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First published online 23 January 2008
doi: 10.1242/dev.017590

Development 135, 839-847 (2008)
Published by The Company of Biologists 2008
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Drosophila Pxt: a cyclooxygenase-like facilitator of follicle maturation

Tina L. Tootle and Allan C. Spradling*

Howard Hughes Medical Institute, Carnegie Institution, Department of Embryology, 3520 San Martin Drive, Baltimore, MD 21218, USA.


Figure 1
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  		Fig. 1. Stage 10B egg chamber maturation in vitro is influenced by Cox1 inhibitors and exogenous PGs. (A) Normal egg chamber at stage 10B, (B) stage 12 or (C) stage 14. (D-K) Extent of development characteristic of stage 10B egg chambers matured in vitro and treated as indicated. (D) Control, develops to stage 14 with only slight reduction in dorsal appendages; (E-I) aspirin blocks maturation in a dose-dependent manner. (J) 150 µM NS-398 blocks in vitro development; (K) partial rescue of developmental block induced by 150 µM NS-398 by PGH2 at 20 µM. Anterior is towards the left in all images. Asterisks indicate nurse cell region; arrows indicate dorsal appendages. (L) Effects of Cox1 inhibitors and exogenous PGs on egg chamber development in vitro. Scale bar: 50 µM. `Stalled' was defined as incomplete nurse cell breakdown in these experiments. t-tests were performed comparing drug treated (blue bar) to drug plus prostaglandin (yellow, green or red). Flu, fluprostenol.

 

Figure 2
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  		Fig. 2. pxt is expressed throughout oogenesis and required for egg maturation. (A) Schematic of the pxt gene, showing the 5' and 3'UTR as white boxes, the exons as gray boxes, the alternative splice exon as a black box, the introns as lines and the insertions as black triangles. (B) RT-PCR and qRT-PCR (numbers below) showing pxt expression in whole ovaries from the indicated genotypes. (C) Female fertility of pxt genotypes relative to wild type 2-5 days (dark gray) or >15 days (light gray) after eclosion. (D) pxtf01000 egg chamber showing a nurse cell dumping defect (asterisk marks nurse cells). (E-I) Whole-mount in situ hybridization using a pxt probe. (E) pxt is expressed weakly early in oogenesis, with an upregulation of expression beginning at S6. (F,G) pxt is expressed in both the germ and somatic cells, including border cell expression (F,F', arrows) and mainbody follicle cells (outlined in G). (H,I) Expression remains strong throughout the rest of oogenesis. The probe concentration is higher in E than in F-I. F' shows DAPI. Scale bar: 50 µM.

 

Figure 3
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  		Fig. 3. pxt mutants are rescued by pxt cDNA, exogenous PGs and mouse Cox1. (A) Fertility of pxtf01000 mutant females (over a 4-day period) is rescued by expression of a UASp-pxt+ transgene driven by heat shock-GAL4 (HS GAL4) or the somatic ovarian driver c587. (B) Rescue of pxtf01000 developmental arrest in vitro by added PGH2 (10 µM) or fluprostenol (Flu) (20 nM). t-tests were performed comparing drug treated (blue) with drug plus prostaglandin (yellow or red). DIC image of a pxtf01000 stage 10B egg chamber developed in vitro without PG (C) showing residual nurse cells (*) or with added 60 µM PGH2 showing rescue (D). (E) Fertility of pxtf01000 mutant females (over 4 days) is rescued by expression of a UASp-MCOX1 transgene driven by heat shock-GAL4 (HS GAL4). (F,G) MCOX1 expression (G) rescues nurse cell dumping (arrows indicate nurse cell nuclei) and dorsal appendage (outlined) formation. Projections of confocal sections of S14s from HS GAL4; Sco/CyO; f01000 (F) and HS GAL4; UAS MCOX1/balancer; f01000 (G). Green is 1B1 (marking membrane) and blue is DAPI. Scale bar: 50 µM.

 

Figure 4
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  		Fig. 4. Loss of pxt and COX inhibitors disrupt actin bundles required for nurse cell dumping. (A-F') Actin bundles in wild-type nurse cells in cytoplasmic (A,A') and subcortical regions (D,D') are greatly reduced in pxt-/- mutants (B,B' and E,E', respectively), and restored by expression of MCOX1 under the control of HS GAL4 (C,C' and F,F'). (G-I) Actin bundles in stage 10B egg chambers developed in vitro until stage 11 in the absence (G) or presence (H) of 1.5 mM aspirin, or with aspirin and 60 µM PGH2 (I). The presence of aspirin disrupts subcortical actin and causes actin aggregation in dumping nurse cells (H), which is partially reversed by PG addition (I). A-F are projections of several confocal sections. A'-F' are zoomed images of one nurse cell from the adjacent image. Red is phalloidin and blue is DAPI. (E-G) Epifluorescent images. Scale bars: 50 µM.

 

Figure 5
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  		Fig. 5. pxt mutants exhibit actin-related defects throughout oogenesis. (A) Normal ovariole and (B) shortened pxt ovariole with missing stages, fused chambers (bracket) and degenerating chambers (star). (C) Multi-nucleate nurse cells in pxt egg chambers as a result of membrane breakdown. Nurse cell nuclei are outlined and arrows indicate actin-rich aggregates containing residual ring canals, compare with S8 in A. (D) Loss of follicular separation and germarium defects in older pxt mutant females. Broken line indicates germarium-stage cysts. (E) Defective border cell migration due to lagging cells (outlined, compare with `BC' in A). (F) Nurse cells further away from the oocyte have membrane instability (outlined in yellow) and actin aggregates (arrow), whereas those closer to the oocyte have more normal actin filament formation (outlined in white). (A-F) Projections of confocal sections. Green is Hts (1B1) and Fas3 (7G10); red is phalloidin and blue is DAPI. A, anterior; P, posterior. Scale bars: 50 µM.

 

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© The Company of Biologists Ltd 2008