The lack of autophagy triggers precocious activation of Notch signaling during Drosophila oogenesis

Abstract

Background: The proper balance of autophagy, a lysosome-mediated degradation process, is indispensable for oogenesis in Drosophila. We recently demonstrated that egg development depends on autophagy in the somatic follicle cells (FC), but not in the germline cells (GCs). However, the lack of autophagy only affects oogenesis when FCs are autophagy-deficient but GCs are wild type, indicating that a dysfunctional signaling between soma and germline may be responsible for the oogenesis defects. Thus, autophagy could play an essential role in modulating signal transduction pathways during egg development.

Results: Here, we provide further evidence for the necessity of autophagy during oogenesis and demonstrate that autophagy is especially required in subsets of FCs. Generation of autophagy-deficient FCs leads to a wide range of phenotypes that are similar to mutants with defects in the classical cell-cell signaling pathways in the ovary. Interestingly, we observe that loss of autophagy leads to a precocious activation of the Notch pathway in the FCs as monitored by the expression of Cut and Hindsight, two downstream effectors of Notch signaling.

Conclusion: Our findings point to an unexpected function for autophagy in the modulation of the Notch signaling pathway during Drosophila oogenesis and suggest a function for autophagy in proper receptor activation. Egg development is affected by an imbalance of autophagy between signal sending (germline) and signal receiving cell (FC), thus the lack of autophagy in the germline is likely to decrease the amount of active ligand and accordingly compensates for increased signaling in autophagy-defective follicle cells.

Figure 1

Lack of ATG1 in FCs affects proper egg development. Heat shock flipase (HS-FLP) mediated generation of FC clones mutant for ATG1 (marked by the lack of GFP) caused a wide array of oogenesis defects. Compound eggs with more than 16 cysts (outlined in yellow) (A, A’) and ovarioles lacking stalk cells (E, E’) were often observed. Some eggs consisted of less than the 16 germline cyst normally present in WT eggs (outlined in yellow) (B, B’). Inverted eggs presenting the oocyte in the wrong position (outlined in yellow) (C, C’) or eggs with two oocytes (outlined in yellow) (D, D’) were less frequently observed. Mature eggs regularly lack DAs (arrowheads) (F, G). Stage 14 egg chambers often contain persisting nurse cell nuclei (PNCN) (arrowheads) (I, I’) that are normally degenerated in control eggs with WT clones (H, H’). Anterior is to the left, posterior to the right, except F, G: anterior to the top, posterior to the bottom, dorsal to the front. Scale bar: 50 μm Genotypes: A-E, G and I: hs flp/+; ATG1^{∆ 3D}FRT80B/FRT80B-UbiGFP. F and H: hs flp/+; FRT80Biso/FRT80B-UbiGFP .

Figure 2

Expression pattern of different GAL4 driver lines. The GAL4 driver lines were tested for the expression of UAS-GFP. A) The e22c-GAL4 line drives GFP expression in the follicular stem cells and thus in all FCs, albeit patchy. B) Slbo-GAL4 expresses strongly in border cells, in stretched FCs and in columnar FCs at the dorsal anterior side (centripetal FCs) and the posterior end and also in posterior FCs at early stage 9. C) Fru-GAL4 drives expression in stalk cells, in anterior and posterior FCs from stage 6/7 on, in border cells, stretched FCs and in the very posterior columnar FCs. D) c355-GAL4 expresses from stage 7 onwards in all cells including border, stretched, and columnar cells, but not in polar cells. E) c306-GAL4 expresses in stalk cells, weakly in anterior- and stronger in posterior FCs, border cells, stretched cells and columnar FCs, similar to slbo-GAL4. F) 109-30-GAL4 drives expression in stalk precursor- and stalk cells. G) Upd-GAL4 expresses exclusively in the polar cells. H) Ey-GAL4 served as a control and does not drive expression in the ovaries. I) Summary of the expression patterns of the GAL4 lines used. J) Schematic drawing showing the position of all cell types specified above during different stages of oogenesis. Anterior/posterior FCs corresponds to the stages 6/7 to early 9, stretched and columnar to stages 9 till stage 10/11. Anterior is to the left, posterior to the right. Abbreviations: a, anterior; p, posterior; d, dorsal; v, ventral; s, stage. Scale bar: 50 μm. Genotypes: A: e22c-GAL4/UAS>GFP, B: slbo-GAL4/UAS>GFP, C: fru(168)-GAL4/UAS>GFP, D: c355-GAL4/+; UAS>GFP/+, E: c306-GAL4/+; UAS>GFP/+, F: 109-30-GAL4/UAS>GFP, G: upd-GAL4/+; UAS>GFP/+, H: ey-GAL4/UAS>GFP .

Figure 3

Autophagy is important in certain FC subtypes for DA formation. Spatially restricted GAL4 driver lines were used to express ATG1, ATG4, ATG5 and ATG8 RNAi and the effect of autophagy knock down on the dorsal appendage (DA) phenotype was quantified. Downregulation of ATG gene expression with the broad e22c-GAL4 driver causes the most severe DA defects (A and C-C”). Knocking down ATG genes with slbo-GAL4 and fru-GAL4 causes similar strong phenotypes (A and D-E’). Overexpression of ATG RNAi with c355-GAL4, c306-GAL4, 109-30-GAL4 and upd-GAL4 only generated a low percentage of eggs with defective DAs (A and F, F’). Similarly, the controls (UAS>lacZ, ey-GAL4) only occasionally showed defective eggs (A and B). A: Error bars show S.D. of the mean, P-values: * P<0.05, ** P<0.01, *** P<0.001. Eggs were collected from 5 females for each genotype, n=3. B-F’: Anterior is to the top, posterior to the bottom, dorsal to the front. Scale bar: 100 μm. Genotypes: GAL4 driver see Figure 2, ATG-RNAi lines see Additional file 2: Figure S2.

Figure 4

Autophagy modulates Notch signaling in Drosophila FCs. Notch signaling activity in eggs containing ATG mutant FC clones was monitored using the downstream targets Cut and Hindsight (Hnt). (A) In HS-FLP induced FC clones mutant for Notch (marked by the lack of GFP, outlined in yellow), Cut is not downregulated post stage 6. In contrast, ATG1 (B) or ATG13 (C) mutant FCs show a precocious downregulation of Cut compared to WT cells. (D) Under normal conditions, the expression of Hnt is upregulated by Notch signaling, which is not accomplished in cells mutant for Notch (marked by the lack of GFP, outlined in yellow). In FC clones mutant for ATG1 (E) or ATG13 (F), Hnt upregulation occurs earlier or stronger than in surrounding WT cells. Anterior is to the left, posterior to the right. Scale bar: 20 μm. Genotypes: A, D: hs flp FRT19A-UbiGFP/N^55e11FRT19A. B, E: hs flp/+; ATG1^{∆ 3D}FRT80B/FRT80B-UbiGFP. C, F: hs flp/+; ATG13^{∆ 74}FRT82/FRT82-UbiGFP .