The dark kinase STK32A regulates hair cell planar polarity opposite of EMX2 in the developing mouse inner ear
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eLife assessment
This important study advances our understanding of the molecular players downstream of the transcription factor Emx2 that establish planar cell polarity in hair cells of the mammalian inner ear. The conclusions, which are supported by compelling evidence, will be of interest to those studying the development and function of the vestibular system and mechanisms of planar cell polarity.
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Abstract
The vestibular maculae of the inner ear contain sensory receptor hair cells that detect linear acceleration and contribute to equilibrioception to 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 in mouse by regulating the distribution of the transmembrane receptor GPR156 at hair cell boundaries in one group of cells. However, the genes regulated by EMX2 in this context were previously not known. Using mouse as a model, 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 expression in hair cells on the opposite side. 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 maculae, with EMX2-mediated repression of Stk32a determining the final position of the LPR.
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eLife assessment
This important study advances our understanding of the molecular players downstream of the transcription factor Emx2 that establish planar cell polarity in hair cells of the mammalian inner ear. The conclusions, which are supported by compelling evidence, will be of interest to those studying the development and function of the vestibular system and mechanisms of planar cell polarity.
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Reviewer #1 (Public Review):
The authors initiated the study motivated by the lack of knowledge about the molecular events downstream of the polarity effector Emx2 in the mammalian inner ear, hypothesizing that some of those molecular players will be found by sequencing cells that normally express Emx2 in ears from Emx2-mutant mice.
The hypothesis is sound, the technologies used are standard and well-established, and the presented data is of high quality. The results largely support the authors' conclusions. However, the authors have not formally demonstrated that Stk32A is a transcriptional target of Emx2. It is clear that it is positioned downstream of the events triggered by Emx2, and that it can reverse Emx2 activity, but the data do not support the claim that the kinase is under direct transcriptional control of Emx2.
The …
Reviewer #1 (Public Review):
The authors initiated the study motivated by the lack of knowledge about the molecular events downstream of the polarity effector Emx2 in the mammalian inner ear, hypothesizing that some of those molecular players will be found by sequencing cells that normally express Emx2 in ears from Emx2-mutant mice.
The hypothesis is sound, the technologies used are standard and well-established, and the presented data is of high quality. The results largely support the authors' conclusions. However, the authors have not formally demonstrated that Stk32A is a transcriptional target of Emx2. It is clear that it is positioned downstream of the events triggered by Emx2, and that it can reverse Emx2 activity, but the data do not support the claim that the kinase is under direct transcriptional control of Emx2.
The revelation that Stk32A has two separate functions in planar polarity is significant.
The results will have a significant impact in the field because it provides one of the more persuasive molecular links between Emx2 and the polarization machinery.
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Reviewer #2 (Public Review):
Previous studies have shown that the transcription factor Emx2 controls mirror-image PCP along the line of polarity reversal (LPR) by regulating the trafficking of an orphan receptor GPR156. However, the underlying mechanism is unknown. Here, the authors provide evidence that Emx2 represses transcription of Stk32a, which, in turn, negatively regulates GPR156 surface expression, thereby coupling cell-intrinsic and tissue-level PCP in the vestibular sensory epithelia.
Overall, the data are clearly presented and largely convincing. Using RNA-seq and ISH and both loss- and gain-of-Emx2 in vivo, the authors show that Stk32a is expressed in a complementary domain to Emx2 via Emx2-mediated repression. Gain- and loss-of-Stk32a experiments demonstrate that Stk32a is required for hair cell PCP in the Emx2-negative …
Reviewer #2 (Public Review):
Previous studies have shown that the transcription factor Emx2 controls mirror-image PCP along the line of polarity reversal (LPR) by regulating the trafficking of an orphan receptor GPR156. However, the underlying mechanism is unknown. Here, the authors provide evidence that Emx2 represses transcription of Stk32a, which, in turn, negatively regulates GPR156 surface expression, thereby coupling cell-intrinsic and tissue-level PCP in the vestibular sensory epithelia.
Overall, the data are clearly presented and largely convincing. Using RNA-seq and ISH and both loss- and gain-of-Emx2 in vivo, the authors show that Stk32a is expressed in a complementary domain to Emx2 via Emx2-mediated repression. Gain- and loss-of-Stk32a experiments demonstrate that Stk32a is required for hair cell PCP in the Emx2-negative regions and is sufficient to reorient PCP in the Emx2-positive region. Moreover, Stk32a negatively regulates GPR156 localization to apical junctions without affecting core PCP proteins or Emx2 expression. However, there are several notable weaknesses, including a) because transcripts of the Stk32a mutant allele were still present, the nature of the Stk32a mutation is unclear; b) Mechanisms by which Emx2 represses Stk32a transcription were not addressed or discussed; c) Mechanisms by which Stk32a regulates GPR156 surface expression were not addressed. Addressing these issues at least partially would provide stronger support for the proposed model and improve the paper's impact.
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Reviewer #3 (Public Review):
The manuscript by Jia, Ratzan et al. is elegant and makes an important contribution to the hair cell and PCP field. Using a subtractive approach involving deep sequencing of the mouse Emx2 mutant and control mice, they identified Stk32a as a candidate gene regulated by EMX2. Next, they made a Stk32a mouse mutant and showed that STK32a is necessary/sufficient to determine hair bundle orientation in the vestibule. Moreover, they show that STK32A governs GPR156. The images are compelling. I have no major concerns.
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