1. Volume 34, Issue 1, Pages 96-107 (July 2015)
RNA Binding Protein Nanos2 Organizes Post-transcriptional Buffering System to Retain Primitive State of Mouse Spermatogonial Stem Cells 
Zhi Zhou, Takayuki Shirakawa, Kazuyuki Ohbo, Aiko Sada, Quan Wu, Kazuteru Hasegawa, Rie Saba, Yumiko Saga 
Developmental Cell 
Volume 34, Issue 1, Pages (July 2015)
DOI: /j.devcel
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2. Developmental Cell 2015 34, 96-107DOI: (10.1016/j.devcel.2015.05.014)
Copyright © 2015 Elsevier Inc. Terms and Conditions

3. Figure 1
Cytoplasmic mRNP Components Are Highly Enriched in Nanos2+ Spermatogonia
(A) A schematic illustration showing the properties of early spermatogonial populations based on gene expression. The x axis shows the differentiation stage. The y axis shows the self-renewal capacity.
(B) A schematic illustration showing the isolation procedure for Nanos2+, Ngn3+, and c-Kit+ spermatogonia from juvenile testis (3–4 weeks) by flow cytometry using transgenic mice (Nanos2+ and Ngn3+) and an anti-c-Kit antibody.
(C) The relative mRNA levels of major mRNP markers in Nanos2+, Ngn3+, c-Kit+ spermatogonia, and GS cells as measured by qPCR analyses. The average relative mRNA levels (±SD) are shown (n = 3), with the mRNA level of Nanos2 assigned a value of 1. Asterisks indicate significant differences between each population by t test (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001).
(D) Representative image of Dcp1a and Rck foci in isolated Nanos2+ cells immunostained with Dcp1a and Rck antibodies. Scale bar, 5 μm. The detailed method is provided in the Supplemental Experimental Procedures.
(E) Statistic analyses of Rck, Dcp1a, and Nanos2 focus counting in different spermatogonial populations. The mean ± SD (n = 15) for each population is shown. The number of foci in the Ngn3+ and c-Kit+ cell populations was compared to those in the Nanos2+ population by t test.
(F) Shown is a representative wild-type testis section (4 weeks) immunostained for Nanos2, Rck, and CDH1, and then counterstained with DAPI. Nanos2 and Rck co-localized to the same sparkle-like granules. Rck is a marker for both PBs and SGs. Scale bar, 10 μm.
(G) Western blot of a coIP experiment in GSCs with an anti-Nanos2 antibody. Cnot9 was used as a positive control for interaction with Nanos2 (Bhandari et al., 2014; Suzuki et al., 2010). Normal rabbit IgG was used as a negative control.
See also Figure S1.
Developmental Cell  , DOI: ( /j.devcel )
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4. Figure 2 mRNPs Components Are Required for SSC Maintenance
(A) Nanos2-conditional knockout (cKO) and conditional overexpressing (cOE) GSCs treated with DMSO (vehicle) or 4-OHT (1 μM) for 48 hr were harvested for western blotting. The filled triangle indicates endogenous Nanos2, and the empty triangle indicates 3 × flag-Nanos2, which is also shown in Figures 3C and 3H.
(B) Nanos2-cKO and Nanos-cOE GSCs were plated in medium containing DMSO or 4-OHT and passaged three times at 5-day intervals. The number of cells was counted to determine cell recovery as an indicator of growth. The mean ± SD (n = 3) is shown. The numbers of Nanos2 cKO and cOE GSCs treated with DMSO or 4-OHT at each time point were compared by t test (∗p < 0.05; ∗∗p < 0.01).
(C) A portion of the GSC populations from (B) was fixed for immunofluorescence (IF) with an anti-Rck antibody, and the number of foci was quantified. The mean (±SD) is shown for each group (n = 20). The foci in the Nanos2 cKO and cOE population incubated with 4-OHT were compared to those incubated with DMSO by t test.
(D) Stable GSCs infected with lentiviruses containing an inducible control shRNA or Rck shRNA were cultured with DMSO or 4-OHT (1 μM) for 48 hr and then harvested for western blotting. The mean ± SD (n = 3) is shown above a representative western blot (see also Figure S2).
(E and F) Rck-cKD GSCs were fixed and used for IF with anti-RCK and Dcp1a antibodies (E), and the number of foci was quantified (F) as shown in (C) (n = 20).
(G) Rck cKD GSCs were cultured as shown in (B) to determine the cell recovery rate. The mean (±SD) is shown (n = 3).
(H) QPCR analysis of the key spermatogonial marker genes in Rck-cKD GSCs treated with 4-OHT for 48 and 96 hr. For each gene, the average mRNA level in GSCs treated with 4-OHT was subtracted from that in GSCs treated with DMSO (n = 3), and is shown as (log2) (±SD). The mRNA levels in Rck-cKD GSCs treated with 4-OHT were compared to the levels in the same cells treated with DMSO at 48 hr and 96 hr by t test (∗p < 0.05; ∗∗p < 0.01).
See also Figure S2.
Developmental Cell  , DOI: ( /j.devcel )
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5. Figure 3
Nanos2 Repressed mTORC1 Signaling by Trapping and Recruiting mTOR to mRNPs
(A and B) Western blotting of the indicated proteins from two Nanos2-cKO GSC lines (independently isolated, named #1 and #2) cultured with DMSO or 4-OHT. Quantification of the relative levels of p-Akt, p-ERK, and p-RPS6 (normalized to the total levels of the respective proteins) are shown in (B).
(C and D) Western blotting of the indicated proteins from Nanos2-cOE cells cultured with DMSO or 4-OHT. Quantification of the mean relative intensities (±SD) of p-RPS6 against total RPS6 is shown in (D).
(E and F) Western blotting of the indicated proteins from Nanos2-cKO cells treated with or without 4-OHT together in the presence or absence of the mTORC1 inhibitor RAPA (20 nM). Quantification of the mean relative intensities (±SD) of p-RPS6 compared to total RPS6 is shown in (F).
(G) Cell recovery assay of control/RAPA− (black), Nanos2-cKO/RAPA− (red), Control/RAPA+ (pink), Nanos2-cKO/RAPA+ (blue) under normal culture conditions. Data shown represent the mean ± SD (n = 3).
(H and I) Nanos2-cOE GSCs were treated with or without 4-OHT for 24 hr and then treated with or without RA (100 nM) for another 24 hr. Samples were collected for mTORC1 signaling analysis. The relative p-RPS6 levels (mean ± SD) are shown (n = 3) in (I).
(J and K) Detection of endogenous mTOR in GSCs. IF staining of mTOR shows that it co-localized with Nanos2 (J) and Rck (K).
(L) Immunoprecipitation of GSC lysates with an anti-Nanos2 antibody. Normal rabbit IgG was used as a negative control.
(M and N) IF staining of Rck and mTOR in Nanos2-cOE cells cultured with DMSO or 4-OHT (M) and the statistical analysis of the number of merged foci (N; n = 17). The mean (±SD) is shown in (N). The numbers of foci in Nanos2-cOE cells treated with 4-OHT were compared to those in cells treated with DMSO by t test.
See also Figure S3.
Developmental Cell  , DOI: ( /j.devcel )
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6. Figure 4 Target mRNA Screening in Nanos2-Containing mRNPs
(A) The expression level changes of key genes induced by Nanos2 overexpression in Nanos2-cOE GSCs. The average mRNA level in Nanos2-cOE GSCs treated with DMSO was subtracted from that in the same cells treated with 4-OHT (n = 3) and is shown as (log2) (±SD).
(B) Schematic illustration showing the overlapping transcripts that are both repressed by Nanos2 and co-immunoprecipitated with Nanos2.
(C) QPCR analyses of the key transcripts in GSCs immunoprecipitated with an anti-Nanos2 antibody. Relative enrichment compared to the input transcript level (assigned a value of 1) (±SD; n = 3) is shown. Enrichment of immunoprecipitated mRNAs was compared to the input levels by t test. Similar calculations were used in (D), (E), and (G).
(D) QPCR analyses of the same transcripts shown in (C) with an anti-Rck antibody.
(E) QPCR analyses of RNA-IP with an anti-Nanos2 antibody from Rck-cKD GSCs treated with DMSO or 4-OHT (cKO). The relative enrichment of each gene in Rck-cKD GSCs treated with 4-OHT compared to that in cells treated with DMSO (control) is shown (±SD; n = 3) and was compared by t test. Similar calculations were used for the data shown in (G). NS, p > 0.05.
(F) Nanos2-cKO GSCs treated with DMSO or 4-OHT for 48 hr were collected for western blotting. The Rck and Dcp1a protein levels were not affected by depletion of Nanos2.
(G) QPCR analyses of an RNA IP of Nanos2-cKO GSCs treated with DMSO or 4-OHT using an anti-Rck antibody. Relative enrichment in cells treated with 4-OHT compared to the levels in cells treated with DMSO (control) are shown (±SD; n = 3). ∗p < 0.05; ∗∗p < 0.01.
See also Figure S4.
Developmental Cell  , DOI: ( /j.devcel )
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7. Figure 5
Regulation of Germ Cell Differentiation-Related Genes by Nanos2-Containing mRNPs
(A) Top panel: subcellular fraction profile of GSCs generated by sucrose gradient centrifugation as a polysome assay. Lower panel: the total protein from each fraction was used for western blotting, and a representative blot is shown (n = 3).
(B) Distribution of Gapdh, sohlh2, Dmrt1, and Dazl transcripts in Nanos2-cKO GSCs treated with DMSO or 4-OHT for 48 hr. The transcript levels in each cell fraction were analyzed by RT-PCR. The percentage of each total transcript present in the major fractions (mRNP, monosomes, and polysomes) is shown (n = 3, also see Figure S5).
(C) Co-immunofluorescence staining of Nanos2 and Sohlh2 proteins in GSCs treated with DMSO (control) or 4-OHT (Nanos2-cKO) for 48 hr. DNA was counterstained with DAPI (blue). Scale bar, 10 μm.
(D and E) Nanos2-cKO GSCs treated with DMSO or 4-OHT for 48 hr were collected for western blotting. Sohlh2, Dmrt1, and Dazl levels were increased with the depletion of Nanos2. The relative intensity of each protein (±SD) is shown (n = 3) in (E). The level of each protein in 4-OHT-treated Nanos2-cKO GSCs was compared to the level in DMSO-treated Nanos2-cKO GSCs (assigned a value of 1).
Developmental Cell  , DOI: ( /j.devcel )
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8. Figure 6
In Situ Detection of a Direct Interaction between Nanos2 and Target mRNAs
(A) Diagram of the method used for the in situ observation of target mRNAs tagged by the MS2 system.
(B) Visualization of the mRNAs by IF using an anti-GFP antibody in GSCs expressing NLS-HA-MS2-VENUS. Sohlh-mRNA was detected in the cytoplasm only when the sohlh2 mRNA contained an MS2-sequence, and the signal co-localized with Nanos2 (red). Scale bar, 5 μm.
(C and D) Representative IF images showing the localization of MS2-Sohlh2 mRNA (green) with Rck protein (red) in Nanos2-cKO GSCs treated with DMSO or 4-OHT for 48 hr (C) and the statistical analysis of merged foci of MS2-Sohlh2 mRNA and Rck (D; n = 20). The data shown are the mean (±SD). The number of foci in Nanos2 cKO GSCs treated with 4-OHT were compared to that in cells treated with DMSO by t test.
(E and F) Comparison of the sohlh2-3′-UTR and BGH-polyA for interaction with Nanos2 protein in wild-type GS cells (n = 20). Representative IF images are shown in (E) and the average data for the merged foci are shown in (F).
Developmental Cell  , DOI: ( /j.devcel )
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9. Figure 7
Unrestrained Expression of Sohlh2 in SSCs Promotes Differentiation
(A) Method used for forced expression of Sohlh2 by eliminating post-transcriptional regulation of sohlh2 in Nanos2+/GFRα2+ population. GFRα1-CreERT2 was used to induce Sohlh2 expression by 4-OHT injection. Samples were collected at 2, 14, and 28 days after injection of 4-OHT.
(B) Induction of Sohlh2 expression (Flag; red) in GFRα1+ cells (green). Whole mount immunostaining of 4-week TG testis tubules at 2 days after injection with oil (control) or 4-OHT.
(C) Total Nanos2+ (Nanos2-enhancer-EGFP) cells were analyzed in Sohlh2 TG mice injected with oil (n = 3) and 4-OHT (n = 3) at the indicated times by FACS. The data shown are the average cell number (±SD). The average number of Nanos2+ cells in 4-OHT-injected mice was compared to that in DMSO-injected mice by t test.
(D) Sohlh2-cOE GSC lines established from TG mice were cultured with DMSO or 4-OHT for 48 hr, and total protein was collected for western blotting.
(E) The relative expression of self-renewal-related mRNAs was examined in Sohlh2-GSCs treated with DMSO or 4-OHT for 48 hr by qPCR. Average mRNA levels (±SD) are shown (n = 3).
(F–H) Testis sections prepared from sohlh2-cOE TG mice at 28 days after injection with oil and 4-OHT were examined by H&E staining (F), IF staining of GFRα1+ (green in G), and Plzf+ (red in H). Scale bars, 50 μm.
(I and J) Comparison of the number of GFRα1+ (I) and Plzf+ (J) cells per seminiferous tubule in control (oil) and sohlh2 TG mice (4-OHT). The data shown are the average of 30 tubules (±SD).
(K) Schematic illustrations of the post-transcriptional regulation mediated by mRNP foci with (top) and without (bottom) Nanos2 in Nanos2+ and NGN3+ spermatogonia.
See also Figure S6.
Developmental Cell  , DOI: ( /j.devcel )
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