Identification of new X-chromosomal genes required for Drosophila oogenesis and novel roles for fs(1)Yb, brainiac and dunce

Abstract

We performed a screen for female sterile mutations on the X chromosome of Drosophila melanogaster and identified new loci required for developmental events in oogenesis as well as new alleles of previously described genes. We present mapping and phenotypic characterization data for many of these genes and discuss their significance in understanding fundamental developmental and cell biological processes. Our screen has identified genes that are involved in cell cycle control, intracellular transport, cell migration, maintenance of cell membranes, epithelial monolayer integrity and cell survival or apoptosis. We also describe new roles for the genes dunce (dnc), brainiac (brn) and fs(1)Yb, and we identify new alleles of Sex lethal (Sxl), ovarian tumor (otu), sans filles (snf), fs(1)K10, singed (sn), and defective chorion-1 (dec-1).

Figure 1

Oogenesis in wild type Drosophila. A germarium with the three different germarial regions (indicated) is shown and a vitellarium with stages 2–6, and a stage 10 egg chamber.

Figure 2

Crossing scheme used to isolate X chromosome female sterile lines. Males of the genotype yw118FRT19A (Bloomington stock B1744) were mutagenized, and F3 female progeny were tested for fertility. SxlM4 is a male lethal allele of Sxl and was used to eliminate unwanted males from the first cross.

Figure 3

New genes required for patterning of the germarium. gl, germline; fcs, follicle cells. (A) Adducin-like localization in fs(1)124/fs(1)124 mutant germarium reveals spectrosome-containing cells and small cysts with branched fusomes. (B) Nuclear staining of an fs(1)259/fs(1)259 egg chamber containing four polyploid nurse cells and one oocyte nucleus (arrow). Scale bar = 10μm. (C) Actin staining of an fs(1)259/fs(1)259 egg chamber containing multiple nurse cell/oocyte cysts. Arrows point to oocytes. (D) Nuclear staining of a late stage 9 wild type egg chamber. (E) Nuclear staining of a late stage 9 fs(1)217/fs(1)217 egg chamber from a 2-d-old female, revealing a reduced number of follicle cells. Scale bar = 50μm. (F) Large fused egg chamber from a 6-d-old fs(1)217/fs(1)217 female. Only a small number of follicle cells surround this egg chamber. Arrows point to follicle cell nuclei.

Figure 4

Requirements for fs(1)Yb (A-F ) and brn (G-I ) in patterning of the germarium. fcs, somatic follicle cells; gc, germ cells; R1: germarial region 1. (A) Actin staining of fs(1)Yb⁷²/fs(1)Yb⁷²mutant germarium revealing multiple cysts within a single egg chamber. Arrows point to oocytes. Scale bar = 50μm. (B,C) Sxl localization in 1-d-old wild type (B) and fs(1)Yb⁷²/fs(1)Yb⁷²(C) mutant germaria. Scale bar = 10μm. (D,E,F) Adducin-like localization in 1-d-old wild type (D), 1-d-old fs(1)Yb⁷²/fs(1)Yb⁷²(E), and 6-d–old fs(1)Yb⁷²/fs(1)Yb⁷²(F) germaria. Arrow in (D) points to a branched fusome. Arrows in (E,F) point to unbranched fusomes (spectrosomes). Scale bars = 10μm. (G) Actin staining of a brn¹⁹⁸/brn¹⁹⁸mutant ovariole which lacks stalk cells, resulting in failure to separate egg chambers. Three egg chambers are labeled with arrowheads. (H) Germline staining with anti-Vasa antibody of a wild type germarium and early-stage egg chambers, revealing the continuously growing germline cells. (I) Vasa staining of a brn²²⁸/brn²²⁸ovariole revealing depletion of germline cells. Scale bar in (H,I) = 50μm.

Figure 5

Genes required for mid-oogenesis. (A) fs(1)186/fs(1)186 mutant's egg chamber labeled for actin to show abnormal aggregation of follicle cells over the oocyte. The arrowheads point out some of the follicle cells that do not contact the germline. (B) Stage 9 egg chamber from fs(1)186/fs(1)186 in which follicle cells have formed a two-layer epithelium over the oocyte (* indicates follicle cell layers). (C) The follicle cell aggregation phenotype is partially suppressed in fs(1)186/fs(1)186; Bic-D^PA66/Df(2L)TW119. (D,E) Actin staining in a stage 5 (D) and a stage 9 (E) fs(1)234 /fs(1)234 mutant egg chamber showing the progressive loss of germline cell membranes. (F) Same egg chamber as in (E) stained for nuclei. The strong actin staining in (E) is due to aggregation of ring canals (arrow in E) and the border cells (arrowhead in E and F). Scale bars in (A,C,D) =  20μm. Nuclear labeling of fs(1)225/fs(1)225 mutant ovaries reveals (G) enlarged nurse cell nuclei and (H) supernumerary nurse cells.

Figure 6

(A -D) Defects in late transport of nurse cell contents in mutants for fs(1)140 and fs(1)3. oo, oocyte; fcs, follicle cells; ncs, nurse cells; ncn, nurse cell nucleus. (A,B) Nomarski views of (A) fs(1)140 and (B) fs(1)3 egg chambers in which nurse cell dumping has failed. (C,D) Double labeling of nuclei (red) and actin (green) in (C) fs(1)140/fs(1)140 and (D) fs(1)3/fs(1)3. In fs(1)140/fs(1)140, actin cables fail to form and the nurse cell nuclei appear to become caught in the ring canals (arrow) during dumping. In fs(1)3/fs(1)3, actin cables form normally (arrows point to actin cables). (E) Nuclear staining of fs(1)164/fs(1)164 reveals pycnotic nuclei (arrows) in stage 8 of oogenesis. (F) Actin staining of fs(1)221a/fs(1)221a reveals failed border cell migration (arrow points to border cells).