Figure 3.
Internalized polar cells do not exhibit signs of death, and their engulfment depends on Rac and Rho. (A–C’) Single sections from confocal images of border cell clusters from stage 10 egg chambers expressing UAS-PLCδPHGFP and the genotypes indicated. Egg chambers were stained with FAS3 (gray), cDCP1 (magenta), and DNA was visualized by DAPI (blue). Arrows in A indicate pyknotic nuclei. White asterisks in B and C indicate non-internalized polar cells, and yellow asterisk in C indicates internalized polar cell. Scale bar: 5 µm. (D–F’) Single sections from confocal images of a dying egg chamber (D and D’) or of border cell clusters from egg chambers in stage 10 (E–F’), expressing UAS-PLCδPHGFP and the genotypes indicated. Egg chambers were stained with FAS3 (gray), LysoTracker dye (magenta) to visualize acidic compartments, and DAPI (blue) to visualize DNA. White arrows in D point to pyknotic nurse cell nuclei. Yellow inset in D is shown in a higher magnification in D’. White asterisks in E indicate non-internalized polar cells, and yellow asterisk in F indicates internalized polar cells. Scale bar is 50 µm for the egg chamber in D and 5 µm for D’ and for border cell clusters in E–F. (G–L) Maximum intensity projection from five slices (G–I’) or nine slices (J–L’; z-step: 0.5 µm for G–H’ and J–K’, 0.16 µm for I, I’, L, and L’) of border cell clusters expressing UAS-PLCδPHGFP and the genotypes indicated. Images in I, I’, L, and L’ were obtained by airyscan imaging to visualize the localization of RAB5 (I and I’) and RAB7 (L and L’) at a high spatial resolution, and are not suitable for comparison of expression levels. Egg chambers were stained for FAS3 (magenta) and for RAB5 (G–I) or RAB7 (J–L; gray). DNA was visualized by DAPI (blue). White asterisks in G and J indicate non-internalized polar cells and yellow asterisks in H–I and K and L indicate internalized polar cells. Scale bar: 5 µm. (G’–L’) Images from G–L showing only RAB5 (G’–I’) or RAB7 (J’–L’) staining in Royal LUT where white represents the highest amount of the indicated protein and black the lowest one. (M–O) Single section (M) or maximum intensity projection from six slides (N and O; z-step: 0.5 µm) of a dying egg chamber (M) or border cell clusters expressing UAS-PLCδPHGFP and the genotypes indicated. Egg chambers were stained for FAS3 (magenta) and ATG8 (gray). DNA was visualized by DAPI (blue). Scale bar is 50 µm for the egg chamber in M and 5 µm for border cell clusters in N and O. (M’–O’) Images from M–O showing only ATG8 staining in Royal LUT. (P–S’’) Single sections from confocal images of border cell clusters from egg chambers at stage 9 or 10 expressing UAS-PLCδPHGFP and the genotypes indicated. Egg chambers were stained with pSRC (gray), F-actin (magenta), and DAPI (blue). White asterisks in P–R indicate non-internalized polar cells. In R, one polar cell is being internalized (yellow asterisk) by one border cell, and in S both polar cells are being internalized (yellow asterisks) by one border cell. White arrows in R’ and S point to pSRC accumulation and yellow arrows in R’’ and S’’ point to F-actin accumulation. Scale bar: 5 µm. (T–V’) Single sections from confocal images of border cell clusters from egg chambers at stage 9 or 10 expressing UAS-PLCδPHGFP and the genotypes indicated. Egg chambers were stained with pMLC (gray), FAS3 (magenta), and DAPI (blue). White asterisks in T and U indicate non-internalized polar cells and the yellow asterisk in V indicates an internalized polar cell. The orange arrow in V’ points pMLC accumulation along with the squeezing of a polar cell nucleus. Scale bar: 5 µm. (W) Quantifications of polar cell internalization events from stage 10 egg chambers of the indicated genotypes. Error bars: mean ± SEM (n = 3). The total number of clusters analyzed (n) is indicated at the top of each bar. Statistical significance was assessed with a Fisher exact test (one-tailed) and P values are shown at the top of the graph. Polar cells in B, C, E, L, and N–V are outlined with a yellow dashed line.

Internalized polar cells do not exhibit signs of death, and their engulfment depends on Rac and Rho. (A–C ’ ) Single sections from confocal images of border cell clusters from stage 10 egg chambers expressing UAS-PLCδPHGFP and the genotypes indicated. Egg chambers were stained with FAS3 (gray), cDCP1 (magenta), and DNA was visualized by DAPI (blue). Arrows in A indicate pyknotic nuclei. White asterisks in B and C indicate non-internalized polar cells, and yellow asterisk in C indicates internalized polar cell. Scale bar: 5 µm. (D–F’) Single sections from confocal images of a dying egg chamber (D and D’) or of border cell clusters from egg chambers in stage 10 (E–F’), expressing UAS-PLCδPHGFP and the genotypes indicated. Egg chambers were stained with FAS3 (gray), LysoTracker dye (magenta) to visualize acidic compartments, and DAPI (blue) to visualize DNA. White arrows in D point to pyknotic nurse cell nuclei. Yellow inset in D is shown in a higher magnification in D’. White asterisks in E indicate non-internalized polar cells, and yellow asterisk in F indicates internalized polar cells. Scale bar is 50 µm for the egg chamber in D and 5 µm for D’ and for border cell clusters in E–F. (G–L) Maximum intensity projection from five slices (G–I’) or nine slices (J–L’; z-step: 0.5 µm for G–H’ and J–K’, 0.16 µm for I, I’, L, and L’) of border cell clusters expressing UAS-PLCδPHGFP and the genotypes indicated. Images in I, I’, L, and L’ were obtained by airyscan imaging to visualize the localization of RAB5 (I and I’) and RAB7 (L and L’) at a high spatial resolution, and are not suitable for comparison of expression levels. Egg chambers were stained for FAS3 (magenta) and for RAB5 (G–I) or RAB7 (J–L; gray). DNA was visualized by DAPI (blue). White asterisks in G and J indicate non-internalized polar cells and yellow asterisks in H–I and K and L indicate internalized polar cells. Scale bar: 5 µm. (G’–L’) Images from G–L showing only RAB5 (G’–I’) or RAB7 (J’–L’) staining in Royal LUT where white represents the highest amount of the indicated protein and black the lowest one. (M–O) Single section (M) or maximum intensity projection from six slides (N and O; z-step: 0.5 µm) of a dying egg chamber (M) or border cell clusters expressing UAS-PLCδPHGFP and the genotypes indicated. Egg chambers were stained for FAS3 (magenta) and ATG8 (gray). DNA was visualized by DAPI (blue). Scale bar is 50 µm for the egg chamber in M and 5 µm for border cell clusters in N and O. (M’–O’) Images from M–O showing only ATG8 staining in Royal LUT. (P–S’’) Single sections from confocal images of border cell clusters from egg chambers at stage 9 or 10 expressing UAS-PLCδPHGFP and the genotypes indicated. Egg chambers were stained with pSRC (gray), F-actin (magenta), and DAPI (blue). White asterisks in P–R indicate non-internalized polar cells. In R, one polar cell is being internalized (yellow asterisk) by one border cell, and in S both polar cells are being internalized (yellow asterisks) by one border cell. White arrows in R’ and S point to pSRC accumulation and yellow arrows in R’’ and S’’ point to F-actin accumulation. Scale bar: 5 µm. (T–V’) Single sections from confocal images of border cell clusters from egg chambers at stage 9 or 10 expressing UAS-PLCδPHGFP and the genotypes indicated. Egg chambers were stained with pMLC (gray), FAS3 (magenta), and DAPI (blue). White asterisks in T and U indicate non-internalized polar cells and the yellow asterisk in V indicates an internalized polar cell. The orange arrow in V’ points pMLC accumulation along with the squeezing of a polar cell nucleus. Scale bar: 5 µm. (W) Quantifications of polar cell internalization events from stage 10 egg chambers of the indicated genotypes. Error bars: mean ± SEM (n = 3). The total number of clusters analyzed (n) is indicated at the top of each bar. Statistical significance was assessed with a Fisher exact test (one-tailed) and P values are shown at the top of the graph. Polar cells in B, C, E, L, and N–V are outlined with a yellow dashed line.

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