Activation of ADF/cofilin mediates attractive growth cone turning toward nerve growth factor and netrin‐1

Proper neural circuitry requires that growth cones, motile tips of extending axons, respond to molecular guidance cues expressed in the developing organism. However, it is unclear how guidance cues modify the cytoskeleton to guide growth cone pathfinding. Here, we show acute treatment with two attractive guidance cues, nerve growth factor (NGF) and netrin‐1, for embryonic dorsal root ganglion and temporal retinal neurons, respectively, results in increased growth cone membrane protrusion, actin polymerization, and filamentous actin (F‐actin). ADF/cofilin (AC) family proteins facilitate F‐actin dynamics, and we found the inactive phosphorylated form of AC is decreased in NGF‐ or netrin‐1‐treated growth cones. Directly increasing AC activity mimics addition of NGF or netrin‐1 to increase growth cone protrusion and F‐actin levels. Extracellular gradients of NGF, netrin‐1, and a cell‐permeable AC elicit attractive growth cone turning and increased F‐actin barbed ends, F‐actin accumulation, and active AC in growth cone regions proximal to the gradient source. Reducing AC activity blunts turning responses to NGF and netrin. Our results suggest that gradients of NGF and netrin‐1 locally activate AC to promote actin polymerization and subsequent growth cone turning toward the side containing higher AC activity. © 2010 Wiley Periodicals, Inc. Develop Neurobiol 70: 565–588, 2010     

Rapid regulation of neuronal growth cone shape and surface morphology by nerve growth factor

Scanning electron microscopy was used to study regulation of growth cone shape and surface morphology by nerve growth factor (NGF). The growth cones of cultured rat sympathetic neurons and neuronally-differentiated PC12 cells were observed under conditions of continuous NGF exposure, NGF withdrawal, and NGF readdition. Growth cones of cells cultured in the continuous presence of NGF were mostly spread in shape and about 60% possessed surface ruffles. Ruffles appeared to be largely restricted to growth cones in that few were observed on cell bodies and neurites. Withdrawal of NGF for 4–5 hr caused most of the growth cones to take on a non-spread or contracted appearance and to lose their ruffles. Readdition of NGF promoted rapid changes in growth cone properties. Within 30 sec, ruffling was again evident on the growth cones and remained prominent there throughout the course of treatment (up to 5 hr). This was in contrast to cell bodies on which, as previously reported, ruffling also occurred following NGF readdition, but only transiently (for less than 15 min). Respreading of growth cones also occurred under these conditions. This was evident within 1 min of NGF readdition and reached the levels observed in continuously-treated cultures within 1–2 hr. Neurites were also examined. Ruffles were only rarely present in the continuous presence of NGF and were absent after NGF withdrawal. NGF readdition elicited ruffling along neurites within 30 sec; the prevalence of such ruffles diminished to that seen in continuously-treated cultures within about an hour. As evidence of the specificity of these NGF effects, epidermal growth factor and dibutyryl cAMP, agents that elicit responses in PC12 cells, but do not promote their neuronal differentiation, had no observable effect on NGF-deprived growth cones. These findings demonstrate that NGF exerts very rapid effects on growth cone shape and surface morphology. Such actions may play roles in regulation of growth cone movement and guidance by NGF.Special Issue dedicated to Dr. E. M. Shooter and Dr. S. Varon.     

Regulation of neurite outgrowth mediated by localized phosphorylation of protein translational factor eEF2 in growth cones

Nerve growth cones contain mRNA and its translational machinery and thereby synthesize protein locally. The regulatory mechanisms in the growth cone, however, remain largely unknown. We previously found that the calcium entry‐induced increase of phosphorylation of eukaryotic elongation factor‐2 (eEF2), a key component of mRNA translation, within growth cones showed growth arrest of neurites. Because dephosphorylated eEF2 and phosphorylated eEF2 are known to promote and inhibit mRNA translation, respectively, the data led to the hypothesis that eEF2‐mediating mRNA translation may regulate neurite outgrowth. Here, we validated the hypothesis by using a chromophore‐assisted light inactivation (CALI) technique to examine the roles of localized eEF2 and eEF2 kinase (EF2K), a specific calcium calmodulin‐dependent enzyme for eEF2 phosphorylation, in advancing growth cones of cultured chick dorsal root ganglion (DRG) neurons. The phosphorylated eEF2 was weakly distributed in advancing growth cones, whereas eEF2 phosphorylation was increased by extracellular adenosine triphosphate (ATP)‐evoked calcium transient through P2 purinoceptors in growth cones and resulted in growth arrest of neurites. The increase of eEF2 phosphorylation within growth cones by inhibition of protein phosphatase 2A known to dephosphorylate eEF2 also showed growth arrest of neurites. CALI of eEF2 within growth cones resulted in retardation of neurite outgrowth, whereas CALI of EF2K enhanced neurite outgrowth temporally. Moreover, CALI of EF2K abolished the ATP‐induced retardation of neurite outgrowth. These findings suggest that an eEF2 phosphorylation state localized to the growth cone regulates neurite outgrowth. © 2012 Wiley Periodicals, Inc. Develop Neurobiol, 2013     

Growth cone behavior and production of traction force

The growth cone must push its substrate rearward via some traction force in order to propel itself forward. To determine which growth cone behaviors produce traction force, we observed chick sensory growth cones under conditions in which force production was accommodated by movement of obstacles in the environment, namely, neurites of other sensory neurons or glass fibers. The movements of these obstacles occurred via three, different, stereotyped growth cone behaviors: (a) filopodial contractions, (b) smooth rearward movement on the dorsal surface of the growth cone, and (c) interactions with ruffling lamellipodia. More than 70% of the obstacle movements were caused by filopodial contractions in which the obstacle attached at the extreme distal end of a filopodium and moved only as the filopodium changed its extension. Filopodial contractions were characterized by frequent changes of obstacle velocity and direction. Contraction of a single filopodium is estimated to exert 50-90 microdyn of force, which can account for the pull exerted by chick sensory growth cones. Importantly, all five cases of growth cones growing over the top of obstacle neurites (i.e., geometry that mimics the usual growth cone/substrate interaction), were of the filopodial contraction type. Some 25% of obstacle movements occurred by a smooth backward movement along the top surface of growth cones. Both the appearance and rate of movements were similar to that reported for retrograde flow of cortical actin near the dorsal growth cone surface. Although these retrograde flow movements also exerted enough force to account for growth cone pulling, we did not observe such movements on ventral growth cone surfaces. Occasionally obstacles were moved by interaction with ruffling lamellipodia. However, we obtained no evidence for attachment of the obstacles to ruffling lamellipodia or for directed obstacle movements by this mechanism. These data suggest that chick sensory growth cones move forward by contractile activity of filopodia, i.e., isometric contraction on a rigid substrate. Our data argue against retrograde flow of actin producing traction force.     

Modeling the role of myosin 1c in neuronal growth cone turning

We addressed the mechanical basis for how embryonic chick dorsal root ganglion growth cones turn on a uniform substrate of laminin-1. Turning is significantly correlated with lamellipodial area but not with filopodial length. We assessed the lamellipodial contribution to turning by asymmetric micro-CALI of myosin isoforms that causes localized lamellipodial expansion (myosin 1c) or filopodial retraction (myosin V). Episodes of asymmetric micro-CALI of myosin 1c (or myosin 1c and V together) caused significant turning of the growth cone. In contrast, repeated micro-CALI of myosin V or irradiation without added antibody did not turn growth cones. These findings argue that lamellipodia and not filopodia are necessary for growth cone turning. To model the role of myosin 1c on growth cone turning, we fitted the measured trajectories from asymmetric micro-CALI of myosin 1c-treated and untreated growth cones to the persistent random walk model. The first parameter in this equation, root-mean-square speed, is indistinguishable between the two data sets whereas the second parameter, the persistence of motion, is significantly increased (2.5-fold) as a result of asymmetric inactivation of myosin 1c by micro-CALI. This analysis demonstrates that growth cone turning results from an increase in the persistence of directional motion rather than a change in speed. Taken together, our results suggest that myosin 1c is a molecular correlate for directional persistence underlying growth cone motility.     

Cyclic nucleotides and calcium in human lymphocytes induced to divide

Intracellular concentrations of cyclic adenosine 3'-5' monophosphate (cAMP) and cyclic guanosine 3'-5' monophosphate (cGMP) were measured in human lymphocytes induced to divide by the addition of lectins, 12-O-tetra-decanoylphorbol-13-acetate (TPA) and the calcium ionophore A 23187. cGMP levels rose within minutes without concomitant alterations in cAMP concentration. The cAMP and cGMP levels rose during the prereplicative and replicative phases respectively. Under calcium depleting conditions, both the fluctuations in cyclic nucleotide levels and the increase in [3H[ thymidine incorporation into DNA were abolished, suggesting a role for calcium ions in the regulation of lymphocyte proliferation.     

Neurite development in vitro. I. The effects of adenosine 3′5′-cyclic monophosphate (cyclic AMP)

Elevated levels of 3′5′ adenosine monophosphate (cyclic AMP) stimulate a wide variety of cellular events including aggregation, differentiation, morphological expression, pigment migration, and secretion. The role of cyclic AMP in these events prompted our present study of embryonic chick dorsal root ganglia. Test substances were applied to cultures during the routine feeding procedure. Their development was quantitatively evaluated on the basis of explant size, length of glial-like outgrowth, distribution of growth, neurite number, length, diameter, and degree of arborization. These parameters were all shown to be independent of each other. The high variability of in vitro neurite development necessitated the use of over 100 cultures per treatment group. Cultures treated with 5′ AMP exhibited no significant differences from controls. Those treated with cyclic AMP, dibutyryl cyclic AMP, or Nerve Growth Factor (NGF) exhibited statistically significant increases in area of outgrowth, the number of neurites per culture, and in diameters, lengths, and degree of neurite arborization. The growth promoting activity of dibutyryl cyclic AMP and NGF were greater than those of cyclic AMP. Electron microscopic study shows neurites formed under the influence of cyclic AMP or its dibutyryl derivative to resemble those grown in NGF. These studies suggest the possibility that cyclic AMP stimulates neurite growth by mediating the process of microtubule (MT) assembly. They further prompt us to speculate that one way NGF enhances neurite development is by stimulating MT assembly via a “Second Messenger System”.     

Capsaicin-Induced Ion Fluxes Increase Cyclic GMP but Not Cyclic AMP Levels in Rat Sensory Neurones in Culture

Capsaicin, which induces fluxes of sodium, calcium, and potassium ions in a subset of both neonatal and adult rat dorsal root ganglion neurones, increased cyclic GMP (cGMP) levels by a factor of 20 (EC50 0.07 microM) to 10-20 pmol cGMP/mg protein in these cells. Cyclic AMP (cAMP) levels were unaffected. Nonneuronal cells derived from rat ganglia, and both neurones and nonneuronal cells from chick were unresponsive to capsaicin. Capsaicin-induced cGMP elevation in rat dorsal root ganglion (DRG) neurones was unaffected by pertussis toxin, lowered by compounds that block voltage-sensitive calcium channels, and was abolished by the removal of extracellular calcium. Calcium, guanidine, and rubidium fluxes were unaffected by treatment of DRG cells with sodium nitroprusside or dibutyryl cGMP. The cGMP response to capsaicin is thus a function of capsaicin-evoked calcium uptake through voltage-sensitive calcium channels. Elevated cGMP levels do not, however, contribute to capsaicin-evoked ion fluxes or to their desensitisation.     

Rapid retraction of neurites by sensory neurons in response to increased concentrations of nerve growth factor

The phenomenon of growth cone (GC) and neurite retraction resulting from a rapid incrase in concentration of the trophic molecule NGF was studied. Neurite outgrowth from explants of 8-d chick embryo dorsal root ganglia was achieved at very low NGF concentrations with heart conditioned medium during overnight culture. Quickly incrasing the NGF concentration in the growth medium dramatically affected GC and neurite morphology: the majority of GCs and neurites collapsed and retracted towards the cell body over a course of approximately 2-5 min. Retraction was elicited by increasing NGF levels from 0 to 0.05 ng/ml to as little as 0.5 ng/ml but did not occur if the NGF concentration during the initial overnight culture period exceeded 0.8 ng/ml, regardless of how much the concentration was elevated. Similar concentration changes of cytochrome c or insulin did nt result in retraction. Neurites that had been separated from their cell bodies by cutting close to their exit from the explant still retracted when NGF levels were raised. Cytochalasin B reversible inhibits retraction, whereas colchicine allows retraction to occur. Observation of cell- substratum adhesion during retraction revealed that some adhesion points remain during retraction and that they correspond to the ends of NGF leels and that it may involve microfilaments in the neurite cytoskeleton. The NGF concentration changes that elicit neurite retraction suggest that a primary event in retraction may be increased occupancy of a high-affinity NGF receptor on neurites.     

Contact guidance of chick embryo neurons on single scratches in glass and on underlying aligned human skin fibroblasts

The influence of substratum topography on the morphology and orientation of neurites of chick embryo neurons was studied.Two series of experiments are reported. One concerned the behaviour of growth cones when the axons become contact-guided by the surface texture. The second studied contact guidance of neurites extending on a compact layer of fixed aligned human skin fibroblasts (HSF).It was observed that when the growth cones of sensory neurons isolated from dorsal root ganglions encountered a single scratch in a glass surface (0.1-2 microm in depth and diameter) they turned and continued movement following the axis of the scratch. These neurons became contact-guided as a result of the sequence of events. The growth cone filopodia recognized the irregularity in the substratum surface, whereas the growth cone lamella stabilized contact with the scratch and moved forward along the scratch axis. Scanning electron microscope revealed that the single scratches 150 nm in width and ca. 100 nm deep growth cone filopodia less than 200 nm in diameter could detect and react by turning into them. These filopodia extensions followed the edge of scratches. However, phase contrast and Nomarski's differential interference contrast appeared insufficient for analysis of primary contact guidance of fine growth cone filopodia which themselves are often less than 200 nm. In neuron cultures on fixed aligned HSF, the neuron aggregates assumed spindle-like shapes, and sparsely seeded individual neurons extended axons along the long axes of the fibroblasts. The axons extended significantly further on the fixed underlying fibroblasts than on collagen-covered glass. In crowded cultures of neurons, the cells extended neurites ignoring both the surface anisotropy (the scratches) and the orientation of the aligned fibroblasts. Immunofluorescence staining of neurons with antibodies against neurofilaments made it possible to analyse their shape and orientation on the fibroblasts. Computer-assisted image analysis permitted the observed alignment of the neurites to be characterized quantitatively.     

Evidence that calcium may control neurite outgrowth by regulating the stability of actin filaments

We investigated the effects of calcium removal and calcium ionophores on the behavior and ultrastructure of cultured chick dorsal root ganglia (DRG) neurons to identify possible mechanisms by which calcium might regulate neurite outgrowth. Both calcium removal and the addition of calcium ionophores A23187 or ionomycin blocked outgrowth in previously elongating neurites, although in the case of calcium ionophores, changes in growth cone shape and retraction of neurites were also observed. Treatment with calcium ionophores significantly increased growth cone calcium. The ability of the microtubule stabilizing agent taxol to block A23187-induced neurite retraction and the ability of the actin stabilizing agent phalloidin to reverse both A23187-induced growth cone collapse and neurite retraction suggested that calcium acted on the cytoskeleton. Whole mount electron micrographs revealed an apparent disruption of actin filaments in the periphery (but not filopodia) of growth cones that were exposed to calcium ionophores in medium with normal calcium concentrations. This effect was not seen in cells treated with calcium ionophores in calcium-free medium or cells treated with the monovalent cation ionophore monensin, indicating that these effects were calcium specific. Ultrastructure of Triton X-100 extracted whole mounts further indicated that both microtubules and microfilaments may be more stable or extraction resistant after treatments which lower intracellular calcium. Taken together, the data suggest that calcium may control neurite elongation at least in part by regulating actin filament stability, and support a model for neurite outgrowth involving a balance between assembly and disassembly of the cytoskeleton.     

Semaphorin 3E/collapsin-5 inhibits growing retinal axons

During development, the formation of neural networks is reflected by the oriented extension of neurites. Using retinal ganglion cells (RGCs) as a model, we identified the yet uncharacterized chick semaphorin Sema3E/collapsin-5 as a repulsive cue for outgrowing axons. Sema3E/collapsin-5 was highly regulated during retinal histogenesis, with peak expression during the period of intraretinal axon growth. Polymerase chain reaction analysis demonstrated Sema3E/collapsin-5 mRNA in retina layers, from which RGC axons are excluded. Neither isolated RGCs nor purified retinal Müller glia cells synthesized Sema3E/collapsin-5. Sema3E/collapsin-5 receptor sites were visualized by alkaline phosphatase fusion proteins in the axon-rich optic fiber layer. Time-lapse video recording of chick in vitro cultures revealed a growth cone collapsing activity of recombinant Sema3E/collapsin-5. This effect was specific for RGCs, since dorsal root ganglia (DRG) neurons of the peripheral nervous system were not affected. Comparison with Sema3A/collapsin-1 displayed a reciprocal specificity, because Sema3A/collapsin-1 hampered exclusively DRG but not RGC growth cones. The collapsing effect was mediated by low cGMP levels, but not cAMP, as revealed by a set of agonists. In summary, the data suggest a possible role of chick Sema3E/collapsin-5 in restricting growth of retinal ganglion cell axons to the optic fiber layer.     

Nerve Outgrowth by Dorsal Root Ganglia in vitro: Stimulation by Inhibitors of DNA Metabolism in the Absence of Exogenous Nerve Growth Factor

Dorsal root ganglia from 8-day chick embryos can be stimulated to extend nerve processes in culture by inclusion of cytosine arabinoside (Ara-C) in the culture medium, in the absence of exogenous nerve growth factor (NGF). The degree of stimulation is dose dependent, and is not mimicked by either free cytosine or free arabinose. Since Ara-C is known to inhibit DNA synthesis, other inhibitors of DNA synthesis were tested. Hydroxyurea, fluorodeoxyuridine, and 3 mM thymidine all stimulated nerve outgrowth in the absence of exogenous NGF. In addition, bromodeoxyuridine also stimulated nerve outgrowth. In all cases, stimulation was observable after 24 h of culture, with maximal outgrowth achieved by 72 h of culture. The experimental response was never as large as the response to NGF, but was up to seven times greater than control outgrowth. In all cultures, nerve processes were characterized by growth cones at their distal tips, colchicine-sensitivity, and a high tubulin content visualized by immunofluorescence with anti-tubulin antibody.     

RhoA-kinase and myosin II are required for the maintenance of growth cone polarity and guidance by nerve growth factor

Growth cones are highly polarized and dynamic structures confined to the tips of axons. The polarity of growth cones is in part maintained by suppression of protrusive activity from the distal axon shaft, a process termed axon consolidation. The mechanistic basis of axon consolidation that contributes to the maintenance of growth cone polarity is not clear. We report that inhibition of RhoA-kinase (ROCK) or myosin II resulted in unstable consolidation of the distal axon as evidenced by increased filopodial and lamellipodial extension. Furthermore, when ROCK or myosin II was inhibited lamellipodia formed at the growth cone migrated onto the axon shaft. Analysis of EYFP-actin dynamics in the distal axon revealed that ROCK negatively regulates actin polymerization and initiation of protrusive structures from spontaneously formed axonal F-actin patches, the latter being an effect attributable to ROCK-mediated regulation of myosin II. Inhibition of ROCK or myosin II blocked growth cone turning toward NGF by preventing suppression of protrusive activity away from the source of NGF, resulting in aborted turning responses. These data elucidate the mechanism of growth cone polarity, provide evidence that consolidation of the distal axon is a component of guidance, and identify ROCK as a negative regulator of F-actin polymerization underlying protrusive activity in the distal axon.     

Nerve growth factor increases the cyclic GMP level and activates the cyclic GMP phosphodiesterase in PC12 cells

Nerve growth factor (NGF) rapidly increases the cyclic GMP (cGMP) level about 2-3-fold and enhances the cGMP phosphodiesterase (PDE) activity about 2-fold in rat pheochromocytoma PC12 cells. No changes in the level of cyclic AMP (cAMP) and in the activity of cAMP PDE were found. GTP and a nonhydrolysable analog of GTP, GMP-PCP, at 100 microM, were able to mimic the effect of NGF on the cGMP PDE activity. These results suggest that the cGMP system may be one of the second messengers of NGF action in PC12 cells.     

Growth of neurites without filopodial or lamellipodial activity in the presence of cytochalasin B

To examine the role in neurite growth of actin-mediated tensions within growth cones, we cultured chick embryo dorsal root ganglion cells on various substrata in the presence of cytochalasin B. Time-lapse video recording was used to monitor behaviors of living cells, and cytoskeletal arrangements in neurites were assessed via immunofluorescence and electron microscopic observations of thin sections and whole, detergent-extracted cells decorated with the S1 fragment of myosin. On highly adhesive substrata, nerve cells were observed to extend numerous (though peculiarly oriented) neurites in the presence of cytochalasin, despite their lack of both filopodia and lamellipodia or the orderly actin networks characteristic of typical growth cones. We concluded that growth cone activity is not necessary for neurite elongation, although actin arrays seem important in mediating characteristics of substratum selectivity and neurite shape.     

Behavior of fish retinal growth cones encountering chick caudal tectal membranes: A time-lapse study on growthcone collapse

In a cross species in vitro assay, growth cones from fish temporal retina elongating on laminin lanes were observed with time-lapse videomicroscopy as they encountered lanes and territories that carried membrane fragments from the chick caudal tectum. Caudal tectal membranes of adult fish and embryonic chick are known to possess a repellent guiding component for temporal retinal axons. The caudal membranes of chick exert a particularly strong influence on fish temporal axons. Contacts with chick caudal membranes by just a few filopodia and parts of the lamellipodia evoked a turning response away from the membrane lane of the entire growth cone. Contacts by filo- and lamellipodia over the entire circumference of the growth cone, however, caused invariably growth cone collapse and retraction. During growth cone turning and collapse and retraction, filopodia remained in contact with the tectal membrane fragments, suggesting strong membrane–filopodia adhesion simultaneous to growth cone repulsion by the repellent guiding component. © 1993 John Wiley & Sons, Inc.     

Growth cones integrate signaling from multiple guidance cues.

Nerve growth factor (NGF) and semaphorin3A (Sema3A) are guidance cues found in pathways and targets of developing dorsal root ganglia (DRG) neurons. DRG growth cone motility is regulated by cytoplasmic signaling triggered by these molecules. We investigated interactions of NGF and Sema3A in modulating growth cone behaviors of axons extended from E7 chick embryo DRGs. Axons extending in collagen matrices were repelled by Sema3A released from transfected HEK293 cells. However, if an NGF-coated bead was placed adjacent to Sema3A-producing cells, axons converged at the NGF bead. Growth cones of DRGs raised in 10(-9) M NGF were more resistant to Sema3A-induced collapse than when DRGs were raised in 10(-11) M NGF. After overnight culture in 10(-11) M NGF, 1-hr treatment with 10(-9) M NGF also increased growth cone resistance to Sema3A. Pharmacological studies indicated that the activities of ROCK and PKG participate in the cytoskeletal alterations that lead to Sema3A-induced growth cone collapse, whereas PKA activity is required for NGF-mediated reduction of Sema3A-induced growth cone collapse. These results support the idea that growth cone responses to a guidance cue can be modulated by interactions involving coincident signaling by other guidance cues.     

RhoA‐kinase and myosin II are required for the maintenance of growth cone polarity and guidance by nerve growth factor

Growth cones are highly polarized and dynamic structures confined to the tips of axons. The polarity of growth cones is in part maintained by suppression of protrusive activity from the distal axon shaft, a process termed axon consolidation. The mechanistic basis of axon consolidation that contributes to the maintenance of growth cone polarity is not clear. We report that inhibition of RhoA‐kinase (ROCK) or myosin II resulted in unstable consolidation of the distal axon as evidenced by increased filopodial and lamellipodial extension. Furthermore, when ROCK or myosin II was inhibited lamellipodia formed at the growth cone migrated onto the axon shaft. Analysis of EYFP‐actin dynamics in the distal axon revealed that ROCK negatively regulates actin polymerization and initiation of protrusive structures from spontaneously formed axonal F‐actin patches, the latter being an effect attributable to ROCK‐mediated regulation of myosin II. Inhibition of ROCK or myosin II blocked growth cone turning toward NGF by preventing suppression of protrusive activity away from the source of NGF, resulting in aborted turning responses. These data elucidate the mechanism of growth cone polarity, provide evidence that consolidation of the distal axon is a component of guidance, and identify ROCK as a negative regulator of F‐actin polymerization underlying protrusive activity in the distal axon. © 2006 Wiley Periodicals, Inc. J Neurobiol, 2006     

cGMP-mediated signaling via cGKIalpha is required for the guidance and connectivity of sensory axons

Previous in vitro studies using cGMP or cAMP revealed a cross-talk between signaling mechanisms activated by axonal guidance receptors. However, the molecular elements modulated by cyclic nucleotides in growth cones are not well understood. cGMP is a second messenger with several distinct targets including cGMP-dependent protein kinase I (cGKI). Our studies indicated that the alpha isoform of cGKI is predominantly expressed by sensory axons during developmental stages, whereas most spinal cord neurons are negative for cGKI. Analysis of the trajectories of axons within the spinal cord showed a longitudinal guidance defect of sensory axons within the developing dorsal root entry zone in the absence of cGKI. Consequently, in cGKI-deficient mice, fewer axons grow within the dorsal funiculus of the spinal cord, and lamina-specific innervation, especially by nociceptive sensory neurons, is strongly reduced as deduced from anti-trkA staining. These axon guidance defects in cGKI-deficient mice lead to a substantial impairment in nociceptive flexion reflexes, shown using electrophysiology. In vitro studies revealed that activation of cGKI in embryonic dorsal root ganglia counteracts semaphorin 3A-induced growth cone collapse. Our studies therefore reveal that cGMP signaling is important for axonal growth in vivo and in vitro.