Self-maintained escort cells form a germline stem cell differentiation niche

Abstract

Stem cell self-renewal is controlled by concerted actions of niche signals and intrinsic factors in a variety of systems. In the Drosophila ovary, germline stem cells (GSCs) in the niche continuously self-renew and generate differentiated germ cells that interact physically with escort cells (ECs). It has been proposed that escort stem cells (ESCs), which directly contact GSCs, generate differentiated ECs to maintain the EC population. However, it remains unclear whether the differentiation status of germ cells affects EC behavior and how the interaction between ECs and germ cells is regulated. In this study, we have found that ECs can undergo slow cell turnover regardless of their positions, and the lost cells are replenished by their neighboring ECs via self-duplication rather than via stem cells. ECs extend elaborate cellular processes that exhibit extensive interactions with differentiated germ cells. Interestingly, long cellular processes of ECs are absent when GSC progeny fail to differentiate, suggesting that differentiated germ cells are required for the formation or maintenance of EC cellular processes. Disruption of Rho functions leads to the disruption of long EC cellular processes and the accumulation of ill-differentiated single germ cells by increasing BMP signaling activity outside the GSC niche, and also causes gradual EC loss. Therefore, our findings indicate that ECs interact extensively with differentiated germ cells through their elaborate cellular processes and control proper germ cell differentiation. Here, we propose that ECs form a niche that controls GSC lineage differentiation and is maintained by a non-stem cell mechanism.

Fig. 1.

ECs at different positions can undergo apoptosis and cell proliferation. (A) c587 drives UAS-GFP expression in ESCs and ECs in regions 1 and 2a, and also in early follicle cells (FCs) in region 2b. GSCs (dashed lines) and differentiated germ cells, including 16-cell cysts, can be identified by presence of a spectrosome (SS) and branched fusome (FS), respectively. (B) PZ1444 can also label ESCs (arrow), ECs (arrowheads) and cap cells (oval) in the germarium in which germ cells are labeled by Vasa-GFP. (C-F) ApopTag-positive ECs are found in region 2a (C,D, arrows) and the 2a/2b boundary (E,F, arrows). (G) An EC at the 2a/2b boundary is positive for BrdU after two hours of BrdU incorporation. (H,I) GSCs (white arrowheads), mitotic cysts and follicle cells in the germarium are positive for BrdU (green) one day after three days of BrdU feeding. Most of the ECs (black arrowheads) are negative for BrdU, but some ECs (arrows) are BrdU-positive. (J-L) After two weeks of chase following three days of BrdU feeding, all the GSCs and mitotic cysts are BrdU-negative, but some ECs (arrows) remain positive for BrdU. ESCs are normally negative for BrdU (black arrowhead, K), but are rarely found to be positive (black arrowhead, L). Scale bars: 10 μm in B-L.

Fig. 2.

ECs are not maintained by ESCs. (A,A′) Quantitative data showing that the percentages of the germaria carrying one or more lacZ-labeled ESCs (A) and the percentages of the germaria carrying only lacZ-labeled ECs, but no lacZ-labeled ESCs (A′) remain largely constant one week, two weeks and three weeks ACI. (B) A lacZ-positive ESC (arrow) contacts cap cells (oval) and a GSC (dashed circle), which is accompanied by a few lacZ-positive ECs (arrowheads). (C) A lacZ-positive EC (arrowhead) is a few cells away from cap cells (oval) but without any lacZ-positive ESCs. (D,D′) Quantitative data showing that the percentages of the germaria carrying one GFP-labeled ESC (D) and the percentages of the germaria carrying only GFP-labeled ECs (D′), remain largely unchanged one week, two weeks and three weeks ACI. These GFP-labeled EC clones are induced by PMML. (E) Only a GFP-positive ESC (arrow) contacts cap cells (oval) and a GSC (dashed circle) but no GFP-positive ECs are detected. (F,G) Only GFP-positive ECs (arrowheads) can be found in region 2a (F) or at the 2a/2b boundary (G). The image in G represents overlaid confocal sections, and the remaining images are single confocal sections. Scale bar: 10 μm.

Fig. 3.

Differentiated germ cells are wrapped by EC cellular processes. (A) Differentiated germ cell cysts (dashed lines) are encased by GFP-positive EC cellular processes. (B) A cryosectioned germarium showing that germ cell cysts (dashed lines) are separated from one another by GFP-positive EC cellular processes. (C-I) Germaria labeled for GFP (green) and β-galactosidase (PZ1444, red, C), Hts (red, D) or Fas3 (red, differentiated follicle cells, E-I) containing a GFP-positive EC (arrow) at the ESC position (C), region 1 (D,E), region 2a (F,G) or the 2a/2b boundary (H,I). The ECs in D and E have short cellular processes encasing the cystoblast (CB) and mitotic cyst, respectively, whereas those in F-I have longer cellular processes wrapping around 16-cell cysts. The images in C, E, F and H are one confocal section, whereas images in D, G and I are overlaid confocal sections. Scale bar: 10 μm.

Fig. 4.

Differentiated germ cells might maintain EC cellular processes. (A) c587;UAS-GFP; bam^Δ86/bam^Δ86 germarium showing that all the ECs (one by arrowhead) fail to form cellular processes penetrating inside tumorous germ cells. (B) c587;UAS-GFP; bam^Δ86/bam^Δ86 cryosection showing that ECs can form some protrusions (arrowhead) that fail to encase germ cells. (C) c587;UAS-dpp/UAS-GFP germarium showing that ECs fail to form long cellular processes wrapping single germ cells (dashed lines). (D) c587;UAS-dpp/UAS-GFP germarium showing that EC cellular processes wrap round a differentiated cyst (arrow) but fail to penetrate inside the clusters (dashed lines) containing single germ cells. Scale bar: 10 μm.

Fig. 5.

Defective Rho signaling in ECs disrupts EC-germ cell interactions and germ cell differentiation. The c587;UAS-Rho^DN/UAS-GFP germaria (single confocal section) in A-C are labeled for GFP, Hts and DNA, whereas c587;UAS-Rho^DN/PZ1444 germaria (overlaid confocal sections) in D-F and H-I are labeled for β-galactosidase (PZ1444), Hts and DNA, and ApopTag, β-galactosidase (PZ1444) and DNA, respectively. (A) The GFP-positive cellular processes of Rho^DN-expressing ECs fail to wrap around a differentiated cyst (dashed lines). (B) Rho^DN expression in ECs leads to a reduction of regions 1 and 2a of the germarium and a defect in wrapping a differentiated cyst (dashed lines) by EC cellular processes. (C) Spectrosome-containing single germ cells and a 16-cell cyst (dashed lines) are clustered together and are not individually wrapped by Rho^DN-expressing EC cellular processes. Arrowheads indicate spectrosomes. (D) Germarium contains a normal number of ECs but excess single germ cells (arrowheads) located anteriorly to differentiated cysts (arrows). (E,F) Germaria show a moderate (E) or severe (F) reduction of ECs and excess spectrosome-containing single germ cells (arrowheads). (G) Rho^DN-expressing germaria have significantly fewer ECs than do control germaria. P-value is indicated. (H) A control germarium contains no apoptotic ECs. (I) A Rho^DN-expressing germarium contains an apoptotic EC (arrowhead). Scale bar: 10 μm.

Fig. 6.

Defective Rho signaling in ECs delays cystoblast differentiation. The germaria in A-C are labeled for GFP, Hts and DNA, those in D-F are labeled for β-galactosidase (Dad), Hts and DNA, and those in G and H are labeled for pERK, Hts and DNA. (A) Two control GSCs (solid circles) expressing low bam-GFP and differentiated cysts expressing high bam-GFP. (B,C) c587;UAS-Rho^DN/bam-GFP germarium carrying many of spectrosome-containing single germ cells, most of which express bam-GFP (cystoblast-like; dashed circles) and the remaining ones show low bam-GFP expression (GSC-like; solid circle). (D) Two control GSCs (solid circles) expressing high levels of Dad-lacZ. (E,F) c587; UAS-Rho^DN/Dad-lacZ germaria carrying spectrosome-containing single germ cells, of which those further away from the niche (dashed circle) express lower Dad-lacZ than do endogenous GSCs (solid circles) contacting the niche. Some single cells away from the niche (solid circle, F) express high levels of Dad-lacZ. (G) Wild-type ECs (two indicated by arrowheads) are positive for pERK staining. (H) Rho^DN-expressing ECs away from (some indicated by arrowheads) or close to (dashed circle) single germ cells remain positive for pERK staining. (I) Rho^DN-expressing ECs have significantly less pERK than control ECs. Error bars represent s.d. Number above horizontal line represents P-value. Scale bar: 10 μm.

Fig. 7.

Rho contributes to the restriction of BMP signaling to the GSC niche. The germaria in A-F are labeled for Hts and DNA. (A,B) Rho^DN expression in ECs leads to the accumulation of excessive spectrosome-containing single germ cells. (C) The percentages of excessive single germ cell-containing germaria in ovaries in which Rho^DN is specifically expressed in the ECs and which are wild type, heterozygous for dpp (dpp^hr4 and dpp^hr56), or co-expressed with RNAi constructs for dally (one on chromosome 2 and one on chromosome 3) and dpp (47A and 47R). In addition to endogenous GSCs, three more additional single cells outside the GSC niche are considered to be excessive single germ cells because a wild-type germarium contains one to two CB-like single germ cells. (D-F) The germ cell differentiation defect induced by Rho^DN expression can be dramatically suppressed by a heterozygous dpp^hr4 mutation (D), by co-expression of dally RNAi (E) or by co-expression of dpp RNAi (F). (G) Working model for EC maintenance and Rho function in ECs. When an EC dies, its neighbor EC divides to generate a new EC to replace the lost one. Rho works in ECs to restrict BMP signaling within the GSC niche by preventing Dpp diffusion via regulation of EGFR-Dally signaling and repressing dpp mRNA accumulation. In addition, Rho signaling is important for EC survival.