Developmental Biology

Meioc-Piwil1 complexes regulate rRNA transcription for differentiation of spermatogonial stem cells

Toshihiro Kawasaki author has email address
Toshiya Nishimura
Naoki Tani
Carina Ramos
Emil Karaulanov
Minori Shinya
Kenji Saito
Emily Taylor
Rene F Ketting
Kei-ichiro Ishiguro
Minoru Tanaka
Kellee R Siegfried
Noriyoshi Sakai author has email address

Department of Gene Function and Phenomics, National Institute of Genetics, SOKENDAI (The Graduate University for Advanced Studies), Mishima, Japan
Department of Genetics, School of Life Science, SOKENDAI (The Graduate University for Advanced Studies), Mishima, Japan
Division of Biological Science, Nagoya University, Nagoya, Japan
Institute of Molecular Embryology and Genetics, Kumamoto University, Kumamoto, Japan
Biology Department, University of Massachusetts Boston, Boston, United States
Institute of Molecular Biology (IMB), Mainz, Germany

https://doi.org/10.7554/eLife.104295.1

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Figures and data

Low translational activity of meioc mutant spermatogonia
(A) Schema of the development of spermatogonial cysts surrounded by Sertoli cells in zebrafish. (B) Histology (HE) and immunostaining against Plzf and spermatocyte markers (Sycp1, 3) in the wildtype and the moto^−/− testes. Scale bar: 10 µm. (C, D) OP-Puro fluorescence analysis (C) and quantification of the signal intensities (D) in wildtype and meioc^mo/mospermatogenic cells. Scale bars: 10 µm. (E-F) Effect of cycloheximide (CHX, 0.2 µM) on differentiation of SSCs in culture. Yellow dotted lines; germ cell clumps. Sycp3; Immunostaining of Sycp3. Arrowheads; examples of a cell with a large nucleolus. The graph (F) presents the percentage of clumps of SSCs and differentiated cells (Differ) shown in Panel E. Scale bar; 50 mm. Data are represented as mean ± SD.

Defect on upregulation of rRNA transcription in meioc^mo/mo spermatogonia
(A) In situ hybridization of 5S, 5.8S, 18S, and 28S rRNA and immunohistochemistry with anti-Rpl15 antibody in spermatogonia (gonia) and spermatocytes (cyte) in wildtype and meioc^mo/mo. Yellow dotted lines indicate 1-2-cell spermatogonia. (B-D) Fluorescent in situ hybridization of 28S rRNA in wildtype. The graphs present quantification of signal intensities of 28S rRNA in nucleoli (C) and in cytoplasm (D) in spermatogenic cells. Arrowheads; nucleoli. (E) qRT-PCR analysis of rRNAs and R2 between wildtype and meioc^mo/mopurified undifferentiated spermatogonia. (F) Bisulfite-sequencing analysis of the tandem repeat region in the IGS region of the 45S-S rDNA locus in purified undifferentiated spermatogonia of wildtype and meioc^mo/mo. Arrows; position of bisulfite primers in the tandem repeat elements (blue, magenta and white boxes), black dots; methylated CpG sites, white dots; unmethylated sites. *p < 0.05, **p < 0.01. Scale bars: 10 µm.

ythdc2 mutant spermatogonia have different defects from meioc^mo/mo.
(A) Histology (HE) and immunostaining against Plzf and spermatocyte markers (Sycp1, 3) in the ythdc2^−/−testes. (B) Representative image of meioc^mo/moand ythdc2^−/− testes sections stained with PAS (periodic acid Schiff) and hematoxylin. Dotted lines; 1-2-cell cyst spermatogonia (black) and 4≤-cell cysts (yellow). (C) The number of 1-2-, 4-, and 8-cell cyst spermatogonia per mm² of sections in wildtype, meioc^mo/mo and ythdc2^−/− testes. (D) Ratio of the number of 4-8-cell cyst spermatogonia to 1-2-cell cysts in wildtype, meioc^mo/mo and ythdc2^−/− testes. (E) In situ hybridization of 5S, 5.8S, 18S, and 28S rRNA and immunohistochemistry with anti-Rpl15 antibody in the ythdc2^−/− testes. Yellow dotted lines; 1-2-cell spermatogonia. *p < 0.05, **p < 0.01. Scale bars: 10 µm.

Meioc directly binds with Piwil1 and affects the localization of Piwil1.
(A) Immunostaining of Ddx4 and Piwil1, Piwil2, Tdrd1 and Tdrd6 in wildtype and meioc^mo/mospermatogonia. The arrowhead; the Piwil1 signal in the nucleolus. (B) Immunostaining against Piwil1 and fibrillarin (left panels) and quantification of nucleolar Piwil1 (right panel) in wildtype and meioc^mo/mospermatogonia. Arrowheads; Fibrillarin positive nucleolus. (C) Immunostaining of Piwil1 and Ddx4 (left panels) and quantification of Piwil1 in germ granules (right panel) in wildtype and meioc^mo/mo spermatogonia. Arrowheads; Ddx4 positive germ granules. (D) Coimmunoprecipitation of Meioc and Piwil1 using testis lysate. Meioc signals were detected in Piwil1 immunoprecipitate and vice versa. Benzonase: addition of benzonase nuclease. **p < 0.01. Scale bars: 10 µm.

Reduction of Piwil1 compensated phenotypes of meioc^mo/mo.
(A, B) In situ hybridization of 28S rRNA in wildtype and meioc^mo/mo;piwil1^+/−spermatogonia (1-2-cell cysts). Graphs (B) show the relative signal intensity in the cytoplasm normalized to the intensity of lobule myoid cells (left) and nucleoli normalized to the intensity of the nucleoplasm (right). (C, D) Differentiated spermatogonia in meioc^mo/mo and meioc^mo/mo;piwil1^+/−testes. Yellow dotted lines; differentiated spermatogonia. Graphs (D) show the number of 16-cell and 32-cell cyst spermatogonia per mm² of sections. ND: not detected. (E-G) meioc^mo/mo and meioc^mo/mo piwil1^+/− testis sections stained with PAS and hematoxylin. Cysts of 1-2-cell spermatogonia (black) and 4≤-cell cysts (yellow) are indicated by dotted lines. Graphs show numbers of 1-, 2-, 4-, and 8-cell cysts per mm² in sections of meioc^mo/mo and meioc^mo/mo;piwil1^+/− testes (F), and ratio of the number of 4-8-cell cysts to 1-2-cell cysts in wildtype, meioc^mo/mo and meioc^mo/mo;piwil1^+/−(G). *p < 0.05, **p < 0.01. Scale bars: 10 µm.

Nucleolar Piwil1 interacted with Setdb1 and causes silenced epigenetic state of rDNA loci
(A) Fold enrichment of pre-rRNA (5’ETS-18S rRNA) in Piwil1 immunoprecipitated RNA relative to the control IgG in wildtype and meioc^mo/mo testes. (B) Immunostaining of Piwil1 (left panels) and the percentage of spermatogonia with detectable nucleolar Piwil1 (right panel) in the meioc^mo/mo testes treated with a-amanitin (Am), actinomycin D (Ac) and BMH-21 (B). Arrows; Piwil1 detectable nucleoli, arrowheads; Piwil1 undetectable nucleoli, C; control without inhibitors, IC; initial control. (C) Immunostaining of Setdb1 and fibrillarin in wildtype and meioc^mo/mo spermatogonia. Arrowheads; nucleoli. (D) Co-IP of Piwil1 and Setdb1 using meioc^mo/mo testes lysate. Piwil1 was detected in Setdb1 IP. (E) Intensities of Setdb1 in nucleoli in wildtype and meioc^mo/mo spermatogonia. (F, G) ChIP-qPCR analysis of H3K9me3 (F) and Piwil1 (G) levels in 45S-rDNA region in wildtype, piwil1^+/−, and meioc^mo/mo testes. Position of primers were indicated in Supplemental Figure S3. Mean ± s.d. are indicated. (H) Immunostaining of HP1α and Piwil1 in wildtype and meioc^mo/mo spermatogonia. Arrowheads; nucleolus. (I) Co-IP of Piwil1 and HP1α using meioc^mo/mo testis lysate. HP1α was detected in Piwil1 IP. (J) Intensities of HP1α in nucleoli in wildtype and meioc^mo/mo spermatogonia. *p < 0.05, **p < 0.01. Scale bars: 10 µm.

Meioc was required for upregulation of 28S rRNA
(A, B) Expression patterns of 28 rRNA (A) and Piwil1 (B) in isolated sox17::egfp spermatogonia, based on the amount of Meioc granules and the localization. Right panels are intensities of 28 rRNA (A) and Piwil1 (B) for each class of the purified wildtype 1-2-cell spermatogonia. 51≤ C and N; 51≤ cytoplasmic and nuclear Meioc granules, respectively. *p < 0.05, **p < 0.01, n: number of analyzed spermatogonia. Scale bars: 10 µm. (C) Graphical abstract. Meioc prevents the nucleolar localization of Piwil1 and its associated Setdb1 and HP1α to upregulate rRNA transcripts that are required for zebrafish SSCs to differentiate.

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