Planar Cell Polarity Signaling Guides Cochlear Innervation

Summary

Core planar cell polarity (PCP) proteins are linked to mechanisms of growth cone turning in response to Wnt ligands and direct the cellular organization and planar polarity of neurons, neuroepithelia and supporting cells, many of which are also the substrates along which growth cones navigate (Onishi et al., 2014; Tissir and Goffinet, 2013). Genetic manipulations in the mouse further demonstrate that PCP proteins along the migratory path can have a non-cell autonomous influence on growth cone turning (Ghimire and Deans, 2019; Ghimire et al., 2018) and neuronal migration (Davey et al., 2016; Glasco et al., 2012; Qu et al., 2010). For example, in the cochlea a distinctive 90-degree turn made by the peripheral axons of type II Spiral Ganglion Neurons (SGN) is dependent upon the PCP protein Vangl2 and the Frizzled receptors Fzd3 and Fzd6. When the corresponding genes are removed from non-neuronal supporting cells at the initial site of axon turning, the outcome of the turn is randomized (Ghimire and Deans, 2019; Ghimire et al., 2018). Together these observations demonstrate that the planar polarized development or organization of the cochlear environment through which these growth cones navigate has a substantive impact on pathfinding and axon projections. The outstanding question remains how planar polarization of cochlear supporting cells contributes to growth cone behavior, axonal trajectories and connectivity. Possibilities include the a priori structural polarization of cells that act as physical guides, the polarized distribution of axon guidance cues within these cells, or direct intercellular PCP signaling between supporting cells and navigating growth cones. The caudal migration of facial branchiomotor neurons (FBMN) is similarly dependent upon the planar polarized distribution of PCP proteins in the neuroepithelial cell substrate along which they migrate (Davey et al., 2016). This raises the broader question of whether planar polarization of cells along a growth cone’s trajectory might contribute as a general mechanism of axon guidance, or whether this mechanism is a specialized adaptation unique to the cochlea.

Outer hair cell innervation requires axon turning towards the cochlear base

The neural basis of audition begins with sensory receptors in the cochlea of the inner ear called hair cells that are arranged in distinct rows running the length of the cochlear spiral. There are two types of auditory hair cells innervated by two different classes of afferent Spiral Ganglion Neurons (SGN) that contribute to distinct auditory functions (Zhang and Coate, 2017). Sounds that are perceived and used for communication or localization are detected by inner hair cells (IHC) that are exclusively innervated by type I SGNs. Outer hair cells (OHC) are the second type and contribute to cochlear amplification mechanisms that modulate auditory function. The OHCs are exclusively innervated by the type II SGNs which have poorly understood function, but resemble nociceptors and are activated by intense sounds and OHC damage, and therefore may be important for maintaining auditory homeostasis (Flores et al., 2015; Liu et al., 2015; Nowak et al., 2021). This could be significant because OHCs are vulnerable to chemical and auditory trauma, and their death is frequently associated with deafness and hearing loss.

The most striking feature of type II SGNs is the morphology of their peripheral axons which make distinctive 90-degree turns towards the base of the cochlear spiral in order to innervate multiple OHCs within a single row (Figure 1) (Berglund and Ryugo, 1987). Although they are post-synaptic afferent neurons, the SGN process contacting hair cells are typically considered peripheral axons because type I SGNs are myelinated and because both types are capable of initiating action potentials (Weisz et al., 2009; Zhang and Coate, 2017). The type II SGN synapse with OHCs also contains synaptic vesicles and may be reciprocal and likely modulatory (Thiers et al., 2008). A compelling aspect of type II SGN peripheral axon turning and anatomy is that it is coordinated with the tonotopic organization of the cochlear spiral. Hair cells in the cochlear base are tuned to detect higher frequency sounds than hair cells located closer to the cochlear apex. As a result, peripheral axon turning towards the base ensures that individual type II SGNs always receive input from hair cells detecting a quarter-octave range of higher frequency sounds than adjacent type I SGNs (Brown, 1987). The guidance events directing this innervation pattern show remarkable fidelity, and there are very few axon turning errors irrespective of where the type II SGN is positioned along the length of the cochlear spiral.

Figure 1: type II spiral ganglion neurons innervate outer hair cells of the mammalian cochlea.

(A) The peripheral axon of the type II SGN turns 90 degrees to project towards the cochlear base while the central axon exits the cochlea through the VIIIth cranial nerve. (B) Enlarged schematic corresponding to the boxed region in ‘A’ illustrating type I SGN innervation of IHCs and type II SGN turning and innervation of the OHCs. (C) Neurofilament 200 (NF200) immunolabeling of type I (arrowheads) and type II SGN (arrows) peripheral axons in the newborn mouse.

Growth cone turning towards the cochlear base requires Intercellular PCP signaling in the environment

Recent studies demonstrate that PCP signaling is required to guide cochlear innervation by the type II SGNs.It is well established that auditory hair cells rely upon PCP signaling with neighboring supporting cells to coordinate the orientation of polarized bundles of stereocilia located on their apical cell surfaces (Curtin et al., 2003; Montcouquiol et al., 2003; Wang et al., 2005; Wang et al., 2006). Stereociliary bundles consists of elongated microvilli called stereocilia that are arranged in rows of increasing height with the tallest adjacent to a laterally displaced cilium called the kinocilium. Polarization of this bundle underlies the mechanotransduction mechanism crucial for the detection of sound (Gillespie and Muller, 2009). Due to the exquisite precision of hair cell and stereociliary bundle patterning this system is an ideal model for studying PCP signaling in vertebrates.

Intercellular polarity signals are relayed between neighboring cells by the core PCP proteins that include the receptor Frizzled (Fzd) and its cytoplasmic effector Dishevelled (Dvl in mammals), the transmembrane protein Van Gogh (Vangl1/2 in mammals) and its associated protein Prickle (Pk1/2 in mammals), and the large cadherin Flamingo/Starry Night (CELSR1/2/3 in mammals). In Drosophila these proteins are asymmetrically localized along the poles of individual cells with complexes of Flamingo, Frizzled and Dishevelled opposite to those of Flamingo, Van Gogh and Prickle. Intercellular signals are relayed at intercellular junctions where the extracellular domains of Flamingo and Frizzled oppose those of Flamingo and Van Gogh in the neighboring cell (Goodrich and Strutt, 2011; Simons and Mlodzik, 2008). These aspects of signaling and interdependent protein localization are well conserved for Vangl1/2, Frizzled3/6 and CELSR1/2/3 in the vertebrate inner ear (Duncan et al., 2017; Stoller et al., 2018; Wang et al., 2006). Intercellular PCP signaling also guides dynamic events such as cell migration and convergent extension movements that precede neural tube closure, though in this context it is more difficult to visualize the polarized distribution of core PCP proteins because they are highly dynamic (Butler and Wallingford, 2017).

The peripheral axon of the type II SGN turns towards the cochlear base in response to PCP signaling between cochlear supporting cells that surround the hair cells and line the axon trajectory. In the absence of Vangl2, or the combined loss of Fzd3 and Fzd6, axonal projections are randomized with an equal proportion of axons projecting towards the cochlear apex and base (Ghimire and Deans, 2019; Ghimire et al., 2018). Although these proteins are also necessary for hair cell PCP, the PCP signals guiding axon turning and hair cell development are likely independent because turning is initiated prior to OHC contact at an inferior position within the cochlea, and turning errors occur when Vangl2 is removed specifically from the supporting cells at this location (Ghimire et al., 2018). Furthermore, turning errors occur in the CELSR1 knockout cochlea which has normally organized stereociliary bundles further indicating that growth cone turning and hair cell polarity are independent (Duncan et al., 2017; Ghimire et al., 2018). PCP function in this context is also non-cell autonomous because spatially restricted conditional knockouts in which vangl2 or Frizzled3/6 are removed from cochlear supporting cells have similar phenotypes as constitutive KOs (Ghimire and Deans, 2019; Ghimire et al., 2018). In contrast, no phenotypes were seen when Vangl2 was selectively removed from SGNs. While genetic redundancy may account for this negative result, that seems unlikely since frequency of turning errors is not increased when both Vangl1 and Vangl2 are targeted (Ghimire et al., 2018). One possibility is that the PCP-dependent guidance mechanism in the cochlea is distinct from commissural axons of the neural tube where Vangl2 is known to function within the neuronal growth cone (Onishi and Zou, 2017; Shafer et al., 2011) alongside CELSR3 and Dvl2 (Onishi et al., 2013; Onishi et al., 2020). The non-autonomous phenotypes in the cochlea are also inconsistent with a model in which the growth cones respond to a gradient of Wnt because that signaling pathway also requires PCP function in the growth cone. Instead, Wnt signaling in the cochlea may act to establish the distribution of PCP proteins within the environment prior to innervation (Ghimire and Deans, 2019).

What is the function of PCP signaling in the environment and how does it impact growth cone behavior?

Current evidence from KO and CKO mouse models demonstrate that a component of PCP signaling in the developing cochlea is non-autonomous, and is required in cochlear supporting cells during type II SGN innervation. The cellular environment through which the peripheral axon projects is structurally complex and diverse, containing seven populations of molecularly and structurally distinct supporting cells (Kolla et al., 2020) that intercalate to form a crystalline array running the length of the cochlear spiral. One possibility is that PCP signaling directs the structural polarization of these cells and that these structures subsequently guide growth cone behavior. The Deiters cells are one class of supporting cell that show distinct planar polarization organized along the length of the cochlear spiral orientated parallel to the type II SGN peripheral axons (Figure 2A). Deiters cells are located beneath the OHCs in a stratified epithelium and extend an apical process towards the apex of the cochlear spiral. As a result, the Deiters cell’s soma and apical surface contact different hair cells. In Vangl2 CKOs, the development of this apical process is disrupted, often failing to project towards the cochlear apex, and demonstrating that supporting cells are planar polarized in a PCP dependent manner (Copley et al., 2013). However, despite this striking polarized structure, it is unlikely that the apical processes directly guide peripheral axon turning because they project in opposite directions. Moreover, growth cone turning requires expression of the transcription factor Proxl in SGNs (Fritzsch et al., 2010) where it could regulate expression of axon guidance receptors. This requirement is more consistent with growth cone turning occurring in response to axon guidance cues rather than structural and organization changes in cochlear supporting cells.

Figure 2: Planar polarization of cochlear supporting cells guides peripheral axon turning.

(A) Deiters cells (DC) sit below OHCs in a stratified epithelia and extend a planar polarized apical process (examples marked by arrowheads) towards the cochlear apex. (B) Peripheral axon turning may be guided by the PCP-dependent distribution of conventional axon guidance cues. A repellent could promote turning towards the cochlear base. (C) Alternatively, PCP signaling could promote turning directly via transient intercellular signaling complexes formed between the growth cone and cochlear supporting cells.

A more likely scenario is that PCP signaling establishes the molecular polarization of supporting cells and thereby directs signaling pathways guiding growth cone behavior. PCP proteins are asymmetrically distributed at the junctions between the basolateral surfaces of neighboring supporting cells (Ghimire and Deans, 2019; Ghimire et al., 2018), in addition to their well described distribution at the apical surface at cell boundaries between hair cells and supporting cells (Deans et al., 2007; Duncan et al., 2017; Montcouquiol et al., 2006; Wang et al., 2006). Remarkably, the polarized distribution of PCP proteins at these non-hair cell junctions are oriented perpendicular to the polarized distribution of PCP proteins at junctions containing hair cells (Ghimire and Deans, 2019; Ghimire et al., 2018; Goodyear et al., 2017). As a result, the PCP axis found in supporting cells is correctly oriented relative to the long axis of the cochlea to distribute conventional axon guidance cues in a pattern that could direct turning (Figure 2B). Such a cue would need to be membrane tethered in order to be maintained along one cell boundary of the supporting cells, but based upon its location it could either an attractant or a repellent. In other contexts, PCP signaling can influence the activity of conventional axon guidance cues, for example CELSR3 is required for EphrinA-EphA dependent innervation of the hindlimb (Chai et al., 2014). Although the examples of this are limited, based upon these observations Ghimire et al. suggested that the molecular polarization of supporting cells distributes a repellent along the side of supporting cells facing the cochlear base thereby promoting growth cone turning towards the base (Ghimire et al., 2018).

An exciting alternative is that PCP signaling between the supporting cells and the growth cone is sufficient to bias the growth cone trajectory as they pass between adjacent supporting cells. This molecular integration could be mediated by Frizzled signaling within the growth cone if it transiently interacts with Vangl2 in the environment. Thus, the growth cone is interpreting the planar polarized patterning of the environment through which it travels via transient PCP signaling events mediated by the core PCP complex (Figure 2C). Consistent with this possibility, facial branchiomotor neurons migrate from rhombomere 4 in the hindbrain posteriorly to rhombomere 6, and neuronal migration is also dependent upon PCP signaling. Furthermore, the direction of migration is disrupted in Vangl2, Frizzled and CELSR mutants in mouse and zebrafish (Glasco et al., 2012; Qu et al., 2010; Wada et al., 2006). Moreover, these processes require PCP proteins in the environment and therefore also have a non-cell autonomous component. More significantly, Davey et al. demonstrated that intercellular PCP signals are directly relayed between filopodia on the migrating cells and neuroepithelia along the migratory path (Davey et al., 2016). Since many aspects of cytoskeletal rearrangement are conserved between migrating cells and growth cones it is possible that similar PCP signaling mechanisms occurring in facial branchiomotor neurons also occur in the type II SGN peripheral axon growth cone. A caveat to this model may be that Vangl2 was not required in the growth cone (Ghimire et al., 2018), although Fzd3 and Pk1 do function in type I SGNs to regulate their central projections (Duncan et al., 2019; Stoner et al., 2021; Yang et al., 2017) and PCP functions in type II SGN peripheral growth cones may be masked by genetic redundancy since the full complement of PCP proteins present in this structure are not known.

Conclusion

Several recent papers from my lab suggest that PCP signaling between supporting cells of the developing cochlea influences cochlear innervation and growth cone turning by polarizing cells along the axonal trajectory (Ghimire and Deans, 2019; Ghimire et al., 2018). Based upon these intercellular PCP signals I have proposed three models which are not mutually exclusive and account for our current understanding of cochlear development. Notably, these models each ensure the precise regulation of turning throughout the cochlear spiral which, given the length of the cochlea would be difficult to accomplish using morphogen gradients alone. The outstanding question that will distinguish these alternatives is the identity of the receptor(s) that function in the growth cone as they navigate through the rich microenvironment of the developing cochlea.