Axonal branching
A cGMP signalling pathway essential for sensory axon bifurcation
A prerequisite to form the complex wiring pattern of the mature nervous system is the multiple ramification of primary axon projections during the period of axonal outgrowth. This principle process enables an individual neuron to innervate several distinct targets allowing the integration of information. Despite intensive research efforts the molecular signalling pathways underlying axonal branching remained poorly understood. We have been therefore interested in the molecular analysis of axonal branching and studied this in sensory axons projecting into the spinal cord.
When dorsal root ganglion axons enter the spinal cord at the dorsal root entry zone (DREZ) they bifurcate in a highly stereotyped manner into a rostral and a caudal arm which both extend longitudinally over several segments. Only later collaterals are generated from these stem axons to penetrate the gray matter establishing lamina-specific projections. Thus, from a structural view, sensory axons display at least two types of ramifications within the cord: (1) bifurcation at the DREZ and (2) interstitial branching from stem axons to generate collaterals (Figure 1).
Figure1:
Schematic drawing of the trajectories of sensory axon projections within the spinal cord. (adapted from Schmidt et al. J. Cell Biol. 179: 331-340, 2007)
Our investigations revealed a cGMP signalling cascade, including the receptor guanylyl cyclase Npr2 and the serine/threonine kinase cGKI, essential for the establishment of axon bifurcation at the DREZ of the spinal cord. In the absence of one of these components sensory axons lack the bifurcation at the DREZ, i.e. the ingrowing axon only turns rostrally or caudally instead (Figure 2). This bifurcation error is maintained to mature stages. In contrast, interstitial branching of collaterals from the stem axons remains unaffected.
Figure2:
Bifurcation of sensory axons at the DREZ in Npr2 loss of function mutants is impaired. To visualize the longitudinal branching of single primary afferent axons whole mounts of E13 spinal cords of wild-type (A) or Npr2 loss of function mutants (B) were labeled with the lipophilic tracer DiI. Lateral is to the bottom, caudal to the right and some bifurcations are indicated by an arrow head in the wild-type. Fluorescence images were inverted. Bar, 50 µm. (adapted from Schmidt et al. J. Cell Biol. 179: 331-340, 2007)
At a functional level, the distorted axonal branching is accompanied by reduced neuronal connectivity in the spinal cord, as revealed by electrophysiological studies.
In a next step we focus on the complete elucidation of the cGMP-signalling pathway within sensory axons, with a special respect to phosphorylation targets of cGKI. We use biochemical approaches, in vitro outgrowth studies as well as genetic mouse models. Finally, we aim to apply our knowledge about cGMP signalling in sensory axons to other relevant neuronal cell populations in the central nervous system expressing Npr2 and cGKI during development.
Figure3:
Scheme summarizing the observed bifurcation errors in the absence of cGMP signalling 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) The cGMP signalling cascade in sensory axon comprises the cGMP producing receptor guanylyl cyclase Npr2 and the serine/threonine kinase cGKI. The mechanism of Npr2 activation and the downstream phosphorylation target(s) of cGKI in sensory neurons are yet to be elucidated. (adapted from Schmidt et al. J. Cell Biol. 179: 331-340, 2007)