Deciphering the genetic interactions between Pou4f3, Gfi1, and Rbm24 in maintaining mouse cochlear hair cell survival

  1. Guangqin Wang
  2. Yunpeng Gu
  3. Zhiyong Liu  Is a corresponding author
  1. Institute of Neuroscience, State Key Laboratory of Neuroscience, CAS Center for Excellence in Brain Science and Intelligence Technology, Chinese Academy of Sciences, China
  2. University of Chinese Academy of Sciences, China
  3. Shanghai Center for Brain Science and Brain-Inspired Intelligence Technology, China
5 figures, 1 table and 4 additional files

Figures

Figure 1 with 2 supplements
RBM24 expression is repressed in cochlear hair cells (HCs) in the absence of POU4F3.

(A) Simple illustration of how Pou4f3-/- strain was generated. (B) One example gel image of tail-DNA PCR used to distinguish WT (Pou4f3+/+), heterozygous (Pou4f3+/-), and homozygous (Pou4f3-/-) …

Figure 1—source data 1

The original agarose gel image in Figure 1B (Pou4f3+/+, Pou4f3+/-, and Pou4f3-/-).

https://cdn.elifesciences.org/articles/90025/elife-90025-fig1-data1-v1.zip
Figure 1—source data 2

File containing Figure 1B and the original agarose gel analysis with highlighted bands and sample labels.

https://cdn.elifesciences.org/articles/90025/elife-90025-fig1-data2-v1.zip
Figure 1—figure supplement 1
RBM24 expression is repressed in cochlear hair cells (HCs) at E16.5 when POU4F3 is inactivated.

(A) Illustration of how Pou4f3-/- mice (Founder 0, F0) were rapidly generated using CRISPR-stop. (B–, C) Sanger sequencing chromatograms of WT (B) and F0 Pou4f3-/- (C) samples. The base ‘C’ (black …

Figure 1—figure supplement 2
Severe degeneration of Pou4f3-/- hair cells (HCs) occurs by perinatal ages.

(A–C’’) Dual staining of POU4F3 and MYO7A in middle-turn regions of cochleae from WT mice (A–A’’) at P1 and Pou4f3-/- mice at P0 (B–B’’) and P1 (C–C’’). The Pou4f3-/- mice were from a …

Figure 1—figure supplement 2—source data 1

File containing raw counts of the cochlear hair cells reported in the graph of Figure 1—figure supplement 2D.

https://cdn.elifesciences.org/articles/90025/elife-90025-fig1-figsupp2-data1-v1.xlsx
Figure 2 with 3 supplements
GFI1 is dispensable for RBM24 expression.

(A) Simple cartoon depicting how Gfi1 was inactivated in one-cell-stage zygotes derived from male Gfi1HA-Cre/+ mice by using the CRISPR-stop approach. (B, C) Using sgRNA-6 as an example, Sanger …

Figure 2—figure supplement 1
Construction of Gfi13◊HA-P2A-Cre/+ (Gfi1HA-Cre/+) mouse model in which HA expression faithfully represents GFI1 expression.

(A–C) Simple cartoon illustrating construction of Gfi1HA-Cre/+ strain. In the WT Gfi1 allele (A), a DNA fragment containing 3×HA-P2A-Cre (B) was inserted immediately before the TGA stop codon (red …

Figure 2—figure supplement 1—source data 1

The original agarose gel image in Figure 2—figure supplement 1D (Gfi1+/+, Gfi1HA-Cre/+, and Gfi1HA-Cre/ HA-Cre).

https://cdn.elifesciences.org/articles/90025/elife-90025-fig2-figsupp1-data1-v1.zip
Figure 2—figure supplement 1—source data 2

File containing Figure 2—figure supplement 1D and the original agarose gel analysis with highlighted bands and sample labels.

https://cdn.elifesciences.org/articles/90025/elife-90025-fig2-figsupp1-data2-v1.zip
Figure 2—figure supplement 2
GFI1 expression is lost in Pou4f3-/- hair cells (HCs).

(A) One-step generation of Pou4f3 mutants by using CRISPR-stop with zygotes derived from Gfi1HA-Cre/+ mice. (B, C) Sanger sequencing chromatograms of tail-DNA samples from control Gfi1HA-Cre/+ (B) …

Figure 2—figure supplement 3
RBM24 expression is normal in the absence of Gfi1.

(A) The DNA fragment between sgRNA-4 and sgRNA-9 within the Gfi1 locus was deleted using the CRISPR/Cas9 method. (B) One example gel image of tail-DNA PCR used to distinguish Gfi1+/+, Gfi1+/-, and Gf…

Figure 2—figure supplement 3—source data 1

The original agarose gel image in Figure 2—figure supplement 3B (Gfi1+/+, Gfi1+/-, and Gfi1-/-).

https://cdn.elifesciences.org/articles/90025/elife-90025-fig2-figsupp3-data1-v1.zip
Figure 2—figure supplement 3—source data 2

File containing Figure 2—figure supplement 3B and the original agarose gel analysis with highlighted bands and sample labels.

https://cdn.elifesciences.org/articles/90025/elife-90025-fig2-figsupp3-data2-v1.zip
Figure 3 with 1 supplement
POU4F3 and GFI1 expressions are normal in the absence of Rbm24.

(A) Simple cartoon showing how CRISPR-stop was used to induce mosaic inactivation of RBM24 in one-cell-stage mouse zygotes harboring Gfi1HA-Cre/+. Notably, the injection was in the late one-cell …

Figure 3—figure supplement 1
GATA3 is downregulated but IKZF2 is upregulated in Rbm24-deficient hair cells (HCs).

(A) Simple illustration of how mosaic Rbm24 mutant mice were generated. (B–C’’) Dual staining of RBM24 and GATA3 in cochlear tissues from control Rbm24+/+ (B–B’’) and Rbm24-/- (C–C’’) mice at P1. (D)…

Three Rbm24 enhancers are sufficient to drive specific EGFP expression in cochlear hair cells (HCs).

(A) Visualization of ATAC-seq (top row, orange) of P1 cochlear HCs (from our previous study, GSE181311) and POU4F3 Cut&Run assay (bottom row, blue) of E17.5 cochlear HCs (from another study, …

Forced expression of RBM24 fails to rescue outer hair cell (OHC) degeneration in Pou4f3-/-mutants.

(A) Generation of Rosa26loxp-stop-loxp-Rbm24*3×HA/+ (Rosa26Rbm24/+) mouse strain. The C-terminus of ectopic RBM24 was fused with three HA tags. (B) One example gel image of tail-DNA PCR used to …

Figure 5—source data 1

The original agarose gel image in Figure 5B (Rosa26+/+ and Rosa26Rbm24/+).

https://cdn.elifesciences.org/articles/90025/elife-90025-fig5-data1-v1.zip
Figure 5—source data 2

File containing Figure 5B and the original agarose gel analysis with highlighted bands and sample labels.

https://cdn.elifesciences.org/articles/90025/elife-90025-fig5-data2-v1.zip

Tables

Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Strain, strain background
(Mus musculus)
Atoh1Cre/+Yang et al., 2010bLin Gan lab
(Augusta University)
A knock-in mouse strain
Strain, strain background
(M. musculus)
Rosa26-loxp-stop-loxp-tdTomato (Ai9)Stock# 007905The Jackson LaboratoryA knock-in mouse strain
Strain, strain background
(M. musculus)
Ikzf2V5/+Stock#
038463
The Jackson LaboratoryA knock-in mouse strain
Strain, strain background
(Mus musculus)
Pou4f3 mutantThis paperAvailable upon request from Liu LabConstructed by CRISPR-stop
Strain, strain background
(M. musculus)
Gfi1 mutantThis paperAvailable upon request from Liu LabConstructed by CRISPR-stop
Strain, strain background
(M. musculus)
Rbm24 mutantThis paperAvailable upon request from Liu LabConstructed by CRISPR-stop
Strain, strain background
(M. musculus)
Pou4f3 nullThis paperAvailable upon request from Liu LabConstructed by CRISPR
/Cas9
Strain, strain background
(M. musculus)
Gfi1 nullThis paperAvailable upon request from Liu LabConstructed by CRISPR
/Cas9
Strain, strain background
(M. musculus)
Gfi13xHA-P2A-CreThis paperAvailable upon request from Liu LabA knock-in mouse strain
Strain, strain background
(M. musculus)
Eh1-EGFP+This paperAvailable upon request from Liu LabA transgenic mouse line
Strain, strain background
(M. musculus)
Eh2-EGFP+This paperAvailable upon request from Liu LabA transgenic mouse line
Strain, strain background
(M. musculus)
Eh3-EGFP+This paperAvailable upon request from Liu LabA transgenic mouse line
Strain, strain background
(M. musculus)
Rosa26CAG-lsl-Rbm24*3xHA/+This paperAvailable upon request from Liu LabA knock-in mouse strain for ectopic Rbm24 expression
AntibodyAnti-RBM24 (rabbit polyclonal)Proteintech18178-1-APIF (1:500)
AntibodyAnti-POU4F3
(mouse monoclonal)
Santa Cruzsc-81980IF (1:500)
AntibodyAnti-POU4F3
(rabbit polyclonal)
Novus BiologicalsNBP1-88349IF (1:500)
AntibodyAnti-HA
(rat monoclonal)
Roche11867423001IF (1:500)
AntibodyAnti-MYO7A
(rabbit polyclonal)
Proteus Biosciences25-6790IF (1:500)
AntibodyAnti-MYO7A
(mouse monoclonal)
DSHBMYO7A 138-1IF (1:500)
AntibodyAnti-CTIP2
(rat monoclonal)
Abcamab18465IF (1:500)
AntibodyAnti-INSM1 (guinea pig)Jia et al., 2015A kind gift from Dr. Carmen BirchmeierIF (1:6000)
AntibodyAnti-GFP
(chicken polyclonal)
Abcamab13970IF (1:500)
AntibodyAnti-GATA3
(goat polyclonal)
R&D SystemsAF2605IF (1:500)
AntibodyAnti-V5
(mouse monoclonal)
Bio-RadMCA1360IF (1:500)

Additional files

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