QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 18 February 2004
doi: 10.1242/dev.01026

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Song, X. Articles by Xie, T. Search for Related Content PubMed PubMed Citation Articles by Song, X. Articles by Xie, T. Social Bookmarking                         What's this?

Bmp signals from niche cells directly repress transcription of a differentiation-promoting gene, bag of marbles, in germline stem cells in the Drosophila ovary

Xiaoqing Song^1,*, Marco D. Wong^1,*, Eihachiro Kawase^1,*, Rongwen Xi¹, Bee C. Ding¹, John J. McCarthy¹ and Ting Xie^1,2,

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 (71K): [in a new window]   Fig. 1. dpp signaling activity is restricted to GSCs and some cystoblasts where bam transcription is actively repressed. (A) Diagram showing GSCs, their differentiated progeny and surrounding somatic cells. In panels B-F, cap cells are highlighted by circles, whereas GSCs are indicated by asterisks. (B) Tip of the Dad-lacZ germarium labeled for nuclear ß-Gal (red), Hts (green, fusomes) and DAPI (blue), showing high Dad expression in GSCs and in a cystoblast (arrow), but not in another cystoblast (arrowhead). (C) Tip of the bam-GFP germarium labeled for GFP (green), Hts (red, fusomes) and DAPI (blue), showing bam expression in a cystoblast (arrowhead) and cysts, but not in GSCs. (D) Tip of the bam-GFP;Dad-lacZ germarium labeled for ß-Gal (red), GFP (green) and DAPI (blue), showing that GSCs and two cystoblasts (arrows) express high Dad but no bam, and that a cystoblast (arrowhead) has low Dad and begins to express bam. (E,F) Tip of the bam-GFP germarium labeled for pMad (red), GFP (green), Hts (blue, fusomes) and DAPI (white, F), showing high pMad accumulation but no bam expression in GSCs, and low pMad but bam expression in a cystoblast (arrowhead, E). TF, terminal filament; GSCs, germline stem cells; SS, spectrosome; Cpc, cap cells; CB, cystoblast; FS, fusome; IGS, inner sheath cells; CS, cysts. All micrographs are shown at the same scale. Scale bar: 10 µm.

View larger version (56K): [in a new window]   Fig. 2. dpp is essential for repressing bam transcription in GSCs. Germaria in panels A-D are labeled for Hts (red, fusomes), GFP (green) and DAPI (blue), whereas the germaria in E-H are labeled for pMad (red), GFP (green), Hts (blue, fusomes) and DAPI (white; F,H). All the GSCs are indicated by asterisks, and cap cells in all the panels are marked by circles. (A) A germarial tip from a bam-GFP female cultured at 29°C for one week, showing no bam expression in GSCs. (B) A germarial tip from a bamGFP dpphr56/dpphr4 female cultured at 29°C for 2 days, showing that one of the two GSCs begins to express bam. (C,D) Germarial tips from bamGFP dpphr56/dpphr4 females cultured at 29°C for 4 (C) or 7 (D) days, showing that the only remaining GSC starts to express bam. (E,F) Germarial tip from a bam-GFP female cultured at 29°C for 4 days, showing high pMad accumulation and no bam expression in GSCs. (G,H) A germarial tip from a bamGFP dpphr56/dpphr4 female cultured at 29°C for 4 days, showing that two mutant GSCs have low pMad levels and begin to express bam. All micrographs are shown at the same scale. Scale bar: 10 µm.

View larger version (69K): [in a new window]   Fig. 3. dpp overexpression is sufficient for repressing bam transcription in single germ cells. Germaria in panels A and C-F are labeled for Hts (red, fusomes), GFP (green) and DAPI (blue), whereas the germarium in B is labeled for Vasa (red, germ cells) and Hts (green, fusomes). Circles highlight cap cells and asterisks indicate GSCs. (A) A germarial tip showing c587-gal4-driven UAS-GFP expression in inner sheath cells but not in cap cells. (B) A c587-gal4;UAS-dpp germarium resulting from dpp overexpression is filled with single germ cells with a spectrosome. The inset shows the tip of the germarium (highlighted by a rectangle in B) at a higher magnification (4x), containing only germ cells with a spectrosome (arrows). (C) Tip of the c587-gal4;bam-GFP;UAS-dpp germarium showing that the accumulated spectrosome-containing germ cells (two indicated by arrows) a few cells away from the tip of the germarium fail to express bam-GFP. (D) Middle portion of the c587-gal4;bam-GFP;UAS-dpp germarium showing that spectrosome-containing germ cells (two indicated by arrows) fail to express bam-GFP. (E) Tip of the hs-gal4;UAS-dpp germarium showing no bam expression in GSCs, but expression in differentiated germ cells without any heat-shock treatments. (F) Tip of the hs-gal4;UAS-dpp germarium showing no bam expression in GSCs and in spectrosome-containing germ cells (two indicated by arrows) distant from the tip after three days of heat-shock treatments. Scale bars: in A, 10 µm for A,C-F; in B, 60 µm.

View larger version (85K): [in a new window]   Fig. 4. gbb is expressed in the somatic cells of the germarium and is essential for maintaining GSCs and for repressing bam transcription in GSCs. (A) A DNA gel with RT-PCR products showing that gbb is expressed in the somatic cells of the germarium but not in GSCs. In this gel, mRNA for whole ovaries, agametic ovaries, inner sheath cells and GSC-like germ cells are marked by templates 1, 2, 3 and 4, respectively. vasa and dpp genes are positive controls, whereas rp49 is an internal control. Germaria in B-E are labeled for Hts (red, fusomes) and DAPI (blue), whereas germaria in F-I are labeled for Hts (red, fusomes), GFP (green) and DAPI (blue). Circles highlight cap cells, whereas asterisks indicate GSCs. (B) Germarial tip from a wild-type bam-GFP female cultured at 29°C for 1 week showing two GSCs. (C-E) Germarial tips from the bam-GFP gbb4/gbbD4 females cultured at 29°C for one week showing one GSC (C), no GSC but 16-cell cysts (one indicated by arrow; D) and no GSCs and no cysts (E). (F) Germarial tip from a bam-GFP gbb4/gbbD4 female cultured at room temperature for 2 days showing that the remaining GSC does not express bam. (G,H) Germarial tips from bam-GFP gbb4/gbbD4 (G) and bam-GFP gbb4/gbbD20 (H) females cultured at 29°C for one week, showing that the remaining single GSC expresses bam. (I) A germarial tip from a c587-gal4/UAS-gbb; bam-GFP female showing a normal number of GSCs, and normal bam-GFP expression in cystoblasts (arrow) and other differentiated germ cells. Scale bar in B: 10 µm for B-I.

View larger version (88K): [in a new window]   Fig. 5. Reduction of pMad is correlated with upregulated bam transcription in gbb mutant GSCs. All the germaria are labeled for pMad (red), GFP (green), Hts (blue, fusomes) and DAPI (white). The panels A'-F' represent the corresponding DAPI images for panels A-F. Circles highlight cap cells, whereas asterisks indicate GSCs. (A) A germarial tip from a bam-GFP female cultured at 29°C for 4 days showing normal pMad expression and no bam-GFP expression in GSCs. (B-F) Germarial tips from either bam-GFP gbb4/gbbD4 (C,F) or bam-GFP gbb4/gbbD20 (B,D,E) females cultured at 29°C for 4 days, showing reduced pMad expression. gbb mutant GSCs with easily detected pMad do not express bam-GFP (B,C; one indicated by an arrow in D), whereas the other GSCs with severely reduced pMad show bam-GFP expression (one indicated by an arrowhead in D). All the micrographs are shown at the same scale. Scale bar: 10 µm.

View larger version (81K): [in a new window]   Fig. 6. punt and Med are required cell-autonomously in GSCs to repress bam transcription. Germaria from punt10460/punt135 mutant females cultured at 18°C for 2 days (A), or at 29°C for 2 (B) or 7 (C,D) days, are labeled for Hts (red, fusomes and somatic follicle cells), bam-GFP (green) and DAPI (blue). Germaria in E-H from the punt10460/punt135 mutant females cultured at 29°C for 4 days are labeled for pMad (red), bam-GFP (green), Hts (blue, fusomes) and DAPI (white). Germaria in I-L are labeled for arm-lacZ (red), GFP (green), Hts (blue) and DAPI (white). F,H,J and L represent corresponding DAPI stainings for E,G,I and K, respectively. Circles highlight cap cells, whereas asterisks indicate GSCs. (A-D) Germarial tips showing two GFP-negative GSCs (A), one GFP-positive and one GFP-negative GSC (B), one GFP-positive GSC (C) and no GSC (D). (E-H) Germarial tips showing two GFP-positive GSCs with severely reduced pMad (E,F) and one GFP-positive GSC with severely reduced pMad (G,H). (IL) Germarial tips showing a bam-GFP-positive marked punt135 GSC (outlined by a dashed line; I) and a bam-GFP-positive marked Med26 GSC (dashed line; K). The marked GSCs are identified by loss of arm-lacZ expression. All micrographs are shown at the same scale. Scale bar: 10 µm.

View larger version (53K): [in a new window]   Fig. 7. Mad and Med bind directly to the bam silencer in vitro. (A) Sequence alignment of the bipartite bam and brk silencers, in which conserved base pairs are boxed. Sites A (red) and B (green) of the bam silencer are as previously described (Chen and McKearin, 2003a). (B) A Cy5 5'-modified oligonucleotide containing the bam silencer and unlabeled competitors in electrophoretic mobility shift assays (competitor A+B, the unlabeled silencer with sites A and B; competitor A, same flanking sequences with site A only; competitor B, same flanking sequences with site B only). (C) Immunoblot analysis of purified recombinant GST-tagged proteins with a mouse anti-GST antibody. Lane 1, 50 ng of GST; lane 2, 200 ng of GST-Mad; lane 3, 200 ng GST-Med. The 30 kDa bands in lane 3 are probably C-terminal degradation products of GST-Med protein. (D) Gel shift assay showing that Mad or Med bind to the bam silencer in vitro. Approximately 10 nmol of protein was used in each binding reaction. The double shift bands for Med may result from partially degraded proteins. The labeled probe without protein (lane 1) or with GST protein (lane 2) serve as negative controls. The labeled probe binds to Mad (lane 3) and Med (lane 7). The unlabeled competitors A+B (lane 4), A (lane 5) or B (lane 6) could effectively compete away Mad binding. The unlabeled competitors A+B (lane 8) or A (lane 9) could effectively compete for Med binding but B (lane 10) could only partially compete. (E) Current model for how Bmp niche signals control GSC identity by directly repressing bam transcription. Bmp signals from cap cells produce the highest levels of pMad, which associates with Med and directly occupies the bam silencer to repress its transcription in GSCs. As a cystoblast moves away from cap cells, levels of pMad are reduced to below the critical threshold level, bam transcription is then derepressed and activated by an unknown activator (indicated by `?').

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter