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1 Supplementary Materials for Cytoneme-Mediated Contact-Dependent Transport of the Drosophila Decapentaplegic Signaling Protein Sougata Roy, Hai Huang, Songmei Liu, Thomas B. Kornberg* *Corresponding author. Published 2 January 2014 on Science Express DOI: /science This PDF file includes: Tables S1 to S5 Figs. S1 to S3 Caption for movie S1 Other supplementary material for this manuscript includes the following: Movie S1
2 Protein Relative intensity (lower/upper layer) Dpp:GFP 2.9±1.16 pmad 2.1±0.351 dad-lacz 2.1±0.115 put-lacz 1.13±0.117 tkv-lacz 0.54±0.075 Table S1: Relative levels of Dpp and Dpp signaling in the layers of ASP. Intensities were measured using Image J, in a rectangle in the mid region of ASP that included cells with the highest levels of Dpp or Dpp signaling; numbers represent ratio of lower/upper. The size and relative position of the rectangle was the same for both the upper and lower level optical sections. Five specimens were measured and the average relative values are presented.
3 Mutants and transgene expression Duplicated ASP Abnormal TC* dpp ts (18 C) 0% (0/20) NS dpp ts (29 C)** 23% (8/35) NS btl>cd8:gfp***, tub-gal80 ts 0% (0/30) 0% (0/30) btl>tkv DN, tub-gal80 ts 17% (6/35) 57% (20/35) btl>cd8:gfp, >Put DN, tub-gal80 ts 9% (3/35) 29% (10/35) btl>cd8:gfp, >putrnai, tub-gal80 ts 80% (16/20) NS btl>cd8:gfp, >Dad, tub-gal80 ts 20% (7/35) 51% (18/35) ap>, tub-gal80 ts, dad-gfp 0% (0/12) NS ap>dpprnai, dad-gfp, tub-gal80 ts 13% (6/50) NS btl>cd8:gfp, >Tkv DN, >Dad, tub-gal80 ts 67% (10/15) NS dpp ts, btl>ngfp, >Tkv DN (29 C)** 75% (9/12) NS btl>cd8:gfp, >Caps DN /+ ; >Caps DN / tub- 27% (8/30) NS btl>cd8:gfp, >capsrnai, tub-gal80 ts 13% (4/30) NS btl>cd8:gfp, caps C28fs, trn 17 / + 0% (0 /30) NS btl>cd8:gfp, >Caps DN /+ ; caps C28fs, trn 17 / + 60% (9/15) NS btl>cd8:gfp, >capsrnai; caps C28fs, trn 17 / + 40% (6/15) NS btl>trnrnaitub-gal80 ts 20% (3/15) NS btl>nrgrnaitub-gal80 ts 59%(16/27)** 11% (2/19) NS Table S2: ASP and TC phenotypes associated with loss of Dpp signaling. The two phenotypes associated with loss of Dpp function were not observed in any animals with normal Dpp signaling, either wild type (not listed), dpp ts at permissive temperature (18 C) or in the presence of ap-gal4. Every genetic condition tested affected ASP morphology; TC morphology was also affected in every condition that was scored. Expression of these knock-down constructs was limited by Gal80 repression during growth at 18 C prior to the L3 period, and was strong only when the larvae were incubated at 29 C for various times (dpp ts, 24 hr; dpprnai, 12 hr; nrgrnai, 12 and 24 hr; others, 6 hr) immediately prior to analysis. The increased penetrance with Tkv DN and Dad together (67%) compared to either alone (17% and 20%, respectively) or with Dpp ts and Tkv DN together (75%) compared to either alone (23% and 20%, respectively) suggests that the knock-down conditions were partial and that the phenotypes that were
4 scored were sensitive to the degree of knock-down under the conditions that were used. Therefore, the incomplete penetrance of the phenotypes is most likely attributable to the short window of knock-down or to incomplete inactivation of Dpp signaling and does not reflect the essential and major role of Dpp signaling in the TC and ASP. Caps DN genotypes had two copies of the UAS-Caps DN transgene. *, abnormal branching in ventral TC of Tr2 **, 24 hr heat shock ***, >denotes Gal4 dependent expression mediated by UAS NS, not scored.
5 Condition Dad>GFP Dad>LacZ pmad Dad N putrnai N Tkv DN Tracheal expression Tkv DN,Dad -6 N N diarnai -2.4 N -1.3 nrgrnai -2.8 N -2.2 Caps DN N caps DN, caps C28fs, trn 17-4 N N capsrnai, caps C28fs, trn 17-3 N N Disc expression ap>dpprnai -2.3 N -1.6 dpp>nrgrnai -1.5 N N Table S3. Levels of Dpp signaling in ASP. Dad-driven GFP and LacZ expression and pmad staining were measured in the mutant genotypes indicated, and fold changes were calculated based on appropriate control. Five samples were examined for each genotype. Heterozygous mutant constructs (as in Table S2) were examined in the following genetic backgrounds: for tracheal expression, btl-gal4, UAS-CD8:Cherry, Dad-GFP, tub-gal80 ts, or btl-gal4, UAS-CD8:GFP, Dad-LacZ, tub-gal80 ts ; for disc expression, ap-gal4 or dpp- Gal4, Dad-GFP, tub-gal80 ts ; for pmad staining, btl-cd8:gfp; tub-gal80 ts. Controls lacked mutant constructs. N, not performed.
6 Genotype # cytonemes/ <25 µm >25 µm btl>gal4, >CD8:GFP/+; tub-gal80 ts /+ 0.54± ±.9 btl>gal4, >CD8:GFP/>nrgRNAi; tub-gal80 ts / ± ±0.02 btl>gal4, >CD8:Cherry/tub-Gal80 ts ; +/ ± ±0.023 btl>gal4, >CD8:Cherry/tub-Gal80 ts ; diarnai/ ± ±0.012 Table S4: Dependence of cytoneme number on neuroglian and diaphanous function. Counts were made of cytonemes in 10 ASP preparations from control (btl-gal4, UAS- CD8:GFP/+; Gal80 ts /+) and nrgrnai-expressing (btl-gal4, UAS-CD8:GFP/UASnrgRNAi; Gal80 ts /+) animals. Larvae were reared at 18 C and shifted to 29 C 12 hours prior to dissection. Cytonemes on the circumference of the ASP were counted and categorized into two size groups (<25 µm and >25 µm). Flies expressing nrgrnai (p < for both types of cytonemes) or diarnai (p= for <25 µm and p< for >25 µm) significantly reduced number of cytonemes compared to the control.
7 Induction (hr) # cytonemes/µm perimeter ASP pmad / wing disc pmad <25µm >25µm Upper layer Lower layer ± ± ± ± / ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±0.02 Table S5. Dependence of cytoneme number and Dpp signaling on Shi function. Third instar larvae that expressed shi ts1 in trachea (btl>gal4 UAS-Shi ts1 ) and that had been raised at 18 C were transferred to 30 C for the indicated times. Cytoneme numbers and levels of pmad were measured and calculated as described in SOM. Based on ANOVA followed by Tukey HSD test, the numbers of cytonemes that were <25 µm or>25 µm significantly declined (compared to 0 hr) within ½ hr after shift to 30 C (p<0.01 for all changes to cytoneme numbers, except for <25 µm cytonemes at ½ hr, p<0.05). In contrast, pmad levels did not change significantly between 0 and ½ hr in either upper or lower layers, but longer incubations reduced pmad levels significantly compared to 0 hr (p<0.01) in both layers.
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11 Movie S1: Movement of co-localized Dpp:GFP and Tkv:Cherry puncta in cytonemes. Tkv:Cherry was expressed in trachea (btl-tkv:cherry); Dpp:GFP was expressed in the dpp domain of the wing disc (dpp-dpp:gfp). Each frame of the movie is a maximum Z-projection that was compiled with ImageJ, and is shown at two frames per second. Each Z-stack 0.4 μm steps) was captured at intervals of two time points per minute at 488 nm and 561 nm using an inverted spinning disc confocal microscope. Scale bar, 40 μm.
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SUPPLEMENTARY INFORMATION
Supplementary Notes Downregulation of atlastin does not affect secretory traffic We investigated whether Atlastin might play a role in secretory traffic. Traffic impairment results in disruption of Golgi
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