Changes in rRNA transcription influence proliferation and cell fate within a stem cell lineage

Abstract

Ribosome biogenesis drives cell growth and proliferation, but mechanisms that modulate this process within specific lineages remain poorly understood. Here, we identify a Drosophila RNA polymerase I (Pol I) regulatory complex composed of Under-developed (Udd), TAF1B, and a TAF1C-like factor. Disruption of udd or TAF1B results in reduced ovarian germline stem cell (GSC) proliferation. Female GSCs display high levels of ribosomal RNA (rRNA) transcription, and Udd becomes enriched in GSCs relative to their differentiating daughters. Increasing Pol I transcription delays differentiation, whereas reducing rRNA production induces both morphological changes that accompany multicellular cyst formation and specific decreased expression of the bone morphogenetic protein (BMP) pathway component Mad. These findings demonstrate that modulating rRNA synthesis fosters changes in the cell fate, growth, and proliferation of female Drosophila GSCs and their daughters.

Fig. 1

Disruption of udd results in reduced GSC proliferation and loss. Negatively marked (A) control, (B) udd¹ and (C) udd^null clones (white dotted lines) dissected 21 days after clone induction stained for GFP (green), Hts (red) and DNA (blue). Arrowheads mark cap cells. (D) Percentage of germaria with GSCs clones over time. (E) Percentage of GSC clones that produce a differentiating cyst over time. (F) Percentage of GSCs positive for the mitotic marker phospho-histone H3 (pH3). Scale bars represent 10 µm.

Fig. 2

Characterization of a Drosophila SL1-like complex. (A) w¹¹¹⁸ germarium stained for endogenous Udd (green), Mod (red) and Vasa (blue). (B) Co-immunoprecipitation of FLAG-tagged TAF1B and TAF1C-like, and HA-tagged Udd from transfected S2 cells. (C) GFP-TAF1B and (D) TAF1C-like-GFP co-localize with endogenous Udd in S2 cells. Arrows mark transfected cells and arrowheads mark non-transfected cells. (E) udd^null mosaics stained for Udd (green), BrUTP (red) and DNA (blue). Heterozygous nurse cells (arrowhead) exhibit Udd expression and BrUTP incorporation, whereas udd^null mutant cells (white-dotted line) show little BrUTP labeling. (F) Northern blot of total ovarian RNA isolated from indicated genotypes probed using a fragment of Internal Transcribed Spacer (ITS). Ethidium Bromide stained mature 28S and 18S rRNA. 5S rRNA was used as a loading control. (G) ChIP-qPCR analysis of da>HA-udd ovaries reveals Udd associates with specific sites within the rRNA promoter and External Transcribed Spacer (ETS), as indicated by arrows and bars. Control represents anti-HA IP from the da-gal4 background. (C, D) Scale bars represent 5 µm. Scale bars represent 10 µm in other panels.

Fig. 3

GSCs and undifferentiated cells maintain high levels of Udd. (A-A’’) Control germarium stained for Udd (green), BrUTP (red) and DNA (blue). GSCs (inset) exhibit high levels of (A’) BrUTP labeling and (A’’) Udd. (B) w¹¹¹⁸ germarium stained for Bam (green), BrUTP (red), DNA (blue). (C, C’) Still images from live cell imaging (movies S1 and S2) showing GFP-tagged Udd (green) and mRFP-tagged histone H2Av (red) in a dividing GSC at the times indicated. Arrows point to the dividing GSC and resulting daughters. (D) No heat-shock (no HS) control hs-bam; bam^∆86 mutant germarium and (E) heat-shocked (HS) hs-bam; bam^∆86 mutant germarium 36 hours after bam induction stained for Udd (green), BrUTP (red) and DNA (blue). (D’, E’) BrUTP labeling alone. Scale bars represent 10 µm.

Fig. 4

Modulating rRNA synthesis influences cyst development and BMP signaling in the germline. (A) bam^∆86 and (B) udd¹ bam^∆86 double mutant germaria stained for Phosphotyrosine (pTyr) (green), Hts (red) and DNA (blue). Arrows point to cysts with branched fusomes. (C) nos-gal4 and (D) nos>Tif-IA stained for Hts (red) and DNA (blue). (E) Quantification of single cells upon Tif-IA over-expression. (F) nos-gal4 and (G) nos>TIF-IA stained for Dad-LacZ. (H) Quantification of Dad-LacZ expression upon Tif-IA over-expression. (I) Western blots of bam^∆86 and udd¹ bam^∆86 ovarian extracts probed for Mad, Medea and histone H2B proteins. (C,D and F,G) Two copies of nos-gal4 were present. Scale bars represent 20 µm. (*) Denotes significance of p<0.0001.