Wnts and Wnt inhibitors do not influence axon outgrowth from chicken statoacoustic ganglion neurons

The peripheral growth cones of statoacoustic ganglion (SAG) neurons are presumed to sense molecular cues to navigate to their sensory targets during development. Based on previously reported expression data for Frizzled receptors, Wnt ligands, and Wnt inhibitors, we hypothesized that some members of the Wnt morphogen family may function as repulsive cues for SAG neurites. The responses of SAG neurons to mammalian Wnts -1, -4, -5a, -6, and -7b, and the Wnt inhibitors sFRP -1, -2, and -3, were tested in vitro by growing SAG explants from embryonic day 4 (E4) chicken embryos for two days in 3D collagen gels. Average neurite length and density were quantified to determine effects on neurite outgrowth. SAG neurites were strongly repelled by human Sema3E, demonstrating SAG neurons are responsive under these assay conditions. In contrast, SAG neurons showed no changes in neurite outgrowth when cultured in the presence of Wnts and Wnt inhibitors. As an alternative approach, Wnt4 and Wnt5a were also tested in vivo by injecting retroviruses encoding these genes into the chicken otocyst on E3. On E6, no differences were evident in the peripheral projections of SAG axons terminating in infected sensory organs as compared to uninfected organs on the contralateral side of the same embryo. For all Wnt sources, bioactivity was confirmed on E6 chick spinal cord explants by observing enhanced axon outgrowth, as reported previously in the mouse. These results suggest that the tested Wnts do not play a role in guiding peripheral axons in the chicken inner ear.

► Chick SAGs are unresponsive to Wnts and Wnt inhibitors in vitro and in vivo.
► Reduced neurite outgrowth in the presence of Sema3E shows explants are responsive.
► Spinal cord assays confirm bioactivity of the tested molecules in chick.
► Results suggest SAG neurites may not use Wnts and sFRPs as guidance cues in vivo.