c-Kit Receptor Maintains Sensory Axon Innervation of the Skin through Src Family Kinases

Abstract

Peripheral somatosensory neurons innervate the skin and sense the environment. Whereas many studies focus on initial axon outgrowth and pathfinding, how signaling pathways contribute to maintenance of the established axon arbors and terminals within the skin is largely unknown. This question is particularly relevant to the many types of neuropathies that affect mature neuronal arbors. We show that a receptor tyrosine kinase (RTK), c-Kit, contributes to maintenance, but not initial development, of cutaneous axons in the larval zebrafish before sex determination. Downregulation of Kit signaling rapidly induced retraction of established axon terminals in the skin and a reduction in axonal density. Conversely, misexpression of c-Kit ligand in the skin in larval zebrafish induced increases in local sensory axon density, suggesting an important role for Kit signaling in cutaneous axon maintenance. We found Src family kinases (SFKs) act directly downstream to mediate Kit's role in regulating cutaneous axon density. Our data demonstrate a requirement for skin-to-axon signaling to maintain axonal networks and elucidate novel roles for Kit and SFK signaling in this context. This Kit-SFK signaling axis offers a potential pathway to therapeutically target in sensory neuropathies and to further explore in other neurobiological processes.

Single-cell analysis of Rohon-Beard neurons implicates Fgf signaling in axon maintenance and cell survival

“…We are particularly interested in the role of receptor tyrosine kinase (RTK) signaling pathways, as these are major regulators of neural development, differentiation, and maintenance. In the case of RBs, the RTKs c-Kit and TrkC have critical roles in neuronal development and axon maintenance (Williams et al 2000; Tuttle et al 2022), but other important RTK pathways in this context have not been identified. Gene Ontology analysis (Ashburner et al 2000) of all three RB subtypes for major RTK pathways revealed expression of genes in the FGF signaling pathway was increased 1.76 fold (number of observed divided by the number of expected genes in this GO category) in RBs.…”

“…5A-B). Using a previously established protocol for characterizing the effect of RTK inhibitors on RB axons (Tuttle et al 2022), we treated RB:GFP larvae beginning at 3 dpf with SU5402 for 48 h. This led to a major loss of innervation in the tail skin in a dose dependent manner (Fig. 5C-E).…”

“…For live high-throughput imaging of caudal tails of wild-type and dovitinib-treated larvae we used the Large Particle (LP) Sampler and VAST BioImager (Union Biometrica) to automate the delivery of zebrafish from 96 multi-well plates to the BioImager microscope platform as previously described (Tuttle et al 2022). Z-stacks of 218 frames with a step size of 1.84 μm were acquired using Zen Blue 2.0 software (Carl Zeiss).…”

“…Caudal tail axon density was measured as previously described (Tuttle et al 2022). Briefly, we imported confocal images of RB:GFP caudal tails into FIJI (Schindelin et al 2012), converted images to 8-bit, and generated maximum intensity Z-projections.…”

“…RBs have become a popular model for screening drug neurotoxicity as well understanding axon damage and axon regeneration (Rieger and Sagasti 2011; Cuevas et al 2013; Doganli et al 2013; Wang et al 2015; Adula et al 2022). In particular, zebrafish RBs have been used to study the cellular and molecular bases of drug induced peripheral neuropathies (DIPNs) (Lisse et al 2016; Cadiz Diaz et al 2022; Tuttle et al 2022). DIPNs are caused by damage to the peripheral sensory nervous system as an unintended, off-target effect from many different types of small molecular drugs to treat cancers, including both cytotoxic and targeted therapeutics ( e.g .…”