DEV020685 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Smt3 expression pattern in L3. Smt3 expression (green) and nuclei (purple) in the indicated tissues. Green and purple channels are shown separately in black and white. Smt3 is expressed ubiquitously in L3 stages in the nuclei of all tissues analysed.

• Supplemental Figure S2 -

  Fig. S2. The structure of the nuclear lamina is compromised in smt3i PG cells. (A,B) Transmission electron microscopy images of PG cells from wild-type (WT; A) and smt3i (B) larvae. The interface between gland cells has an elaborated array of interdigitations in the extracellular space (ECS) in WT larvae (A) that is often reduced or absent in smt3i larvae (B). The appearance of large aggregates of VLPs and electron-dense parallel bands (inset in B) is characteristic of smt3i nuclei (Nu). The VLPs appear highly packed and the nucleolus is not longer visible. Cy, cytoplasm. Scale bars: 2 µm. (C,D) Single confocal micrographs showing PG cells stained with anti-lamin Dm0 antibodies (green) and DAPI (purple). The thickening of the lamin layer shown by EM was confirmed by immunostaining (arrow in D).

• Supplemental Figure S3 -

  Fig. S3. Innervation from the brain appears not to be impeded in smt3i larvae. (A,D) Schematic of the neuronal innervation of WT (A) and smt3i (D) ring glands based on multiple confocal sections of anti-HRP-stained glands. (B,E) Anti-HRP (green) shows innervation of the PG cells by neurosecretory neurons. Nuclei are labelled with DAPI (purple). Despite the morphological changes in the plasma membrane of smt3i larvae, nervous terminals appear to reach most of the PG cells and seem to be able to establish contacts with knockdown cells, as shown by formation of the boutons that are used for the secretion of peptides. Whether these boutons are able to transmit the signal remains to be determined. (B′,E′) Green channels showing HRP staining alone in black and white. (C,F) Transmission electron microscopy images of PG cells from WT (C) and smt3i (F) larvae. Varicose nerve terminals containing the electron-dense vesicles (arrows) and the membrane surrounding them (arrowheads) characteristic of neurosecretory endings are similar in WT and knockdown larvae. Scale bars: 500 nm.

• Supplemental Figure S4 -

  Fig. S4. The number of filipin-positive droplets is reduced in smt3i PG cells. (A) Confocal single plane micrographs showing filipin staining in WT or smt3i ring glands, taken with the 40× objective at two different zoom magnifications as indicated. (B) Lipid droplets of each micrograph in A were labelled manually with black dots and are represented separately. (C) The number of dots per panel was counted using the ‘Analyze particle’ tool from ImageJ software, transferred to an Excel sheet and represented graphically. smt3i PG cells show a significantly reduced number of lipid droplets.