Bifurcation of sensory axons at the DREZ is impaired in the constitutive cGKI knockout. (A) Schematic drawing of the trajectories of sensory axon projections within the spinal cord (D, dorsal; V, ventral). (B) Sensory growth cones in the process of bifurcation at the DREZ in wild-type mice. Bar, 20 μm. (C and D) Dorsal views of DiI-labeled axons of wild-type or cGKI-deficient mice are shown. Fluorescence images are inverted, lateral is at the bottom and caudal is at the right. Arrowheads in B and C indicate bifurcations in the wild-type. Bar, 50 μm. (E) Quantification of T-shaped branches, single rostral, or caudal turns in wild-type or cGKI-deficient mice. The numbers of counted single axons are given in brackets for different trunk levels for each genotype. In total, 25 wild-type and 17 cGKI-deficient embryos were analyzed blind with regard to the genotype. To test whether the preference for rostral turns is statistically significant, the binomial test was used and showed that the distribution is other than 50:50 (P = 0.05).

The receptor GC Npr2 is selectively expressed in developing DRG neurons. (A) RT-PCR analysis of cGMP-dependent protein kinases, NOSs, sGCs, or pGCs in mouse E12 spinal cord or DRG. S, spinal cord; D, DRG; −, no template in the reaction mixture. The sizes of the PCR amplification products are given in parenthesis. (B–E) Expression pattern of Npr2 and cGKIα transcripts in transverse sections of mouse E12 spinal cord by in situ hybridizations. Sense probe served as control. Bar, 200 μm.

Bifurcation of sensory axons at the DREZ in Npr2 loss-of-function mutants is impaired. (A) Schematic representation of microscopic analysis of bifurcation errors at the DREZ. The star indicates a single highlighted sensory neuron within a DRG. (B and C) To visualize the longitudinal branching of single primary afferent axons, whole mounts of E12–14 spinal cords were labeled with the lipophilic tracer DiI (Honig and Hume, 1989). Lateral is to the bottom and caudal to the right. Some bifurcations are indicated by an arrowhead in the wild type. Fluorescence images were inverted. (D) Quantification of T-shaped branches, single rostral (R-Turns) or caudal turns (C-Turns) in wild-type or cn/cn mice. The numbers of counted single axons for each genotype are given in brackets for different trunk levels. In total, 13 wild-type and 8 mutant embryos were analyzed blind with regard to the genotype. To test whether the preference for rostral turns is statistically significant, the binomial test was used and indicated a 50:50 distribution. (E) Quantification of the relative area of the developing dorsal funiculus in transverse sections stained by anti-trkA antibodies. The values are means of 16 transverse sections for wild-type and cn/cn embryos, each at thoracic (64.1%; SD 5.3%; ***, P < 0.001, Mann-Whitney U test) or lumbar (59%; SD 11.1%; ***, P < 0.001, Mann-Whitney U test) levels. (F and G) Dorsolateral regions of transverse sections of the spinal cord stained by antibodies to trkA revealed premature ingrowth of axon fascicles (arrowheads) into the dorsal horn in the direction of the central canal. (H–K) Transverse sections of embryonic spinal cord stained by antibodies to TAG-1 or to L1 revealed that the pattern of TAG-1–positive axons or of L1-positive axon tracts is not affected in Npr2-inactive mutants. Arrowheads in K indicate misguided axon fascicles in the dorsal horn also observed by anti-trkA staining (G). Bars: (B, C, F, and G) 50 μm; (H–K) 100 μm.

Collaterals of sensory axons are generated in the absence of cGKI or in Npr2 loss-of-function mutants. (A–F) Staining of cross sections by antibodies to trkA, parvalbumin, or peripherin to visualize collaterals of sensory axons. Arrowheads indicate nociceptive or proprioceptive collaterals. Bar, 50 μm. (G–I) Single axons labeled by DiI and their developing collaterals are shown from E14 embryos (arrowheads). A dorsolateral view is presented. Bar, 20 μm. (J and K) Quantification of the distances between two collaterals in microscopic view fields of wild-type and mutant mice at E14. Frequency cumulation plots are shown for distances >5 μm. Distributions of distance lengths are significantly different for mutant mice compared with the corresponding wild-type data (P = 0.019 for Npr2; P = 0.001 for cGKI; Mann-Whitney U test). The numbers of measured distances were 54 for cGKI-deficient mice (five embryos) and 48 for the corresponding wild type (five embryos), and 63 for cn/cn mice (four embryos) and 78 for the wild type (11 embryos). Note that cn/cn and cGKI mutant mice were from a different genetic background.

The failure of sensory axons to bifurcate persists in mature cGMP-signaling mutant mice. (A–C) A Thy-1–EGFP allele (Feng et al., 2000) was crossed into cn/cn or cGKI-deficient mice. Mice were analyzed at P21 and dorsal views are shown. Arrowheads indicate bifurcations in the wild type. (D) Quantification of T-shaped branches, single rostral, or caudal turns in the wild type, cGKI-deficient mice, or Npr2 loss-of-function mutants. The numbers of counted single axons are given in brackets for each genotype. 12 animals were analyzed in total. (E–H) A very small percentage of sensory axons showed an unusual growth behavior at the DREZ in mutant mice of the cGMP signaling cascade. Arrowheads indicate short processes (E and F) and an axon that grew in one direction then looped backwards at the DREZ (H). Bars, 100 μm.

Connectivity with second order neurons in the superficial dorsal horn of the spinal cord is reduced in cn/cn mice. (A–F) The overall laminar organization in the dorsal horn is not affected in cn/cn mice as depicted by isolectin B4 staining or anti-CGRP and anti-VGLUT1 antibodies. Bar, 100 μm. (G) Relative number of capsaicin-responding and nonresponding cells is reduced in cn/cn in comparison with wild-type mice. (**, P < 0.01, χ² test). The numbers of cells measured are indicated within the columns. mEPSCs were recorded from neurons in the superficial dorsal horn in the presence of 1 μM strychnine, 100 μM picrotoxin, and 100 μM APV to block glycinergic and GABAergic input as well as NMDA receptor–mediated currents. 1 μM tetrodotoxin was used to block action potential–dependent neurotransmitter release. (H) Example current traces of mEPSCs at resting conditions and after application of 10 μM capsaicin in wild-type and cn/cn mouse. (I) Application of capsaicin leads to a significant increase in the frequency of mEPSC in wild-type as compared with cn/cn neurons (*, P = 0.032, Mann-Whitney U test). Under resting conditions, a reduced but not significantly different frequency was observed in capsaicin-responding neurons of cn/cn mice (P = 0.055, Mann-Whitney U test). The frequency of capsaicin nonresponding cells is not affected by the absence of Npr2 activity. (J) The amplitude and decay time constant of mEPSCs in current remains unchanged in Npr2 mutant mice. Numerical data are reported as mean ± SEM.

Scheme summarizing the observed bifurcation errors in the absence of cGMP signaling of cGKI or Npr2 mutant mice. (A) In wild-type mice sensory axons bifurcate as soon as they enter the spinal cord and stay in the oval bundle of His, whereas in mutant mice only turns into rostral or caudal directions were observed. A small proportion of axons were found to grow directly to the central canal in both genotypes. (B) For the activation mechanism of the receptor GC Npr2 and downstream phosphorylation targets of cGKIα, see Discussion.