cGKIα is selectively expressed in developing DRG axons. (a) Western blot analysis demonstrates the presence of the α, but not the β, isoform of cGKI in embryonic DRGs. Lane 1, antibodies to both cGKI isoforms (c, common); lane 2, antibodies to the α isoform; lane 3, antibodies to the β isoform. For comparison, a blot of antibodies to L1 is shown. (b–h) Immunohistochemical detection of cGKI. (b–d) cGKIα distribution in the E11–E13 spinal cord. cGKIα is expressed in the DREZ and the developing dorsal funiculus as well as weakly and transiently on motoneuron columns (arrowheads) and in preganglionic neurons. Bar, 100 μm. (e) cGKIα expression in proprioceptive collaterals in the dorso-medial spinal cord at E14. Bar, 50 μm. (f) cGKIα expression in nociceptive collaterals in the dorso-lateral spinal cord at E14. (g) cGKIα distribution in the E15 spinal cord. Bar, 100 μm. (h) cGKI is distributed throughout a cultivated DRG growth cone. Bar, 5 μm. Sections were either stained by antibodies recognizing both isoforms of cGKI (E11, E12, and E13) or only the α isoform (E14 and E15). Both antibody preparations gave identical results. Tissue sections of cGKI-deficient mice did not stain with antibodies against cGKI (unpublished data). D, drg; R, dorsal root; Z, DREZ.

Pathfinding of nociceptive neurons is impaired in the DREZ of cGKI-deficient mice. trkA-positive axons were analyzed in transverse or horizontal sections of wild-type (+/+) and cGKI-deficient mice (−/−) at E12–E14. (a, b, f, g, h, i, j, k, l, and m) Transverse sections; (c, d and e) horizontal sections. The plane of transverse section used in a and b is shown in a cartoon in n. The plane of horizontal sections in c is indicated in o (the most ventral part of the DREZ), and the plane in d and e is indicated in p (center of the DREZ). (j–m) Staining of transverse sections using polyclonal antibodies to L1 as a more general axon marker (j and k) or a mAb to TAG-1 to label commissural axons (l and m). These stainings indicate that the pattern of the lateral as well as the ventral funiculus and the growth of the commissural axons to the floor plate are not affected by the absence of cGKI. Arrowheads in e, g, i, and k point to axons or axon fascicles leaving the primordium of the dorsal funiculus of cGKI-deficient embryos to extend directly to the central canal. In transverse sections, dorsal is up, and in horizontal sections, rostral is up. Bars: (a–e) 10 μm; (f and g) 50 μm; (h–m) 100 μm. C, central canal.

Impairment of the VRP of the spinal cord in the absence of cGKI. (a) In vitro–hemisected spinal cord preparation to measure monosynaptic and polysynaptic reflexes evoked by primary afferents. A scheme of a cross section of the spinal cord is shown and stimulating and recording electrodes are indicated. Dorsal is up. (b) VRPs of wild-type and cGKI-deficient neonatal mice. Electrical stimulation of dorsal root fibers generates a potential that can be recorded at the ventral root. The VRP is composed of a fast component evoked by A-fibers and a slower one activated by C-fibers (Heppenstall and Lewin, 2001). (c and d) Quantification of the VRP in wild-type and cGKI-deficient mice. The integrated area of the C-fiber area is significantly reduced in cGKI-deficient mice (P < 0.05; n = 6 per group). The integrated area of the A-fiber response was reduced but did not reach significance (P = 0.06). (e) Wind-up in wild-type and cGKI-deficient neonatal mice. Repetitive electrical stimulation of dorsal root fibers (20 times, 1 Hz) evokes an increase in the potential recorded from the ventral root. (f) Quantification of wind-up in wild-type and cGKI-deficient mice. Wind-up, measured as the percentage increase in the potential, is not different between wild-type and cGKI-deficient mice (P = 0.7; n = 6 per group).

Longitudinal growth of sensory axons is impaired in the spinal cord of cGKI-deficient mice. (a) Schematic drawing of the three-dimensional morphology of a single sensory neuron within the spinal cord. (b) To visualize the longitudinal branching, primary afferents in preparations of E12 or E13 embryos were labeled with the lipophilic axonal tracer DiI (Honig and Hume, 1989). Whole mounts were analyzed laterally with a fluorescence microscope. Whereas wild-type (+/+) DRG axons branch equally in rostral and caudal direction, cGKI-deficient (−/−) DRG axons have a strong preference for growing primarily in rostral direction (to the right). Fluorescence images were inverted. Bar, 100 μm. (c) Three adjacent confocal images of DiI-labeled axons of wild-type (+/+) and mutant mice (−/−) within the DREZ are shown. Note that mutant axons turn preferentially rostrally as bundles. Bar, 100 μm. D, DRG; R, dorsal root.

Quantitative analysis of proprioceptive and nociceptive axons within the developing spinal cord. (a–d) Less proprioceptive and nociceptive axons and their collaterals are observed in transverse sections of E15 cGKI-deficient than wild-type mice using parvalbumin (a and b) or trkA staining (c and d). Fluorescence images were inverted. In c and d, only the dorsal region of the spinal cord is shown. Arrowheads in a and b indicate parvalbumin-positive collaterals. In d, the two arrows indicate dorso-medial regions not populated by trkA-positive axons. Bars, 100 μm. (e) Quantification of trkA staining in transverse and horizontal sections at different levels of the spinal cord, which was taken as measure for axons growing in the developing dorsal funiculus at E13. The mean of the total pixel intensity of sections from wild-type embryos at the thoracic level was taken as 100%. In horizontal sections, the staining intensity of serial sections of the total developing dorsal funiculus in three neighboring regions was summed. The intensity of staining in wild-type embryos was taken as 100%. The last two columns indicate quantitative measurements of four fields in the E15 dorsal superficial horn, the termination zone for nociceptive C-fibers. Error bars indicate standard deviations. (f) Sum of cell numbers in L4 and L5 DRG at E13 and E14 (black, wild type; white, cGKI-deficient embryos). The number of analyzed DRGs and embryos is indicated above each column. Error bars indicate standard deviations. (g) Western blot analysis of DRG extracts from wild-type and cGKI-deficient E13 embryos using polyclonal antibodies to trkA or, for comparison, to the Ig cell adhesion protein L1. The 145-kD component of trkA and the 200-kD doublet of L1 are shown.

cGKI counteracts semaphorin 3A–induced growth cone collapse in vitro. (a) Growth cone collapse assay in the presence of 8-pCPT-cGMP or 8-Br-cAMP using DRG explants from litter-matched wild-type (+/+) or cGKI-deficient mice (−/−). The concentration and the presence of semaphorin 3A is indicated below the columns. The percentage of collapsed versus uncollapsed growth cones is presented and the number of counted collapsed growth cones and DRGs is given above the columns. The effect of semaphorin 3A alone was calculated as 100%. Error bars refer to SEM. Asterisk indicates significant difference from the samples without pretreatment with 8-pCPT-cGMP, P < 0.001 (t test). (b) cGKI protein could be detected in Western blots of DRG of wild-type (+/+) but not in DRGs of cGKI-deficient (−/−) embryos using polyclonal antibodies directed to both isoforms of cGKI. (c) Growth cones from wild-type (+/+) or cGKI-deficient (−/−) mice in the presence of semaphorin 3A alone or semaphorin 3A and 0.5 mM 8-pCPT-cGMP. Neurons were stained by mAb A2B5 for visualization and fluorescence images were inverted. DRGs from heterozygous (+/−) mice behaved as wild-type DRGs (unpublished data). Bar, 100 μm. Note that growth cones in the bottom images are shown at a higher magnification than in the top images. Bar, 10 μm. (d) Outgrowth of DRG explants of cGKI-deficient mice (−/−) is indistinguishable from those of wild-type mice (+/+). Explants from lumbar regions of litter-matched E13 embryos grown for 24 h on laminin-1 were silver stained (Rager et al., 1979).