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First published online 18 February 2004
doi: 10.1242/dev.01025

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Gbb/Bmp signaling is essential for maintaining germline stem cells and for repressing bam transcription in the Drosophila testis

¹ Stowers Institute for Medical Research, 1000 East 50th Street, Kansas City, MO 64110, USA
² Department of Anatomy and Cell Biology, University of Kansas School of Medicine, 3901 Rainbow Boulevard, Kansas City, KS 66160, USA

View larger version (99K): [in a new window]   Fig. 1. Dpp and Gbb function cooperatively to maintain GSCs in the Drosophila testis. (A) A diagram of the testis tip including GSCs and SSCs. Normally, seven to ten GSCs (three are shown here for demonstration; round red cells) and somatic stem cells (also known as cyst progenitor cells; red elliptical cells) directly contact the hub cells (gray cells). The gonialblast, which is encapsulated by two differentiated somatic cyst cells, moves away from the hub cells and divides to produce a two-cell, four-cell, eight-cell or eventually a 16-cell cluster, which can be identified by the branched fusome (green lines). The testes in B-E are labeled for FasIII (red, hub cells), Hts (green, spectrosomes and fusomes) and DAPI (blue). The testis in F is labeled for Hts (green) and DAPI (blue). The hub cells are labeled red by FasIII in B-E and highlighted by a circle in F, whereas the GSCs are highlighted by broken lines in B-F. (B) The tip of a wild-type testis showing seven GSCs that contact the hub cells. (C,D) The tips of two gbb4/gbbD20 mutant testes showing two remaining GSCs (C) or no GSCs (D) close to the hub cells. (E) A tip of dpphr4/dpphr56 mutant testis showing seven GSCs near hub cells. (F) The tip of a dpphr4/dpphr56 gbbD20 mutant testis showing three remaining GSCs near the hub cells. All the images are shown at the same magnification. Scale bar: 10 µm.

View larger version (126K): [in a new window]   Fig. 2. punt is required for maintaining GSCs in the Drosophila testis. All the testes are punt10460/punt135 mutants and are labeled for Hts (green) and DAPI (blue) except nuclei of dying cells are labeled red in D. The hub cells are highlighted by circles, whereas GSCs are identified by broken lines. (A) The tip of a punt mutant testis one week after being cultured at 22°C showing eight GSCs. (B,C) The tips of two punt mutant testis one week after being cultured at 29°C showing one remaining GSC (B) or no GSCs (C). (D) The tip of a punt mutant testis 4 days after being cultured at 29°C showing that no GSCs undergo apoptosis except a few late somatic cyst cells (arrow). All the images are shown at the same magnification. Scale bar: 10 µm.

View larger version (93K): [in a new window]   Fig. 3. GSCs and gonialblasts but not other differentiated germ cells are responsive to Bmp signaling in the testis. The hub cells and the GSCs in A, B and D-F are highlighted by circles and broken lines, respectively. (A) The tip of a Dad-lacZ/+ testis labeled for nuclear lacZ (red), Hts (green) and DNA (blue), showing that all five GSCs and all gonialblasts (arrowhead) express Dad. Arrows indicate that somatic cyst cells also express Dad. (B) The tip of a gbb4/gbbD20; Dad-lacZ/+ testis labeled for nuclear lacZ (red), Hts (green) and DNA (blue), showing that three remaining GSCs and all the gonialblasts do not have detectable Dad expression. (C) The tip of a testis overexpressing Dad specifically in the germ cells labeled for Hts (green) and DAPI (blue), showing no germ cells in the testis. (D) The tip of a wild-type testis labeled for pMad (red), Hts (green) and DAPI (blue), showing pMad accumulation predominantly in GSCs. (E) The tip of a gbb4/gbbD4 mutant testis labeled for pMad (red), Hts (green) and DAPI (blue), showing no detectable pMad accumulation in GSCs. (F) The tip of a punt10460/punt135 mutant testis labeled for pMad (red), Hts (green) and DAPI (blue), showing no detectable pMad accumulation in the remaining GSC. All the images are shown at the same magnification. Scale bar: 10 µm.

View larger version (120K): [in a new window]   Fig. 4. Bmp downstream components are required in GSCs for their maintenance in the Drosophila testis. All the testes are labeled for lacZ (red), Hts (green) and DNA (blue). Marked wild-type or mutant GSCs (highlighted by broken lines) are identified as the cells that contain a spectrosome, directly contact hub cells and lack lacZ expression, whereas unmarked GSCs are lacZ positive (red). (A) The tip of a testis carrying a marked 2-day-old wild-type GSC. (B) The tip of a testis carrying two marked two-week old GSCs. (C) The tip of a testis carrying a marked 2-day-old tkv mutant GSC. (D) A testis that has lost a marked tkv GSC clone two weeks after clone induction. (E) The tip of the testis carrying a marked 2-day-old mad mutant GSC. (F) The tip of a testis that has lost a marked mad GSC clone 2 weeks after clone induction. All the images are shown at the same magnification. Scale bar: 10 µm.

View larger version (87K): [in a new window]   Fig. 5. Gbb signaling is essential for repressing bam transcription in GSCs in the Drosophila testis. The testes in A-C are labeled for GFP (green), Hts (red) and DNA (blue); the testis in D is labeled for lacZ (red), GFP (green), Hts (white) and DNA (blue); the testes in E and F are labeled for Hts (green) and DAPI (blue). The hub cells are highlighted by circles, whereas some GSCs are highlighted by broken lines. (A) The tip of a bam-GFP wild-type testis showing no bam expression in GSCs (arrowhead) and gonialblasts (arrow). (B) The tip of a gbb4/gbbD20 mutant testis (after being cultured at 29°C for one week) showing elevated bam-GFP expression in GSCs. (C) The tip of a punt10460/punt135 mutant testis (after being cultured at 29°C for a week) showing elevated bam-GFP expression in GSCs. (D) The tip of a testis carrying a marked Med mutant GSC (arrowhead, lacZ negative) and unmarked wild-type GSCs (arrow, lacZ positive), showing elevated bam-GFP expression in the mutant Med GSC. (E) The tip of a hs-bam testis showing three remaining GSCs 1 week after heat-shock treatments. (F) The tip of a nos-gal4VP16;UAS-bam testis showing no GSCs. All the images are shown at the same magnification. Scale bar: 10 µm.

View larger version (177K): [in a new window]   Fig. 6. Overexpression of dpp but not gbb completely represses bam transcription in the testis. The testes in A and B are labeled for FasIII (red), Hts (green) and DNA (blue), whereas the testes in C and D are labeled for GFP (green), Hts (red) and DAPI (blue). The hub cells are identified by FasIII staining (red) in A and B, and are highlighted by circles in C and D. (A) The tip of a dpp-overexpressing testis showing more hub cells and slightly more single germ cells with a spectrosome (arrows) two or three cells away from the hub cells. (B) The tip of a gbb-overexpressing testis showing a normal hub and normal germ cell development. (C) The tip of a dpp-overexpressing testis showing that late differentiated germ cells (a two-cell cluster indicated by an arrow; a 16-cell cluster indicated by an arrowhead) fail to express bam-GFP. (D) The tip of a gbb-overexpressing testis showing that bam-GFP expression in the two-cell clusters (arrows) is delayed but is expressed in late differentiated germ cells (arrowhead). All the images are shown at the same magnification. Scale bar: 10 µm.

View larger version (68K): [in a new window]   Fig. 7. Two Bmp like molecules, Dpp and Gbb, are expressed in the somatic cells in the Drosophila testis. The testes in A-C are labeled for Hts (red), GFP (green) and DAPI (blue), and their hub cells are highlighted by circles. (A) The tip of a upd-gal4;UAS-GFP testis showing GFP-labeled hub cells. (B) The tip of a c587-gal4;UAS-GFP testis showing GFP-labeled somatic stem cells and somatic cyst cells. (C) The tip of a vasa-GFP testis showing GFP-labeled germ cells including GSCs. (D) A DNA gel with RT-PCR products showing that gbb and dpp mRNAs are primarily present in the somatic cells of the testis. In this gel, mRNAs from the whole testes, purified germ cells, somatic cyst cells/somatic stem cells and hub cells are marked as templates 1, 2, 3 and 4, respectively. vasa serves as a positive control, while rp49 is an internal control. For dpp (10x), approximately 10-fold more RNA template was used because of its low abundance. A-C are shown at the same magnification. Scale bar: 10 µm.

View larger version (70K): [in a new window]   Fig. 8. A current working model for how Bmp signals maintain GSCs in the Drosophila testis. In this model, Upd from hub cells, Gbb/Dpp from hub cells/somatic stem cells are important for GSC maintenance. An unknown signal initiated by EGFR/Raf signaling from somatic cyst cells is important for the proper differentiation of gonialblasts. Dpp/Gbb signaling also helps repress bam expression in GSCs and in gonialblasts (GBs). Two-cell germ cell clusters distant from hub cells/somatic stem cells receive less Bmp signaling and begin to express bam and promote further differentiation.

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