Depletion of 43-kD growth-associated protein in primary sensory neurons leads to diminished formation and spreading of growth cones.

The 43-kD growth-associated protein (GAP-43) is a major protein kinase C (PKC) substrate of growing axons, and of developing nerve terminals and glial cells. It is a highly hydrophilic protein associated with the cortical cytoskeleton and membranes. In neurons it is rapidly transported from the cell body to growth cones and nerve terminals, where it accumulates. To define the role of GAP-43 in neurite outgrowth, we analyzed neurite regeneration in cultured dorsal root ganglia (DRG) neurons that had been depleted of GAP-43 with any of three nonoverlapping antisense oligonucleotides. The GAP-43 depletion procedure was specific for this protein and an antisense oligonucleotide to the related PKC substrate MARCKS did not detectably affect GAP-43 immunoreactivity. We report that neurite outgrowth and morphology depended on the levels of GAP-43 in the neurons in a substrate-specific manner. When grown on a laminin substratum, GAP-43- depleted neurons extended longer, thinner and less branched neurites with strikingly smaller growth cones than their GAP-43-expressing counterparts. In contrast, suppression of GAP-43 expression prevented growth cone and neurite formation when DRG neurons were plated on poly- L-ornithine. These findings indicate that GAP-43 plays an important role in growth cone formation and neurite outgrowth. It may be involved in the potentiation of growth cone responses to external signals affecting process formation and guidance.

Abstract. The 43-kD growth-associated protein (GAP-43) is a major protein kinase C (PKC) substrate of growing axons, and of developing nerve terminals and glial cells. It is a highly hydrophilic protein associated with the cortical cytoskeleton and membranes. In neurons it is rapidly transported from the cell body to growth cones and nerve terminals, where it accumulates. To define the role of GAP-43 in neurite outgrowth, we analyzed neurite regeneration in cultured dorsal root ganglia (DRG) neurons that had been depleted of GAP-43 with any of three nonoverlapping antisense oligonucleotides. The GAP-43 depletion procedure was specific for this protein and an antisense oligonucleotide to the related PKC substrate MARCKS did not detectably affect GAP-43 immunoreactivity.
We report that neurite outgrowth and morphology depended on the levels of GAP-43 in the neurons in a substrate-specific manner. When grown on a laminin substratum, GAP-43-depleted neurons extended longer, thinner and less branched neurites with strikingly smaller growth cones than their GAP-43expressing counterparts. In contrast, suppression of GAP-43 expression prevented growth cone and neurite formation when DRG neurons were plated on poly-Lornithine.
These findings indicate that GAP-43 plays an important role in growth cone formation and neurite outgrowth. It may be involved in the potentiation of growth cone responses to external signals affecting process formation and guidance.
T rIE growth of axons during development and regeneration is associated with the expression of specific growth-associated proteins (GAPs) ~ that are thought to play essential roles in the growth process (Skene, 1989;Skene and Willard, 1981). Prominent among the GAPs is the highly hydrophilic, cortical cytoskeleton-and membraneassociated 43-kD-protein GAP-43 (Skene, 1989;Benowitz and Routtenberg, 1987;Skene and Virag, 1989;Moss et al., 1990;Meiri and Gordon-Weeks, 1990). GAP-43 is a major protein kinase C (PKC) substrate of the axonal growth cone and nerve terminal (Meiri et al., 1986;Skene et al., 1986). This protein is an early marker for postmitotic neurons and its expression correlates with axonal growth during development and regeneration (Skene and Willard, 1981;Basi et al., 1987;Skene, 1989;Coggins and Zwiers, 1991). Downregulation of GAP-43 during development frequently coincides with the activity-sensitive elimination of collaterals, and with the establishment of the adult pattern of synaptic connectivity (Neve and Baer, 1989;Caroni and Beker, 1992). However, in certain areas of the central nervous system 1. Abbreviations used in this paper: CNS, central nervous system; DRG, dorsal root ganglion; GAP, growth-associated protein; IR, immunoreactivity; PKC, protein kinase C.
In neurons, GAP-43 associates with Golgi membranes shortly after synthesis and is rapidly transported to growth cones and nerve terminals, where it accumulates. In vitro, GAP-43 binds to calmodulin in the absence of calcium (Andreason et al., 1983), and to the GTP-binding protein Go in a palmitoylation-sensitive manner (Strittmatter et al., 1990;Sudo et al., 1992). Intracellular application of antibodies to GAP-43 inhibits evoked neurotransmitter release (Dekker et al., 1989), suggesting that it may play a role in exocytosis. Finally, transfection experiments carried out in non-neuronal cell lines have shown that GAP-43 affects morphogenetic processes at the cell surface (Zuber et al., 1989;Widmer and Caroni, 1993). Spreading cells expressing high levels of GAP-43 were strikingly rich in filopodia, and the effects of GAP-43 on cell spreading and morphology could be modulated by mutating the PKC phosphorylation site of GAP-43 (Widmer and Caroni, 1993).
Analysis of a PC12 mutant cell line that does not express GAP-43 suggested that GAP-43 is not required for neurite outgrowth in these cells (Baetge and Hammang, 1991). In cultured hippocampal neurons, suppression of kinesin expression after delivery of antisense oligonucleotide prevented the transport of GAP-43 from the cell body to the neurites, but did not seem to alter axonal outgrowth (Ferreira et al., 1992). These experiments therefore indicate that the presence of GAP-43 in neurites may not be essential for their outgrowth. On the other hand, however, experiments with specific antibodies against GAP-43 in neuroblastoma cells (Shea et al., 1991), and with antisense oligonucleotides in PC12 cells (Jap Tjoen San et al., 1992) have suggested that GAP-43 enhances neurite outgrowth rates in these cells.
To define the possible role(s) of GAP-43 in neurite outgrowth in primary neurons, we analyzed neurite regeneration in cultured chick dorsal root ganglion (DRG) neurons that had been dissociated in the presence of antisense oligonucleotides to GAP-43. Using this approach it was possible to reduce specifically the expression of GAP-43 in ,~40% of the cultured neurons to levels below the limit of detection by immunocytochemistry.
We report that in the absence of detectable GAP-43, neurite elongation of DRG neurons grown on laminin/poly-Lornithine in the presence of NGF was in fact accelerated. However, neurons depleted of GAP-43 extended thin, infrequently branched neurites with extremely small growth cones. On the other hand, when grown on polyornithine, GAP-43-depleted DRG neurons failed to form growth cones and hence neurites failed to emerge from the initial symmetric lamellae.
Our results reveal that GAP-43 plays an important role in spreading, branching, and possibly formation of growth cones. GAP-43 may be a local nodulator of axonal cortex and growth cone activity to transduce external signals into the formation and guidance of neurites.

Oligonucleotides
For the GAP-43 experiments three different oligonucleotide pairs were used. Antisense oligonucleotide-1 (-8+9) consisting of the sequence GCACAG-CATGATCGTAT is the inverse complement of ancleotide -8 to +9 (Baizer et al., 1990) and overlaps the start codon AUG. Antisense oligonucleotide-2 (+9+26) has the sequence TTTGTTCTTCTCATACAG. Antisense oligonucleotide-1 and -2 were chosen for their possible disruption of the translation start. Antisense oligonucleotide 3 (+643+659) is a 17-mer of the sequence ACAGCATCTGTCTTCTC. It is the inverse complement of nucleotide +643 to +659, coveting a 35-base-long unmatched stretch of a bifurcation loop as suggested by the RNA fold program of Zuker and Stiegler (Zuker and Stingier, 1981). For the MARCKS experiment the antisense oligonucteotide had the sequence CACCCATC~TTA (-9 to +7 of chicken MARCKS; Graffet al., 1989). The corresponding sense oligonucleotides were the inverse complements of the antisense oligonucleotides. Biotinylated oligonucleotides were generated by adding six Biotin-Phosphorarnidite residues (Amersham International, Buckinghamshire, England) to the 5' end. The oligonucleotides were synthesized on an Applied Biosystems 380B synthesizer, ethanol precipitated, desalted over an NAP~-25 column (Pharmacia LKB Biotechnology AB, Uppsala, Sweden) and taken up in glass-distilled water to a concentration of 10/~g//tl.

Cell Culture and Application of Oligonucleotides: Trituration Method
The method used to introduce the oligonucleotides into neurons was based upon the trituration method described by Borasio et al. (1989). Two to four chick lumbar embryonic day (E)16 DRCrs were trypsinized for 35-40 rain at 37°C (0.05% trypsin [GIBCO-BRL, Gaithersburg, MD] in Ca2+/Mg 2+free Hanks buffer). The ganglia were washed once with DME/10% FCS and then with Ca2+/Mg2+-free Hanks buffer. Between wash steps ganglia were allowed to settle down. The medium was then aspirated while leaving a residual volume of 45-50 ~1.5/~1 of an oligonucleotide solution (10 ~g/~tl in H20) were then added and the ganglia were dissociated by trituration (20-25 cycles through a Gllson yellow tip). The suspension was then left for 5 rain at room temperature after which the cells were suspended in 2 ml DME/10% FCS and preplated for 2 h in a 35-ram tissue culture dish (Coming Inc., Coming, NY) at 37°C/5% CO2 in a humidified atmosphere (McCarthy and Partlow, 1976). Nonadherent cells were collected, pelleted for 5 rain at 120 g, resuspended in DME/10% FCS containing 20 ng/rni 2.5 S NGF (Harlan Bioproducts for Science, Inc., Indianapolis, IN), and plated on coated glass coverslips (18 ram) at a cell density of ,'~1,500 cells per 200 Itl and coverslip. Cells were grown at 37°C/5% CO2 in a humidified incubator.
For coating, coverslips were first treated with 1 N HC1 for 6 h at 65°C (Tanaka and Kirschner, 1991)

Evaluation of GAP-43 Staining
All cells from randomly selected fields were analyzed. Cells were distributed into three categories: cells with strong, intermediate, and no specific GAP-43 immunoreactivity (IR). No GAP-43 immunoreactivity (GAP-43 LR) is a stringent criterium, and corresponds to the background staining of non-neuronal cells present in the culture. The assignment of a cell to strong or intermediate GAP-43 IR was controlled by monitoring with a S/W-CCD-Camera (AVT-Horn) and a KV-M1430B TV (SONY): a fixed brightness level was selected to include ,o40% of the GAP-43 expressing cells in mock-treated cultures, and cells with GAP-43 signal not detected on the screen at that brightness level were attributed to the intermediate GAP-43 IR category.

Morphometric Analysis
After processing for immunocytochemistry, cells were selected randomly in the phase channel, photographed in phase (10x) and analyzed using the NIH Image 1.41 program fed with a PHV-A7E Photo Video Camera (SONY). Neurite length was determined by measuring the length from the cell soma to the tip of the longest neurite. Branching behavior was scored by counting the branchpoints of a cell's longest neurite compared with its length. The diameter of the longest neurite was measured in the vicinity of the growth cone, using photomicrographs (100x) of the integrin signal. Statistical analysis of neurite length was performed by applying the Mann-Whitney Utest, because the distribution of the values was skewed. All other data were analyzed with t test.

Depletion of GAP-43 in Cultured DRG Neurons by Antisense Oligonucleotides
To characterize the effect of GAP-43 on neurite outgrowth we generated cultures consisting of neurons that were devoid of GAP-43 from the onset of neurite regeneration in vitro. To achieve an efficient introduction of oligonucleotides into dissociated neurons, El6 chick DRG neurons were triturated in the presence of high concentrations of the oligonucleotides. After tfituration the neurons were preplated for 2 h on a tissue culture plastic substratum in the absence of NGF to deplete the culture of rapidly adhering non-neuronal cells.
1,000-2,000 neurons per DRG survived the procedures preceding plating, as estimated by counting of Trypan blue excluding cells after the preplating step. Experiments with biotinylated oligonucleotides showed that 4 h after final plat-ing, "070% of the neurons still contained significant amounts of oligonucleotides (data not shown). 18 h after plating on a laminin substratum in the presence of NGF 30-50 % of the neurons had formed processes that were longer than their cell diameter, irrespective of whether they had been treated with no-, sense-, or antisense oligonucleotides.
As shown in Fig. 1, DRG neurons extended neurites in the absence of GAP-43. Comparable numbers of GAP-43depleted neurons were detected 1 and 24 h after plating of the neurons on laminin with NGF, and substantial extension of neurites from these DRG neurons was detected as early as 1 h after plating of the neurons on a laminin-coated substratum in the presence of NGF (Fig. 1 e). Therefore, under these experimental conditions, regeneration of neurites by DRG neurons does not require the presence of detectable GAP-.43.
As shown in Fig. 2 a, the antisense treatment produced a substantial number of DRG neurons with undetectable levels of GAP-43 in their cell bodies and neurites. GAP-43 depletion was observed when the aimration was performed in the presence of either of three different antisense oligonucleotides, but not in the presence of the corresponding sense oligonucleotides (Fig. 2 a). A quantitative analysis of GAP-43 immunoreactivity contents in populations of DRG neurons treated with the three antisense and sense oligonucleotides is presented in Fig. 2 a. Contents of neurons displaying high, intermediate, and undetectable levels of GAP-43 were comparable for the sense oligonucleotides, and for untreated or mock-treated cultures. All these cultures contained comparable amounts of neurons with high and intermediate signal levels, and a very small proportion (1-5%) of neurons with no detectable GAP-43 IR. Treatment with any of the three antisense oligonucleotides dramatically shifted these proportions, resulting in lower numbers of neurons ('010%) with high levels of GAP-43, and ,040% of the neurons with no detectable GAP-43 signal (Fig. 2 a). Fig. 2 b shows that no differences in the levels of GAP-43 IR were detected 3 d after plating in neurons treated with antisense or sense oligonucleotide, indicating that the effect of the antisense oligonucleotides on DRG GAP-43 IR was reversible. Fig. 2 c shows that the antisense, but not the sense oligonucleotides specifically detected GAP-43 mRNA on Northern blots of chick DRG poly(A) ÷ RNA (two mRNA species, see Baizer et al., 1990). Finally, Fig. 2 d demonstrates that the treatment resulted in a strong reduction in the total contents of GAP-43, as determined by immunoblot analysis. However, the corresponding levels of the neurofilament protein NF-160 did not change in a comparable manner (Fig. 2 d). Our data indicate that the effect of the antisense oligonucleotides was specific for GAP-43. Thus, three specific antisense oligonucleotides, but not the corresponding sense oligonucleotides, had comparable effects on the levels of GAP-43 in the DRG neurons (Fig. 2 a). In addition, integrin innrmnoreactivity signals in neurons with high or low levels of GAP-43 were comparable (Fig. 1). Similarly, levels of the cortical cytoskeleton-associated protein CAP-23 were apparently unchanged (Fig. 3, a and b).
Since the latter is a PKC substrate of neurons and developing tissues with subcellular distribution and structural properties similar to those of GAP-43 (Widmer and Caroni, 1990), this observation suggests that in these acute experiments compensatory reactions leading to changes in the lev- L-ornithine/laminin-coated coverslips, fixed and stained with the mAb 5F10 against GAP-43 and with the antiserum against/~l-integrin. The neurons shown in a, b, and d were representative of those expressing high levels of GAP-43. Note thin, longer neurites in GAP-43-depleted neuron (c). Also note that already during the initial phase of neurite outgrowth antisensetreated neurons were depleted of GAP-43 IR and displayed the characteristic pattern of neurite outgrowth as detected in older cultures (e). Bar, 50 t~m. els of at least one related protein associated with the cortical cytoskeleton did not occur. To rule out the possibility that the effects of the antisense oligonucleotides on GAP-43 IR and neurite outgrowth were simply due to the formation of an R N A -D N A hybrid in the neurons, we triturated the ganglia in the presence of an antisense oligonucleotide to MARCKS (Aderem, 1992). This protein is a PKC substrate with biochemical properties similar to those of GAP-43. As shown in Fig. 3, c and d, the antisense, but not the corresponding sense oligonucleotide significantly reduced MARCKS IR in the neurons. On the other hand, no detectable changes in GAP-43 IR or neurite morphology (see also  Table II) were detected in the presence of the antisense oligonucleotide to MARCKS, indicating that the effects of the GAP-43 oligonucleotides were specific for this protein. Therefore, these combined observations indicate that s0e- to the coding sequence of chick GAP-43. This probe visualizes the two chick GAP-43 mRNA species, as described (Baizer et al., 1990). (d) Immunoblot analysis of GAP-43 in DRG neurons treated as described in a. Equal amounts of total protein combined from six separate triturations was analyzed. After GAP-43 detection, the same blot was reprobed for neurofilament-160. The band that migrates slightly more slowly than GAP-43 in the lower panel corresponds to a major species in the homogenate, and the signal is not specific for the GAP-43 antibody. Lane labeled brain: 15 #g of homogenate protein from chicken E7 brain. Densitometric quantitation of the immunoblot data is shown in the graph. Values were normalized to the intensity of the unspecific band mentioned above.
cific alterations in GAP-43 contents were not accompanied by comparable changes in other major components of neurites and growth cones, and that the effects of the GAP-43 antisense oligonucleotides on GAP-43 IR and neurite morphology were specific for this protein.

DRG Neurons Depleted of GAP-43, Extend Thin Neurites, with Infrequent Branching and Very Small Growth Cones
The most dramatic difference between DRG neurons regenerating neurites on a laminin-coated substratum in the presence or absence of GAP-43 was observed when growth cones were compared. Fig. 4 shows that in the absence of GAP-43 the growth cones of DRG neurons were extremely small. In most cases, a few filopodia pointing in the direction of neurite extension could be observed, but the growth cone region was not significantly wider than the average neurite diameter, and the characteristic well-spread appearance of growth cones growing on laminin was essentially never observed. The panels in Fig. 4 are from 18 h cultures and were representative of all the growth cones from neurons expressing high, respectively undetectable levels of GAP-43. A quantitative analysis of the relationship between growth cone surface area and GAP-43 levels is presented in Table I. Interestingly, variations in growth cone size were much more pronounced among the neurons expressing intermediate levels of GAP-43 (Table I), suggesting that high or undetectable levels of GAP-43 have a dominant effect on growth cone formarion and behavior.
Similar observations were made when growth cone sizes were analyzed during the initial phase of neurite elongation  also Table II). Cells were treated with 200 #M sense (c) or antisense (d) MARCKS oligonucleotide, fixed and stained with an antiserum against chick MARCKS and with mAb 5F10 against GAP-43. Bar, 50 /zm. on laminin (data not shown). However, during the first 1-2 h in culture, when neurite lengths were comparable with cell diameters, GAP-43-depleted neurons did display slightly larger growth cones (see Fig. 1 e). This residual growth cone activity may be due to a greater tendency to form large growth cones during the very initial phases of neurite regeneration.
The GAP-43-depleted neurons of Figs. 1 and 3 have thinner, longer and more numerous neurites than their GAP-43-expressing counterparts. A quantitative analysis of neuritic morphological parameters as a function of the levels of GAP-43 immunoreactivity is shown in Fig. 5. This analysis shows that in the absence of GAP-43 DRG neurites extending on laminin are particularly long, thin, and with little branch  Table I). As in bI, growth cones in b2-b5 displayed no detectable GAP-43 IR. Bar, 25 #m. Experimental conditions were as described in Fig. 1. All cells from randomly selected fields with process(es) longer than two cell diameters were scored, and the size of the cell's largest growth cone was estimated, x > 250 #m2: growth cones larger than that shown in Fig. 4, a4; x < 30 #m2: growth cones smaller than that shown in Fig.4, b2. IR levels were defined as in Fig. 2. n is the total number of cells that were analyzed per type of experiment. Each of these total numbers of cells was set as 100% in the sense and antisense experiments. The data indicate the presence of a strong correlation between growth cone size and GAP-43 IR levels.
points. For each separate experiment, antisense-and corresponding sense-oligonucleotide-treated neurons were generated and analyzed, and the neuritic morphology parameters displayed the same dependency on the levels of GAP-43 immunoreactivity, irrespective of whether the analysis encompassed a single set of experiments or all experiments were combined. Significantly, as in the growth cone size analysis, parameter values did not depend on the presence of oligonucleotides during the dissociation procedure, but only on the final levels of GAP-43 IR in the DRG neurons. This observation suggests that dissociation of the DRGs in the presence of antisense oligonucleotides to GAP-43 affected the relative contents of neurons with high, intermediate, and extremely low levels of GAP-43 immunoreactivity, and it was the levels of GAP-43 that produced the differences in growth cone and neuritic morphology. This relationship between a specific shift by the GAP-43 antisense oligonucleotides towards neurons with lower or undetectable GAP-43 IR and the distribution of neuritic morphologies in the culture was confirmed by the morphometric analysis of neurons in the control-and antisense-treated cultures ir-respective of GAP-43 IR levels (Table II, see also Table I).
The data of Table II also demonstrate that neuritic morphology was specifically affected by antisense oligonucleotides to GAP-43, but not by antisense oligonucleotides to the related PKC substrate MARCKS. These data support our interpretation that variations in growth cone size, neurite length, and branching correlated with GAP-43 IR levels in the neurons, and that the effect of the antisense oligonucleotides was to shift the GAP-43 content distribution towards lower levels of this protein. This interpretation is further supported by the data of Fig. 6: both sense-and antisense-treated cultures contained neurons with high, intermediate and low-toundetectable GAP-43 IR levels, and GAP-43 contents correlated with neuritic morphology in both types of cultures.

Impaired Formation of Neurites by GAP-43-depleted DRG Neurons Grown on a Polyornithine Substratum
Laminin is a potent promoter of neurite outgrowth for DRG neurons (Rogers et al., 1983), and downstream reactions to  Experiments were carried out as described in Fig. 1. Ceils from randomly selected fields with processes longer than two cell diameters were scored, and subdivided into GAP-43 IR levels as described in Fig. 2 a. Data from two independent experiments were pooled for each type of culture. (a) Neurite length (length of the longest neurite). * P < 0.02; ** P < 0.0005. (b) Branchpoints per 100/~m of neurite length as a function of GAP-43 IR. ns, not significant; * P < 0.001. (c) Neurite diameter. * P < 0.0005; ** P < 0.00005. Significance of differences as compared with strong the interaction of the growth cone with laminin involve the activation of PKC (Bixby, 1989). GAP-43 is a major PKC substrate of the growth cone, and it can interact with and modulate the function of components involved in signal transduction, such as calmodulin, Go and PIP-kinase (Andreason et al., 1983;Strittmatter et al., 1990;van Dongen et al., 1985). To determine whether the characteristic morphological changes in GAP-43-depleted neurons were restricted to the laminin substratum, we plated these neurons in the same culture medium, but on polyornithine without laminin. We further reasoned that if laminin was providing the neurons with a strong stimulus to grow, the absence of such a stimulus may better reveal possible competence impairments in GAP-43-depleted neurons. As shown in Fig. 7, GAP-43-depleted DRG neurons failed to form neurites when plated on polyornithine in the presence of NGF. Clearly, in the absence of laminin neurite outgrowth was also reduced in neurons expressing high levels of GAP-43 (Fig. 7, a, b, and e). However, and in contrast to the outcome on a laminin substratum, in the absence of GAP-43 no growth cone or neurite emerged from the initial lamellar configuration (Fig. 7, c, d,f, and g). Similar observations were made when the neurons were examined 2, 4, or 8 h after plating. Total numbers of fixed neurons at the end of the various experiments were undistinguishable in the sense-and antisense oligonucleotide-treated cultures. Thus, failure to form neurites in the absence of GAP-43 was not due to overall reduced adhesiveness in these neurons. In addition, amounts of GAP-43-depleted neurons did not differ from those expressing GAP-43 when cell attachment was compared on laminin, polyornithine, or glass (data not shown). It therefore appears that GAP-43-depleted neurons did respond to laminin with neurite outgrowth, but that in the absence of laminin an intrinsic impairment in their ability to form growth cones and neurites was revealed. As a consequence, GAP-43-depleted neurons formed lamellae with rudimentary asymmetries and filopodia, but no growth cones nor neurites emerged.

Discussion
We have depleted El6 chick DRG neurons of GAP-43 by an antisense oligonucleotide approach. This allowed us to analyze neurite regeneration by cultured primary neurons in the absence of detectable levels of GAP-43 immunoreactivity. The analysis reveals, to our knowledge for the first time, that the formation, morphology, and growth of growth cones and neurites are affected by the levels of GAP-43 in the neurons. These findings suggest that levels of GAP-43 in growth cones, axons and nerve terminals may affect the local responsiveness of these structures to morphogenetic stimuli, thus modulating their competence to grow.
GAP-43 IR group are given for the three morphological parameters. For neurite length, the significance was determined by the Mann-Whitney U test because the distribution of the values was skewed. The remaining significance values were determined by t test. Values are means +SD. Numbers in the columns represent the number of cells analyzed. Note that antisense oligonucleotides only affected the relative contents in the culture of neurons with high, intermediate, and undetectable levels of GAP-43 IR. Experiments were carried out as described in Fig. 1. Data are from 18-h cultures. These data support the conclusion that shifts in neuritic morphologies in cultures treated with antisense GAP-43 oligonueleotides were due to GAP-43 depletion in these cultures. Note both, neuron expressing high levels of GAP-43 (arrow) and neuron expressing very low levels of GAP-43 in same field of sensetreated culture (c). Also note that in neurons with intermediate signal levels GAP-43 IR tended to be lower in neurites than in cell bodies. Bar, 50 t~m.

Production of GAP-43-depleted Primary Neurons
The DRG trituration method described in this study was adapted from a procedure developed to introduce the oncogenic form of the ras protein in cultured chick DRG neurons (Borasio et al., 1989). In that study, an efficiency of transfer of close to 100% was estimated, and sufficient amounts of a 21-kD protein could be introduced into the neuronal cytosol to rescue significant numbers of the neurons in the absence of NGF. Our results suggest that the treatment with antisense oligonucleotide against GAP-43 may have affected GAP-43 protein levels in most DRG neurons. To estimate the efficiency of the method we triturated the DRGs in the presence of biotin-coupled oligonucleotide. After 4 h in culture >70 % of the ceils contained fluorescently labeled oligonucleotide in their cell body and nucleus (data not shown). In addition, the data from the quantitative analysis of Fig. 2 are consistent with the interpretation that the antisense oligonucleotide treatment specifically produced a general shift of GAP-43 levels in most neurons in the culture, possibly leading from high to intermediate and from intermediate to undetectable signal values.
It is not usual to culture DRG neurons from El6 chicks. Our selection of this late stage, as opposed to neurons from younger embryos, that regenerate neurites in culture with greater efficiency was dictated by the difficulties that we encountered in depleting younger chick DRG neurons from GAP-43. Application of the same experimental procedure to E8-E10 DRGs yielded a similar shift in the distribution of GAP-43 signal levels in cultured neurons, but almost no neu-rons could be depleted of GAP-43 immunoreactivity. This observation is probably due at least in part to the higher initial contents of GAP-43 in these neurons. In addition, preliminary pulse-chase experiments with 35S-labeled amino acid precursors suggested that the metabolic half-life of GAP-43 in E8-E10 chick DRG neurons (>6 h) is longer than in older neurons. It is therefore possible that an additional difficulty in rapidly depleting young DRG neurons of GAP-43 by an antisense approach is the comparably high metabolic stability of the protein in these neurons. As a consequence, we have restricted our presentation to the data with El6 neurons, but a brief discussion of the comparable phenotypic effects caused by reduced GAP-43 levels on E8 DRG neurons is presented below.
Depletion of GAP-43 IR was specifically due to interaction of the antisense oligonucleotides with GAP-43 mRNA (Fig.  2 c), and an oligonucleotide complementary to MARCKS mRNA affected MARCKS IR, but not GAP-43 IR (Fig. 3 d).
In addition, the antisense oligonucleotide procedure specifically affected GAP-43 levels, without obvious modifications of the signal levels for Bl-integrin, CAP-23 or neurofilament-160. Integrin-B1 is a subunit of cell surface receptors for extracellular matrix proteins that affect neurite outgrowth (Horwitz et al., 1985;Tomaselli et al., 1986;Letourneau and Shattuck, 1989); CAP-23 is, like GAP-43, a PKC substrate of the cortical cytoskeleton (Widmer and Caroni, 1990); and neurofilament-160 is a major component of the neuritic cytoskeleton. Therefore, although the levels of other neuronal proteins were not assessed, it is clear that the antisense procedure not only specifically affected the lev- Figure 7. GAP-43-depleted neurons fail to form growth cones and neurites when plated on poly-L-ornithine. Experiments were carded out as described in Fig. 1, except that the poly-L-omithine substratum had not been additionally coated with laminin. (a-d) 15-h culture; (e-g) 23-h culture. (a, b, and e) Sense-3 oligonucleotide; (c, d, f, and g) antisense-3 oligonucleotide. Bar, 50 #m. els of GAP-43, but probably did not cause generalized alterations in proteins involved in neuritic outgrowth.

Role of GAP-43 in Neurite Outgrowth
The properties of El6 chick DRG neurites regenerating on a laminin substratum correlated with the levels of GAP-43 in the neurons. In the presence of high levels of GAP-43 neurites were of larger diameter, had larger growth cones, and branched more as compared to those having low amounts of GAP-43. In the absence of detectable levels of GAP-43 IR neurites were longer, very thin, with nearly undetectable growth cone structures and little branching points. The quantitative analysis of Fig. 2, Table I, and Table II indicates that the distribution of neuritic morphologies in the culture correlated with that of neurons with strong, intermediate and undetectable GAP-43 IR levels, and that both were affected by the antisense treatment in a similar manner. These data support the interpretation that the relationship described here between the pattern of neurite outgrowth and GAP-43 levels is a direct outcome of GAP-43 contents in the neurons. These experiments therefore indicate that GAP-43 levels affect growth cone and neurite formation and morphology in primary neurons.
Most GAP-43-sensitive properties of the neurites may be rationalized assuming that the main site of action of GAP-43 is the growth cone. Thus, reduced branching and longer neurites in the absence of GAP-43 are likely to be due to reduced growth cone exploratory activity. This may lead to reduced hindrance of the pushing activity by the microtubule-based elongation apparatus (Letourneau et al., 1987). Time-lapse video microscopic analysis of DRG neurites regenerating in the presence and absence of GAP-43 should indicate whether this interpretation is correct. In the absence of detectable GAP-43 IR, neurites of strikingly thin diameter were already observed during the initial phases of process outgrowth on laminin (Fig. 1 e). This may either be a consequence of reduced contents of neurite-forming components in small growth cones, of a diminished laminin-mediated stimulus for neurite formation, or of reduced adhesiveness in GAP-43depleted neurites. Here again, time-lapse video microscopic analysis may provide indications on whether the reduced neurite diameter of GAP-43-depleted neurons is related to growth cone activity.
We detected a similar relationship between GAP-43 contents and neuritic morphology in E8 DRG neurons growing on laminin in the presence of NGE Neurons with low contents of GAP-43 displayed an initial tendency to elaborate large extended lamellae that failed to form the characteristic point of transition between growth cone and neurite. Neurites growing in the presence of low levels of GAP-43 were thin, little branched, longer than average, with frequent lateral lamellae and unpolarized growth cones (data not shown). Therefore, like in El6 neurons, low levels of GAP-43 appeared to interfere with growth cone formation and activity in the E8 DRG neurons.
When grown on polyornithine, GAP-43-depleted neurons failed to form growth cones and elongate neurites. This may possibly have been due to the absence of a strong neurite outgrowth stimulus from the substratum. These observations are consistent with recent reports on the outgrowth of neuroblastoma (Shea et al., 1991) and PC12 neurites (Yankner et al., 1990;Jap Tjoen San et al., 1992) in the presence of low or elevated GAP-43 activity. In neuroblastoma cells, introduction of an antibody to GAP-43 into the cytosol by lipofection interfered with the formation of neurites (Shea et al., 1991). Since these cells may have a comparatively low potential of extending neurites, the effect of the antibody may be related to our observations with GAP-43-depleted neurons on polyornithine. Transfected PC12 lines expressing elevated levels of GAP-43 in a constitutive manner responded markedly more vigorously to stimulation of neurite formation by NGF (Yankner et al., 1990). Therefore, like in our experiments, high initial levels of GAP-43 may enhance a neuron's responsiveness to a neurite outgrowth stimulus. Similar conclusions on a possible role of GAP-43 in mediating responsiveness of PC12 cells to the neurite-promoting activity of NGF were reached in a recent depletion study with antisense oligonucleotides (Jap Tjoen San et al., 1992).

Possible Mechanisms
One possible interpretation of our data is that levels of GAP-43 affect growth cone and neurite adhesiveness. Thus, GAP-43-depleted neurons regenerating neurites on laminin may display smaller growth cones, reduced branching, and thinner neurites due to reduced attachment to the substratum (Bray, 1982). Arguing against a simple reduction of the effects of GAP-43 to changed adhesiveness, however, are the observations that, first, attachment of DRG neuronal cell bodies on laminin, polyornithine or glass was not affected by GAP-43 levels, and second, that on a very adhesive substrate like polyornithine growth cone and neurite, but not lamellae formation was impaired in the absence of GAP-43. These results are therefore more consistent with the hypothesis that GAP-43 may affect the responsiveness of the growth cone, and possibly also of the neurite machinery to growth signals. Accordingly, GAP-43 may be an amplifier of local stimuli to the growth cone and neurite (see also Strittmatter et al,, 1992). This interpretation is consistent with our observations on the effects of GAP-43 and its phosphorylation-site mutants on spreading and local surface morphology of nonneuronal cell lines (Widmer and Caroni, 1993). In these transfection experiments, GAP-43 appeared to promote the formation of local actin-containing surface structures like filopodia and pseudopods. This effect of GAP-43 was espe-cially prominent during cell spreading and was superimposed onto the intrinsic tendency of different cell types to form such structures. In addition, phosphorylation-site mutants of GAP-43 had opposite effects on cell spreading and morphology: a Ser42 to Ala42 mutant interfered with cell spreading, whereas a Ser42 to Asp42 mutant promoted cell spreading. It is therefore tempting to speculate that GAP-43 may amplify local signals involved in local surface morphogenetic activity, and this function of GAP-43 may be modulated by PKC-mediated phosphorylation. The appearance of neurites regenerating from DRG neurons on laminin in the absence of GAP-43 may be due to altered responsiveness of the growth cone to some of the neurite-promoting activities induced by laminin. Similarly, reduced competence to elaborate neurite-associated structures in GAP-43depleted neurons may prevent the formation of growth cones and neurites on a polyornithine substratum.
What growth cone components are affected by GAP-43? In neurons and in transfected ceils, GAP-43 levels affected the organization of actin-based cell surface structures, at the tip of the growth cone and pseudopods. In addition, during spreading of transfected cells, co-accumulation of GAP-43 and F-actin at surface structures was detected (Widmer and Caroni, 1993). Conceivably, therefore, GAP-43 may affect the local activity of actin-based structures at the cell surface, possibly by interacting with molecular components that control the dynamic properties of these structures. It will therefore be important to determine whether GAP-43 and proteins of potentially related function like CAP-23 (Widmer and Caroni, 1990) and MARCKS (Aderem, 1992) interact with proteins involved in microfilament dynamics.

Function of GAP-43
Our observations can be related to the pattern of expression of GAP-43 during development and regeneration to generate hypotheses on the possible biological role of this protein.
The proposed role for GAP-43 in amplifying local environmental signals into growth cone activity implies that this protein may enhance competence in axonal outgrowth and guidance, and its downregulation at the time of the elimination of collaterals may coincide with a reduced susceptibility of axons to local growth stimuli (see also Skene, 1989). Similarly, reinduction of GAP-43 may potentiate regeneration, and its presence in selected adult CNS regions may indicate an elevated susceptibility for local growth. A similar hypothesis was proposed recently by Strittmatter et al. (1992). Further, it should be mentioned in this context that the expression of GAP-43, and presumably its range of action, is not restricted to neurons, as differentiating astrocytes, oligodendrocytes, and Schwann ceils express GAP-43 (Vitkovic et al., 1988;da Cunha and Vitkovic, 1990;Deloulme et al., 1990;Curtis et al., 1991;Curtis et al., 1992;Woolf et al., 1992). Like in neurons, GAP-43 expression in glial cells coincides with phases of elevated morphogenetic and motile activity. This relation between GAP-43 expression and morphogenetic activity is particularly striking in terminal Schwann cells at denervated neuromuscular junctions, where reinduction of GAP-43 coincides with a dramatic expansion of this cell's processes in the perisynaptic space (Woolf et al., 1992). In conclusion, GAP-43 may be an important amplifier of local morphogenetic processes at the cell cortex in both neurons and in glial cells.