The conserved RNA helicase YTHDC2 regulates the transition from proliferation to differentiation in the germline
- Stanford University School of Medicine, United States
- Academic Medical Center, University of Amsterdam, Netherlands
Figures
Figure 1 with 1 supplement
A DExH box helicase conserved from Drosophila to mammals is expressed in male germ cells entering meiosis.
(A) Cladogram of Bgcn-related proteins in Drosophila melanogaster (dm), Mus musculus (mm), Homo sapiens (hs) and Caenorhabditis elegans (ce). Protein sequences from the NCBI protein database. (B) …
Figure 1—figure supplement 1
YTHDC2 expression during the first wave of spermatogenesis.
(A-C′) Immunofluorescence images of wild-type seminiferous tubule cross-sections from (A, A′) P10, (B, B′) P12, and (C, C′) P14 testes stained for YTHDC2 (white above, green below), DDX4 (red) and …
Figure 2 with 2 supplements
Adult Ythdc2 mutant male germ cells fail to properly execute meiosis.
(A, B) Whole mount images of (A) wild-type and (B) Ythdc2-/- testes from 12-week-old male mice. (C) Testis weights from 8-week-old adult wild-type and Ythdc2-/- mice (n = 6 testes per group; t test, …
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Figure 2—source data 1
Testis weights in adult wild-type and Ythdc2-/- mice.
- https://doi.org/10.7554/eLife.26116.007
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Figure 2—source data 2
Number of TUNEL + cells per tubule cross-section in wild-type and Ythdc2-/- testes.
- https://doi.org/10.7554/eLife.26116.008
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Figure 2—source data 3
Percentage of P30 tubule cross-sections containing indicated cell types in wild-type and Ythdc2-/- testes.
- https://doi.org/10.7554/eLife.26116.009
Figure 2—figure supplement 1
Generation of the Ythdc2 knockout mouse.
(A) Diagram of the Ythdc2 targeted trap allele (tp) showing the location of the targeted gene trap cassette between exons 5 and 6 (top), and the generation of the null allele (-) though excision of …
Figure 2—figure supplement 2
Phenotypic characterization of Ythdc2 mutant mice.
(A, B) Average total weight of wild-type and Ythdc2-/- (A) male and (B) female mice over time. (n ≥ 20 mice per time point). Error bars: SEM. (C, D) Cross-sections of epididymis from 8-week-old (C) …
Figure 3
YTHDC2-deficient male germ cells do not properly progress through meiotic prophase during the first wave of spermatogenesis.
(A–H) Testis seminiferous tubule cross-sections counterstained with PAS from wild-type (top) and Ythdc2-/- (bottom) mice during the first wave of spermatogenesis: (A, B) P8; (C, D) P10; (E, F) P12; …
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Figure 3—source data 1
The most advanced cell stage in P12 wild-type and Ythdc2-/- testis tubules.
- https://doi.org/10.7554/eLife.26116.011
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Figure 3—source data 2
Percentage of P12 wild-type and Ythdc2-/- testis tubules containing cells with condensed chromosomes.
- https://doi.org/10.7554/eLife.26116.012
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Figure 3—source data 3
Number of chromosomes in Ythdc2-/- metaphase-like nuclei.
- https://doi.org/10.7554/eLife.26116.013
Figure 4 with 1 supplement
Defects in expression of some meiotic markers in Ythdc2 mutant male germ cells.
(A–D) Immunofluorescence images of testis tubule cross-sections. (A, B) Testes from P10 (A) wild-type and (B) Ythdc2-/- mice stained for STRA8 (green) and DAPI (blue) (n = 3 mice per group). (C, D) …
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Figure 4—source data 1
Percentage of tubules containing DMC1 + germ cells in wild-type and Ythdc2-/- P12 testes.
- https://doi.org/10.7554/eLife.26116.016
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Figure 4—source data 2
Expression of Spo11 mRNA in wild-type and Ythdc2-/- testes.
- https://doi.org/10.7554/eLife.26116.017
Figure 4—figure supplement 1
Expression of meiotic markers in Ythdc2 mutant males.
(A, B) Immunofluorescence images of (A) leptotene and (B) zygotene germ cells from P14 wild-type and Ythdc2-/- mice stained for SYCP3 (green), γ-H2AX (red) and DAPI (blue). (C, D) …
Figure 5 with 2 supplements
YTHDC2 is required for meiotic prophase in the fetal ovary.
(A, B) Whole mount ovaries from 8-week-old adult female (A) wild-type and (B) Ythdc2-/- mice (n = 3 wild-type and n = 5 Ythdc2-/- mice). (C, D) PAS-stained ovary sections from P21 (C) wild type (8/8 …
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Figure 5—source data 1
Number of DDX4 + cells in wild-type and Ythdc2-/- P0.5 ovaries.
- https://doi.org/10.7554/eLife.26116.021
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Figure 5—source data 2
Percentage of DDX4 + cells that are pH3 + in wild-type and Ythdc2-/- E16.5 ovaries.
- https://doi.org/10.7554/eLife.26116.022
Figure 5—figure supplement 1
Ythdc2 mutant females have defects in the fetal ovary.
(A, B) PAS-stained sections of ovaries from P60 (A) wild-type and (B) Ythdc2-/- mice. (C, D) H and E-stained ovary sections from P5 (C) wild-type and (D) Ythdc2-/- mice. (E, F) Immunofluorescence …
Figure 5—figure supplement 2
MEIOC coimmunoprecipitates with YTHDC2.
(A) Immunoprecipitation with α-YTHDC2 from wild-type testis extract. Western blot probed with α-YTHDC2 or α-MEIOC. (B) MEIOC protein expression in P12 wild-type and Ythdc2-/- testes. Western blot …
Figure 6 with 1 supplement
Ythdc2-/- male germ cells do not properly up-regulate meiotic transcripts at the mitosis to meiosis transition.
(A, B) Heatmaps representing transcripts differentially expressed in Ythdc2-/- compared to wild type in (A) P12 and (B) P14 testes (adjusted p-value<0.05), with corresponding expression levels for …
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Figure 6—source data 1
Differentially expressed transcripts in P12 Ythdc2-/- testes compared to wild type.
- https://doi.org/10.7554/eLife.26116.025
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Figure 6—source data 2
Differentially expressed transcripts in P14 Ythdc2-/- testes compared to wild type.
- https://doi.org/10.7554/eLife.26116.026
Figure 6—figure supplement 1
P12 and P14 RNA-sequencing dataset analysis.
Clustering analysis of RNA-sequencing from P12 and P14 wild-type and Ythdc2 mutant (-/-) testes.
Figure 7 with 1 supplement
YTHDC2-deficient meiotic germ cells fail to turn off expression of Cyclin A2.
(A-B′′) Immunofluorescence images of testis cross-sections from P12 (A-A′′) wild-type and (B-B′′) Ythdc2-/- mice stained in parallel for Cyclin A2 (green), SYCP3 (red) and DAPI (blue). Arrow: …
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Figure 7—source data 1
Cyclin A2 protein expression in wild-type and Ythdc2-/- leptotene spermatocytes.
- https://doi.org/10.7554/eLife.26116.029
Figure 7—figure supplement 1
Cyclin A2 protein expression in wild-type and Ythdc2 mutant preleptotene cells.
(A-B′′) Immunofluorescence images of testis cross-sections from P12 (A-A′′) wild-type and (B-B′′) Ythdc2-/- mice stained in parallel for Cyclin A2 (green), SYCP3 (red) and DAPI (blue). Arrow: …
Figure 8 with 2 supplements
YTHDC2 binds specific RNA targets.
(A) Top GO and Reactome (R) -terms for mRNAs enriched by immunoprecipitation of YTHDC2 from wild-type P12 testes. (B) RT-qPCR analysis of RNA transcripts following fRIP for YTHDC2 (blue) or IgG …
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Figure 8—source data 1
RNAs enriched by fRIP for YTHDC2 from P12 testes.
- https://doi.org/10.7554/eLife.26116.033
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Figure 8—source data 2
Validation of YTHDC2-bound RNAs by fRIP-qRT-PCR from P12 testes.
- https://doi.org/10.7554/eLife.26116.034
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Figure 8—source data 3
Analysis of expression levels of YTHDC2-bound RNAs by qRT-PCR in P12 wild-type and Ythdc2-/- testes.
- https://doi.org/10.7554/eLife.26116.035
Figure 8—figure supplement 1
RNA expression levels of YTHDC2-bound RNAs in Ythdc2 mutant testes relative to wild type.
Volcano plots representing fold change differences in mRNA expression in Ythdc2 mutant testes relative to wild type at P12 and P14. YTHDC2-bound RNAs, determine by immunoprecipitation of YTHDC2 …
Figure 8—figure supplement 2
The m6A to A ratio is similar in wild-type and Ythdc2 mutant testes.
(A) Ratio of m6A to A levels in poly-A selected mRNAs from P12 wild-type and Ythdc2-/- testes based on HPLC. Error bars: STD. (B) Metagene representation of m6A modification sites in mRNAs of …
Figure 9 with 1 supplement
YTHDC2 localizes to RNA granules.
(A-A′′) High-magnification immunofluorescence images of spermatocytes from an adult mouse testis cross-section stained for YTHDC2 (green) DCP1A (red) and DAPI (blue). (B) High-magnification image of …
Figure 9—figure supplement 1
MYBL1 expression in Ythdc2 mutant testes.
MYBL1 (A-MYB) protein expression in P12 wild-type and Ythdc2-/- testes. Western blot probed with α-MYBL1 or α-Actin.
Additional files
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Supplementary file 1
Breeding data for Ythdc2 heterozygous (Ythdc2+/-) mice showing that Ythdc2 null mutant mice are born at the expected Mendelian ratio.
- https://doi.org/10.7554/eLife.26116.038
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Supplementary file 2
Breeding data for Ythdc2 homozygous mutant (Ythdc2-/-) mice showing that Ythdc2-/- mice are infertile.
- https://doi.org/10.7554/eLife.26116.039
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Transparent reporting form
- https://doi.org/10.7554/eLife.26116.040
