DOI: 10.1038/ncb3267 Supplementary Figure 1 A group of genes required for formation or orientation of annular F-actin bundles and aecm ridges: RNAi phenotypes and their validation by standard mutations. a-h, Confocal projection of phalloidin staining showing the airway F-actin bundles of mid 2nd instar larvae (65 h AEL) (left) and light microscope view of the ECM morphology of 3rd instar larvae (around 74h AEL) (right). Compared to the DT of the control (a), E-cadherin (DE-cad)-RNAi (b), apkc-rnai (c), par6-rnai (d) cause disorientation of annular-tcp landmarks while cdc42-rnai (e), profilin (chic)-rnai (f), DAAM-RNAi (g) and Act5C-RNAi (h) cause defective F-actin polymerization and short or irregular ECM ridges. Arrows in b indicate gaps between neighbouring cells. (i), Confocal projections of DT cells of mid 2nd instar larvae. rab11-rnai expressing cells are marked with CD8- GFP (green or yellow dots). F-actin bundles are not formed in rab11-rnai expressing cells and the bundles of adjacent wild-type cells are misaligned (arrow). j,k, Confocal views of the DT at 65 h AEL mid 2nd instar of the control (j) or apkc-rnai (k). Left, single sections of transmitted light image. Right, projection views of phalloidin staining. Arrowheads mark the apical lining of airway cells. Tube diameter expansion has occurred normally (left, arrowheads) till this stage while apkc-rnai causes disorientation of annular F-actin bundles compared to controls (right).l-t, Light microscope views of aecm ridges in early 2nd instar DT (at around 50 h AEL) (l-n) or early 3rd instar DT (at around 74 h AEL) (o-t). Compared to the control (l, o), mutants of cdc42 (m), DAAM (n) or apkc (p) show disorientation of the aecm ridges. q-t, Clones marked with CD8-GFP (green, lower panels). Compared to the control (q), mutant clones of profilin (chic) (r), par6 (s) or E-cadherin (DEcad) (t) show disruption or disorientation of the aecm ridges. Yellow dots mark clone borders. u-w, Light microscope views of the aecm ridges of early 3rd instar DT (at around 74 h AEL). The control (u), ft maternal and zygotic mutants (v) or ds zygotic mutant (w) have comparable regular aecm ridges. Scale bars: 20 mm. Fluorescent images in a-h are representative of at least 10 larvae from 5 independent experiments. Light microscope views in a-h are representative of at least 5 larvae from 2 independent experiments. Images in I are representative of at least 6 larvae from 2 independent experiments. Images in j and k are representative of at least 10 larva from 2 independent experiments. Images in l-p are representative of at least 4 larvae from 3 independent experiments. Images in q-t are representative of at least 5 larvae from 2 independent experiments. Images in u-w are representative of at least 6 larvae from 2 independent experiments. WWW.NATURE.COM/NATURECELLBIOLOGY 1
Supplementary Figure 2 Dynamic localization of Rab11-GFP during airway remodelling. a-d, Rab11-GFP staining at different time points during the second instar stage. Top: confocal projection views of DT. Middle: single sections of the lateral view for Rab11-GFP (middle) and bottom: transmitted light views. Yellow and black arrows mark the apical cell surface that becomes progressively separated from the old aecm lining. Rab11-GFP homogeneously distributes in the cytoplasm at 57 h AEL (a). Rab11-GFP accumulates apically by 62 h AEL, when lumen expansion had started (b) and shows a transient enrichment at longitudinal junctions (blue arrow) compared to transverse junctions (blue arrowhead) at 65 h AEL (c). The apical enrichment of Rab11-GFP is lost by 67 h AEL when tube expansion was completed (d). e, A schematic of the typical branches in a single metameric unit of the respiratory network. DT or transverse connectives (TC) branches are marked. f-h, Single confocal sections of the DT at 65 h AEL mid 2 nd instar (f), or the TC, TC(I) (g) and TC(II) (h). From left to right, transmitted light image, Rab11-GFP staining (green), E-cadherin (DE-cad) staining (magenta) and merge of Rab11-GFP and E-cadherin (DE-cad). Rab11-GFP is enriched at the longitudinal junctions in all branches (arrow). Scale bars: 20 mm. Images in a-d are representative of at least 10 larvae from 2 independent experiments. Images in f are representative of at least 10 larvae from 2 independent experiments. Images in l-p are representative of at least 4 larvae from 3 independent experiments. Images in g and h are representative of at least 3 larvae from 2 independent experiments. 2 WWW.NATURE.COM/NATURECELLBIOLOGY
Supplementary Figure 3 Phenotypic validation of 2 candidate annular-tcp genes from the protein interaction network and anisotropic localization of key annular-tcp regulators during tube remodelling. a-f, Validation of cip4 and PP1α-87B function in annular-tcp. Phalloidin staining of the DT at mid 2nd instar (65 h AEL) (a-c,f) or mid 1st instar (41 h AEL) (d,e). Compared to the controls (a, d), Cip4 overexpression (b) compromised F-actin bundle formation like RNAi of its interaction partner cdc42 (c) or DAAM (Supplementary Fig. 1g) while PP1α-87B-RNAi (e) caused disorientation of F-actin bundles like RNAi of its interaction partner par6 (f). g-l, Anisotropic localization of key annular- tube TCP regulators during remodelling. g, A summary showing the time course of localization of various TCP regulators at different stages of tube remodelling and expansion during 2nd instar larvae. Initiation of diametric tube expansion becomes evident when the old aecm lining detaches from the apical cell surface, which is followed by successive anisotropic localization of TCP regulators. T- and L- means transverse and longitudinal, respectively. Note that 2 molting cycles occur every 24 hours at 25 C. The different phases of tube remodelling and the anisotropic localization of TCP regulators occur at each molt. h-n, A confocal projection (h) or single sections (i-n) of the DT at 2nd instar larvae in the wild type. h, apkc and Par6 show similar localization. Note that at this stage, in addition to their well-documented accumulation along the apical junctions, diffuse cytoplasmic localization occurs preferentially along the transverse junctions. i-l, At 63-64 h AEL, RhoGEF2 amount is low near the transverse junctions, where Par6-GFP accumulates (blue arrows in i, j) while RhoGEF2 becomes enriched at longitudinal junctions (yellow arrowheads in j). At 63.5-64.5 h AEL, longitudinal localization of RhoGEF2 (k) precedes Rab11- GFP enrichment (l) (arrowheads). m-n, RhoGEF2 signals (m) accumulate in the cytoplasm and are largely excluded from the apical regions marked with phalloidin (yellow arrows). In contrast, pmlc (n) predominantly localizes in apical regions (yellow arrows). Asterisks indicate lumens. Scale bars: 20 mm. Images in a-f are representative of at least 3 larvae from 2 independent experiments. Images in h are representative of at least 8 larvae from 4 independent experiments. Images in i-l are representative of at least 4 larvae from 2 independent experiments. Images in m and n are representative of at least 6 larvae from 3 independent experiments. WWW.NATURE.COM/NATURECELLBIOLOGY 3
Supplementary Figure 4 Effects of manipulations of RhoA activity on TCP and rab11 interaction with RhoA signalling. Confocal projections of the DT at 2nd instar larvae at 64-66 h AEL upon expression of a constitutive active form (a-c) or a dominant negative form (d-f) of RhoA. Flip-out clones using the Act5c>y>gal4 construct are marked by cytoplasmic GFP or CD8- GFP (green). DE-cad marks all junctions. RhoA V14 expression increases levels of pmlc (a) or phalloidin labelled F-actin cables (b) (arrows) while RhoA N19 expression decreases levels of pmlc (d) or phalloidin labelled F-actin cables (e) (arrows). In either situation, Rab11 is also mislocalised at the transverse junctions (c,f, arrows). We note that single cell clones of RhoA N19 do not change directions of annular-tcp (e compare with f), suggesting that neighbouring wild type cells can communicate the direction of annular-tcp. g, In rab11-rnai expressing clones, preferential pmlc accumulation at longitudinal junctions is lost. Quantification is shown in h. (n=29 for L and 20 for T junctions from 6 rab11-rnai larva, NS, not significant by unpaired two-tailed Student s t test). Error bar, s.e.m. See also Supplementary Table 3 for scatterplots. Scale bars: 20 mm. Images in a-c are representative of at least 4 larvae from 2 independent experiments. Images in d-f are representative of at least 3 larvae from 2 independent experiments. Data in g and h are aggregated from 3 independent experiments. 4 WWW.NATURE.COM/NATURECELLBIOLOGY
Supplementary Figure 5 Transverse apkc localization upon knock down of chic, RhoA or Rab11. Uif involvement in annular-tcp. Confocal projections of the DT of 2nd instar larvae (63 h AEL) showing the transverse apkc localization (arrows) in the wild type (a), chic-rnai (b), RhoA N19 (c) or rab11-rnai. Flipped out clones of the Act5c>y>gal4 construct are marked with CD8-GFP (bottom) in c and d. e-g, Quantification of relative intensity of apkc along longitudinal (L) or transverse junctions (T) in chic-rnai (e), RhoA N19 (f) or rab11-rnai (g) (n=43 for L and 37 for T junctions from 6 chic-rnai larvae, ****P<0.0001, n=31 for L and 37 for T junctions from 6 RhoA N19 larvae, ****P<0.0001, n=26 for L and 17 for T junctions from 4 rab11-rnai larvae, ***P=0.0009 p-values were calculated using the unpaired two-tailed Students t-test). Error bar: s.e.m. Analysis of source data is also shown in Supplementary Table 3. h-i, Rab11-GFP localization of 2nd instar larvae (65 h AEL) in the control (h) or upon DE-cad-RNAi (i). j-k, Confocal projections of phalloidin staining of the DT at mid-1st instar (42 h AEL) (left) or light microscopic views of aecm ridges at early L2 (around 50 h AEL) (right). Compared to the control (j), uif-rnai (k) causes aecm ridge disorientation, which is especially evident at cell junctions. l, A single confocal section of DT stained for Uif (green) and E-cadherin (DEcad, magenta) at 64 h AEL. In addition to the broad apical staining, Uif is preferentially detected along the longitudinal junctions (arrowhead) compared to the transverse junctions (arrow). In addition to strong junctional signals, DEcad antibody weakly stains the cytoplasm 33. Scale bars: 20 mm. Images in a are representative of at least 10 larvae from 4 independent experiments. Data in b-g are aggregated from 2 independent experiments. Analysis of source data is also shown in Supplementary Table 3. Images in h and i are representative of at least 6 larvae from 2 independent experiments. Fluorescent images in j and k are representative of 6 larvae from 2 independent experiments. Light microscope views in j and k are representative of at least 10 larvae from 4 independent experiments. Images in l are representative of 6 larvae from 2 independent experiments. WWW.NATURE.COM/NATURECELLBIOLOGY 5
Supplementary Table Legends Supplementary Table 1 A genome-wide, tissue-specific RNAi screening identifies a group of annular-tcp genes. Part A: The primary screen results for each RNAi line tested. A list of RNAi lines and their airway phenotypes in the primary screen. The phenotypic classification score of the btl-gal4 screen was designated as follows; 10: liquid clearance defective, 20: lethal/adult morphology phenotype, 90: no significant phenotype. For each line, results from the mef2-gal4 line 20 or pnr-gal4 line 19 if available were merged. Phenotype scores with the pnr-gal4 screen are simple sums of the assigned scores for phenotype strength (minimum 0 and maximum 10) of their different categories. Part B: Candidate genes required for airway morphogenesis and function according to the primary screen. A list of genes showing the liquid clearance phenotype or lethal/adult morphology phenotype. The stronger phenotype was chosen if more than 2 RNA lines were tested. The phenotypic classification score was designated as in part A. Part C: Reproducibility for liquid clearance defective genes. Liquid clearance defective genes, which were tested with more than one strain were selected from all the tested RNAi lines (part A). Part D: Comparison of the 3 genome wide tissue specific RNAi screens (btl-gal4, mef2-gal4 and pannier-gal4). From each screen, positive genes that were also tested in both of the other 2 screens with any one of RNAi lines were selected. For btl-gal4, 3131 genes from 3777 genes (liquid clearance defective or adult phenotypes). Similarly, 2195 genes and 3036 genes were selected from the mef2-gal4 screen and the pannier-gal4 screen, respectively. A Venn diagram was drawn with these genes. For 3131 genes from the btl-gal4 screen, the primary screen results were extracted from part A and aligned according to common positive hits/btl-gal4 specific positive hits. Part E: Validation of the RNAi screen quality. Liquid clearance defects and lethality were assessed for 30 randomly picked genes from each of the 3 categories: 1) genes required for airway maturation 2) genes required for airway specification and branching and 3) genes dispensable for Drosophila development. When multiple RNAi lines were tested, those that showed the severest phenotype were selected. Part F: GO enrichment of the positives of the primary screen. Supplementary Table 2 Secondary screening identifies 12 phenotypic categories. A-Q, For each RNAi line, phenotypes were scored for each of the 12 phenotypic categories. When phenotypes could be scored at embryonic stages, it was scored and is presented in different columns. Significant phenotypes were assigned the value 1 in each phenotypic class. Part A: A full list of the secondary screen results for each RNAi line tested. Part B-M: Candidate regulators and the corresponding RNAi lines for each of the 12 phenotypic categories. Part N-Q: One group, larval ectopic ANF-GFP accumulation (part M) was classified into sub-groups according to the GFP localization patterns. The following phenotypic categories were established. For tube morphology, narrow tubes (highlighted in part B), winding tubes (part C), wider tubes (part D), lumen integrity (part E), apical ECM structure (part F), apical ECM annular orientation (part G), tube pigmentation (part H). For cell morphology; apical membrane structure (part I), cell shape/size (part J). For GFP localisation; reduced luminal ANF-GFP (part K), protein clearance defect (part L) and ectopic cytoplasmic ANF-GFP (part M). As sub-categories of ectopic cytoplasmic ANF-GFP (part M): intracellular puncta (part N), perinuclear (part O), diffuse cytoplasmic (part P), and localized cytoplasmic (part Q). Supplementary Table 3 Scatterplots of the quantification source data. Part A-D, Scatterplots of relative intensities of Rab11, RhoGEF2, pmlc, apkc in each indicated genotype and at each indicated stage according to the longitudinal (L) and transverse (T) junctions. Part E, A scatterplot of the deviation angle of the ECM orientation relative to the TT axis is presented according to the relative longitudinal (L) or transverse (T) orientations of the mutant cells in the clones. The solid lines show the median difference. 6 WWW.NATURE.COM/NATURECELLBIOLOGY