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Fig. S1. Integrins and Integrin linked kinase (Ilk) are expressed in the larval peripheral nerve. Antibodies for different integrin subunits were used to immunolabel third instar nerves expressing Ilk endogenously tagged with GFP (Ilk-GFP). (A,E) Single 0.2 µm z-sections show βPS (A, red), αPS2 (E, red), αPS3 (E, blue), Ilk-GFP (green) and DAPI (A, blue) labeling. The boxed regions in A and E are digitally expanded in B,C and in F, respectively. The green lines in A and E show the positions of the orthogonal sections in D and G, respectively. (A-D) Ilk-GFP (green) and βPS (red) immunolabeling overlap in the peripheral nerve (arrows). Arrowheads point to Ilk-GFP that is associated with βPS labeling in the different focal planes shown in B (focal plane Z=31) and C (focal plane Z=34). (E-G) Ilk-GFP (green) is associated with αPS2 (red, arrowheads), αPS3 (blue, arrows). Scale bars: 10 µm in A,E; 5 µm in B-D,F,G.
Fig. S2. Talin forms adhesion complexes with βPS integrin in the larval peripheral nerve. Antibodies for βPS (red) and HRP (blue) were used to immunolabel third instar nerves from larvae with Talin endogenously tagged with GFP (green). (A,D) Single 0.2 µm z-sections. The boxed regions in A and D are digitally expanded in B and E, respectively. The green lines in A and D show the positions of the orthogonal sections in C and F, respectively. (A-C) Talin-GFP (green) and βPS immunolabeling (red) are often found associated with each other (arrows). (D-F) Expression of Talin-RNAi in the perineurial glia (46F::talin-RNAi). CD8-GFP was co-expressed and labeled PG (green). The PG failed to wrap around HRP-labeled axons (blue), similar to the phenotype seen with βPS-RNAi. Strong βPS immunolabeling (red) was still observed in the internal glial layers of the nerve (arrows) and only weak immunolabeling was observed in the perineurial outer membrane (arrowheads). Scale bars: 10 µm in A,D; 5 µm in B,C,E,F.
Fig. S3. Expression of αPS2 and αPS3 integrin RNAi in glial cells of the peripheral nerve. Immunolabeling for specific integrin subunits in third instar larval peripheral nerves. repo-GAL4 was used to express UAS-CD8-GFP (A) and UAS-integrin-RNAi (B,C). All panels are projections of the entire z-stack. (A) Repo::CD8-GFP. Control showing αPS2 (red) and αPS3 (blue) immunolabeling in peripheral nerves (N). αPS3 labeling was also observed in trachea (T). (B) Repo::UAS-αPS2-RNAi. αPS2 (red) but not αPS3 (blue) labeling was decreased in the peripheral nerve. αPS2 labeling was still present in the body wall muscles (M). (C) Repo::UAS-αPS3-RNAi. αPS3 (blue) but not αPS2 (red) labeling was decreased in the peripheral nerve. αPS3 labeling was still observed in the trachea (T). Scale bars: 5 µm.
Fig. S4. Expression of βPS-RNAi in glial cells does not change the neural lamella in peripheral nerves. (A-F) Repo-GAL4 was used to express βPS-RNAi in all three glial layers and the NL was labeled with Perlecan-GFP (green) and by γ-Laminin immunolabeling (red). (A,D) Single 0.2 µm z-sections of the nerves. (B,F) Projections of the entire z-stack for each. Boxed regions were digitally expanded as shown in B and E. βPS labeling was greatly decreased in Repo:: βPS-RNAi nerves (blue in D-F) as compared with that in control nerves (blue in A-C). βPS was also found in trachea (T) and muscles (M), and this immunolabeling was not changed in the RNAi larvae. Perlecan-GFP and γ-Laminin labeling appeared to be the same in control and RNAi nerves suggesting no change to the NL when βPS integrin was reduced. Scale bars: 10 µm in A,D; 5 µm in B,C,E,F.
Fig. S5. Overexpression of Mmp2 in perineurial glia changes the morphology of the central nervous system. The perineurial glial coverage of the nervous system was labeled using 46F::CD8-GFP. The length of the CNS (arrow) increased greatly in larvae expressing Mmp2 in the perineurial glia (46F::Mmp2; B) as compared with a control larva (A). PN, peripheral nerve.
Movie 1. A control nerve in which 46F::CD8-GFP-labeled perineurial glial membrane (green) forms a seamless layer around the nerve. DAPI staining (red) is used to label glial nuclei.
Movie 2. A 46F::βPS-RNAi nerve in which CD8-GFP-labeled perineurial glial membrane (green) fails to wrap around the internal HRP-labeled axons (blue). A perineurial glial nucleus is shown by DAPI staining (red).
Movie 3. A control nerve in which numerous Nrv2::CD8-GFP-labeled membrane processes (green) come from the DAPI-labeled wrapping glial nucleus (red, center). A perineurial or subperineurial glial nucleus is also labeled by DAPI (red, bottom left).
Movie 4. An Nrv2:: βPS-RNAi nerve in which only a major CD8-GFP-labeled membrane process (green) is sent out from the DAPI-labeled wrapping glial nucleus (red, center). DAPI labeling also shows a perineurial or subperineurial glial nucleus (red, bottom right).
