Transcription factor Emx2 controls stereociliary bundle orientation of sensory hair cells
- University of Maryland, United States
- National Institute on Deafness and Other Communication Disorders, National Institutes of Health, United States
Figures
Figure 1 with 1 supplement
Regional expression of Emx2 in the maculae.
(A) Medial view of a mouse left inner ear with its six sensory organs (grey). Enlarged are the utricle and saccule showing their subdivisions, hair bundle polarity (arrows), LPR (yellow line), and …
Figure 1—figure supplement 1
Expression of Emx2 in sensory organs of the mouse inner ear.
(A–B) Whole mount in situ hybridization of the utricle showing Emx2 hybridization domain (A; n = 3) lateral to that of the β-tectorin-positive striola (B; n = 3). Neither genes are expressed in the …
Figure 2 with 2 supplements
The Emx2 lineage domain is present in Emx2 functional null maculae.
(A–C) and (I–K) are the same specimens as (C–E’) and (F–H’) in Figure 1. (A–C) In Emx2cre/+;RosatdT/+ utricles, the border of the Emx2 lineage domain (cyan line) coincides largely with LPR (yellow …
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Figure 2—source data 1
Sensory area quantifications for Emx2 mutant maculae.
- https://doi.org/10.7554/eLife.23661.005
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Figure 2—source data 2
Total hair cell number in utricles of loss- and gain-of-function Emx2 mutants.
- https://doi.org/10.7554/eLife.23661.006
Figure 2—figure supplement 1
Emx2 immunostaining in the Emx2Cre/+ maculae correlates with the reversed hair bundle polarity.
In the utricle (A–F”) and saccule (G–L”), the lineage (A,D,G,J, blue) and expression (A’,D’,G’,J’, green) domains of Emx2 are both restricted to the LES and IR, respectively. (C,F,I,L) show Emx2 …
Figure 2—figure supplement 2
Mutant phenotypes of Emx2Cre/Cre and Emx2Cre/- embryos are similar to Emx2 -/- mutants.
(A–B) The kidneys below the adrenal glands (dotted lines) are absent in Emx2Cre/Cre (B) compared to Emx2Cre/+ embryos (A). (C) The size of the cortex is smaller in Emx2Cre/Cre than Emx2Cre/+ …
Figure 3
Regional differences in timing of cell cycle exit are maintained in the Emx2-/- utricle.
(A–C’) E18.5 Emx2+/- utricles injected with EdU at E11.5 (A-A’; n = 3), E14.5 (B-B’; n = 3), or E16.5 (C-C’; n = 3) and harvested at E18.5 (G) were processed for anti-Myosin VIIa (HC marker), …
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Figure 3—source data 1
Timing of terminal mitosis in utricular HCs of Emx2 mutants.
- https://doi.org/10.7554/eLife.23661.010
Figure 4
Ectopic Emx2 reverses hair bundle polarity in the utricle.
Compared to the medial side of the LPR in controls (A–C), hair bundle polarity (arrows) in the medial region of Sox2CreER/+;RosaEmx2/+ (D-F, tamoxifen given at E12.5 and E13.5, gain-of-function …
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Figure 4—source data 1
Quantification of HC density in Emx2 gain-of-function mutants.
- https://doi.org/10.7554/eLife.23661.012
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Figure 4—source data 2
Quantification of apical surface area of HCs in Emx2 gain-of-function mutants.
- https://doi.org/10.7554/eLife.23661.013
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Figure 4—source data 3
Quantification of hair bundle orientation in Emx2 gain-of-function mutants.
- https://doi.org/10.7554/eLife.23661.014
Figure 5
Ectopic Emx2 reverses hair bundle polarity in the saccule and cristae but not organ of Corti.
(A–D) Saccule, (E–J) anterior and lateral cristae (AC and LC), as well as (K–N) organ of Corti of RosaEmx2/+ (A–B, E–G, K–L) and Gfi1Cre/+;RosaEmx2/+ (C–D, H–J, M–N) inner ears at E18.5 (n = 7). In …
Figure 6 with 2 supplements
Hair bundle polarity reversal is associated with reversed Gαi but not Pk2 localization in Emx2 mutants.
(A) Schematic diagram illustrates the distribution of Pk2 and Gαi in HCs across the LPR of a control utricle. (B–C) In control utricles, Pk2 staining (green) is concentrated on the medial side of …
Figure 6—figure supplement 1
Distribution of Vangl2 and Par6 immunoreactivites in apical HCs is not changed in Emx2 mutant utricles.
(A–B) In controls (n = 3), Vangl2 immunostaining (green) are restricted, stronger between adjacent supporting cells and weaker between HC and supporting cell junction. The staining pattern of Vangl2 …
Figure 6—figure supplement 2
Distribution of Pk2 immunoreactivites in apical HCs is maintained in Emx2 GOF-SE late mutant utricles.
In the GOF-SE late utricle (A–B"), hair bundles can be normal (black arrows), reversed (red arrows), or misoriented (blue arrows). However, the immunoreactivities of Pk2 is maintained on the medial …
Figure 7
Inhibition of Gαi reverses hair bundle orientation in the Emx2-positive domain of maculae.
(A–B’) In control utricles, hair bundles are pointing toward medial and lateral in the respective LES and MES (arrows), and Gαi staining (A’,B’) is associated with the kinocilium (A,B; n = 3). (C–D’)…
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Figure 7—source data 1
Quantification of hair bundle orientation in Ptx mutants.
- https://doi.org/10.7554/eLife.23661.020
Figure 8
Emx2-mediated hair bundle polarity reversal requires heterotrimeric G-proteins.
(A–H’) Compared to RosaPtx/+ controls (A–B), some hair bundles show reversed polarity in the LES (C–C’) but not the MES (D–D’) of Gfi1Cre/+;RosaPtx/+ utricles. Ectopic Ptx and Emx2 in Gfi1Cre/+;RosaE…
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Figure 8—source data 1
Quantification of hair bundle orientation in utricles of Ptx single and Ptx and Emx2 compound mutants.
- https://doi.org/10.7554/eLife.23661.022
Figure 9
Conserved expression of Emx2 in the three chicken maculae.
(A–C’) Anti-Emx2 immunostaining of the chicken utricle (A,A’), saccule (B,B’) and lagena (C,C’) at E12. (A–C) Anti-spectrin staining (red) shows opposite hair bundle orientation across the LPR …
Figure 10 with 1 supplement
emx2 regulates hair bundle polarity in neuromasts.
(A) Surface view of an A-P and D-V oriented neuromast and a sagittal view of the cellular architecture of a neuromast in the lateral line of zebrafish. (B) HCs with opposite orientation are in a 1:1 …
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Figure 10—source data 1
Quantification of hair bundle orientation in neuromasts of emx2 loss- and gain-of-function mutants.
- https://doi.org/10.7554/eLife.23661.025
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Figure 10—source data 2
Quantification of calcium-activated HCs with oriented stimuli in emx2 gain-of-function neuromasts.
- https://doi.org/10.7554/eLife.23661.026
Figure 10—figure supplement 1
Polarity phenotypes in the utricle and lateral crista of emx2 loss- and gain-of function zebrafish mutants.
(A) Two targeted sites of deletion (red site 1 and green site 2) in exon1 of emx2 using CRISPR/Cas9 technology. Guide targets (blue), PAM sequences (red), and examples of deletions found are shown. …
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