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is required for the guidance and connectivity of sensory axons
Address correspondence to Fritz G. Rathjen, Medical Research Council, Robert-Rössle-Str. 10, D-13092 Berlin, Germany. Tel.: 49-30-9406-3709. Fax: 49-30-9406-3730. E-mail: rathjen{at}mdc-berlin.de
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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.
Key Words: cGMP signaling; axonal pathfinding; cGMP-dependent protein kinase I; sensory axons; nociceptive flexion reflex
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In many cell types, cGMP is a widely used second messenger that has several distinct targets (Hofmann et al., 2000; Lucas et al., 2000), including activation of cGMP-dependent protein kinase I (cGKI)* and cGKII, regulation of cAMP levels through the activation or inhibition of cAMP-dependent phosphodiesterases, and opening of cyclic nucleotide-gated cation channels. cGMP is generated by soluble and particulate guanylyl cyclases (Gibson and Garbers, 2000) and degraded by phosphodiesterase V. The serine/threonine kinases cGKI and II, of which cGKI is known to be expressed in the developing spinal cord (Qian et al., 1996), are composed of an NH2-terminal domain, a regulatory segment, and a catalytic domain. In mammals, cGKI is expressed in two alternatively spliced isoforms, termed and ß, that differ in their NH2-terminal domains (Pfeifer et al., 1999). These NH2-terminal stretches regulate homodimerization, cGMP affinity and activation of the catalytic domain, and the substrate binding/anchoring of the isoenzymes. cGKI, which has been investigated extensively in the context of regulation of smooth muscle tone, has a variety of pleiotropic cellular regulatory functions, including calcium release (Schlossmann et al., 2000) and myosin phosphatase activation (Surks et al., 1999). Here, we have analyzed whether cGKI is implicated in growth cone steering in vitro and in vivo. Our results indicate that cGKI is important for growth cone extension in the dorsal root entry zone (DREZ) of the spinal cord and counteracts semaphorin 3A–induced growth cone collapse in vitro. We therefore demonstrated that cGMP signaling is important for axonal pathfinding in vivo and that cGMP signaling is mediated via cGKI. Furthermore, physiological investigations indicated that pathfinding errors of sensory axons caused by the absence of cGKI resulted in a significant impairment of the nociceptive flexion reflex.
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, but not cGKIß, is selectively expressed in sensory axons of the developing spinal cord isoform of cGKI is expressed whereas the ß isoform remains undetectable (Fig. 1 a). Immunostaining of cryostat sections with these three different antibody preparations revealed a very restricted pattern of expression of cGKI. cGKI is expressed in cell bodies and growing axons of DRG neurons (Fig. 1, b–g), and sensory axons at the DREZ were especially heavily labeled for cGKI. At more advanced stages, the entire dorsal funiculus and the descending collaterals of the proprioceptive and, more weakly, of the nociceptive neurons were also stained at E14 (Fig. 1, e and f). The labeling intensity declined in the dorsal funiculus at more advanced developmental stages (Fig. 1 g). Spinal neurons and axonal tracts were not cGKI positive, however weak transient labeling was observed in motoneurons at earlier developmental stages (Fig. 1, b and c, arrowheads) and in preganglionic neurons. In addition, cGKI appeared ideally situated to transduce signals in growing sensory axons, as punctate cGKI immunoreactivity could be observed in the filopodia and lamellipodia of DRG neuron growth cones in culture (Fig. 1 h). This very restricted spatio-temporal expression profile of cGKI in sensory axons suggested that cGKI might regulate growth and pathfinding of DRG axons in specific regions in vivo, in particular at the DREZ.
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is restricted to sensory axons at early developmental stages, we analyzed the trajectory of sensory axons within spinal cords of cGKI-deficient embryos (Wegener et al., 2002) (see Fig. 6 b) in whole mounts and in transverse as well as horizontal sections. Once sensory axons arrive at the DREZ of the developing spinal cord, each axon bifurcates into a rostral and caudal branch extending over several segments without growing into the gray matter (Davis et al., 1989; Eide and Glover, 1995; Mirnics and Koerber, 1995, 1997; Ozaki and Snider, 1997). After a waiting period, collaterals grow out from these longitudinal axons and form lamina-specific projections in the gray matter (Fig. 2 a). As expected from the localization, the absence of cGKI in cGKI-deficient mice did not affect the overall structure of the spinal cord and the DRGs or their dorsal roots (unpublished data). To visualize the growth in rostral/caudal direction within the spinal cord, we used the lipophilic axonal tracer DiI (Honig and Hume, 1989) to label primary afferents in preparations of E12 or E13 embryos fixed in paraformaldehyde. Whole mounts were analyzed after 2, 3, and 4 d of incubation with a fluorescence microscope. Whereas wild-type DRG axons extended a rostral and caudal branch with an equal frequency (24 DRGs analyzed), DRG axons in mutant mice had a strong preference for growing primarily in a rostral direction (67% of 24 DRGs from 13 mutant embryos) (Fig. 2 b; Table I). A confocal microscopic analysis of these DiI-labeled preparations revealed that wild-type fascicles of dorsal roots were split into a very fine meshwork within the DREZ, as would be expected from the formation of T-like branches that are laying on top of each other (Fig. 2 c, left). In contrast, in mutant embryos, thick fascicles of axons turning either caudally or, preferentially, in a rostral direction were observed (Fig. 2 c, middle and right). In summary, these observations indicate a defect of the mutant DRG axons within the DREZ in rostral/caudal growth.
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was also expressed in the collaterals of proprioceptive axons (Fig. 1 e), we followed the development of the proprioceptive axon collaterals in transverse serial sections using parvalbumin as a marker. Although pathfinding appeared unaffected in mutant embryos, fewer parvalbumin-positive proprioceptive collaterals arrived in ventral spinal cord to innervate motoneurons (Fig. 4, a and b).
Functional connectivity of spinal reflexes are impaired in cGKI-deficient mice
The longitudinal branching error, the decreased anti-trkA staining, which might reflect a reduced axon number in the dorsal funiculus, and the reduced innervation of the nociceptive superficial dorsal horn by trkA-positive sensory fibers in cGKI mutant mice prompted us to examine the functional connectivity of sensory afferents in mutant mice. We did this by using an in vitro spinal cord preparation (Fig. 5 a; Heppenstall and Lewin, 2001) to measure the integrated ventral root potential in isolated postnatal day 5 (P5)–P9 spinal cord. We found that the fast motoneuron potential evoked by electrical stimulation of large diameter afferents was reduced in magnitude in cGKI mutant mice, but this reduction of 40% did not reach statistical significance (Fig. 5 c; t test, P = 0.06). At higher stimulation intensities, a nociceptor-specific long-lasting ventral root potential is evoked that represents activation of the nociceptive flexion reflex. This potential was substantially (50%) and significantly reduced in cGKI mutant mice compared with wild-type controls (Fig. 5, b–d) (P values and percentages refer to integrated area of A-fiber and C-fiber response). We also measured the activity-dependent facilitation of the ventral root potential evoked by repetitive (1 Hz) stimulation of nociceptive axons, a phenomenon termed wind-up. The magnitude of the facilitation (percentage increase from baseline) was unaltered in mutants, indicating that this post-synaptic plasticity was largely intact (Fig. 5, e and f). Thus, the defect in cGKI-mediated regulation of axon guidance at the DREZ early in development leads to a loss of functional connectivity that is selective for a pain-related spinal reflex. The striking correlation we observed between the anatomical and functional lack of sensory connectivity suggested that the magnitude of the flexion reflex response is limited by nociceptor connectivity within the superficial spinal cord.
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as a component of the sensory axon growth cone machinery that transduces endogenous cGMP changes required for correct sensory axon guidance within the developing DREZ of the spinal cord. Tracing with DiI and anti-trkA staining demonstrated that sensory axons are unable to identify their correct path at the entrance zone of the spinal cord and, as a consequence, are unable to generate a rostral and caudal branch or, alternatively, are unable to maintain one of the two branches. Evidence for mistargeting of sensory axons in cGKI mutant mice was observed by anti-trkA staining, as many axon fascicles grow directly into the dorsal horn inappropriately in the direction of the central canal most likely without the formation of a rostral or caudal axon. It is not possible with present methods to distinguish whether cGKI-deficient sensory axons are unable to branch at the DREZ or sustain two branches indefinitely. As a consequence of this branching or retention failure, the developing dorsal funiculus of mutant mice was found to be smaller, and less collaterals were observed that can project to lamina-specific targets in the dorsal and ventral horn. The longitudinal growth error is most likely not due to a lack of axonal defasciculation in the DREZ, because sensory nerves of mutant mice do not appear more fasciculated outside the cord and in vitro–cultivated DRG axons do not generate thicker fascicles. The preference for rostral growth within the DREZ in the absence of cGKI remains currently speculative. However, a striking correlation was found between our anatomical and functional investigations, and therefore, perhaps as a result of early errors in sensory axon targeting in the DREZ, the amplitude of the nociceptive flexion reflex is selectively and substantially reduced in mutant mice. Thus axon targeting errors have clear functional consequences for spinal reflex circuit connectivity. The reduction in the sensory neuron to dorsal horn connectivity we observed functionally probably indicates that the reduction in the innervation of lamina II by nociceptive sensory neurons is not compensated by an increased number of synapses per sensory neuron. Interestingly, plasticity of the nociceptive flexion reflex, termed wind-up, is preserved, indicating that this largely post-synaptic phenomenon (Herrero et al., 2000) is unaffected by the reduced anatomical connectivity of C-fiber afferents found in cGKI mutant mice. Early in development, cGKI shows also a very restricted localization within the spinal cord, and, consequently, pathfinding of other axon systems was not affected by the absence of cGKI.
Previous in vitro assays indicated that cGMP can antagonize the semaphorin 3A–induced growth cone collapse (Song et al., 1998). Our analysis of growth cone collapse using cGKI-deficient DRGs indicated that the activation of cGKI mediates, in part, this counteracting effect of cGMP. Because growth cones of cGKI-deficient DRGs collapse as efficiently as wild-type DRGs, it can also be concluded that cGKI is not part of the semaphorin 3A signaling pathway that results in the collapse of sensory growth cones. This contrasts with conclusions drawn from in vitro studies using exclusively pharmacological reagents (Dontchev and Letourneau, 2002). Although semaphorin 3A has been described as an in vitro branching factor for Xenopus retinal growth cones but not for rat E17 DRG neurons (Wang et al., 1999; Campbell et al., 2001), a possible counteracting effect by activation of cGKI might not play a role for sensory pathfinding at the DREZ, as studies on semaphorin 3A– or neuropilin-1–deficient mice have not reported sensory neuron guidance defects similar to those we describe here (Kitsukawa et al., 1997; Taniguchi et al., 1997).
The guidance signals within the DREZ and the responding guidance receptor(s) on sensory axons whose activation elevates intracellular cGMP and subsequently cGKI remain to be determined. Slit2, a large extracellular matrix glycoprotein that acts as an axonal guidance signal for several axon systems (Erskine et al., 2000; Niclou et al., 2000; Ringstedt et al., 2000; Bagri et al., 2002; Plump et al., 2002), has also been described as an in vitro branching factor for DRG neurons growing on collagen (Wang et al., 1999). Although the more general limitation of monolayer cell cultures does not allow a definitive conclusion, most likely collateral branching and not the formation of T-like branches were analyzed under the experimental conditions described by Wang et al. (1999), because relatively advanced DRG neurons from E17 rats were assayed. A recent study also suggested that cGMP might modulate Slit2 signaling via cGKs (Nguyen-Ba-Charvet et al., 2001). However, in these investigations, the pharmacological reagent KT5823 was used, which does not function as an inhibitor of cGKI in living cells, in contrast to in vitro conditions (Burkhardt et al., 2000).
The analysis of downstream targets of cGKI might also help to define the exact function of cGKI in sensory growth cone steering at the DREZ further. In other cell types outside the nervous system, cGKI lowers [Ca2+]i by interfering with intracellular calcium release (Schlossmann et al., 2000) and/or activation of maxi KCa channels (Sausbier et al., 2000), and may decrease myosin light chain phosphorylation by interacting with the myosin targeting subunit of myosin phosphatase (Surks et al., 1999) or by phosphorylation of RhoA, leading to its inactivation (Sauzeau et al., 2000). Phosphorylation/dephosphorylation of the myosin light chain regulates the cytoskeletal organization in many cell types (Hall, 1998; Burridge, 1999) and could affect filopodial extension in growth cones by modulating retrograde flow of actin filaments (Lin et al., 1996). In addition, organization of cytoskeletal actin by cGKI in growth cones of sensory axons might also occur by affecting the activity of the actin organizing proteins VASP (Reinhard et al., 2001) or CRP2 (Huber et al., 2000). Further studies will reveal which, or whether a combination of these multiple downstream targets is regulated by cGKI in the DREZ.
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Immunodetection, DiI tracing, and DRG cell counting
For Western blotting, DRGs from E13 mice were collected, solubilized in SDS-PAGE sample buffer, and separated by 10% SDS-PAGE. Equal loading of lanes was confirmed by staining with antibodies to L1 (Rathjen and Schachner, 1984). Rabbit antibodies specific to both isoforms, to the or ß isoform of cGKI, were generated as detailed elsewhere (Schlossmann et al., 2000). For immunohistochemical detection, cryostat sections of formaldehyde-fixed embryos were stained by indirect immunofluorescence using rabbit antibodies to cGKI (1:500 to 1:1,000), trkA (1:2,000; provided by L. F. Reichardt, University of California San Francisco, San Francisco, CA), parvalbumin (1:1,000; Calbiochem), TAG-1 (Dodd et al., 1988), or L1 (Rathjen and Schachner, 1984) followed by goat anti–rabbit or rabbit anti–mouse antibodies coupled to Cy3 (Dianova). To detect intracellular localized cGKI, cultivated DRG neurons were fixed and permeabilized with 0.1% Triton X-100 before incubation with antibodies. For DiI (Molecular Probes) tracing, mouse embryos were immersion fixed in 4% paraformaldehyde in PBS and stored in fixative until the application of axon tracer. DiI dissolved in 100% ethanol was injected into single DRGs. The preparations were then incubated in 4% paraformaldehyde buffer at 37°C for 2–4 d. Whole mounts were viewed laterally using a confocal fluorescence microscope (Bio-Rad Laboratories).
The intensity of trkA staining was quantified in serial horizontal sections at E13 in three different defined fields in thoracic regions of the spinal cord using NIH image software. The pixel intensity of each field of the complete primordium of the dorsal funiculus (eight to nine serial sections of 20 µm thickness) was integrated, and the mean calculated and presented as relative units. In transverse sections, the trkA-stained area was delineated with a mouse cursor to measure the area and the intensity in throracic and lumbar regions. Data from four to five adjacent sections of each level were collected and the mean was calculated.
Cells from L4 and L5 DRG of E13 and E14 embryos were counted in serial cryostat sections following methods previously described (ElShamy and Ernfors, 1996; Farinas et al., 1996; Riethmacher et al., 1997). Sections (14 µm) were stained by the fluorescent Hoechst 33258 nuclear stain (Spaltmann and Brummendorf, 1996). The maximum diameter observed by this stain was 10 µm, suggesting that the actual cell numbers might be underestimated.
Growth cone collapse assay
For the growth cone collapse assay, we followed methods developed by Raper and Kapfhammer (1990) using E13 DRG explants from the lumbar region grown on poly-L-lysine/laminin-1 (GIBCO BRL) for 24 h in F-12 (GIBCO BRL) supplemented with hNGF-ß (20 ng/ml; Boehringer), N2 supplement (GIBCO BRL), and bovine pituitary extract (200 µg/ml; GIBCO BRL) (Luo et al., 1993). Explants were pretreated with the indicated concentrations of 8-pCPT-cGMP, 8-Br-cGMP, or 8-Br-cAMP (BioLog) for 30 min followed by incubation with semaphorin 3A–containing culture supernatants of transiently transfected COS7 cells for 1 h. Culture supernatants were titrated to induce a collapse of 90–100% of growth cones after 1 h. Glutaraldehyde-fixed explants were stained by mAb A2B5 (Developmental Studies Hybridoma Bank) for visualization. cDNA encoding chick semaphorin 3A (collapsin-1) (Luo et al., 1993) was obtained by RT-PCR and subcloned without the signal sequence into a modified pSG5 vector (Stratagene) containing the signal sequence of neurofascin (Volkmer et al., 1992) followed by a myc and his tag. The cDNA sequence of the construct was verified by DNA sequencing. A cDNA construct obtained from J.A. Raper (University of Philadelphia, Philadelphia, PA) served as a control in some experiments.
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Footnotes |
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* Abbreviations used in this paper: cGKI, cGMP-dependent protein kinase I; DREZ, dorsal root entry zone; DRG, dorsal root ganglion; E, embryonic day; P, postnatal day; VRP, ventral root potential.
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Acknowledgments |
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This work was supported by grants from Deutsche Forschungsgemeinschaft, Boehringer Ingelheim Stiftung, VolkswagenStiftung, and Fond der Chemischen Industrie.
Submitted: 10 July 2002
Revised: 23 September 2002
Accepted: 24 September 2002
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