Papss mutants show defects in SG lumen expansion.

(A) Stage 16 SGs immunostained for Stranded at Second (SAS). (B) In situ hybridization using an antisense probe for Papss mRNA in WT embryos. (C-H) Stage 14 and stage 16 SGs immunostained for Ecad and CrebA. Yellow asterisks indicate regions of irregular, non-uniform expansion (D, E, H) or over-expansion (G) of the SG lumen. (I, I’) Quantification of different SG lumen phenotypes. (J) Quantification of SG lumen length. n=7-11 SGs for each genotype. See the Materials and Methods for the sample size of each genotype. One-way ANOVA test with multiple comparison (*, p<0.05; ns, non-significant). (K, K’) Quantification of the SG lumen diameter (K) and the coefficient of variation for each SG (K’). The diameter is measured in three different regions of the lumen. The average diameter, as well as the individual data points, are shown. The same samples are used for the quantifications in J-K’. One-way ANOVA test with multiple comparison (***, p<0.001; ns, non-significant) (L) Quantification of SG lumen area. n=7-16 SGs for each genotype. One-way ANOVA test with multiple comparison (***, p<0.001; ****, p<0.0001; ns, non-significant). Horizontal lines indicate mean values.

Mutations in Papss result in mislocalized Crb and disruption of the aECM architecture.

(A) Cartoon showing the subcellular localization of Crb in SG cells. (B, C) Stage 16 SGs immunostained for Crb and Ecad. Magnified images are shown for yellow dotted boxed regions. (D) Quantification of the Crb signal intensity ratio between the SAR and apical-medial regions of SG cells. (n=7, WT; n=6, Papss2 (4-8 cells per SG)). Student’s T-test with Welch’s correction (***, p<0.001). (E-G) Stage 16 SGs stained for Ecad and wheat germ agglutinin (WGA). Magnified images are shown for the boxed regions. Arrows, WGA localization at the apical membrane in WT (yellow) and Papss mutant (cyan) SGs. Yellow arrowhead in E, WGA localizes as thin filaments in the lumen of WT SG. Cyan arrowheads, WGA localizes as a highly condensed structure in the thin luminal region of the Papss SG. White arrowheads, WGA localizes as a condensed, filamentous structure in the expanded luminal region of the Papss SG. Yellow arrowheads in G, WGA localizes as a mildly condensed filament in the lumen of sage>Papss-PE; Papss2 SG. (H, I) TEM images of stage 15 SGs. SGs are pseudo-colored in magenta. Magnified images of the luminal areas (boxed regions) are shown in the bottom. Yellow asterisk in H, electron-dense luminal material in WT SG. Cyan asterisks in I, condensed electron-dense luminal material in the Papss mutant SG. Green asterisk, a large luminal area lacking electron-dense material in the Papss mutant SG.

Loss of Papss results in disorganized Golgi structures and defects in intracellular trafficking components.

(A-D) Stage 16 SGs stained with WGA. Magnified images are shown for the boxed regions. Yellow arrows, WGA signals as cytoplasmic puncta. Cyan arrows, WGA signals in the nuclear envelope. (E-H) Three-dimensional (3D) reconstruction of cytoplasmic WGA puncta in stage 16 SGs. (I, I’) Quantification of the number of WGA puncta (I) and signal intensity (I’) within SG cells. n=5 SGs for all genotypes, except WT in G (n=6). One-way ANOVA test with multiple comparisons (**, p<0.01; ***, p<0.001; ns, non-significant). Horizontal lines indicate mean values. (J, K) Stage 16 SGs stained for GFP (for ManII-GFP) and WGA. Magnified images are shown for the boxed regions. Yellow arrows in J, ManII-GFP and WGA puncta colocalize in WT SG. Asterisks in K, dispersed ManII-GFP signals in Papss mutants. Cyan arrows in K, little colocalization of ManII-GFP and WGA in the Papss SG. (L, L’) Line intensity profile showing the signal intensity of ManII-GFP (green) and WGA (magenta) along the yellow and cyan dotted lines in J and K, respectively. (M, N) TEM images of the Golgi. Yellow arrows in M, Golgi structures in WT SG cells. Magenta arrows in N, Golgi structures in the Papss SG. Cyan arrows in N, electron-dense structures in the Golgi of Papss mutant SG cells. (O, P) Stage 16 SGs immunostained for Rab11 and Ecad. Magnified images are shown for boxed regions. Rab11 signals at the apical membrane are increased in Papss mutants (cyan arrowheads in P) compared to WT (yellow arrowheads in O), with occasional large punctate signals mislocalized to the basolateral domain (cyan arrow in P). (Q, R) Stage 16 SGs immunostained for Sec15 and Ecad. Compared to strong apical Sec15 signals in the WT SG (yellow arrowheads in Q), Sec15 signals are reduced in Papss mutants (cyan arrowheads in R). (S, T) Quantification of the intensity of Rab11 (S) and Sec15 (T) at the apical membrane regions in WT and Papss mutant SGs. Welch’s t-test (**, p<0.05). Horizontal lines indicate mean values. (U, V) TEM images for the apical region of SG cells. Yellow arrowheads, electron-dense vesicles in both genotypes. Red arrowheads, empty, larger vesicle-like structures in Papss mutants. (W) Quantification of the electron-dense vesicle numbers per cell slice. Welch’s t-test (**, p<0.05). Horizontal lines indicate mean values.

Papss mutants show premature SG cell death.

(A, B) Stage 16 SGs immunostained for CrebA, DCP-1 and Ecad. Cross-sectional images are shown for the yellow dashed line in B. Yellow arrowheads, DCP-1 signal is detected in the SG in Papss mutants. White dashed lines, the basal boundary of the SG. (C) TEM image of stage 15 SG cells, including a dying cell (yellow arrow). Cyan arrows, nuclei of normal living cells. (D) Quantification of the number of acellular regions in stage 16 SGs. (n=8 SGs, WT; n=10, Papss2; n=15, sage>Papss-PD; Papss2; n=8, sage>Papss-PDK193A,F593P; Papss2) (E) Quantification of the number of SG cells at stage 16 (n=10 SGs, WT; n=10, Papss2; n=8, Papss2/Df; n=8, sage>p35; Papss2; n=10, sage>Papss-PE; Papss2; n=8, sage>Papss-PDK193A,F593P; Papss2). One way ANOVA test (****, p<0.0001; ns, non-significant). (F, G) Stage 16 SGs immunostained for CrebA, Ecad, and mCherry (for Hemo3x-mCh). Cross-section images are shown for the yellow dashed lines. White dashed lines, the basal boundary of the SG. Yellow arrowhead in F, hemocyte signals outside of the control SG. White arrowhead in G, hemocyte signals are present inside the SG in Papss mutants. (H, I) Stage 16 SGs immunostained for DAPI, mCh (for Hemo3x-mCh), and Ndg. Magnified views are shown for boxed regions. Yellow dashed lines, the basal boundary of the SG. (H) In WT, hemocytes are found outside of the SG (yellow arrowheads), with Ndg forming a barrier at the basal boundary of the SG (yellow arrows). (I) In Papss mutants, hemocytes invade the SG epithelia (white arrowheads), where Ndg signals are irregular or absent (white arrows).

Loss of Papss disrupts the localization of Dpy and Pio in the aECM.

(A) Stage 16 SGs stained for a chitin-binding protein (CBP) and Ecad. (B) Cartoon of the mCh-Pio fusion protein showing the region for the antibody and protease cleavage sites. (C, D) Stage 14 and stage 16 SGs immunostained for mCh (for mCh-Pio) and GFP (for Dpy-YFP). The mCh-Pio (white arrowheads) and Dpy-YFP (cyan arrowheads) signals are largely absent from the expanded region of the lumen. Along one side of the lumen, weak mCh-Pio signals colocalize with condensed Dpy-YFP signals (yellow arrows). (E-H’’’) Stage 14 and stage 16 SGs immunostained for Pio, GFP, and Ecad. Magnified images of boxed regions in E-H and shown in E’-H’’. F’’ and H’’’ are cross-sectional images of the SG lumen for regions indicated by white lines F’ and H’’. Yellow arrowheads, Pio and Dpy localized to the apical membrane. Cyan arrowheads, Dpy and Pio localized to the SG lumen. Note a higher proportion of Dpy and Pio localized to the lumen in the Papss mutant SG at stage 14 (cyan arrowheads in G’). White arrowheads in H’-H’’’, Dpy accumulates towards one side of the lumen in the stage 16 Papss SG. Yellow asterisks, Pio is absent from the lumen at stage 16 in the Papss SG.

Pio is essential for uniform SG lumen diameter by maintaining Dpy in the lumen.

(A, B) SGs at stages 13-16 stained for WGA and Ecad. Yellow arrowheads in A, filamentous luminal WGA signals. Cyan arrowheads in B, WGA signals at the apical membrane. Yellow asterisks, loss of lumina WGA signals. White arrowheads, constricted lumen. (C) Stage 16 SGs immunostained for Ecad and CrebA. Cross sectional images for the yellow and green dotted lines are shown on the right. Yellow arrowhead, flat protrusion of the lumen. Green arrowhead, constricted lumen. (D) Stage 16 SGs stained for Ecad and GFP (for Dpy-YFP). Projection images contain merged z-sections, from the apical surface of the SG cells to midway through the SG. Single section, one z-section at the approximate midpoint of the lumen width. Yellow arrowhead, area of constricted lumen. Yellow asterisk, luminal area with no Dpy-YFP signals. (E) Single z-sections of confocal images of SGs at stages 15 and 16 stained for Ecad, Pio and WGA. White arrowheads, area of constricted lumen. Yellow asterisks, no luminal signals of WGA and Pio. (F) Quantification of the number of constrictions in the SG lumen at stage 16 (n=7 for both genotypes). (G, G’) SGs at stages 14-16 stained for Ecad, WGA and Pio. Higher magnification of the yellow boxed regions is shown in G’. (H, I) Stage 16 SGs overexpressing NpWT (H) or Npmut (I) that are immunostained for GFP (for Dpy-YFP and Np-GFP), Pio, and mCh (for mCh-Pio). NpWT-overexpressing SGs show strong, uniform mCh-Pio signals in the lumen (cyan asterisks in H), whereas Npmut-overexpressing SGs shows no detectable signals in the lumen (cyan asterisk in I). In NpWT-overexpressing SGs, the luminal mCh-Pio signals are so bright that the cytoplasmic, punctate mCh-Pio signals are rarely detectable alongside the luminal signals. Increasing the brightness to visualize the cytoplasmic punctate signals shows very strong luminal mCh-Pio signals (inset in H). NpWT overexpression shows high levels of Pio (magenta arrowheads in H) and dpy-YFP (green arrowheads in H) signals in both the apical membrane and lumen. In contrast, Npmut overexpression shows strong Pio signals in the apical membrane and various levels of Pio in the lumen. (I’, I’’) Higher magnification of the yellow boxed regions. The lumen diameter of the region with undetectable luminal Pio levels is slightly larger (magenta asterisk) compared to the region with clear luminal Pio signals (magenta arrowheads).

A targeted Df screen revealed Papss as a key enzyme for SG lumen expansion.

Fly strains used.

Antibodies Used.

A proposed model.

(A) Cartoon diagram showing normal lumen expansion in WT SG and defective luminal morphology in Papss, pio, and Np mutants. (B) Organization of the ZP domain protein-containing aECM in the SG. In WT, the ZP-C fragment of Pio (which is cleaved by Np and furin proteases) forms a complex with Dpy to build a filamentous scaffold within the lumen. In Papss mutants, this fragment is absent from the lumen at stage 16, and the Dpy-containing aECM structure becomes aggregated and highly condensed. The absence of luminal Pio in pio and Np mutants results in the loss of luminal Dpy and lumen constrictions. This figure was created using BioRender.com/g1oi56b.