During neural circuit formation, axons navigate several choice points to reach their final target. At each one of these intermediate targets, growth cones need to switch responsiveness from attraction to repulsion in order to move on. Molecular mechanisms that allow for the precise timing of surface expression of a new set of receptors that support the switch in responsiveness are difficult to study in vivo. Mostly, mechanisms are inferred from the observation of snapshots of many different growth cones analyzed in different preparations of tissue harvested at distinct time points. However, to really understand the behavior of growth cones at choice points, a single growth cone should be followed arriving at and leaving the intermediate target. Existing ex vivo preparations, like cultures of an “open‐book” preparation of the spinal cord have been successfully used to study floor plate entry and exit, but artifacts prevent the analysis of growth cone behavior at the floor plate exit site. Here, we describe a novel spinal cord preparation that allows for live imaging of individual axons during navigation in their intact environment. When comparing growth cone behavior in our ex vivo system with snapshots from in vivo navigation, we do not see any differences. The possibility to observe the dynamics of single growth cones navigating their intermediate target allows for measuring growth speed, changes in morphology, or aberrant behavior, like stalling and wrong turning. Moreover, observation of the intermediate target—the floor plate—revealed its active participation and interaction with commissural axons during midline crossing.

cGMP signaling elicited by activation of the transmembrane receptor guanylyl cyclase Npr2 (also known as guanylyl cyclase B) by the ligand CNP controls sensory axon bifurcation of DRG and cranial sensory ganglion (CSG) neurons entering the spinal cord or hindbrain, respectively. Previous studies have shown that Npr2 is phosphorylated on serine and threonine residues in its kinase homology domain (KHD). However, it is unknown whether phosphorylation of Npr2 is essential for axon bifurcation. Here, we generated a knock-in mouse line in which the seven regulatory serine and threonine residues in the KHD of Npr2 were substituted by alanine (Npr2-7A), resulting in a nonphosphorylatable enzyme. Real-time imaging of cGMP in DRG neurons with a genetically encoded fluorescent cGMP sensor or biochemical analysis of guanylyl cyclase activity in brain or lung tissue revealed the absence of CNP-induced cGMP generation in the Npr2 7A/7A mutant. Consequently, bifurcation of axons, but not collateral formation, from DRG or CSG in this mouse mutant was perturbed at embryonic and mature stages. In contrast, axon branching was normal in a mouse mutant in which constitutive phosphorylation of Npr2 is mimicked by a replacement of all of the seven serine and threonine sites by glutamic acid (Npr2-7E). Furthermore, we demonstrate that the Npr2 7A/7A mutation causes dwarfism as described for global Npr2 mutants. In conclusion, our in vivo studies provide strong evidence that phosphorylation of the seven serine and threonine residues in the KHD of Npr2 is an important regulatory element of Npr2-mediated cGMP signaling which affects physiological processes, such as axon bifurcation and bone growth.

Genetic investigations have demonstrated that a specific form of axonal branching - the bifurcation of afferents from dorsal root ganglia (DRG), cranial sensory ganglia (CSG) and mesencephalic trigeminal neurons (MTN) – is regulated by a cGMP-dependent signaling pathway. This cascade is composed of the ligand C-type natriuretic peptide (CNP), the receptor guanylyl cyclase Npr2, and the cGMP-dependent protein kinase Iα (cGKIα). In the absence of any one of these components, axons no longer bifurcate, instead they turn in either an ascending or a descending direction. To gain further mechanistic insights into the process of axon bifurcation we applied different cell culture approaches to decipher downstream activities of cGKI in somatosensory growth cones. We demonstrate that CNP induces an enlargement of DRG growth cones via cGKI which is considered as the priming step of axon bifurcation in the spinal cord. This growth cone remodeling was both blocked by pharmacological inhibitors of S-palmitoylation and potentiated by blocking de-palmitoylation. cGKI colocalizes with the palmitoylome and vesicular structures including the endoplasmic reticulum, early endosomes, lysosomes primarily in the central domain of the growth cone as well as with the Golgi apparatus at the level of the soma. Interestingly, an acyl-biotin-exchange chemistry-based screen indicated that 8pCPT-cGMP-induced signaling induces S-palmitoylation of a restricted pool of proteins in the DRG-derived cell line F11. Overall, our data indicate that CNP-induced cGMP signaling via cGKI affects growth cone morphology of somatosensory afferents. Moreover, it also suggests that S-palmitoylation might play a role in this process.

Bifurcation of axons from dorsal root ganglion (DRG) and cranial sensory ganglion (CSG) neurons is mediated by a cGMP-dependent signaling pathway composed of the ligand C-type natriuretic peptide (CNP), the receptor guanylyl cyclase Npr2 and the cGMP-dependent protein kinase I (cGKI). Here, we demonstrate that mesencephalic trigeminal neurons (MTN) which are the only somatosensory neurons whose cell bodies are located within the CNS co-express Npr2 and cGKI. Afferents of MTNs form Y-shaped branches in rhombomere 2 where the ligand CNP is expressed. Analyzing mouse mutants deficient for CNP or Npr2 we found that in the absence of CNP-induced cGMP signaling MTN afferents no longer bifurcate and instead extend either into the trigeminal root or caudally in the hindbrain. Since MTNs provide sensory information from jaw closing muscles and periodontal ligaments we measured the bite force of conditional mouse mutants of Npr2 (Npr2flox/flox;Engr1Cre) that lack bifurcation of MTN whereas the bifurcation of trigeminal afferents is normal. Our study revealed that the maximal biting force of both sexes is reduced in Npr2flox/flox;Engr1Cre mice as compared to their Npr2flox/flox littermate controls. In conclusion sensory feedback mechanisms from jaw closing muscles or periodontal ligaments might be impaired in the absence of MTN axon bifurcation.

Axon navigation depends on the interactions between guidance molecules along the trajectory and specific receptors on the growth cone. However, our in vitro and in vivo studies on the role of Endoglycan demonstrate that in addition to specific guidance cue – receptor interactions, axon guidance depends on fine-tuning of cell-cell adhesion. Endoglycan, a sialomucin, plays a role in axon guidance in the central nervous system of chicken embryos, but it is neither an axon guidance cue nor a receptor. Rather Endoglycan acts as a negative regulator of molecular interactions based on evidence from in vitro experiments demonstrating reduced adhesion of growth cones . In the absence of Endoglycan, commissural axons fail to properly navigate the midline of the spinal cord. Taken together, our in vivo and in vitro results support the hypothesis that Endoglycan acts as a negative regulator of cell-cell adhesion, in commissural axon guidance.