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First published online September 30, 2004
doi: 10.1242/10.1242/dev.01391

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Germline stem cells in the Drosophila ovary descend from pole cells in the anterior region of the embryonic gonad

Miho Asaoka^* and Haifan Lin

Department of Cell Biology, BOX 3709, Duke University Medical Center, Durham, NC 27710, USA

View larger version (47K): [in a new window]   Fig. 1. Anterior somatic cells in the embryonic gonad appear to be the precursors of niche cells in larval and adult gonads. The lacZ staining (blue) of enhancer trap 3914 is specific to anterior somatic cells in embryonic gonad (A), to nascent terminal filament (TF) and cap cells in the late third instar larval ovary (B), as well as to terminal filament (TF), cap cells (CC), and inner germarial sheath cells (IGS) in the adult ovary (C). In the adult testis (D), 3914 is specifically expressed in hub cells (H), somatic cyst progenitor cells (SCP) and somatic cyst cells (SCC). All panels are oriented with the anterior side of the gonad up and the posterior side down. Pole cells in A are stained with the anti-Vasa antibody (brown). Note that germ cells in all panels, such as GSCs in the adult testis (D), are negative for lacZ staining. Scale bars: 10 µm.

View larger version (123K): [in a new window]   Fig. 2. Pole cells located in the anterior half of the embryonic gonad develop into GSCs in the adult ovary. (A-D) Embryonic gonads viewed as superimposed DIC and EGFP images, showing representative locations of an EGFP-marked pole cell in the embryonic gonad. All gonads are oriented with anterior to the left and posterior to the right. The outline of the gonad is evident in DIC, with arrowheads pointing to the gonadal border to assist viewing. A marked pole cell (white spheres) is located at the anterior tip (A), anterior (B), posterior (C), or posterior tip (D). Dotted lines show the midline of the gonads. (E,G) An ovariole with one EGFP-labeled GSC (white arrow in G), as shown in DIC (E) and corresponding EGFP (G) images, from a female developed from an embryo with a marked pole cell located in the anterior half of the gonad. The EGFP-labeled GSC has produced multiple labeled germline cysts and egg chambers, which form an alternating pattern of labeled germline cysts and egg chambers in the ovariole. (F,H) An ovariole with both GSCs labeled by EGFP (white arrows in H), as shown in DIC (F) and corresponding EGFP (H) images, from a female developed from an embryo with a marked pole cell located in the anterior half of the gonad. The EGFP-marked GSCs have produced a continuously labeled string of labeled germline cysts and egg chambers in the ovariole. Scale bars: in A, 5 µm for A-D; in E, 20 µm for E-H.

View larger version (71K): [in a new window]   Fig. 3. Pole cells located in the posterior half of the embryonic gonad directly differentiate into cystoblasts. (A,C) An ovariole with an EGFP-labeled germline cyst in the germarium, as shown in DIC (A) and corresponding EGFP (C) images, from a female that developed from an embryo with a marked pole cell located in the posterior half of the gonad. Note that GSCs (white arrow in C) are not labeled by EGFP. The labeled cystoblasts have by now developed into germline cysts. (B,D) An ovariole with three EGFP-labeled developing egg chambers in DIC (B) and corresponding EGFP (D) images, from a female that developed from an embryo with a marked pole cell located in the posterior half of the gonad. Note that GSCs (white arrow in D) are not labeled by EGFP, thus the labeled egg chambers must have derived from three labeled PGCs that have directly differentiated into cystoblasts. Scale bar in A: 20 µm for A-D.

View larger version (60K): [in a new window]   Fig. 4. PGCs derived from anterior pole cells preferentially associate with the nascent cap cells. (A,A') A late third instar larval ovary derived from an embryonic gonad containing a labeled anterior pole cell viewed under low and high magnification, respectively. The ovary, with the anterior pole up, is stained with anti-1B1 antibody (red) to outline somatic cells and spectrosomes in germ cells, and with anti-GFP antibody (green) to identify PGCs derived from the marked pole cell. Note that the PGC marked by an arrow is in direct contact with the base of two forming terminal filaments (TF). (B,B') A late third instar larval ovary derived from an embryonic gonad containing a labeled posterior pole cell viewed under low and high magnification, respectively. The ovary, with the anterior pole up, is stained with anti-1B1 (red) and anti-GFP (green) antibodies. Note that the PGC marked by an arrow is approximately six cells away from the base of the forming terminal filaments (TF). (C) Summary of the location of PGCs (green) derived from marked anterior or posterior pole cells (green). The fractional numbers at the lower right corner of each third instar larval or prepupal (3°L/PP) ovary indicates the number of ovaries with shown location of PGCs versus the total number of this class of 3°L/PP ovaries examined. Yellow cells are nascent TFs. Scale bars: in A, 20 µm for A,B; in A', 10 µm for A',B'.

View larger version (72K): [in a new window]   Fig. 5. E-Cadherin is not required for PGCs to enter nascent stem cell niches. (A) A clone of PGCs derived from a single, labeled posterior pole cell in the embryonic gonad are labeled with anti-GFP antibody (green). The PGCs are distributed widely along both anteroposterior and mediolateral axes in this late third instar larval ovary. The cells of the ovary are outlined by anti-1B1 staining (red). The dotted line traces the forming terminal filament/cap cells (TF/CpC). (B) A late third instar larval ovary containing shg-null PGCs and their wild-type siblings. All cells in the ovary are outlined by anti-1B1 antibody staining (blue). All PGCs are labeled by anti-Vasa antibody (green). The shg-null PGCs (outlined by a thick white line) show no ß-Galactosidase expression, whereas their wild-type sibling PGCs (thin white line) show elevated expression of ß-Galactosidase (red). Note that a shg-null PGC (arrow) is in contact with nascent TF/CpCs (dotted line, TF). (C,D) A late third instar larval ovary triple-labeled for E-cadherin (green), Vasa (red), and 1B1 (blue). The dotted lines in D show forming TF/CpCs. In all PGCs (arrowheads in C; red in D), either in the anterior or posterior half of the ovary, E-Cadherin is concentrated on cell surface, including at the interface between CpCs and the contacting PGCs, irrespective of whether the spectrosome in the PGCs is apposed to CpCs (PGC pointed by a black arrow) or not (PGC pointed by a white arrow). (E) The shg-null PGCs (red) and their wild-type siblings (orange) show similar distributions in the late third instar larval ovaries, except that more wild-type PGCs are in contact with the posterior somatic cells (P<0.005, t test); the biological significance of which is not understood. Note that, more shg-null than wild-type PGCs are in contact with CpCs.

View larger version (46K): [in a new window]   Fig. 6. PGCs proliferate with random divisional orientation. (A-D) Late third instar larval ovaries containing dividing PGCs (outlined by thick dotted lines). The ovaries were double stained with anti-Vasa antibody (green) to label all PGCs, and anti-1B1 antibody (red) to visualize spectrosome orientation in PGCs and to outline somatic cells. Dividing PGCs in contact with TF/CpCs (outlined by thin dotted lines) are oriented perpendicularly (A,C) or in parallel (B), or approximately 45° (D) to the cap cell-PGC interface. PGCs in contact with posterior somatic cells also divide perpendicularly (C), or in parallel (A,B), to the soma-germline interface. (E) A quantitative comparison of the divisional orientation of PGCs contacting CpCs, of those in the anterior half but not contacting cap cells (anterior PGCs), and of those in the posterior half (posterior PGCs). The CpC-PGC interface is assigned a value of 90°. Each red dot in the diagram represents an individual sample (n=63).

View larger version (80K): [in a new window]   Fig. 7. The fates of EGFP-marked single pole cells in larval and adult testes. (A,B) A late third instar larval testis derived from an embryonic gonad containing a labeled anterior pole cell, viewed under low and high magnifications, respectively. The testis, with its anterior pole to the left, is stained with anti-1B1 antibody (red) that outlines somatic cells, as well as spectrosomes and fusomes (F), in germ cells, and with anti-GFP antibody (green) to identify germ cells derived from the marked pole cell. Note that GSCs, such as the one marked by an arrow in B, are in direct contact with the hub (H; arrowhead), and have produced multiple germline cysts. (C) EGFP image of an adult testis showing a similar marking of GSCs (arrow) and their progeny. (D,E) A late third instar larval testis derived from an embryonic gonad containing a labeled posterior pole cell, viewed under low and high magnifications, respectively. The testis, with its anterior pole to the left, is stained with anti-1B1 (red) and anti-GFP (green) antibodies. Note that no GFP-labeled germ cells are near the hub (H). (F) EGFP image of an adult testis showing that the GSC position (white thin arrow) is not marked, while a two-cell germline cyst (white thick arrow) is marked by EGFP, together with several other more advanced cysts. Scale bars: in A, 100 µm for A,B,D,E; in C, 500 µm for C,F.

View larger version (29K): [in a new window]   Fig. 8. The somatic induction model for GSC fate determination. For details, see text.

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