Comprehensive transcriptome analysis of cochlear spiral ganglion neurons at multiple ages
- Chinese Academy of Sciences, China
- Howard Hughes Medical Institute, Janelia Research Campus, United States
- Shanghai Center for Brain Science and Brain-Inspired Intelligence Technology, China
Figures
Figure 1 with 1 supplement
Genetic model, bulk RNA-Seq methodology, and qPCR quality check.
(A–A’) SGNs were endogenously labeled with tdTomato at P1 (or other ages after E15.5) in the mouse line ShhCre/+; Rosa26-CAG-loxp-stop-loxp-tdTomato/+. Arrow (in A) indicates SGN fibers with weak …
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Figure 1—source data 1
Neuronal gene was enriched, HC and glial genes were depleted in SGNs at P8.
- https://cdn.elifesciences.org/articles/50491/elife-50491-fig1-data1-v2.xlsx
Figure 1—figure supplement 1
qPCR analysis of Mafb, Sox10, and Myosin-VI in SGNs at E15.5, P1, P14, and P30.
(A–A’) Mafb was significantly enriched in SGNs at all four ages, similar to SGNs at P8 (described in Figure 1D). In agreement with the finding that Mafb expression decreased during aging, younger …
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Figure 1—figure supplement 1—source data 1
Neuronal gene was enriched, HC and glial genes were depleted in SGNs at E15.5, P1, P14 and P30.
- https://cdn.elifesciences.org/articles/50491/elife-50491-fig1-figsupp1-data1-v2.xlsx
Figure 2 with 3 supplements
SGN-specific genes showing constant and dynamic expression.
(A) Our selection criteria for constant expression were met by 21 SGN-specific genes, which were expressed in SGNs at all ages but not expressed in HCs or glia. (B) Dynamic expression was exhibited …
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Figure 2—source data 1
Scrt2 and Celf4 expression level by RNA-Seq and q-PCR analyisis.
- https://cdn.elifesciences.org/articles/50491/elife-50491-fig2-data1-v2.xlsx
Figure 2—figure supplement 1
Summary of our bulk RNA-Seq data.
(A) Total number of reads per sample. (B) Percentage of reads mapped to the mm10 genome. (C) Number of genes with TPM (transcripts per million) values of >1. Same sample names are used for panels A, …
Figure 2—figure supplement 2
Post-sequencing quality check by using markers for multiple cell types.
We used markers for 10 cell types including SGNs, HCs, and glial cells. Moreover, to verify RNA quality and integrity, we checked to ensure the expression in all samples of the housekeeping genes Gap…
Figure 2—figure supplement 3
RNA in situ hybridization of SGN-specific genes.
RNA in situ hybridization at P1 was used for validation of SGN-specific genes (four each) showing (A–D) constant expression (Elavl4, Esrrg, Scrt1, and Scrt2) and (E–H) dynamic expression (Stmn3, Shox…
Figure 3
Computational analysis SGN genes without considering whether they are SGN-specific or not.
(A) Cartoon depicting definition of up-regulation (u), down-regulation (d), and unchanged expression (-) during gene transitions between two neighboring ages (arrows). (B) The unchanged category …
Figure 4 with 3 supplements
Scrt2 is expressed in SGNs but not HCs or glia at E15.5 and P1.
(A) Illustration of the genetically modified Scrt2 locus. Detailed information please refer to Figure 4—figure supplement 1. (B–C) Double-labeling for HC-marker Myosin-VI and tdTomato in cochlear …
Figure 4—figure supplement 1
Generation of Scrt2-P2A-tdTomato (Scrt2-tdTomato) knockin mouse strain.
(A–C) Illustration of wild-type Scrt2 allele (A), gene-targeting vector (B), and final Scrt2 allele after correct gene targeting (C). (D–E) Southern blotting with external 3ʹ probe and genomic DNA …
Figure 4—figure supplement 2
Scrt2 is not expressed in neuroblasts at E10.5 but is expressed in SGNs at E13.5.
(A–A’’) Double-labeling of for Sox2 and tdTomato in cryosection tissues from Scrt2-tdTomato/+ mice at E10.5; tdTomato+ cells were detected in the hindbrain, but not in the otocyst (ot), an early …
Figure 4—figure supplement 3
Scrt2 expression is maintained in SGNs at P8, P14, and P30.
(A–B’’’) Confocal cryosection images of inner ear tissues from Scrt2-tdTomato/+ mice at P8, visualized at (A) low resolution, based on tdTomato fluorescence; and (B–B’’’) high resolution, based on …
Figure 5 with 1 supplement
Celf4 is detected in SGNs at E15.5 and further increased at P1 and P30.
(A) Illustration of the genetically modified Celf4 locus. Detailed information please refer to Figure 5—figure supplement 1. (B) Cochlear cryosectioned tissues from Celf4-3xHA-P2A-iCreER-T2A-EGFP/+ …
Figure 5—figure supplement 1
Generation of Celf4-3xHA-P2A-iCreER-T2A-EGFP (Celf4-iCreER) knockin mouse strain.
(A–C) Illustration of wild-type Celf4 locus (A), gene-targeting vector (B), and final Celf4 allele after correct gene targeting (C). (D–E) Southern blotting performed using internal probe (D) and …
Figure 6 with 2 supplements
Fate-mapping analysis of Celf4-iCreER/+; Rosa26-CAG-loxp-stop-loxp-tdTomato/+ mice across multiple ages.
(A) Illustration of fate-mapping analysis method. (B–D’’) Tamoxifen was injected at P1 and P2, and analysis was performed at P11. We detected tdTomato+ cells in SGN and VGN areas based on tdTomato …
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Figure 6—source data 1
Percentage of tdtomato+ SGNs when tamoxifen was given at postnatal ages.
- https://cdn.elifesciences.org/articles/50491/elife-50491-fig6-data1-v2.xlsx
Figure 6—figure supplement 1
Besides SGNs, mesenchymal cells in the cochlea express Celf4.
(A) Celf4-iCreER/+; Rosa26-CAG-loxp-stop-loxp-tdTomato/+ mice were administered tamoxifen at P1 and P2 and analyzed at P11 by using the whole-mount approach, with scanning at different layers …
Figure 6—figure supplement 2
Fate-mapping analysis of Celf4-iCreER/+; Rosa26-CAG-loxp-stop-loxp-tdTomato/+ mice at E10.5.
(A–C) Co-labeling for tdTomato and Myosin-VI in whole-mount cochlear samples from Celf4-iCreER/+; Rosa26-CAG-loxp-stop-loxp-tdTomato/+ mice that were administered tamoxifen at E10.5 and analyzed at …
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Figure 6—figure supplement 2—source data 1
Percentage of tdtomato+ SGNs when tamoxifen was given at E10.5.
- https://cdn.elifesciences.org/articles/50491/elife-50491-fig6-figsupp2-data1-v2.xlsx
Figure 7 with 1 supplement
Scrt2 and Celf4 are expressed in both Type I and Type II SGNs in adult mouse cochlea.
(A–B’’) Co-labeling of tdTomato and Gata3 in cryosection cochlear samples from Scrt2-P2A-tdTomato/+ mice at P30 (A–A’’) and Celf4-iCreER/+; Rosa26-CAG-loxp-stop-loxp-tdTomato/+ mice that were …
Figure 7—figure supplement 1
Scrt2 and Celf4 are also expressed in adult brain.
(A–E’’’) We detected tdTomato+ cells in the olfactory bulb (A), cortex (B), hippocampus (C), and cerebellum (D) of Celf4-iCreER/+; Rosa26-CAG-loxp-stop-loxp-tdTomato/+ mice that were administered …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (Mus musculus) | ShhCre/+ | Liu et al., 2010 | Jackson Lab | stock #: 005622 |
| Strain, strain background (Mus musculus) | Atoh1-CreER+ | Chow et al., 2006 | MMRRC | stock #: 029581-UNC |
| Strain, strain background (Mus musculus) | vGlut3-iCreER/+ | Li et al., 2018 | Available upon request from Liu Lab | Please contact (zhiyongliu@ion.ac.cn) |
| Strain, strain background (Mus musculus) | Scrt2-P2A-tdTomato/+ | It is a knockin mouse line where tdTomato can reflect Scrt2 mRNA expression pattern | Jackson Lab | stock #: 034390 (will be deposited soon) |
| Strain, strain background (Mus musculus) | Celf4-3xHA-P2A-iCreER-T2A-EGFP/+ | It is a knockin mouse line where HA is tagged at Celf4 protein c terminus. In addition, iCreER and EGFP are controlled by Celf4 promoter/enhancer | Jackson Lab | stock #: 034391 (will be deposited soon) |
| RNA extraction kit | PicoPure RNA Isolation Kit | Thermo Scientific | Cat#: KIT0204 | Extracting RNA from isolated cells |
| cDNA generation kit | Ovation RNA-Seq V2 | Tecan Genomics | Cat#:7102–32 | Converting RNA to cDNA |
| Library construction kit | Ovation Rapid DR Multiplex System | Tecan Genomics | Cat#: 0319–32 | Generating sequencing library with eight samples multiplexing |
| Digestion enzyme | Protease | Sigma | Cat#: P5147 | Digesting inner ear tissues |
Additional files
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Supplementary file 1
SGN genes with different dynamic patterns.
- https://cdn.elifesciences.org/articles/50491/elife-50491-supp1-v2.xlsx
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Supplementary file 2
Genotyping and q-PCR primers.
- https://cdn.elifesciences.org/articles/50491/elife-50491-supp2-v2.xlsx
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Supplementary file 3
Sequences of 8 RNA probes.
- https://cdn.elifesciences.org/articles/50491/elife-50491-supp3-v2.docx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/50491/elife-50491-transrepform-v2.docx
