Wnt9a Can Influence Cell Fates and Neural Connectivity across the Radial Axis of the Developing Cochlea

Abstract

Vertebrate hearing organs manifest cellular asymmetries across the radial axis that underlie afferent versus efferent circuits between the inner ear and the brain. Therefore, understanding the molecular control of patterning across this axis has important functional implications. Radial axis patterning begins before the cells become postmitotic and is likely linked to the onset of asymmetric expression of secreted factors adjacent to the sensory primordium. This study explores one such asymmetrically expressed gene, Wnt9a, which becomes restricted to the neural edge of the avian auditory organ, the basilar papilla, by embryonic day 5 (E5). Radial patterning is disrupted when Wnt9a is overexpressed throughout the prosensory domain beginning on E3. Sexes were pooled for analysis and sex differences were not studied. Analysis of gene expression and afferent innervation on E6 suggests that ectopic Wnt9a expands the neural-side fate, possibly by re-specifying the abneural fate. RNA sequencing reveals quantitative changes, not only in Wnt-pathway genes, but also in genes involved in axon guidance and cytoskeletal remodeling. By E18, these early patterning effects are manifest as profound changes in cell fates [short hair cells (HCs) are missing], ribbon synapse numbers, outward ionic currents, and efferent innervation. These observations suggest that Wnt9a may be one of the molecules responsible for breaking symmetry across the radial axis of the avian auditory organ. Indirectly, Wnt9a can regulate the mature phenotype whereby afferent axons predominantly innervate neural-side tall HCs, resulting in more ribbon synapses per HC compared with abneural-side short HCs with few ribbons and large efferent synapses.

The acquisition of positional information across the radial axis of the cochlea

“…At this dose, several known Wnt target genes are upregulated, suggesting that Wnts can promote specification of medial sensory identity to both the IHCs and their associated supporting cells . When Wnt9a is overexpressed in the chicken cochlea, there is a similar increase in the tall hair cells (homologous to IHCs in the medial domain) and a nearly complete loss of the short hair cells (homologous to OHCs in the lateral domain) . The loss of these short hair cells is consistent with the effects brought about by Wnt activation in the mouse.…”

“…Both Kremen1 and GSK‐3β could prevent an excess number of IHCs and pillar cells; however, Wnt inhibition does not imply that Kremen1 and Gsk‐3β play a role in specifying hair cells/pillar cells but merely regulate their numbers. Therefore, the hypothesis that Wnts help specify IHCs/tall hair cells is still valid, even though Wnts are unlikely to be the only regulator of Atoh1 expression (Figures and ). As the field progresses, new regulators of Atoh1 will be identified.…”

“…28 When Wnt9a is overexpressed in the chicken cochlea, there is a similar increase in the tall hair cells (homologous to IHCs in the medial domain) and a nearly complete loss of the short hair cells (homologous to OHCs in the lateral domain). 99 The loss of these short hair cells is consistent with the effects brought about by Wnt activation in the mouse. Together these data suggest that there is evolutionary conservation in the ability of Wnts to promote a medial sensory fate.…”

Expression of class III Semaphorins and their receptors in the developing chicken (Gallus gallus) inner ear

“…However, we also expected Sema3D to show a similar distribution to Sema3F , and that Nrp2 would be in cochlear ganglion neurons. We further hypothesized that the down regulation of both genes in the presence of exogenous Wnt9a contributed to the increase in afferent innervation previously described (Munnamalai et al, ). In accordance with our RNA sequencing data, in situ hybridization shows a severe reduction of both Sema3D and Nrp2 in Wnt9a‐infected BPs (Figure e,g).…”

“…Neuronal cell adhesion molecule (NrCAM) has been shown to associate with Nrp2 (Julien et al, ) and recent work in the mouse inner ear has shown that it is involved in limiting ribbon synapses in the HCs, likely through inhibiting synapse formation or promoting pruning (Harley et al, ). RNA sequencing data failed to detect NrCAM transcripts in the chicken BP at E6 (Munnamalai et al, ), although this time point precedes synapse formation in this organ.…”