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First published online 7 March 2007
doi: 10.1242/dev.02810

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Stonewalling Drosophila stem cell differentiation by epigenetic controls

¹ Department of Molecular Biology, University of Texas Southwestern Medical Center at Dallas, Dallas, TX 75390-9148, USA.
² Graduate School of Biomedical Sciences, University of Texas Southwestern Medical Center at Dallas, Dallas, TX 75390-9148, USA.

View larger version (86K): [in this window] [in a new window]   Fig. 1. Stwl is required for GSC maintenance. (A-C) Ovaries from heterozygous control animals (A) were dissected 10 days post-eclosion. Germ cells were labeled with the germ cell marker anti-Vasa (green), a fusome marker (anti-Hts, red) and the nuclear dye Hoechst (blue). Ovaries from stwlZ1/stwl95 mutant animals were dissected at 4 days (B) and 10 days (C) post-eclosion. (D-G) GSC clones were induced by heat-shock in adult animals and identified by the absence of nuclear GFP. (D) Clones induced in control wild-type genotype. Arrow points to the GSC. (E,F) The presence of clonally-related mutant germ cell clusters at 7 days post-heat-shock (outlined) indicated that each germ cell cluster was derived from a mutant stem cell. The stem cell region of this germarium, however, contained only wild-type stem cells (arrows). (G) Graph showing the frequency of negatively-marked stem cell clones from a wild-type FRT chromosome (blue) or two stwl-null chromosomes (yellow, pink) over a 2-week period.

View larger version (65K): [in this window] [in a new window]   Fig. 2. Ectopic germline expression of Stwl induced an expansion of germline stem cells. (A) Germarium from Nos-Gal4 control animals contained a dividing stem cell that can be recognized by the elongated fusome (arrow) connecting the germline stem cell (GSC)-cystoblast pair of cells that were negative for bam-GFP (red, anti-Hts; blue, Hoechst; green, GFP). (B) Projection of three confocal sections from Nos-Gal4>stwlEY animals. Arrow shows GSC-like cells. (C) Graph showing the average number of GSCs/germarium in various genotypes: Nos-Gal4 alone (light purple), Nos-Gal4 driving CG3919 (a gene neighboring stwl, blue), Nos-Gal4 driving stwl (green), Nos-Gal4 driving stwl/nosRC (pink), Nos-Gal4 driving stwl/Df(nos) (dark purple) or Nos-Gal4 driving stwl/pumET9 (yellow). bam-GFP-negative cells containing single, spherical fusomes were scored as GSCs (Xie and Spradling, 1998; Chen and McKearin, 2003a). The difference in the number of GSC-like cells in Nos-Gal4>stwlEY (Nos->Stwl in C) ovaries was statistically significant by unpaired t-test (P<0.001) when compared individually to the number of GSC-like cells scored in the other genotypes presented in C.

View larger version (54K): [in this window] [in a new window]   Fig. 3. stwl mutants are dominant suppressors of position-effect variegation. (A) Eye of DX1 flies showing strong variegation. (B,C) Eyes of DX1 flies also heterozygous for stwl alleles showing suppressed variegation. (D) The DX1 variegated phenotype was dominantly suppressed by a mutation in the histone methyltransferase encoded by the Su(var)3-9 locus. (E) Graph of optical density of extracted pigments from flies of the indicated genotypes.

View larger version (115K): [in this window] [in a new window]   Fig. 4. stwl was not required for bam silencing and suppressed differentiation independently of bam. Spectrosomes/fusomes are labeled red in each panel. (A) bam transcriptional reporter (green) activity in wild-type germarium. (B) bam transcriptional reporter activity in stwl bam double mutants. Arrows and arrowheads indicate GSCs and cytoblasts, respectively. (C) Germ cells (stained green with anti-Vasa) in bam mutant ovaries contained spherical spectrosomes (red). (D) Elongated fusomes indicative of cyst formation (encircled) in stwl bam mutant ovaries. DNA is blue in C,D.

View larger version (65K): [in this window] [in a new window]   Fig. 5. Expression of Bruno and Lola in stwl mutant cells. (A,B) Bruno (Bru) expression (red) in stwl mutant GSCs (marked by the absence of GFP, green, and outlined) was similar to that of neighboring wild-type germ cells. (C-F) Bru expression (red) was uniform in bam mutant germaria (C,D) but was elevated in stwl bam ovaries (E,F) as differentiating germ cell clusters formed (arrows). Spectrosomes/fusomes (green) were labeled in C and E to show the degree of cyst development. (G,H). Germline clones of stwl mutant cells (marked by the absence of GFP, green, and outlined) contained higher levels of Lola (red) than neighboring cells.

View larger version (47K): [in this window] [in a new window]   Fig. 6. A model for Stwl function in repressing the transcription of germ cell differentiation-promoting factors. In wild-type GSCs (top), transcriptional quiescence of bam, via the Dpp signaling cascade, allows full Nos-Pum translational repression of differentiation-promoting mRNAs. Stwl represses the transcription of these mRNAs, probably through interaction with and/or modification of histones. In stwl- GSCs (bottom), the absence of Stwl function upregulates transcription, thereby increasing the pool of differentiation-promoting mRNAs and promoting cystoblast differentiation despite full Nos-Pum activity.