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First published online 9 November 2005
doi: 10.1242/dev.02151

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Pelota controls self-renewal of germline stem cells by repressing a Bam-independent differentiation pathway

¹ Stowers Institute for Medical Research, 1000 E. 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 (75K): [in a new window]   Fig. 1. pelo is required for controlling ovarian GSC self-renewal. All germaria or ovarioles in this and subsequent figures represent one confocal section and are shown with anterior towards the left. (A) A schematic diagram of the germarium. Red circles indicate spectrosomes (i.e. GSCs and cystoblasts) and branched red structures indicate fusomes (i.e. germ cell cysts). Ovarioles from pelo1 homozygous mutant (C-E) and heterozygous control (B) flies labeled for Vasa (red, germ cells) and for Hts (green, spectrosomes and fusomes). (B-E) Germaria contain two GSCs (B) (indicated by arrowheads), two GSCs (C), one GSC (D) and no GSCs (E). (F) A pelo1 mutant germarium labeled by TUNEL (red), showing that the GSC (arrowhead) was not apoptotic but there are some dying follicle cells (red) in the posterior. Scale bars: 10 µm.

View larger version (57K): [in a new window]   Fig. 2. pelo functions in the cytoplasm of GSCs to control their self-renewal. The tips of the ovarioles (A-D) are labeled for lacZ (green) and Hts (red); the marked GSCs (broken lines) are identified by loss of lacZ expression and presence of a spectrosome on their anterior side. (A,B) Germaria showing the presence of 3-day-old (A) and 24-day-old (B) marked wild-type GSCs. (C) A germarium carrying a marked 3-day-old pelo1 homozygous GSC. (D) A germarium showing loss of a marked pelo1 mutant GSC evidenced by the presence of a marked germline cyst (arrowhead) in an egg chamber 17 days ACI. (E) Quantitative analyses of pelo1 GSC loss and phenotypic rescues by different pelo transgenes, which carry wild-type or mutant (nls*) epitope-tagged pelo. The x-axis shows different genotypes, while the y-axis indicates the average GSC number with a standard error. The blue bars indicate GSC numbers of the 2-day-old germaria of different genotypes, while the red bars represent GSC numbers of 7-day-old germaria. For each genotype at either time point, 40-130 germaria were examined. (F) A germarium with two GSCs (arrowheads) labeled for Hts (green) and DAPI (blue, nuclei) showing that germline expression of pelo is sufficient to rescue GSC loss in pelo1 mutants. (G,H) Mutant germaria with one (G) or no (H) GSC labeled for hh-lacZ (red) and Hts (green), showing the normal number of cap cells. Scale bars: 10 µm.

View larger version (114K): [in a new window]   Fig. 3. Pelo is primarily localized in the cytoplasm of germ cells. (A-D) A nos-gal4VP16; UAS-FLAG-Pelo; Sec61a-GFP germarium labeled for FLAG (red, A) and GFP (green, B). The merged image (C) shows FLAG-tagged Pelo is localized primarily in the cytoplasm but is not particularly rich in ERs (green). (D) The enlarged area of a GSC marked in C. Scale bars: 10 µm.

View larger version (103K): [in a new window]   Fig. 4. pelo modulates dpp pathway activity, but is dispensable for bam suppression in GSCs. Germaria (A-D) are labeled for Hts (red) and DAPI (nuclei, blue). (A) A germarium showing that dpp overexpression causes accumulation of GSC-like cells. (B) A germarium showing that removal of one copy of functional pelo results in partial suppression of the GSC-like tumor phenotype induced by dpp overexpression, which is evidenced by the presence of egg chambers. (C,D) Germaria showing that removal of both copies of pelo partially (C) or completely (D) suppresses the tumor phenotype. (E,E') A germarium labeled for GFP (green) and lacZ (red), showing that a marked pelo mutant GSC (broken lines, black in E) downregulates dad-lacZ expression (red, E') in comparison with its neighboring wild-type GSC (solid circle, green). (F,F') A germarium labeled for GFP (green) and lacZ (red), showing that a marked pelo mutant GSC (broken lines, black in F) does not upregulate bam-GFP expression (F') in comparison with its neighboring wild-type GSC (solid circle, red). Scale bars: 10 µm.

View larger version (95K): [in a new window]   Fig. 5. A mutation in pelo can allow bam mutant cystoblast-like cells to differentiate into cystocytes. Germaria are labeled for Hts (red, A,B) or Orb (red, C,D) and DAPI (nuclei, blue). (A) A germaria homozygous for bam86 and heterozygous for pelo1, showing undifferentiated single germ cells as evidenced by spectrosomes. (B) A germarium double homozygous for pelo1 and bam86 showing the formation of cystocytes, as evidenced by branched fusomes (arrowhead). In addition, many persistent spectrosomes (arrow) are abnormally enlarged. (C) A wild-type germarium showing preferential accumulation of Orb in the newly formed oocyte (arrowhead). (D) A double pelo and bam homozygous germarium showing no oocyte formation, which is suggested by no preferential Orb accumulation in germ cells. Scale bars: 10 µm.

View larger version (101K): [in a new window]   Fig. 6. pelo mutant ovaries exhibit age-dependent egg chamber degeneration. (A) Part of a 2-day-old pelo1 mutant ovariole labeled for Hts (red, follicle cells) and DAPI (nuclei, blue), showing normal nurse cell morphology and the oocyte (arrowhead). (B) Part of a 7-day-old pelo1 mutant ovariole labeled for HtsRC (green, ring canals) and DAPI (nuclei, blue), showing condensed nuclei of degenerating nurse cells in the egg chamber, in which the oocyte is indicated by an arrowhead. (C) Part of a 7-day-old pelo1 mutant ovariole labeled for Hts (red, follicle cells) and DAPI (nuclei, blue), showing several degenerating and growth-arrested egg chambers (arrowheads). (D) Part of an ovariole labeled for lacZ (green) and DAPI (nuclei, blue), showing a degenerating (unusually condensed nurse cell nuclei) and growth-arrested pelo1 mutant germline cyst (arrowhead, no lacZ expression). Scale bars: 10 µm.

View larger version (12K): [in a new window]   Fig. 7. Current working models to explain how Pelo is involved in controlling GSC self-renewal. Bmp signaling directly represses a Bam-dependent differentiation pathway and thereby controls GSC self-renewal. Pelo and Pum function separately to repress different Bam-independent pathways (X and Y in A) or function together to repress a Bam-independent pathway (X in B).