Deciphering the genetic interactions between Pou4f3, Gfi1, and Rbm24 in maintaining mouse cochlear hair cell survival
- Institute of Neuroscience, State Key Laboratory of Neuroscience, CAS Center for Excellence in Brain Science and Intelligence Technology, Chinese Academy of Sciences, China
- University of Chinese Academy of Sciences, China
- Shanghai Center for Brain Science and Brain-Inspired Intelligence Technology, China
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-/-) …
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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
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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 …
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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 …
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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
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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…
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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
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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)…
Figure 4
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, …
Figure 5
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 …
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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
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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 resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (Mus musculus) | Atoh1Cre/+ | Yang et al., 2010b | Lin Gan lab (Augusta University) | A knock-in mouse strain |
| Strain, strain background (M. musculus) | Rosa26-loxp-stop-loxp-tdTomato (Ai9) | Stock# 007905 | The Jackson Laboratory | A knock-in mouse strain |
| Strain, strain background (M. musculus) | Ikzf2V5/+ | Stock# 038463 | The Jackson Laboratory | A knock-in mouse strain |
| Strain, strain background (Mus musculus) | Pou4f3 mutant | This paper | Available upon request from Liu Lab | Constructed by CRISPR-stop |
| Strain, strain background (M. musculus) | Gfi1 mutant | This paper | Available upon request from Liu Lab | Constructed by CRISPR-stop |
| Strain, strain background (M. musculus) | Rbm24 mutant | This paper | Available upon request from Liu Lab | Constructed by CRISPR-stop |
| Strain, strain background (M. musculus) | Pou4f3 null | This paper | Available upon request from Liu Lab | Constructed by CRISPR /Cas9 |
| Strain, strain background (M. musculus) | Gfi1 null | This paper | Available upon request from Liu Lab | Constructed by CRISPR /Cas9 |
| Strain, strain background (M. musculus) | Gfi13xHA-P2A-Cre | This paper | Available upon request from Liu Lab | A knock-in mouse strain |
| Strain, strain background (M. musculus) | Eh1-EGFP+ | This paper | Available upon request from Liu Lab | A transgenic mouse line |
| Strain, strain background (M. musculus) | Eh2-EGFP+ | This paper | Available upon request from Liu Lab | A transgenic mouse line |
| Strain, strain background (M. musculus) | Eh3-EGFP+ | This paper | Available upon request from Liu Lab | A transgenic mouse line |
| Strain, strain background (M. musculus) | Rosa26CAG-lsl-Rbm24*3xHA/+ | This paper | Available upon request from Liu Lab | A knock-in mouse strain for ectopic Rbm24 expression |
| Antibody | Anti-RBM24 (rabbit polyclonal) | Proteintech | 18178-1-AP | IF (1:500) |
| Antibody | Anti-POU4F3 (mouse monoclonal) | Santa Cruz | sc-81980 | IF (1:500) |
| Antibody | Anti-POU4F3 (rabbit polyclonal) | Novus Biologicals | NBP1-88349 | IF (1:500) |
| Antibody | Anti-HA (rat monoclonal) | Roche | 11867423001 | IF (1:500) |
| Antibody | Anti-MYO7A (rabbit polyclonal) | Proteus Biosciences | 25-6790 | IF (1:500) |
| Antibody | Anti-MYO7A (mouse monoclonal) | DSHB | MYO7A 138-1 | IF (1:500) |
| Antibody | Anti-CTIP2 (rat monoclonal) | Abcam | ab18465 | IF (1:500) |
| Antibody | Anti-INSM1 (guinea pig) | Jia et al., 2015 | A kind gift from Dr. Carmen Birchmeier | IF (1:6000) |
| Antibody | Anti-GFP (chicken polyclonal) | Abcam | ab13970 | IF (1:500) |
| Antibody | Anti-GATA3 (goat polyclonal) | R&D Systems | AF2605 | IF (1:500) |
| Antibody | Anti-V5 (mouse monoclonal) | Bio-Rad | MCA1360 | IF (1:500) |
Additional files
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Supplementary file 1
List of sgRNA sequences.
- https://cdn.elifesciences.org/articles/90025/elife-90025-supp1-v1.xlsx
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Supplementary file 2
DNA sequences of ssDNA donor and Rbm24 enhancers.
- https://cdn.elifesciences.org/articles/90025/elife-90025-supp2-v1.xlsx
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Supplementary file 3
DNA sequences of genotyping primers.
- https://cdn.elifesciences.org/articles/90025/elife-90025-supp3-v1.xlsx
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MDAR checklist
- https://cdn.elifesciences.org/articles/90025/elife-90025-mdarchecklist1-v1.docx
