1. Volume 21, Issue 11, Pages 3178-3189 (December 2017)
The Germline Linker Histone dBigH1 and the Translational Regulator Bam Form a Repressor Loop Essential for Male Germ Stem Cell Differentiation 
Albert Carbonell, Salvador Pérez-Montero, Paula Climent-Cantó, Oscar Reina, Fernando Azorín 
Cell Reports 
Volume 21, Issue 11, Pages (December 2017)
DOI: /j.celrep
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2. Cell Reports 2017 21, 3178-3189DOI: (10.1016/j.celrep.2017.11.060)
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3. Figure 1 The Pattern of dBigH1 Expression during Spermatogenesis
(A) Immunostaining of the tip region of a wild-type testis with αdBigH1 antibodies (in red) and αvasa (in green). Regions corresponding to TA spermatogonial cells and spermatocytes are indicated.
(B) As in (A), but for αTj (in green).
(C) As in (A), but for phalloidin (in green), a high-affinity F-actin probe that marks the somatic hub cells (arrowhead).
(D) Immunostaining of a wild-type testis expressing an esg-LacZ reporter with αdBigH1 (in red), αLacZ (in green), and αFasciclin III antibodies (also in green). αFasciclin III antibodies mark the hub (arrowhead). Asterisks indicate αLacZ-positive cells that express dBigH1 and are not attached to the hub.
(E) Immunostaining with αdBigH1 antibodies (in red) of a wild-type testis expressing GFP under the control of the bam promoter (bamP-GFP). GFP was visualized by direct fluorescence (in green). Regions corresponding to TA spermatogonial cells and spermatocytes are indicated.
(F) Magnification of wild-type spermatocytes immunostained with αdBigH1 antibodies (in red).
(G) Magnification of the transition from spermatocytes to round spermatids in a wild-type testis immunostained with αdBigH1 antibodies (in red).
DNA was stained with DAPI (in blue). Scale bars correspond to 25 μm. See also Figure S1.
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4. Figure 2
Bam Depletion Increases dBigH1 Expression in TA Spermatogonial Cells
(A) Testes from control bam-GAL4/+ and knockdown bam > bamRNAi males immunostained with αdBigH1 antibodies (in red) and, to label the contour of each cyst, with αArmadillo (Arm) antibodies (in green). Cysts containing >16 cells are indicated.
(B) As in (A), but for individual GBs and 2-, 4-, 8-, and 16-cell cysts. Several z sections are presented to visualize and count the total number of cells in the cysts as indicated.
DNA was stained with DAPI (in blue). Scale bars correspond to 25 μm (A) and 6 μm (B). See also Figure S2.
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5. Figure 3 Bam Downregulates dBigH1 Expression
(A) Immunostaining of the tip region with αdBigH1 antibodies (in red) and αArmadillo (Arm) antibodies (in green) of testes from nos > Bam::GFP males, in which expression of a Bam::GFP fusion was ectopically induced in GSCs and GBs with nos-GAL4. GFP was visualized by direct fluorescence (also in green). Arm antibodies mark the hub (arrowhead). DNA was stained with DAPI (in blue).
(B) Immunostaining with αdBigH1 antibodies (in red) of testes in which expression of a control GFP construct (left) and a Bam::GFP fusion (right) was ectopically induced in early spermatocytes with chif-GAL4. GFP was visualized by direct fluorescence (in green). DNA was stained with DAPI (in white for better visualization). Primary spermatocytes showing faint DAPI staining and expressing GFP/Bam::GFP are indicated.
(C) Immunostaining with αdBigH1 (in red) and αHA antibodies (in green) of the tip region of testes expressing a tagged dBigH1::HA construct under the control of the vasa promoter (vasaP) in control wild-type (left) and heterozygous bamΔ86 males (right). The TA region is indicated. DNA was stained with DAPI (in blue).
Scale bars correspond to 25 μm. See also Figures S2 and S3.
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6. Figure 4 dBigH1 Depletion Affects GSC Maintenance and Differentiation
(A) Immunostaining of testes from control nos-GAL4/+ and knockdown nos > bigH1RNAi males with αdBigH1 antibodies (in red), αAdducin (Add) antibodies to stain the fusome (in green), and αFasciclin III antibodies to mark the hub (also in green). In the nos > bigH1RNAi panel, a pair of testes is presented, of which one is strongly undeveloped (in the red box); the second testis is included for comparison.
(B) Magnifications of the tip regions of the control nos-GAL4/+ testis in (A) and of two knockdown nos > bigH1RNAi testes. The nos > bigH1RNAi testis in the bottom panel corresponds to the one shown in (A) (red box). Notice the lack of GCs in the nos > bigH1RNAi testis in the center panel and the absence of fusome (stained with αAdd) in GCs of the nos > bigH1RNAi testis in the bottom panel. The positions of the TA region and the hub are indicated.
(C) Percentage of testes showing a lack of GCs (no testis) or a drastic loss of GCs and lack of spermatocytes (undeveloped) in control nos-GAL4/+ (N = 100) and knockdown nos > bigH1RNAi males (N = 89).
(D) Fertility tests of control nos-GAL4/+ and knockdown nos > bigH1RNAi males. The total progeny of single males of the indicated genotypes mated with single wild-type females is presented (see Supplemental Experimental Procedures for details). Data are presented as mean ± SEM (n = 10).
DNA was stained with DAPI (in blue). Scale bars correspond to 100 μm (A) and 25 μm (B). Results correspond to bigH1RNAi line BL (Bloomington Drosophila Stock Center). See also Figures S2, S4, and S5.
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7. Figure 5 dBigH1 Depletion Impairs Spermatocyte Differentiation
(A) Immunostaining of testes from control bam-GAL4/+ and knockdown bam > bigH1RNAi males with αdBigH1 (in red).
(B) Percentage of testes showing a severely enlarged TA region (as that shown in A) or mildly enlarged TA region (as that shown in D) in control bam-GAL4/+ (N = 103) and knockdown bam > bigH1RNAi (N = 34) males.
(C) Fertility tests of control bam-GAL4/+ and knockdown bam > bigH1RNAi males. The total progeny of single males of the indicated genotypes mated with single wild-type females is presented (see Supplemental Experimental Procedures for details). Data are presented as mean ± SEM (n = 10).
(D) Immunostaining with αdBigH1 antibodies (in red) of testes from control and knockdown bam > bigH1RNAi males expressing a Bam::GFP fused protein under the control of the bam promoter (bamP-Bam::GFP). GFP was visualized by direct fluorescence (in green).
(E) Immunostaining of testes from control bam-GAL4/+ and knockdown bam > bigH1RNAi males with αdBigH1 (in red) and, to label the contour of each cyst, with αArmadillo (Arm) antibodies (in green).
(F) Quantitative analysis of the results shown in (E). The percentages of cysts containing ≤8, 16, and ≥32 spermatocytes are presented for control bam-GAL4/+ and knockdown bam > bigH1RNAi testes (N = 12 testes analyzed per condition).
DNA was stained with DAPI. The positions of the TA region are indicated. Scale bars correspond to 25 μm. Results correspond to bigH1RNAi line BL (Bloomington Drosophila Stock Center). See also Figures S2, S4, and S6.
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8. Figure 6 dBigH1 Silences Gene Expression in Spermatocytes
(A) Lorenz curves showing coverage inequality for IP and input samples of dBigH1 ChIP-seq analyses in precellular embryos (left) and testes (right).
(B) The expression level in spermatocytes (log2RMA) is plotted as a function of the dBigH1 content (MAnorm log2fold change (FC) IP/input) for each gene.
(C) Normalized dBigH1 log2 coverage ratio (IP/input) around the TSS is presented for genes of the 1st, 2nd, and 3rd tertiles of dBigH1 content.
(D) The expression levels of 11 genes with high dBigH1 content are determined by qRT-PCR in control bam-GAL4/+ and knockdown bam > bigH1RNAi testes. Expression levels were determined with respect to Act5C expression and normalized with respect to control.
(E) dBigH1 occupancy at the bam promoter is determined by ChIP-qPCR in control bam-GAL4/+ and knockdown bam > bigH1RNAi testes.
(F) The levels of dBigH1 in extracts prepared from control S2 cells (left) and stable S2 cells carrying a Cu2+-inducible dBigH1::FLAG construct (right) are determined by WB using αdBigH1 antibodies after induction with 1 mM CuSO4 (+) or not (−). αH4 antibodies are used for normalization. Increasing amounts of extracts are analyzed (lanes 1 and 2).
(G) dBigH1 expression was induced in a stable S2 cell line carrying a dBigH1::FLAG construct. At the left, dBigH1 occupancy at the bam promoter is determined by ChIP-qPCR after inducing dBigH1 expression and in control uninduced cells. In the right, bam mRNA levels are determined by qRT-PCR after inducing dBigH1 expression and in control uninduced cells.
Data are presented as mean ± SEM (n = 2). See also Figures S6 and S7 and Table S1.
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9. Figure 7
dBigH1 and Bam Form a Double-Repressor Loop that Regulates Entrance into Spermatocyte Differentiation
Bam accumulation during the TA divisions represses dBigH1 expression post-transcriptionally, while dBigH1 is required to silence bam transcription in spermatocytes. This forms a double-repressor loop that is required for spermatocyte differentiation. Bam expression begins when GBs leave the niche, in which Dpp/BMP signals repress bam in GSCs. Then, Bam progressively accumulates during the TA divisions, inhibiting dBigH1 expression. After reaching a maximum at the 8-cell stage, Bam levels decrease by the action of several post-transcriptional mechanisms. Consequently, dBigH1 expression resumes reinforcing transcriptional silencing of bam, which is required to enter the spermatocyte differentiation program (see text for details).
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