layers 5 and 6 have been generated. The axons of layer 5 and 6 neurons later follow a similar pathway to form permanent subcortical projections to the thalamus and tectum, and thereafter the vast majority of subplate neurons die. These results have generated the hypothesis that subplate axons may actually be required for the axons of layer 5 and 6 neurons to innervate their appropriate subcortical targets. The complexity of growth cones has previously been correlated with axonal decision making: differences in growth cone morphologies have been noted in comparisons of leading versus following axons (LoPresti, Macagno, and Levinthal, 1973; Nordlander, 1987; Yaginuma, Homma, Kunzi, and Oppenheim, 1991), and at choice points along axon pathways (Raper, Bastiani, and Goodman, 1983; Tosney and Landmesser, 1985; Caudy and Bentley, 1986a,b; Bovolenta and Mason, 1987; Holt, 1989; Bovolenta and Dodd, 1990; Yaginuma et al., 1991). Thus, as a first step toward addressing the question of whether the axons of deep-layer neurons simply follow subplate axons to their targets, we have studied the morphology of cortical growth cones at various points along the corticothalamic pathway and at different stages of development. We examined the brains of fetal ferrets and cats at ages ranging from embryonic days (E) 24 to E50, using the fluorescent lipophilic tracer 1,1-dioctadecyl-3,3,3',3'-tetramethyl indocarbocyanine perchlorate (DiI) to reveal the axons and growth cones of cortical neurons. Growth cones were drawn, and quantitative measurements of their complexity were made by counting filopodia and calculating their surface area. No morphological differences were found among growth cones at different points along the corticothalamic pathway at a given age. However, growth cones belonging to early-generated cells (likely to be subplate neurons) are significantly larger and more complex than are the growth cones of later-generated cortical neurons. This evidence is consistent with the suggestion that subplate growth cones actively pioneer the corticothalamic pathway, and that the axons of layer 5 and 6 neurons follow it.     

Distribution and possible interactions of actin-associated proteins and cell adhesion molecules of nerve growth cones

Actin filaments and their interactions with cell surface molecules have key roles in tissue cell behaviour. Axonal pathfinding during embryogenesis, an especially complex cell behaviour, is based on the migration of nerve growth cones. We have used fluorescence immunocytochemistry to examine the distribution in growth cones, their filopodia and lamellipodia of several actin-associated proteins and nerve cell adhesion molecules. The leading margins of chick dorsal root ganglion nerve growth cones and their protrusions stain strongly for f-actin, filamin, alpha-actinin, myosin, tropomyosin, talin and vinculin. MAP2 is absent from DRG growth cones, and staining for spectrin fodrin extends into growth cones, but not along filopodia. Thus, organization of the leading margins of growth cones may strongly resemble the leading lamella of migrating fibroblasts. The adhesion-mediating molecules integrin, L1, N-CAM and A-CAM are all found on DRG neurites and growth cones. However, filopodia stain relatively more strongly for integrin and L1 than for A-CAM or N-CAM. In fact, the 180 X 10(3) Mr form of N-CAM may be absent from most of the length of filopodia. DRG neurones cultured in cytochalasin B display differences in immunofluorescence staining which further emphasize that these adhesion molecules interact differentially with the actin filament system of migrating growth cones. Several models for neuronal morphogenesis emphasize the importance of regulation of the expression of adhesion molecules. Our results support hypotheses that cellular distribution and transmembrane interactions are key elements in the functions of these adhesion molecules during axonal pathfinding.     

Spinal neurite reabsorption and regrowth in vitro depend on the polarity of an applied electric field

Retraction and regrowth of frog neural tube neurites have been studied in vitro in control cultures and in the presence of a small, continuously applied electrical field. In control cultures, some degree of retraction was seen in 39% of neurites while 7% were reabsorbed completely. Reabsorption of anodal-facing neurites was at least twice as common, with 67% showing some retraction and 17% almost totally reabsorbed. Cathodal-facing neurites were spared from retraction. Following extreme reabsorption of anodal-facing neurites, reversal of the electric field promoted regeneration in 47% (9/19) of cases studied. growth cone morphology also was determined by the polarity of the applied field. Anodal-facing growth cones had fewer filopodia than cathodal-facing growth cones sharing the same cell body. Field reversal induced a polarity-specific change in filopodia number on individual growth cones: a shift from anodal to cathodal increased filopodia numbers and vice versa. Some possible mechanisms involved and the significance of these results are discussed.     

Elimination of a brain tract increases errors in pathfinding by follower growth cones in the zebrafish embryo

The early zebrafish brain contains a simple axon scaffold of longitudinal tracts connected by commissures. Neurons in the nucleus of the posterior commissure (nuc PC) project growth cones along a specific route in this axonal scaffold, raising the possibility that specific axons in the early scaffold guide nuc PC growth cones. We tested this possibility by analyzing the behavior of nuc PC growth cones in embryos in which a portion of the scaffold, normally traversed by nuc PC growth cones, was surgically prevented from forming. Under these conditions nuc PC growth cones extended along both normal and aberrant pathways. This suggests that specific axons do provide guidance cues, since their removal leads to errors. However, these cues are not obligatory, since some growth cones still followed normal pathways.     

Longleaf pine cone–radial growth relationships in the southeastern U.S.A.

Longleaf pine (Pinus palustris Mill.) cones have been counted annually by the United States Forest Service (USFS) at eleven locations throughout the species’ range since 1958. These data have been useful for understanding spatiotemporal patterns in longleaf pine cone production, and are beneficial in timing regeneration efforts. Variations in annual mast (i.e. seed crop) are known to influence ring widths in numerous tree species, yet this relationship is poorly understood for longleaf pine. This research examines the relationship between longleaf pine cone data and tree-ring growth from trees sampled in the multi-decadal USFS cone-crop study. We examined cone–radial growth relationships using individual tree-ring data and proprietary cone data for each tree from six sites in four locations in the southeastern USA. We found that longleaf pine cones were correlated with basal area increment growth (BAI) over the three-year cone-development cycle. Low BAI years were more frequently associated with above-average cone crop and BAI during years that coincided with the largest cone-crop class (bumper, > 100 cones per tree) were statistically less than any other cone class. We prepared linear models that predicted radial growth using PDSI and cones as predictors, and found that including cones in the models did not improve adjusted R² values. We conclude that while cone production is inversely related to radial growth, the combination of infrequent bumper years and the concentration of cone production by a few trees per stand, creates an environment where radial-growth chronologies assembled from longleaf pine for dendroclimatic purposes are unlikely to be significantly influenced by reproductive strain.     

Axonal guidance in the chick visual system: posterior tectal membranes induce collapse of growth cones from the temporal retina

Membranes from posterior and anterior thirds of the chick optic tectum were added to explants from nasal and temporal retina. Posterior membranes, and to a lesser extent anterior membranes, cause temporal growth cones to collapse and their axonal processes to retract. Neither tectal source has an effect on nasal growth cones. We interpret these results to mean that there is a tectal activity, stronger in the posterior than the anterior region of the tectum, which helps guide growth cones during the development of the retinotectal map. We believe that in vivo this activity helps to steer temporal growth cones away from the posterior tectum. Nasal growth cones, which must map to the posterior tectum, are resistant to it. In vitro, when posterior membranes contact temporal growth cones over their surface, filopodia and lamellipodia withdraw rapidly. This leads to loss of contact between the growth cone and the substrate, followed by collapse.     

Xenopus temporal retinal neurites collapse on contact with glial cells from caudal tectum in vitro.

Nasal and temporal retinal neurites were confronted in culture with glial cells from the rostral and caudal parts of the optic tectum and with glial cells from the diencephalon. Twenty of each of the six classes of encounter between individual growth cones and isolated glial cells were analysed by time-lapse videorecording. The results show that growth cones from the temporal retina collapse when they contact glial cells from the caudal tectum, but do not collapse when they contact glia from other areas. Growth cones of nasal retinal fibres do not collapse on contact with any of the glial types examined. This suggests that the inhibitory phenomena described by others are in part due to the cell surface characteristics of glial cells, and that there are differences between glia from the front and back of the optic tectum.