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doi: 10.1242/10.1242/dev.00499

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Clonal expansion of ovarian germline stem cells during niche formation in Drosophila

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 (114K): [in a new window]   Fig. 1. Related developmental timing between GSC establishment and TF/cap cell formation during ovarian development. The female gonads in A-C were labeled for Hts (red) and GFP (green). D was labeled for Bam (red) and GFP (green). E and F were labeled for lacZ (red) and GFP (green). All the images represent one confocal section and the anterior end is towards the left. (A) A bam-GFP gonad at the late third-instar larval stage showing no significant GFP expression in PGCs. The arrowhead indicates an anterior PGC and the arrow indicates a posterior PGC. (B) A bam-GFP gonad at the larval-pupal transition stage showing sporadic GFP-positive PGCs. The arrowhead indicates one anterior PGC negative for GFP, while the arrow indicates a posterior PGC positive for GFP. (C) A bam-GFP gonad at the early pupal stage showing that all posterior PGCs (arrow) are positive for GFP and anterior PGCs (arrowhead) are negative for GFP. (D) A vasa-GFP gonad at the early pupal stage showing that some of the posterior PGCs (arrow) are BamC-positive and anterior PGCs (arrowhead) are BamC-negative. vasa-GFP labels all germ cells. (E) A hh-lacZ;bam-GFP gonad at the late third instar larval stage showing TF stacks (brackets) forming in a progressive manner across the gonad and no bam-GFP expression in PGCs (arrow). The TF stack indicated by the bottom bracket has more cells than the ones indicated by the upper brackets. (F) A hh-lacZ;bam-GFP gonad at the early pupal stage showing that posterior PGCs (arrow) are bam-GFP-positive and anterior PGCs close to TFs (brackets) and cap cells (arrowhead) are bam-GFP-negative. Scale bars: 20 µm.

View larger version (120K): [in a new window]   Fig. 2. The establishment of GSCs during the early pupal stage. All images represent one confocal section. Hh-lacZ is expressed in the nuclei of TFs/cap cells, whereas Hts staining highlights fusomes in germ cells and membranes of TFs/cap cells and other somatic cells. The lacZ-positive disc-like cells that are packed against each other in a line with their longitudinal axis are TFs, whereas the posterior lacZ-positive cells that do not align with TFs are cap cells. The gonads in A-D are labeled for lacZ (red) and Hts (green); the gonads in E and F are labeled for lacZ (blue), GFP (green) and Hts (red). (A,B) A hh-lacZ gonad at the larval-pupal transition stage showing only TFs. B shows two tightly packed hh-lacZ-positive TFs (brackets) without the appearance of cap cells at a higher magnification in the boxed area in A. (C) Part of a hh-lacZ female gonad at the transitional stage from larva to pupa showing finished TF cells (bracket) and newly formed cap cells. The fusome (arrow) in one of the PGCs moves close to the interface (white line) with the adjacent cap cell (arrowhead). (D) Part of a hh-lacZ female gonad also at the transitional stage showing two different PGC division patterns. In the top part, only one of the two PGC daughter cells (unbroken outline) contacts cap cells (arrowhead); in the lower part, both of the PGC daughters (broken outline) contact cap cells (arrowheads). (E,F) hh-lacZ;bam-GFP gonads showing that the two-cell clusters close to cap cells (arrowheads) are not two-cell cysts but two PGC/GSC daughters. Only one of the two GSC daughters (unbroken lines) is in juxtaposition to TFs/cap cells (arrowhead) in E, whereas both the GSC daughters (broken lines) are in close contact with TFs/cap cells (arrowheads) in F. Scale bars: 20 µm in A; 10 µm in B-F.

View larger version (93K): [in a new window]   Fig. 3. Two mechanisms used by GSCs for populating their niches. (A) An explanatory diagram for two potential mechanisms populating GSC niches: GSCs in one niche come from one PGC (top); GSCs in one niche come from multiple PGCs (bottom). PGCs are marked by gain of lacZ (nuclear) expression (B,C) or loss of lacZ (nuclear and cytoplasmic) expression (D,E) before the late third instar larval stage. (B-E) Germaria are labeled for lacZ (red), Hts (green) and DNA (blue). (B) A germarium with one lacZ-positive marked GSC (broken outline) and a lacZ-negative unmarked GSC (unbroken outline) (i.e. only some of the GSC progeny are marked by lacZ expression). (C) A germarium with three GSCs (their spectrosomes indicated by arrowheads) and their progeny are marked by lacZ expression (an overlaid image). (D) A germarium with one lacZ-negative marked GSC (broken outline) and one lacZ-positive unmarked GSC (unbroken outline) (i.e. only a fraction of differentiated germ cells are marked by loss of lacZ expression). (E) A germarium with three GSCs (broken outline) marked by the loss of lacZ expression, in which all differentiated germ cells are lacZ negative. Scale bar: 10 µm.

View larger version (99K): [in a new window]   Fig. 4. dpp directly signals to anterior PGCs at the early pupal stage of ovarian development. All female gonads were isolated from early pupa. (A,B) A wild-type gonad labeled for BrdU (green, nuclei) and Vasa (red, cytoplasm), showing that the PGCs close to TFs/cap cells have a higher tendency to be BrdU-positive than the other PGCs. Several BrdU-positive somatic cells in the germ cell zone are indicated by arrows. (B) Area in A outlined by broken lines, showing anterior BrdU-positive PGCs (arrowheads) and a BrdU-positive somatic cell (arrow). (C,D) A gonad labeled for dpp mRNA (red) and Hts (green), showing the expression of dpp mRNA in anterior somatic cells. (D) Boxed area in C at higher magnification, showing dpp mRNA in TFs and cap cells (arrowhead) but not in PGCs indicated by spectrosomes (arrow). (E,F) A Dad-lacZ/+ gonad labeled for lacZ (red) and Hts (green), showing that Dad is preferentially expressed in the PGCs close to TFs/cap cells. (F) Boxed area in E at higher magnification, showing a Dad-positive PGC (outlined by an unbroken line) adjacent to TFs/cap cells and a Dad-negative PGC lying more posterior (outlined by a broken line). Scale bars: 20 µm for A,C,E.

View larger version (102K): [in a new window]   Fig. 5. dpp is capable of stimulating PGC proliferation during early ovarian development. (A,B) A C587-gal4/UAS-GFP gonad labeled for GFP (green) and Hts (red), showing the expression pattern of a target gene controlled by the GAL4 line in the majority of the somatic cells. (B) The boxed area in A, showing that GFP is only expressed in most of the somatic cells (arrowhead) but not in the PGCs (outlined) that are identified by the presence of spectrosomes (arrow). (C,D) A C587-gal4/UAS-dpp; Dad-lacZ/+ gonad labeled for lacZ (red) and Hts (green), showing that overexpressed dpp induces expression of Dad-lacZ in all the PGCs as well as somatic cells. (D) The boxed area in C, showing that all the PGCs (some PGCs not adjacent to TFs/cap cells are outlined) and somatic cells (arrowhead) express Dad. (E) An early pupal wild-type gonad labeled for dead cells by ApoTag (green, nucleus) and Vasa (red, cytoplasm), showing a dying germ cell (arrow) and dying somatic cells in the anterior (arrowhead). (F) A C587-gal4/UAS-dpp gonad at the early pupal stage labeled for BrdU (green, nucleus) and Vasa (red, cytoplasm), showing that in the dpp-overexpressing gonad BrdU no longer preferentially labels anterior PGCs (arrowhead) and also labels more posterior PGCs (arrows) than in the control. Scale bars: 20 µm for A,C,E,F.

View larger version (89K): [in a new window]   Fig. 6. The requirement of tkv for clonal expansion of GSCs during niche formation. Both germaria are labeled for lacZ (red), Hts (green) and DNA (blue). lacZ-negative mutant tkv PGCs were generated before the late third instar larval stage, and mutant tkv GSCs in the adult ovary were subsequently identified by loss of lacZ expression. (A) A germarium carrying three lacZ-positive wild type GSCs (outlined by unbroken lines) and lacZ-negative tkv mutant germline cysts (outlined by a broken line). The mutant tkv cysts were most likely derived from a lost mutant tkv GSC. (B) A germarium carrying a lacZ-negative tkv mutant GSC (outlined by a broken line), a lacZ-positive wild type GSC (outlined by an unbroken line), and a mutant tkv cyst (arrow). Scale bar: 10 µm.