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First published online 7 February 2007
doi: 10.1242/dev.003392

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Notch signaling controls germline stem cell niche formation in the Drosophila ovary

Xiaoqing Song¹, Gerald B. Call^1,*, Daniel Kirilly^1,2 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 Medical Center, 3901 Rainbow Boulevard, Kansas City, KS 66160, USA.

View larger version (65K): [in this window] [in a new window]   Fig. 1. Forced expression of an activated N can sufficiently expand niche sizes and generate ectopic niches. (A) Schematic of a crosssection of the anterior part of the germarium. (B,B') A confocal section of a c587-gal4; UAS-GFP female gonad at the larval-pupal transition stage labeled for Hts (red), GFP (green) and DNA (blue), showing that the c587-gal4 driver expresses GFP in most, but not all, somatic cells of the gonad including TFs and IGS precursors. (B') Schematic of the region highlighted by a rectangle in B. Panels C,E,G represent one confocal section of the anterior portion of the germaria labeled for ß-gal (red) and Hts (green), while D,F represent overlayed images. (D'-F') Schematic presentations of the areas highlighted by ovals in D-F, respectively; (G') schematic presentation of G. (C) A hh-lacZ/+ germarial tip showing cap cells (oval) and two GSCs indicated by spectrosomes (arrows). ß-gal-positive TFs are not shown on this confocal section. Arrowhead indicates a branched fusome. (D,D') A c587-gal4/+; UAS-Nint/+; hh-lacZ/+ germarial tip showing over 40 lacZ-positive cap cells at the normal location close to the TF (oval), 15 GSCs evidenced by the presence of spectrosomes (two indicated by arrows) and differentiated germ cell cysts evidenced by the presence of branched fusomes (one indicated by an arrowhead). (E,E') A c587-gal4/+; UAS-Nint/+; hh-lacZ/+ germarial tip showing that a group of 12 lacZ-positive cap cells (oval) located away from the tip support six GSCs (one spectrosome denoted by an arrow and one elongated spectrosome by an arrowhead). (F,F') A c587-gal4/+; UAS-Nint/+; hh-lacZ/+ germarial tip showing that a group of 24 lacZ-positive cap cells (oval) surrounded by follicle cells (FC) support eight GSCs (one spectrosome indicated by an arrow). (G,G') A c587-gal4/+; UAS-Nint/+; hh-lacZ/+ germarial tip showing that lacZ-positive cap cells covering the surface of the anterior half of the germarium support the contacting GSCs indicated by spectrosomes, and a differentiated germ cell cyst evidenced by the presence of a branched fusome (arrowhead). Scale bars: 10 µm. CPC, cap cell; DC, developing cyst; ESC, escort stem cell; FC, follicle cell; FS, fusome; GSC, germline stem cell; IGS, inner germarial sheath cell; IGSP, IGS precursor; SS, spectrosome; TF, terminal filament cell; PGC, primordial germ cell.

View larger version (69K): [in this window] [in a new window]   Fig. 2. Ectopic GSCs associated with ectopic niches behave like normal GSCs. The panels A-C and E-H represent one confocal section, whereas D represents an overlayed image of multiple confocal sections. The germaria in A-E are labeled for GFP (green), Hts (red) and DNA (blue), while the ones in F-H are labeled for ß-gal (red), Hts (green) and DNA (blue). (A) A wild-type germarial tip showing that two GSCs (white arrowheads) in contact with cap cells (oval) do not express bam-GFP (black arrowhead). (B) Part of a c587-gal4/+; UAS-Nint/+; bam-GFP/+ germarium showing that two spectrosome-containing GSCs (white arrowheads) that are associated with ectopic cap cells (oval) fail to express bam-GFP. Black arrowheads indicate branched fusomes in differentiated cysts. (C) A middle portion of a c587-gal4/+; UAS-Nint/+; bam-GFP/+ germarium showing that ectopic cap cells (oval) support one bam-GFP-negative spectrosome-containing GSC (arrowhead). (D) The tip of a c587-gal4/+; UAS-Nint/+; bam-GFP/+ germarium showing that the expanded cap cells (indicated by unbroken outline) repress bam expression not only in GSCs (white arrowheads) but also in the spectrosome-containing single germ cells lying more than one cell diameter away (black arrowheads). (E) c587-gal4/+; UAS-Nint/+; bam-GFP/+ germarium showing that the ectopic cap cells (indicated by unbroken outline) repress bam expression not only in GSCs (white arrowheads) but also in the spectrosome-containing single germ cells lying more than one cell diameter away (black arrowhead). (F) The tip of a Dad-lacZ/+ germarium showing that two GSCs (arrowheads) are in contact with cap cells (oval) and express high levels of Dad-lacZ. (G) The tip of a c587-gal4/+; UAS-Nint/+; Dad-lacZ/+ germarium showing that an increased number of cap cells (indicated by unbroken outline) induced by the expression of an activated N support an increased number of GSCs (arrowheads) and cystoblasts that are positive for Dad-lacZ. (H) A middle portion of a c587-gal4/+; UAS-Nint/+; Dad-lacZ/+germarium showing that a group of ectopic cap cells (indicated by unbroken outline) close to follicle cells also support ectopic GSCs (arrowheads) that are also positive for Dad-lacZ. Scale bar: in A, 10 µm for all images.

View larger version (109K): [in this window] [in a new window]   Fig. 3. GSCs in the expanded or ectopic niche are mitotically active and are able to generate differentiated germ cells. The germaria in A-F are labeled for BrdU (green), ß-gal (red) and Hts (blue). A,B,D,E represent a confocal section, whereas C,F are overlayed confocal images. (A) A hh-lacZ/+ germarium showing that two GSCs (broken lines) close to the cap cells (oval) are BrdU-positive after 3 days of BrdU feeding. (B) A hh-lacZ/+ germarium showing that two GSCs (broken lines) close to the cap cells (oval) have lost their BrdU label 3 weeks after BrdU feeding. (C,C') A germarial tip showing that an expanded cap cell cluster (oval) supports nine GSCs (arrowheads), six of which are BrdU-positive (arrows). (D) The tip of a c587-gal4/+; UAS-Nint/+; hh-lacZ/+ germarium after 3 days of BrdU feeding, showing that a cap cell cluster (oval and white arrowhead) near TF support three GSCs (broken lines), two of which are BrdU-positive, and an ectopic cap cell cluster (oval and black arrowhead) supporting two GSCs (broken lines), one of which is BrdU-positive. (E) The tip of a c587-gal4/+; UAS-Nint/+; hh-lacZ/+ germarium showing that GSCs in an expanded cap cell cluster (unbroken outline indicated by a white arrowhead) and an ectopic cap cell cluster (oval and black arrowhead) are BrdU-negative 3 weeks after BrdU feeding. (F) The tip of a c587-gal4/+; UAS-Nint/+; hh-lacZ/+ germarium showing that one (broken line) of six GSCs (arrowheads) in an expanded cap cell cluster (oval) remains BrdU-positive 3 weeks after BrdU feeding. Scale bar: 10 µm.

View larger version (98K): [in this window] [in a new window]   Fig. 4. N signaling controls niche formation in a developmental stage-dependent manner. The panels in A-J represent one confocal section of the adult germaria (A,B,H-J) or early female gonads (C-G) that are labeled for ß-gal (red, A-E,H), Hts (green, C,H), N (green, E), Dl (green, D), CD2 (green, F,G), Vasa (red, G), Lamin C (green, I,J) and DNA (blue, A-J). (A',B',H') Schematic presentations of the areas highlighted by ovals in A,B,H, respectively; (C'-G') Schematic presentations of C-G, in which a yellow line indicates the borderline between TFs and the PGC zone. (A,A') The tip of a UAS-Dl/+; hs-gal4/hh-lacZ germarium overactivating N signaling at the early pupal stage, showing three GSCs (arrows in A and green dots in A') and the increased number of ß-gal-positive cap cells (oval in A and CPC in A'). (B,B') The tip of a UAS-Dl/+; hs-gal4/hh-lacZ germarium overexpressing activated N at the early pupal stage, showing three GSCs (arrows in B and green dots in B') and a group of ß-gal-positive ectopic cap cells (circle in B and CPC in B'). (C,C') A part of a Dl-lacZ/+ female gonad at the larvalpupal transitional stage showing ß-galpositive TFs and ß-gal-negative PGCs (circles with a green dot for spectrosome in C'). (D,D') A part of a hh-lacZ/+ female gonad at the late third-instar larval stage showing that ß-gal-positive TFs and surrounding anterior somatic cells (green ovals filled with blue in D') express Dl protein. (E,E') A part of a hh-lacZ/+ female gonad at the larval-pupal transitional stage showing that ß-gal-positive TFs and IGS precursors (broken lines in PGC zone of E and green lines in PGC zone of E') express N protein. (F,F') A part of an E(spl)mß-CD2 female gonad at the larval-pupal transitional stage showing that TFs (white broken lines in F and black broken line in F') and newly formed cap cells (asterisks in F and arrow in F') express CD2. (G,G') A part of an E(spl)mß-CD2 female gonad overexpressing the activated N at the larval-pupal transitional stage showing that some somatic cells (arrowhead in G and green lines in PGCZ of G') express E(spl) in addition to TFs and newly formed cap cells (asterisk in G). (H,H') A tip of a N264-39/+; hh-lacZ/+ germarium showing two cap cells (arrowhead in H) and one GSC (its spectrosome indicated by an arrow in H). (I-J') The 2-day-old ovoD1rS1 mutant germaria showing that many germaria have cap cells (broken lines) lying adjacent to TFs (brackets) (I, and the lower one in J), but some do not have cap cells (the upper one in J). Scale bar: 10 µm. IGSP, IGS precursor; PGCZ, PGC zone.

View larger version (28K): [in this window] [in a new window]   Fig. 5. Model explaining how N signaling controls GSC niche formation in the Drosophila ovary. (A) Newly formed TFs (purple) express Dl protein and activate N (N*) signaling in neighboring somatic cells and induce them to form cap cells (red oval) supporting two GSCs (light blue circle), while the rest of the somatic cells that are not in contact with TFs form ESCs or IGS cells (brown). PGCs are depicted as dark blue round cells, while differentiated germ cells, including cystoblasts, are yellow round cells. N signaling remains active in cap cells of the adult ovary and is required for their maintenance. (B) When N signaling is expanded to the somatic cells that do not contact TFs but are adjacent to the somatic cells destined to become cap cells, these somatic cells will also become cap cells (and possibly ESCs) and thus increase the niche size in the normal location and the GSC number. (C) When N signaling is ectopically activated in the somatic cells a few cells distant from TFs, these somatic cells differentiate into cap cells (and possibly ESCs). Thus, ectopic niches that are surrounded by IGS cells or follicle cells are formed. (D) When N signaling is active in most, if not all, somatic cells of the gonad, the somatic cells that have active N signaling generate cap cells (and possibly ESCs), forming niche cells, which are able to sustain GSC self-renewal throughout the germaria.