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Science 7 June 2002:
Vol. 296. no. 5574, pp. 1855 - 1857
DOI: 10.1126/science.1069871

Germline Stem Cells Anchored by Adherens Junctions in the Drosophila Ovary Niches
Xiaoqing Song, Chun-Hong Zhu, Chuong Doan, and Ting Xie

Supplementary Material

One of many possible explanations for GSC loss caused by shg and arm mutations is that mutant GSCs cannot compete with wild-type GSCs for contact with cap cells because of defective cell adhesion. Consistent with this interpretation, a wild-type GSC in a germarium carrying a mutant GSC sometimes expanded its contact area with cap cells (fig. S1A). Additionally, in the absence of wild-type GSCs in a germarium (all GSCs were mutated for either arm⁴ or shg^R69), marked GSCs were persistent even 3 weeks after clone induction (fig. S1B), suggesting that the identity of mutant GSCs can be maintained as long as they remain in their niches. We conclude that DE-cadherin-mediated adhesion is essential for anchoring GSCs in their niches but may not directly determine GSC identity in the Drosophila ovary.

Cell adhesion mediated by DE-cadherin is involved in regulating other intercellular signaling events in many different systems (S1). It has been shown that signals from surrounding somatic cells are important for GSC division (S2-S5). To determine whether DE-cadherin-mediated adhesion is important for GSC division, we measured the relative division rates (rdr) for shg mutant GSCs. The rdr for a given genotype was determined by dividing the number of marked cysts per marked GSC by the number of unmarked cysts per unmarked GSC in a given germarium. In comparison with marked wild-type GSCs (rdr = 1.05, n = 132), shg¹⁰⁴⁶⁹ had no obvious effect (rdr = 0.98, n = 161), whereas shg^R69 had a weak effect (rdr = 0.82, n = 26). This indicates that disrupting DE-cadherin-mediated cell adhesion appears to have only a weak effect on GSC division.

Materials and Methods

Drosophila stocks and genetics. The following fly stocks were used in this study and are described in Flybase: yw, hh-lacZ/TM3 Sb, FRT_42D shg^R69, FRT_42D shg¹⁰⁴⁶⁹, FRT_42D +, FRT_18A arm², FRT_18A arm³, FRT_18A arm⁴and FRT_18A arm⁸. All Drosophila stocks were maintained at room temperature on standard cornmeal + molasses + agar media.

Generating mutant shg and arm germ line stem cell clones. Clones of mutant cells were generated by FLP-mediated mitotic recombination, as described previously (S2). To generate mutant shg GSC clones, FRT_42D +, FRT_42D shg¹⁰⁴⁶⁹/CyO and FRT_42D shg^R69/CyO males were mated with virgin females yw hs-FLP; FRT_42D arm-lacZ, respectively. To generate arm GSC clones, FRT_18A arm²/FM7, FRT_18A arm³/FM7, FRT_18A arm⁴/FM7 and FRT_18A arm⁸/FM7 virgin females were mated with males FRT_18A arm-lacZ; hs-FLP, respectively. One- or 2-day-old adult females carrying an arm-lacZ transgene in trans to the mutant-bearing chromosome were heat-shocked four times at 37°C for 1 hour separated by 8 to 12 hours. The females were transferred to fresh food every day at room temperature, and ovaries were removed 1 week, 2 weeks, or 3 weeks after the last heat-shock treatment, and then further processed for antibody staining. To determine stem cell maintenance, the percentages of the ovarioles carrying a marked SSC clone at different time points were calculated by dividing the number of germaria carrying marked follicles by the number of total germaria examined.

Generating mutant shg primordial germ cells to test germ line stem cell recruitment. To generate the stocks for marked PGCs, FRT_42D +, FRT_42D shg¹⁰⁴⁶⁹/CyO and FRT_42D shg^R69/CyO males were mated with virgin females yw hs-FLP; FRT_42D arm-lacZ, respectively. The culture tubes containing the progeny from these crosses that had not reached the late third-instar stage were heat shocked twice for 1 hour with an interval of 6 hours. One- or 2-day-old adult non-CyO females carrying an armadillo-lacZ transgene in trans to the mutant-bearing chromosome were dissected and processed for immunostaining. To determine the efficiency of stem cell recruitment, the percentages of the ovarioles carrying a marked GSC clone for a given genotype were calculated by dividing the number of germaria carrying marked GSCs by the number of total germaria examined.

Immunohistochemistry. The following antisera were used: monoclonal anti-Hts antibody 1B1 [1:4, Developmental Studies Hybridoma Bank (DSHB)], monoclonal antibody anti-Armadillo N7A1 (1:4; DSHB), rat antibody against DE-cadherin (1:100) (S6) and rabbit polyclonal anti- Greek Letter Beta -galactosidase (1:300; Molecular Probes) antibodies.

Ovaries were dissected in Grace's media and fixed in PBS with 4% formaldehyde for 12 to 15 min, then washed with PBT (PBS and 0.1% Triton X-100) 5 times for 15 min each wash. The ovaries were incubated in 0.5% goat serum diluted with PBT for 1 hour. Appropriate primary antibodies were added to PBS and incubated at 4°C overnight, then washed with PBT for 5 times for 15 min or more each wash. Lastly, appropriate secondary antibodies were added and incubated overnight, then washed with PBT 5 times for 15 min each wash. After the last wash, the stained ovaries were mounted in Vectashield mounting media (Vector). All micrographs were taken using a Leica SPII confocal microscope.

To determine whether both contacting membranes of GSCs and cap cells express DE-cadherin proteins, we used a PBT solution containing 0.4 to 0.6% of Triton X-100 to allow the separation of two contacting membranes in adherens junctions under this high detergent condition. The staining procedures and image collection were the same as described above.

Supplemental Figure 1. DE-cadherin-mediated signaling does not affect GSC identity. (A and B) Germaria labeled for LacZ (red), Hts (green), and nuclei (blue). Both panels indicate GSCs (outlined with solid arrowheads), unmarked GSCs (outlined with open arrowhead), cap cells (outlined and marked with CPC), and some marked cysts (outlined only). (A) A germarium carrying a wild-type GSC demonstrates that the wild-type GSC has expanded its contact with multiple cap cells. (B) One of the few and rare 2-week-old double arm⁴ GSC clones that may have been derived from loss of wild-type GSCs followed by replacement with mutant GSCs. All the germaria are shown at the same scale. Scale bar in (B) represents 10 Greek Letter Mu m.