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 first 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 h 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 μm. 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.