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The Histone Variant His2Av is Required for Adult Stem Cell Maintenance in the Drosophila Testis

PLoS Genetics (2013) 9(11)
DOI: 10.1371/journal.pgen.1003903
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Abstract

Many tissues are sustained by adult stem cells, which replace lost cells by differentiation and maintain their own population through self-renewal. The mechanisms through which adult stem cells maintain their identity are thus important for tissue homeostasis and repair throughout life. Here, we show that a histone variant, His2Av, is required cell autonomously for maintenance of germline and cyst stem cells in the Drosophila testis. The ATP-dependent chromatin-remodeling factor Domino is also required in this tissue for adult stem cell maintenance possibly by regulating the incorporation of His2Av into chromatin. Interestingly, although expression of His2Av was ubiquitous, its function was dispensable for germline and cyst cell differentiation, suggesting a specific role for this non-canonical histone in maintaining the stem cell state in these lineages. © 2013 Morillo Prado et al.

Figures

  • Figure 1. His2Av is required cell autonomously for GSC maintenance. (A–A9): Apical tip of wild-type testis immunostained with anti-His2Av (red), anti-Vasa (green), and anti-Fas3 (blue). GSCs (arrows) identified as Vasa positive cells adjacent to the hub (yellow dashed line). (B–B9): Testes expressing Sa-GFP (green) to mark spermatocytes, immunostained with anti-His2Av (red), anti-GFP (green), and anti-Fas3 (blue). Inset shows spermatocyte nucleus with Sa-GFP marking the nucleolus and His2Av localized to the chromosomes; hub (yellow dashed line). (C–D9): Apical tip of wild-type testes immunostained with anti-His2Av (red, C–D9), anti-Tj (green, C) to mark CySCs (arrows, C, C9) adjacent to the hub (yellow dashed line) or anti-Eya (green, D) to mark cyst cells (arrowheads, D, D9). (E–E90): Testes day 3 PCI immunostained with anti-GFP (green) to identify homozygous His2Av810 mutant GSCs (arrows), anti-His2Av (red), anti- Fas3 and Tj (blue) and DAPI (E90); hub (yellow dashed line). Scale bars: 10 mm (F): Percentage of testes with His2Av810 mutant (red) or FRT 82B control (blue) GSCs scored at indicated times PCI.(G): Percentage of testes with His2Av810 mutant spermatocyte cysts in a genetic background either with (blue line, rescue) or lacking (red line, no rescue) a His2Av-mRFP genomic rescue transgene. Data shows average 6 S.D. doi:10.1371/journal.pgen.1003903.g001
  • Figure 2. RNAi knockdown of His2Av function in early germ cells results in GSC loss. (A–D): Phase images of testes with RNAi knockdown of His2Av in GSCs and early germ cells using the NG4VP16 driver (B, D) and sibling control (A, C) at day 4 (A, B), day 12 (D) and day
  • Figure 3. His2Av is not required for germ cell differentiation. (A–C9): Phase (A, B, C) and GFP (A9, B9, C9) images of GFP-negative His2Av810 spermatocyte (A, A9), round spermatid (B, B9), and elongating spermatid (C, C9) cysts at day 8 PCI. Mutant clones (yellow dashed line). (D, E): Phase images of testes with RNAi knockdown of His2Av in spermatocytes using the Bam-Gal4 driver (E) and sibling control (D) at day 8 after RNAi induction. Arrows point to elongated spermatids. Scale Bars (A–E): 50 mm (F–F0): Immunostaining with anti-Bam (F, red and F0), anti-GFP (F, green and F9), anti-ECad and TJ (F, blue) at day 5 PCI. His2Av810 mutant GSC (white arrow), His2Av810 mutant gonialblast (yellow arrow), His2Av810 heterozygous GSC (white arrowhead), His2Av810 mutant 4-cell spermatogonial cyst (yellow dashed line) and hub (white dashed line). Scale bar: 25 mm (G): Number of spermatocytes per cyst of His2Av810 heterozygous (blue) and mutant (red) clones counted at day 8 PCI. doi:10.1371/journal.pgen.1003903.g003
  • Figure 4. His2Av is required cell autonomously for CySC maintenance but not for somatic cyst cell differentiation. (A, B): Percentage of testes with His2Av810 mutant (red line) or FRT 82B control (blue line) CySCs (A) or cyst cells (B) scored at indicated time points PCI. Data shows average 6 S.D. (C–C0): Apical tip of testes 3 days PCI immunostained with anti-GFP (C, green and C0), anti-Zfh-1 (C, red and C9), and anti-Fas3 and TJ (blue). His2Av810 mutant CySCs (arrow), His2Av810 heterozygous CySCs (arrowhead) and hub (yellow dashed line). Scale bar: 12.5 mm (D, D9): Testes from 8 day PCI immunostained with anti-GFP (green) and anti-Eya (red). His2Av810 mutant (arrow) and heterozygous (arrowhead) cyst cells. Scale bar: 10 mm (E–G): Analysis of Drosophila eyes derived from heterozygous GMR-hid/+ (E), homozygous His2Av810 (F), and homozygous FRT control (G) precursor cells. doi:10.1371/journal.pgen.1003903.g004
  • Figure 5. Loss of His2Av function did not cause defects in STAT dependent GSC characteristics. (A–A0): His2Av810/+ testes at 5 days PCI and expressing E-Cadherin-GFP under the control of the nanos-Gal4 driver immunostained with anti-GFP (green), anti-b-galactosidase (LacZ, red) and anti-Fas3 and TJ (blue). His2Av810 mutant GSCs (arrows), His2Av810 heterozygous GSC (arrowhead) and hub (star). Scale bar: 25 mm (B): Percentage of
  • Figure 6. Domino function is required for the association of His2Av with chromatin. (A, B): Percentage of testes with domk08108 mutant (red) or FRT 42D control (blue) GSCs (A) and CySCs (B) scored at indicated times after clonal induction. Data shows average 6 S.D. (C–C90): Apical tip of domk08108/+ testes at day 6 PCI immunostained with anti-GFP (C, green and C9) and anti-His2Av (C, red and C0) and counterstained with DAPI (blue, C and C90). domk08108 mutant GSCs (arrows), domk08108 heterozygous GSCs (arrowheads) and hub (yellow dashed line). Scale bar: 12.5 mm (D): Average ratio of His2Av intensity per unit area in GSC homozygous for FRT 42D, domk08108 or ISWI2 to neighboring GSCs heterozygous for FRT 42D, domk08108 or ISWI2, respectively. Data shows average 6 S.D. P-values of Student’s t-test (2-tailed) are shown. (E): Percentage of testes with His2Av810 (blue) or His2Av810 ;domk08108/+ (red) GSCs scored at indicated times after clonal induction. Data shows average 6 S.D. (F–G90): Apical tip of ISWI2/+ (F–F90) testes at day 6 PCI or His2Av810/+ (G–G90) testes at day 5 PCI immunostained with anti-GFP (F, G, green and F9, G9) and anti-His2Av (F, red and F0) or anti-ISWI (G, red and G0) and counterstained with DAPI (blue, F, G and F90, G90). ISWI2 (F–F90) or His2Av810 (G–G90) mutant GSCs (arrows), ISWI2 (F–F90) or His2Av810 (G–G90) heterozygous GSCs (arrowheads) and hub (yellow dashed line). Scale bars: 12.5 mm (H): Percentage of testes with His2Av810 (blue) or His2Av810 ;ISWI2/+ (red) GSCs scored at indicated times after clonal induction. Data shows average 6 S.D. doi:10.1371/journal.pgen.1003903.g006
  • Figure 7. His2Av mutant GSCs do not exhibit dramatic changes in epigenetic markers of transcriptional state. (A–B90): His2Av810/+ testes 5 days PCI immunostained with anti- GFP (A, B, green and A9, B9), anti-H3K4me3 (A, red and A0) or anti-H3K27me3 (B, red and B0) and counterstained with DAPI (A, B, blue and A90, B90). His2Av810 mutant GSCs (arrows), His2Av810 heterozygous GSCs (arrowhead) and hub (yellow dashed line). Scale bar: 10 mm (A) and 12.5 mm (B). (C–D90): scny02331/+ testes 6 days PCI immunostained with anti- GFP (green, C, D and C9, D9), anti-His2Av (red, C and C0) or anti-H3K4me3 (red, D and D0) and counterstained with DAPI (blue, C, D and C90, D90). scny02331 mutant GSCs (arrows), scny02331 heterozygous GSCs (arrowheads) and hub (yellow dashed line). Scale bar: 25 mm (C) and 12.5 mm (D). doi:10.1371/journal.pgen.1003903.g007

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CITATION STYLE

APA

Prado, J. R. M., Srinivasan, S., & Fuller, M. T. (2013). The Histone Variant His2Av is Required for Adult Stem Cell Maintenance in the Drosophila Testis. PLoS Genetics, 9(11). https://doi.org/10.1371/journal.pgen.1003903

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