{gamma}-Tubulin function during female germ-cell development and oogenesis in Drosophila -- Data Supplement - HTML Page - 1925SuppText.htslp -- Proceedings of the National Academy of Sciences

Supporting Text
Oogenesis Progression Is Not Affected by Mutations in Either g -Tubulin Gene, but Simultaneous Depletion of the Two g -Tubulin Gene Products Results in Agametic Ovaries.
To address the role of g -tubulin in oogenesis, we have studied the behavior of microtubules in WT flies or flies homozygous for either g Tub23C_PI (1, 2) or fs(2)TW1₁ (3–5), which are the most severe mutant alleles available for g Tub23C and g Tub37C, respectively (see Materials and Methods). We could not detect any difference in microtubule distribution between WT and mutants during stages 1–6 of oogenesis by immunofluorescence with an antibody against g -tubulin (data not shown), or after the localization of a nod-kinesin-lacZ reporter, which accumulates at the minus end of microtubules (6) (Fig. 7 A–C, arrows). Also unaffected is the localization of Vasa in freshly laid embryos (data not shown), indicating that microtubule function at late stages of oogenesis is not generally affected (7–9). In conclusion, the microtubule cytoskeleton appears to be overall properly organized during oogenesis in female mutants for either of the two g -tubulins, suggesting that the two protein products are redundant during oogenesis. To test this hypothesis we generated flies mutant for both g -tubulins.

We have studied two different combinations of g -tubulin double mutant flies. Both of them carry g Tub23C_PI (1, 2) in combination with either fs(2)TW1₁, a severe loss of function of g Tub37C ,or fs(2)TW1_RU34, a hypomorph allele of the same gene (4, 5, 10).Homozygous g Tub23C_PI fs(2)TW1₁ females are agametic with rudimentary ovaries (Fig. 7D). The agametic phenotype is fully rescued by transposons carrying a WT copy of either of the two g -tubulins. In contrast to this dramatic effect in oogenesis, the only alterations observed in spermatogenesis are postmitotic and identical to those produced by the g Tub23C_PI mutation on its own (2). Thus maintenance and/or differentiation of the female germ line in Drosophila requires g -tubulin. Moreover, unlike in other tissues and developmental stages, which specifically require one of the two g -tubulin products (refs. 1–4 and S. Llamazares, personal communication), either of them can provide the function required for female germ-line differentiation.

WT Germ Cell Behavior at the Late Third-Instar Larva and Pupa Stages.
We followed germ cell behavior during development in WT and g Tub23C_PI fs(2)TW1₁ females. In late third-instar larvae the WT female gonad is a small (60-m m diameter) round mass of somatic cells (Fig. 8A; ref. 11) and germ cells of homogeneous dimensions (8–10 m m) are present in a coalescent group in the middle of the ovary (11). The position of the centrosome (data not shown) and of the spectrosome (12, 13) within the cell is random with respect to the antero-posterior axis of the gonad (Fig. 8D, arrow points to a spectrosome). At the prepupa stage (stages P1–4 according to ref. 14) the most anterior germ cells are divided in small groups that are each associated with a terminal filament (Fig. 8B, bracket indicates a terminal filament). These cells likely represent the primordia of the germ-line component of adult germaria. About 20 h after puparium formation (Fig. 8C, stage P4/5) two populations of germ cells are present in each germarium (Fig. 8F). Anteriorly, there is a group of three to six larger germ cells (8-10 m m; Fig. 8F, arrowhead), partially in contact with the terminal filament. A larger group of small cells is present in the posterior portion of each germarium (Fig. 8F, arrow), likely differentiating cystocytes. About 50 h after puparium formation (stage P8) the germaria are very similar to adult ones, and some stage 1 and 2 egg chambers are present (11). Materials and Methods
Drosophila
Cultures and Stocks. fs(2)TW1₁ and Df(2L)VA23 are described in ref. 3. fs(2)TW1_RU34(15) is a hypomorph (4, 5, 10) and fs(2)TW1₁ a loss-of-function (5) allele of g Tub37C. g Tub23C_PI is a loss- of-function allele of g Tub23C (1). NZ143.2 was kindly provided by I. Clark (6) (Princeton University, Princeton, NJ) andVas^PD/SM6; P[Vas-GFP] by A. Ephrussi (16) (European Molecular Biology Laboratory, Heidelberg, Germany). Recombinants between fs(2)TW1₁, fs(2)TW1_RU34, and g Tub23C_PI were obtained by standard genetic procedures. g Tub23C_PI b fs(2)TW1₁ px sp was balanced over T(2:3)TSTL, Tb (J. Casal and C.G., unpublished work) to distinguish homozygous larvae and pupae. Two stocks were analyzed for the weaker combination: g Tub23C_PI fs(2)TW1_RU34cn px sp (stock 33) and g Tub23C_PI fs(2)TW1_RU34 cn c px sp (stock 55). For rescue experiments we used the construct P[g Tub37C⁺] (4), or the construct P[g Tub23C⁺] carrying the entire g Tub23C gene (S. Llamazares, personal communication). Cytology.
For g -tubulin (DM1A, Sigma, 1:400) and b -galactosidase (anti-b -galactosidase, Cappel, 1:2,000) staining ovaries were dissected in PBS at room temperature and fixed for 10 min in methanol at –20° C. Before blocking and antibody incubation, ovarioles were separated and permeabilized in PBS with 1% Triton X-100 for 1 h. DNA was counterstained with TOTO-3 (Molecular Probes, 1:800) or DAPI (Sigma, 5 m g/ml). DAPI staining of adult ovaries was performed as in ref. 4. Staining of ring canals with anti-phosphotyrosine antibodies (Transduction Laboratories, Lexington, KY, 1:100) and germ cell staining with rat (1:1,500) or rabbit (1:200) anti-Vasa antibodies (kindly provided by A. Ephrussi and P. Lasko, McGill University, Montreal) were performed as in ref. 17. In situ hybridizations were performed as in ref. 4, using as probe a SacI restriction fragment from the osk full-length cDNA (a gift from A. Ephrussi). Embryos from a stock carrying g Tub23C_PI b fs(2)TW1₁ px sp / ftz-lacZ Cy were fixed as in ref. 18. Pupae were staged by following ref. 14. The gonads were fixed following the protocol described in ref. 17. Additional primary antibodies were rabbit anti-CNN (kindly provided by T. Kaufman, Indiana University, Bloomington, 1:400), rabbit anti-b -spectrin (kindly provided by L. Goldstein, University of California at San Diego School of Medicine, La Jolla, 1:200), and Rb1011 against g Tub37C (ref. 4; 1:200). Rb1015 (1:200) was raised in rabbits against a truncated form of g Tub23C lacking the first 19 amino-terminal amino acids (1) and recognizes both g Tub37C and g Tub23C (data not shown). Secondary antibodies (Jackson ImmunoResearch Laboratories) were diluted 1:400. Samples were imaged with an upright DRBE fluorescence Leica microscope and a laser scanning confocal microscope (TCS-NT, Leica). Images were processed with NIH IMAGE and PHOTOSHOP 3.0 Adobe Systems, Mountain View, CA).

1. Sunkel, C. E., Gomes, R., Sampaio, P., Perdigao, J. & Gonzalez, C. (1995) EMBO J. 14, 28–36.

2. Sampaio, P., Rebollo, E., Varmark, H., Sunkel, C. E. & Gonzalez, C. (2001) Curr. Biol. 11, 1788–1793.

3. Lindsley, D. L. & Zimm, G. G. (1992) The Genome of Drosophila melanogaster (Academic, San Diego).

4. Tavosanis, G., Llamazares, S., Goulielmos, G. & Gonzalez, C. (1997) EMBO J. 16, 1809–1819.

5. Llamazares, S., Tavosanis, G. & Gonzalez, C. (1999) J. Cell Sci. 112, 659–667.

6. Clark, I. E., Jan, L. Y. & Jan, Y. N. (1997) Development (Cambridge, U.K.) 124, 461–470.

7. Hay, B., Jan, L. Y. & Jan, Y. N. (1990) Development (Cambridge, U.K.) 109, 425–433.

8. Brendza, R. P., Serbus, L. R., Duffy, J. B. & Saxton, W. M. (2000) Science 289, 2120–2122.

9. Cha, B. J., Koppetsch, B. S. & Theurkauf, W. E. (2001) Cell 106, 35–46.

10. Wilson, P. G. & Borisy, G. G. (1998) Dev. Biol. 199, 273–290.

11. King, R. C. (1970) Ovarian Development in Drosophila melanogaster (Academic, New York).

12. Lin, H. & Spradling, A. C. (1997) Development (Cambridge, U.K.) 124, 2463–2476.

13. Deng, W. & Lin, H. (1997) Dev. Biol. 189, 79–94.

14. Bainbridge, S. P. & Bownes, M. (1981) J. Embryol. Exp. Morphol. 66, 57–80.

15. Schupbach, T. & Wieschaus, E. (1989) Genetics 121, 101–117.

16. Breitwieser, W., Markussen, F. H., Horstmann, H. & Ephrussi, A. (1996) Genes Dev. 10, 2179–2188.

17. Verheyen, E. & Cooley, L. (1994) Methods Cell Biol. 44, 545–561.

18. Gonzalez, C. & Glover, D. M. (1993) in The Cell Cycle: A Practical Approach, eds. Fantes, P. & Brook, R. (IRL, Oxford), pp. 163–168.