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DVDY_22508_sm_SuppFigS1.tif2418KSupp. Fig. S1. uzip is overexpressed in elav>mCD8::GFP; EP-C04101 embryos. A–D: Stage 15 embryos were prepared for whole-mount in situ hybridization with an uzip antisense probe. Compared with wild-type, the transposon insertion line EP-C04101 directed by the elav-Gal4 driver caused overexpression of uzip in the peripheral nervous system (PNS) and central nervous system (CNS; square brackets). E: The embryo extracts were immunoblotted using a mouse anti-Uzip antibody. An increased protein level of Uzip was also detected in elav>mCD8::GFP; EP-C04101 (square bracket).
DVDY_22508_sm_SuppFigS2.tif16259KSupp. Fig. S2. Uzip is conserved among the insects. A: The Swiss-Port accession number Q4PKR8 of Apis mellifera (APIME) was aligned with four of its expressed sequence tag (EST) sequences including DB778253, DB760136, DB760322, and DB780068. The last 26 amino acid residues of Q4PKR8 are not supported by the EST sequences. B: The proteins most similar to Uzip in other insects, including Drosophila pseudoobscura (DROPS), Anopheles gambiae (ANOGA) and Apis mellifera were aligned with the Uzip protein sequence by ClustalW2 analysis. Q4PKR8-EST_APIME is the protein sequence, which replaces the last 46 amino acids of DB778253 with the last 26 amino acid residues of Q4PKR8 (A).
DVDY_22508_sm_SuppFigS3.tif2754KSupp. Fig. S3. Most Uzip proteins expressed in S2 cells are the glycosylphosphatidyl inositol (GPI) -anchored form. Uzip-His and nontagged Uzip were transfected into S2 cells separately and Western blotting was performed following phosphoinositide phospholipase C (PI-PLC) enzymatic digestion assays. Both Uzip-His and nontagged Uzip were cleaved by PI-PLC and secreted into the supernatant. After PI-PLC assays, a small amount of Uzip was detected in the membrane fraction, suggesting the possibility of a transmembrane form of the protein.
DVDY_22508_sm_SuppFigS4.tif8067KSupp. Fig. S4. The development of glia and neurons is not affected in uzipD43 mutant. A–H: In stage 16 embryos, all axons were stained with anti–horseradish peroxidase (HRP) antibody. The glia and neuron developments were normal in uzipD43 as in wild-type. A,E: The reporter strain of LG loco-lacZ was revealed by anti–β-gal staining. B,F: The midline glia were marked by anti-Wrapper antibody. C,G: The motor neurons were marked by anti–Even-skipped (Eve). D,H: In the repo>mCD8::GFP strain, membrane-anchored GFP was produced in the LG. The LG overlaid the axonal tracts and extend processes to enwrap the axonal tracts (D′ and H′).
DVDY_22508_sm_SuppFigS5.tif6132KSupp. Fig. S5. Overexpression of uzip in glia slightly affects development of axons and longitudinal glia (LG). A,B: The three Fasciclin II (FasII) fascicles in the embryonic ventral nerve cord (VNC) at stage 17 were stained using the 1D4 antibody. The lateral pathway was defective occasionally in the uzip gain-of-function embryos (4.8%, n = 126 hemisegments). C–E: In late stage 15 embryos, the anterior LG were stained using an anti-Prospepo (Pros) antibody (green) and axons were stained with anti–horseradish peroxidase (HRP; red). C,D: Compared with the wild-type embryos (C), the number of Pros-positive cells per hemisegment (square brackets) was increased in the repo>EP-C04101 strain (D). E: The percentages of hemisegments with different numbers of Pros-positive cells were counted. The number above each bar indicates the number of hemisegments counted.
DVDY_22508_sm_SuppFigS6.tif5766KSupp. Fig. S6. The expression and rescued results of another Uzip–cyano fluorescent protein (CFP) in CadN uzipD43. A–F: The stage15 embryos were immunostanined by mouse anti-Uzip (A–D), and the stage 17 embryos were stained by 1D4 antibody (E,F). A–F: Another transgene Uzip-CFP-2 was ectopically expressed in CadN uzipD43. The Uzip distribution and the rescued 1D4 phenotype is comparable to that in the double mutant ectopically expressing Uzip-CFP-1 (Fig. 7K). G: The percentages of hemisegments with defective longitudinal pathways are shown as a bar graph. ***P < 0.005 using a two-tailed Fisher's exact test. The numbers of hemisegments counted are shown above the bars.
DVDY_22508_sm_SuppFigS7.tif1232KSupp. Fig. S7. Uzip-expressing cells do not aggregate to N-cadherin (CadN) -expressing cells. Uzip-His and CadN were transfected into Drosophila S2 cells separately and subjected to aggregation assays. The CadN-expressing cells were labeled with 1,1′, di-octadecyl-3,3,3′,3′,-tetramethylindo-carbocyanine perchlorate (DiI). The experiment was repeated at least three times with similar results.
DVDY_22508_sm_SuppFigS8.tif4794KSupp. Fig. S8. Glial development in the CadN uzipD43 and wnt5 uzipD43 double mutants. A–D: Glia were labeled using repo>mCD8::GFP strain (A,B) or stained with anti-Repo antibody (C,D). Axonal tracts were recognized using anti–horseradish peroxidase (HRP) antibody (A′–D′). longitudinal glia (LG) were absent (square brackets) or exhibited disorganized arrangement in the segments with axonal defects (square brackets) or in the adjacent segments. The glial sheath in the CadN uzipD43 appeared to extend as in the wild-type embryos (arrowhead).
DVDY_22508_sm_SuppTableS1.doc38KSupporting Table S1
DVDY_22508_sm_SuppInfo.doc30KSupporting Information

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