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doi: 10.1242/10.1242/dev.00325

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A discrete transcriptional silencer in the bam gene determines asymmetric division of the Drosophila germline stem cell

Department of Molecular Biology, University of Texas-Southwestern Medical Center, Dallas, TX 75390-9148, USA

View larger version (69K): [in a new window]   Fig. 1. Dynamics of bam expression. Schematic view of the germarium to demonstrate the dynamic pattern of bam mRNA and protein distribution. bam transcripts are undetectable in germline stem cells (GSCs) by RNA in situ hybridization but a small amount of Bam protein is produced and decorates the GSC fusome. In the cystoblast, bam mRNA accumulates to its highest levels. As cysts mature, the concentration of bam transcripts declines and are again undetectable in eight-cell cysts. During the period from cystoblasts through eight-cell cysts, Bam protein concentrations increase dramatically in the cystoplasm and expanding fusomes, reaching their highest levels in eight-cell cysts. Bam protein disappears rapidly from cystocyte cytoplasm once the 16-cell cyst forms.

View larger version (35K): [in a new window]   Fig. 2. The 5' deletion series to refine the minimal bam promoter. Schematic representations of the various deletion constructs are shown in A. Each construct's expression in germline stem cells (GSCs) and cystoblasts (Cb) is indicated in columns beside the appropriate construct schematic. The ability of the [Bam:GFP] constructs to rescue bam mutant flies was scored and is indicated in an additional column. Red and green rectangles represent the bam 5'-UTR and GFP, respectively. (B) Representative confocal images of the GFP expression pattern produced by individual constructs. Each panel contains a single optical section of a germarium in the plane of the terminal filament from animals expressing reporter from the indicated bam promoter fragment. The germarium in A was reacted with antibodies against Hts (red) and GFP (green) to show reporter expression in GSCs and cystoblasts. The arrowhead indicates the stem cell fusome located next to the contact point with cap cells, and the arrow points to the fusome located internally in a cystoblast. Asterisks indicate position of GFP-negative GSCs in A-E. (A) –898/+133-Bam:GFP; (B) –198/+133-Bam:GFP; (C) –96/+133-Bam:GFP; (D) –47/+133-GFP; (E) –168/+133-GFP; (F) –140/+133-GFP. gfp, green fluorescent protein.

View larger version (58K): [in a new window]   Fig. 3. The 3' deletion series. (A) Deletion constructs in schematic form which indicate if the truncated promoter caused GFP expression in germline stem cells (GSCs) and cystoblasts. (B) Confocal sections of the indicated constructs. Asterisks indicate position of GFP-negative GSCs. Arrows indicate the position of the base of terminal filaments when GSCs are GFP positive. (A) –799/+133-GFP; (B) –799/+55-GFP; (C) –799/+4-GFP; (D) –799/-20-GFP; (E) –799/-47-GFP.

View larger version (89K): [in a new window]   Fig. 4. Silencer activity is necessary for germline stem cell maintenance. (A-D) Ovarioles from females expressing Bam inappropriately in GSCs. A and C are DIC images, whereas B and D are DAPI-stained fluorescent micrographs. The ovariole in A and B was taken from a female expressing Bam:GFP from a nos promoter. The ovariole in C and D is from a female carrying 2 copies of a transgene expressing Bam:GFP from a (–799/+4) bam promoter fragment that lacks the sequences required for GSC silencer activity. Note that in both examples, the ovariole contains well-developed cysts with nurse cells but the germarium (arrow) is shriveled.

View larger version (90K): [in a new window]   Fig. 5. Refining the position of silencer sequences. Each panel shows a germarium recovered from females expressing green fluorescent protein (GFP) reporter from transgenes driven by the named promoter fragments. Arrows indicate the position of the terminal filament base, whereas asterisks mark GSCs when these cells are GFP-negative in (A) –799/+55-GFP; (B) –799/+34-GFP; (C) –799/+55(+27-+37)-GFP; (D) –799/–20(+27-+37)-GFP; (E) –799/+150(+37-+39)GFP; (F) –799/–20(+25-+45)-GFP. (G) An example of an ovariole with germline clones from brk[M68] P{FRT}18A/P{Ubi-GFP} P{FRT}18A flies that were heat-shocked 14 days prior to ovary dissection. The arrow points to the fusome in a GFP-negative GSC; several cysts with GFP-negative cystocytes are present.

View larger version (54K): [in a new window]   Fig. 6. Testing post-transcriptional effects of the bam silencer. (A) A schematic of the structure of a transgene in which a fragment of the bam region containing putative silencer sequences was joined to the nos promoter. The relative positions of oligonucleotides used for PCR primers are also shown above the schematic. (B) An example of the GFP expression pattern from the [nosP-bam5'-GFP-bam3'] is shown in a confocal optical section. The cell immediately adjacent to the base of the terminal filament, indicated by the arrow, is GFP-positive. (C) An agarose gel of products generated by RT-PCR of RNA isolated from [nosP-bam5'-GFP-bam3'] using oligonucleotides shown in A. The primer pairs used in the reactions are indicated on the right-hand side of the image.

View larger version (72K): [in a new window]   Fig. 7. Silencer activity is position dependent. A reporter construct (A) was constructed to place the silencer element upstream of a bam promoter that contained only the positively acting elements. GFP expression from the transgene included GSCs. (B) GFP expression from a construct that replaces the putative basal bam promoter element with a P-element minimal promoter element. (C) GFP expressed in the native bam pattern from a promoter fragment that includes the region from –402 to +133. (D,E) GFP expression patterns of promoters derived from the same fragment as in C but carrying deletions of –86 to –61 (D) and –68 to –61 (E) are shown. Arrows point to position of terminal filament base in A and B; asterisk marks GSC in C.

View larger version (50K): [in a new window]   Fig. 8. Transcriptional control elements in the bam promoter. The 5'-UTR and proximal upstream sequences are shown with various landmarks indicated. The 5'-end of the smallest promoter with full rescuing activity tested in the studies is indicated as `–198'. Regions containing transcriptional control elements are presented in large, upper case fonts and distinguishing colored backgrounds. The region that contains germ cell (GC)-specific enhancer element(s) (–86 to –61) is highlighted in blue. The GC-rich sequence that is essential for bam transcription and matches the consensus binding site for Mad protein is underlined. The sequences outlined in gray could be replaced by basal promoter elements from the P-element promoter, whereas the transcriptional start site (+1) is highlighted with green. The bipartite silencer element in the bam 5'-UTR is outlined in two colors, with A in purple and B in yellow. The initiating methionine codon is in red.

View larger version (71K): [in a new window]   Fig. 9. Regulation of elements controlling bam transcription. The transcriptional silencer in the bam 5'-UTR is active in the cells in contact with cap cells, preventing bam expression. This effectively prevents GSCs from differentiating into cystoblasts and maintains them as a self-renewing population. Dpp signaling, and perhaps other signals delivered by diffusion and gap junction connections, are probably important positive regulators of silencer element-binding proteins (SEBPs). In the stem cell daughter, GSC maintenance signaling would be abrogated, the silencer would be inactive and bam would be produced under the control of the enhancer elements that drive transcription in germ cells. During the transition from active-to-inactive silencer, GSC daughters would be `pre-cystoblasts' until bam expression reached critical levels for full cystoblast differentiation. The switch from active-to-inactive silencer would also represent the biochemical mechanism for the asymmetry of the GSC division.