The dark kinase STK32A regulates hair cell planar polarity opposite of EMX2 in the developing mouse inner ear

Abstract

The vestibular maculae of the inner ear contain sensory receptor hair cells that detect linear acceleration, contribute to equilibrioception, and thereby coordinate posture and ambulatory movements. These hair cells are divided between two groups, separated by a line of polarity reversal (LPR), with oppositely oriented planar-polarized stereociliary bundles that detect motion in opposite directions. The transcription factor EMX2 is known to establish this planar polarized organization by regulating the distribution of the transmembrane receptor GPR156 at the hair cell surface in one group of cells, however those genes regulated by EMX2 in this context were previously not known. We have identified the serine threonine kinase STK32A as a downstream effector negatively regulated by EMX2. Stk32a is expressed in hair cells on one side of the LPR in a pattern complementary to Emx2 due to transcriptional repression. Stk32a is necessary to align the intrinsic polarity of the bundle with the core planar cell polarity (PCP) proteins in EMX2-negative regions, and is sufficient to reorient bundles when ectopically expressed in neighboring EMX2-positive regions. We demonstrate that STK32a reinforces LPR formation by regulating the apical localization of GPR156. These observations support a model in which bundle orientation is determined through separate mechanisms in hair cells on opposite sides of the LPR, with EMX2-mediated repression of Stk32a determining the position of the LPR.

Emx2Lineage Tracing Reveals Antecedent Patterns of Planar Polarity in the Mouse Inner Ear

“…EMX2 is not found in the semi-circular canals or their associated cristae (Fig.1A). These patterns of expression have been previously shown by endogenous protein and gene expression and also genetic labeling with Emx2 -Cre (Kimura, Suda et al 2005, Holley, Rhodes et al 2010, Jiang, Kindt et al 2017, Stoller, Roman et al 2018, Jia, Ratzan et al 2023).…”

“…Using Cre-ERt2 mediated genetic lineage tracing we demonstrate that the planar polarity transcription factor EMX2 is expressed in the prosensory domain of the developing inner ear prior to hair cell specification and before segregation of the utricle and saccule into discrete sensory organs. In addition, we show that the lineage of Emx2-expressing cells from this stage is restricted to one side of the LPR in the mature sensory Stoller, Roman et al 2018, Jia, Ratzan et al 2023. However, unlike Emx2-Cre, the Emx2-CreERt2 line does not disrupt EMX2 function as demonstrated by the lack of mutant phenotypes when the transgene is homozygosed.…”

“…These observations were made using a new mouse line in which CreERt2 is expressed and regulated by endogenous Emx2 gene regulatory elements. Based upon patterns of Cre-mediated genetic labeling, the expression of Emx2 -CreERt2 matches Emx2 -Cre and patterns of Emx2 expression determined by immunolabeling and ISH (Holley, Rhodes et al 2010, Jiang, Kindt et al 2017, Stoller, Roman et al 2018, Jia, Ratzan et al 2023). However, unlike Emx2 -Cre, the Emx2 -CreERt2 line does not disrupt EMX2 function as demonstrated by the lack of mutant phenotypes when the transgene is homozygosed.…”

“…EMX2 function is similar in the lateral line neuromasts of zebrafish where Emx2 is only expressed in half the hair cells and these have the opposite stereociliary bundle orientations as the Emx2 -negative hair cells (Jiang, Kindt et al 2017, Lozano-Ortega, Valera et al 2018, Kozak, Palit et al 2020). Despite these well characterized effects on hair cell differentiation, Emx2 expression occurs in the otic vesicle at developmental stages before hair cell specification, and bundle orientation is dependent the polarity effectors GPR156 (Kindt, Akturk et al 2021) and STK32A (Jia, Ratzan et al 2023) that are expressed exclusively in hair cells and function downstream of EMX2. The early and broad pattern of Emx2 expression led us to hypothesize that EMX2 pre-determines the position of the LPR by patterning the prosensory domain shortly after its formation in the otic vesicle, and prior to segregation of the utricle and saccule.…”

Inhibitory G proteins play multiple roles to polarize sensory hair cell morphogenesis

“…EMX2 activates the GPR156 receptor by triggering its polarized enrichment at the apical HC junction, where downstream GNAI/O signaling appears to reverse how core PCP cues are interpreted and to repel the basal body (Figure 9 , role b) (Kindt et al, 2021 ). EMX2 achieves this goal by preventing expression of the kinase STK32A that suppresses apical enrichment and polarization of the GPR156 protein (Jia et al, 2023 ). It is important to note that HC lacking GPR156 have a normal apical cytoskeleton, including a normally formed hair bundle, and only show orientation defects (Kindt et al, 2021 ).…”