The axon guidance gene lola is required for programmed cell death in the Drosophila ovary

Abstract

longitudinals-lacking (lola) was identified in Drosophila as a gene encoding several alternatively spliced transcription factors involved in axon guidance. Here we report that lola also plays a critical role in programmed cell death in the ovary. lola mutant germline clones show a high percentage of egg chambers with nurse cell nuclei persisting past stage 13, indicating a block in developmental nurse cell death. Mutants also show a disruption in the induced programmed cell death that occurs during mid-oogenesis in response to starvation. Further characterization revealed that lola germline clones exhibit abnormal nuclear organization which becomes increasingly severe with age. Chromatin appears diffuse and fails to condense properly or undergo DNA fragmentation in dying nurse cells. Masses of nuclear material accumulate in the ovaries of older flies containing lola germline clones. We propose that lola is necessary for complete chromatin condensation which occurs during programmed cell death in the ovary. Alleles differed in the strength of their phenotypes but interestingly, the severity of their ovarian phenotypes was independent of the strength of their neuronal phenotypes, suggesting a differential requirement for individual lola isoforms in the ovary and nervous system.

Fig. 1

lola alleles vary in the severity of their GLC phenotypes. All egg chambers have been DAPI-stained. (A) Control, yw stage 14 egg chambers do not contain any remaining nurse cell nuclei. (B–H) Representative stage 14 egg chambers containing GLCs of the indicated lola alleles show that most cause moderate to severe defects during oogenesis including dumpless egg chambers. However, lola^4E4 (C) looks wild-type.

Fig. 2

Intragenic complementation analysis. (A) Complementation results with different lethal alleles of lola helped narrow down the location of the lethal mutation in lola⁶²⁹. Offspring from the F1 generation of the indicated cross were counted. Percent viability is calculated as 100 x (observed number of straight winged flies/ expected number of straight winged flies). Expected number of straight winged flies = 1/2(observed number of curly winged flies) (Horiuchi et al., 2003). (B) Locations of P-element insertions in lola^4E4, lola⁰⁰⁶⁴², and lola^5D2, and EMS-induced mutations in lola^ORE120, lola^ORC4, and lola^ORE50. Only relevant exons are depicted and drawing is not to scale (Horiuchi et al., 2003).

Fig. 3

Quantification of ovarian phenotypes of different lola alleles in order of increasing severity. Stage 14 egg chambers were classified as one of four indicated categories. Those classified as other had dorsal appendage defects or were cup-shaped but did not contain persisting nurse cell nuclei and were not dumpless. FRTG13 germline clones were analyzed as a control. n= the total number of stage 14 germline clone egg chambers classified.

Fig. 4

Lola isoform K expression is necessary for normal oogenesis. (A) Extracts from embryos of the genotypes indicated above were separated by SDS-PAGE and subjected to western analysis using an antibody that recognizes the constant region of Lola. The positions of standard protein molecular weight markers are indicated to the right. Compared with yw control embryos, those heterozygous and homozygous for lola⁶²⁹, as well as lola^ORC4, and lola^ORE50 embryos express truncated versions of isoform K (arrows). lola⁶²⁹, lola^ORC4, lola^ORE50, and lola^5D2 homozygotes lack expression of the normal sized isoform K while lola^4E4 retains expression (arrowhead). (B) The same membrane used for (A) was probed with an antibody for Lamin Dm₀ to verify that comparable amounts of protein were added to each lane. (C) Protein extracts from ovaries of the genotypes indicated above were subjected to western analysis as in (A). Ovaries from lola⁶²⁹ heterozygotes and those containing GLCs of lola⁶²⁹ express a truncated version of isoform K (arrow), while only the GLCs lack expression of the normal sized isoform (arrowhead).

Fig. 5

lola GLCs do not undergo DNA fragmentation during checkpoint or developmental PCD. TUNEL analysis (dark purple) and DAPI staining (blue) was performed on all egg chambers. (A) Heterozygous mid-stage egg chambers from nutrient deprived females undergo checkpoint PCD and show TUNEL positive nurse cell nuclei. (B) As an internal control, ovo^D tissue, surrounding lola^ORC4 GLCs, did contain TUNEL positive nuclei (arrows). (C) Mid-stage degenerating egg chambers containing lola^ORC4 GLCs are not TUNEL positive. Arrows indicate three separate egg chambers. (D) Late stage 12 and (E) early stage 13 heterozygous control egg chambers display TUNEL positive nurse cells during developmental PCD. (F) lola^ORC4 GLCs in a dumpless stage 14 egg chamber are not TUNEL positive.

Fig. 6

Nurse cell nuclear membranes do not break down in lola⁶²⁹ GLCs. (A–D) y w hsflp control and (E) lola⁶²⁹ GLC egg chambers contain the BB127 enhancer trap encoding nuclear β-galactosidase and are X-gal stained (blue). (A) A stage 10 yw egg chamber has intact nurse cell nuclear membranes. (B) By stage 10B, the nurse cell nuclear membranes break down and X-gal staining leaks into the cytoplasm. (C) A stage 12 yw egg chamber has undergone cytoplasmic transfer and X-gal staining is seen in the oocyte. (D) By stage 14, no nurse cells remain and all the X-gal staining is seen in the oocyte. (E) A dumpless egg chamber containing lola⁶²⁹ GLCs retains X-gal staining in the nurse cell nuclei.

Fig. 7

Abnormal actin structure is seen in degenerating lola⁶²⁹ GLC egg chambers. Panels depict DAPI staining (blue) on the left and rhodamine-phalloidin staining (red) on the right of the same egg chambers. All images were taken at the same magnification and rhodamine-phalloidin pictures were taken at the same exposure. (A–B) A heterozygous mid-stage egg chamber undergoing checkpoint PCD, induced through nutrient deprivation, contains condensed nurse cell nuclei, small actin clumps (B, arrows) and follicle cells with cellular membranes still intact (arrowheads). (C–F) lola⁶²⁹ GLC egg chambers undergoing checkpoint PCD, without starvation, contain abnormal actin structures (D and F, white arrows), degenerating follicle cells (D, arrowheads), and uncondensed nurse cell nuclei with intact cellular membranes (E and F, yellow arrows). (G–H) A stage 10B heterozygous egg chamber shows formation of organized actin bundles in all nurse cells. (I–J) A stage 10B egg chamber containing lola⁶²⁹ GLCs exhibits actin bundle formation that is disorganized and not uniform throughout the nurse cells. (K–P) Dumpless egg chambers containing lola⁶²⁹ GLCs continue to show disorganized actin structure. Some contain small actin clumps like those seen during mid-stage PCD (N, arrows).

Fig. 8

Variable levels of caspase activity are seen during checkpoint and developmental PCD in lola⁶²⁹ and lola^ORC4 GLCs. Cleaved Caspase-3 antibody staining (green) depicts caspase activity and propidium iodide staining (red) shows nuclei. All images were taken at the same exposure and magnification. (A) A heterozygous mid-stage egg chamber undergoing checkpoint PCD, induced by nutrient deprivation, shows condensed nurse cell nuclei and high levels of caspase activity throughout the egg chamber. (B) Mid-stage egg chambers containing lola^ORC4 or (C) lola⁶²⁹ GLCs, undergoing checkpoint PCD, also show high levels of caspase activity, but it is not uniformly found throughout the entire egg chamber. Nurse cells are not always condensed (B and C, arrows) and a lack of caspase activity is seen both with (B, arrowheads) and without (B and C, arrows) nuclear condensation. (D) A heterozygous stage 12 egg chamber undergoing developmental PCD does not show significant levels of caspase activity with this antibody. (E–G) Dumpless egg chambers containing lola^ORC4 GLCs show either high levels of caspase activity (E), none (F), or very low levels (G). Lack of proper nuclear condensation can also be seen in some of these dumpless egg chambers (G).

Fig. 9

lola⁶²⁹ GLCs exhibit chromatin defects that increase with age of the female. Egg chambers in (A–G) were stained with DAPI and (H) was stained with Cleaved Caspase-3 antibody. (A) lola⁶²⁹ GLC tissue from a one day old female appears normal. (B–D) By two-three days, abnormally-shaped nuclei, along with nuclear condensation defects can be seen. (E) At four days, masses of nuclear material accumulate between egg chambers (arrow) and (F) by seven-eight days, these masses become larger (arrow). (G–H) A group of egg chambers stained with DAPI (G) and Cleaved Caspase-3 antibody (H) show loss of caspase activity after the follicle cells die and the nurse cell cytoplasm disperses (arrows). Remaining nuclear material in (G) may contribute to the masses in (E–F). (A), (C), and (E–F) were taken at the same magnification. (B), (D), and (G–H) were taken at higher magnification.

Fig. 10

lola⁶²⁹ and lola^ORC4 GLCs exhibit abnormal nuclear lamina morphology. Egg chambers in (A–B) were double-labeled with lamin Dm₀ ADL84.12 antibody (green) and rhodamine-phalloidin (red). (A) Heterozygous control egg chambers exhibit normal nuclear lamina morphology. (B) A lola^ORC4 GLC egg chamber exhibits a protrusion of the nurse cell nuclear lamina through the ring canal (arrow, higher magnification inset). Egg chambers in (C–H) were stained with lamin Dm₀ ADL101 antibody (green, C, E, and G) and DAPI to visualize DNA (blue, D, F, and H). (C) Nuclear lamin Dm₀ surrounds the nuclei in heterozygous control egg chambers (higher magnification inset of nucleus indicated by arrow). (E and G) lola⁶²⁹ and lola^ORC4 GLCs exhibit mislocalization of lamin Dm₀ to the cytoplasm ((higher magnification insets of nuclei indicated by arrows). (F and H) No abnormal chromatin morphology is seen in the egg chambers with mislocalized lamin Dm₀ (compare with neighboring egg chambers and heterozygous controls in D).