Supplementary Figure S1: TIPF reporter validation in the wing disc.

Supplementary Figure S1: TIPF reporter validation in the wing disc. a,b, Test of put RNAi. a, In wildtype discs the Dpp target gene Sal (red) is expressed in a broad stripe in the centre of the ventral and dorsal compartments (arrowheads) of the wing pouch b, Transgene mediated knockdown of the type II BMP receptor Punt (put) using ap::gal4 tub::gal80 ts at 25 C reduces Dpp signalling especially in the dorsal compartment. The Sal signal is reduced (arrow) and the dorsal compartment becomes smaller. Wing pouches outlined by dashed lines, dorsoventral compartment boundaries by dotted lines, cell boundaries marked with FasIII (blue) and nuclei with DAPI (light blue). c, TIPF fluorescence depends on the type II receptor Punt. In wing discs of ap::gal4 tub::gal80 ts ; UAS::hp-Put L3 larvae raised at 25 C TIPF fluorescence is reduced in the dorsal compartment (arrow) where ap::gal4 is active. d, ubi::tipf is ubiquitously expressed in the wing disc. anti-gfp immunostaining (green) reveals even TIPF expression throughout the disc. Specifically, there is no evidence of the downregulation along the compartment boundary as seen for endogenous Tkv. Cells outlined with FasIII (red). Scale bars 50 µm except c, 25 µm. -1-

Supplementary Figure S2: Image planes and junction arrangements in testis. Schematic representation of a single hub cell (green) and GSC (blue) pair, outlining the orientation of the image planes used in all testis images. In apical transverse sections (left), the observer looks down (red arrow) through the GSC onto the interface with the hub cell. While the junctions connecting the hub cells apically with the GSC and their neighbours are in focus, most of the GSC cytoplasm and more basal parts of the hub cells are not imaged. In central sections (right), the interior of hub cells and GSCs is visualized, but most of the junctions and associated structures lie above or below the image plane. -2-

Supplementary Figure S3: Germline TIPF expression under nos::gal4- VP16 control. a, Tkv overexpression is sufficient to cause germ cells tumours. In nos::gal4-vp16; UAS::tkv-Cherry testes Tkv-Cherry (red) is overexpressed in the entire germline marked with Vasa (green). DAPI staining (light blue) reveals the presence of excess early germline cells far away from BMP source in the hub (dashed outline), leading to bloating of the testis tip and a loss of differentiated sperm cells. b, Endogenous Tkv is expressed throughout the testis tip. Immunostaining against the type I BMP receptor Tkv (green) reveals expression in the hub (marked with FasIII, red), GSCs (approximately outlined by dashed line), and more distant cells within the testis. Image deconvolved max. intensity Z projection of multiphoton optical slices spanning 10µm. Scale bars a, 50 µm, b, 5 µm. -3-

Supplementary Figure S4: DE-Cadherin-tRFP localization and ubiquitin promotor specificity a, Ubiquitously expressed DE-Cadherin-tRFP labels adherens junctions. In central (top panels) or peripheral (bottom panels) sections through a stage 9 egg chamber of a ubi::de-cadherin-trfp transgenic female the apical (arrowheads) adherens junctions of the follicular epithelium are marked by red fluorescence. Insets magnified views of boxed areas. b, The ubiquitin promoter has low germline activity. In testes from ubi::gfp FRT40A flies, GFP fluorescene (green) is largely excluded from germline cells marked by Vasa (blue) but strongly active in the hub (marked by DE-Cadherin, red). Scale bars a, 25 µm, b, 10 µm. -4-

Supplementary Figure S5: Adherens junctions in the hub. a, Adherens junctions between neighbouring hub cells and towards GSC contain typical adhesion molecules. In transgenic flies expressing tagged DE-Cadherin in the hub (ubi::de-cadherin-trfp, red) both punctate junctions between hub and GSCs (arrowheads) and the apicolateral adhesion belt (arrows) recruit -Catenin (green) and armadillo (blue). b, Junctions between hub and GSCs (arrowsheads) are marked by both DE-Cadherin-GFP expressed in the germline under nos::gal4vp16 control and ubi::de-cadherin-trfp in the somatic cells of the gonad. Note the patchy expression of DE-Cadherin-GFP in only a subset of germline cells. c, Schematic representation of two hub cells and their associated GSCs. Adjacent hub cells are attached in standard epithelial fashion via an apical belt of adherens junctions. Additional junctions connect the hub cells to the overlying GSCs. While some of these junctions appear in optical sections to be connected to the apical belt (arrow), others appear to be independent (arrowheads). Whether this distinction is real or an optical artefact caused by the curved surface of the hub cannot be resolved with certainty by confocal microscopy due to its limited Z-resolution. Scale bars 10 µm. -5-

Supplementary Figure S6: Validation of the trfp-dpp construct. The trfp-dpp fusion protein retains signalling function. Wing discs from UAS::tRFP- Dpp; dpp::gal4 L3 larvae show the characteristic Dpp-dependent increase in width seen also following equivalent Dpp or GFP-Dpp overexpression in the endogenous domain. Cells outlined with FasIII (blue), trfp-dpp in red. Scale bar 50 µm. -6-

Supplementary Figure S7: Knockdown of exocyst components in the hub. a, Sec8 RNAi driven by upd::gal4, tub::gal80 ts (29 C for 7d) causes hub disintegration with intracellular FasIII accumulation (red, arrow). Some germline cells (arrowheads) lose GSC fate and transcribe Bam while contacting the hub (Bam::GFP in green, germline marked with Vasa, blue). b, Sec8 RNAi in the hub causes DE-Cadherin (red, arrowheads) mislocalization to enlarged REs marked by YFP-Rab11 (green). c, Exocyst RNAi in the hub does not increase apoptosis levels. Sec8 RNAi leads to DE-Cadherin mislocalization (blue) and disintegration of the hub (left panel, arrowheads). Hub cells were negative for the apotosis marker activated Caspase-3 (red). Occasionally, dying cells were observed outside the hub in both experimental and control testes (arrow, right panels), confirming that apoptotic cells would not have escaped detection if common in the disintegrating hubs. Scale bars 10 µm. -7-

Supplementary Figure S8: Exocyst and Dpp secretion in the hub. a, GFP-Dpp (green, arrowheads) colocalizes with Sec15-Cherry (red) in upd::gal4, tub::gal80 ts ;; UAS::Sec15-Cherry / UAS::GFP-Dpp hub cells (max. intensity Z projection of three adjacent 1 µm optical slices). b, GFP-Dpp (green, arrowheads) is found adjacent to but does only rarely (14.6±10.5%, n=89) overlap with Sec5 vesicles (red, arrows) near the apicolateral adherens junctions (DE-Cadherin, blue). c, In upd::gal4, tub:: Gal80 ts ;; UAS::Sec15-Cherry testes only roughly one quarter of Sec15- Cherry (red) vesicles colocalize with Sec5 (green, arrow), while the remainder (arrowheads) localize to adjacent areas with low Sec5 levels (24.1±4.2% of Sec15- Cherry punctae, n=54). Hub cells labeled with FasIII (blue). Hubs marked by dashed white outlines, GFP-Dpp induction 3 days at 30 C, all scale bars 5 µm. -8-

Supplementary Figure S9: Exocyst and Gef26 participate in Dpp secretion from the hub. Colocalization of trfp-dpp expressed in the hub by upd::gal4, tub::gal80 ts with YFP- Rab11. a, Following Sec8 RNAi, the recycling endosome becomes expanded and trfp- Dpp (red) punctae show increased colocalization (arrows) with Rab11 endosomes (green). b, In Gef26 dizzy1 / Gef26 6 mutant testes, colocalization of trfp-dpp and YFP- Rab11 marked endosomes becomes more common. c,d, Pixelwise quantification of Dpp / Rab11 colocalization. Intensity in the green (YFP-Rab11) channel is plotted against trfp-dpp fluorescence, heat map indicates pixel density. In Sec8 RNAi testes (c) 65% and in Gef26 dizzy1 / Gef26 6 mutant testes (d) 58% of trfp-dpp positive pixels are also above background for YFP-Rab11 (upper right quadrant). Scale bars 5 µm. -9-

Supplementary Figure S10: Segmentation based colocalization analysis. To control for false positive colocalization scores due to diffuse mislocalization of YFP- Rab11 we determined colocalization of local intensity maxima. a-d, Confocal images of control (a), upd::gal4 Sec6 RNAi (b), upd::gal4 Sec8 RNAi (c), and Gef26 3 / Gef26 6 mutant (d) testes before (top panel) and after (bottom panel) segmentation of channels into binary bitmaps. Colocalization fractions are calculated from the segmented images by automated counting areas and their overlap. e, Quantification of trfp-dpp / YFP- Rab11 colocalization: Sec6 RNAi 79.1±9.9%, n=6 testes; Sec8 RNAi 61.1±7.4%; n=6; Gef26 3 /Gef26 6 59.1±6.3%; n=6; controls 18.6±3.3%; n=6. Differences significant, p<0.0001, one way ANOVA with Tukey s HSD. Error bars indicate SD. -10-

Supplementary Figure S11: Exocyst components in the wing disc. a, Sec5 and GFP-Dpp do not colocalize. In XZ optical sections through Dpp::Gal4 UAS::GFP-Dpp wing discs GFP-Dpp (green, arrows) is mostly found just below the apical region of high Sec5 staining (red, arrowheads) that coincides with the adherens junctions marked by DE-Cadherin (blue). Consequently, colocalization is low (4.3±1.3%, n=576). b, Sec5 immunostaining (green) and Sec15-Cherry (red) expressed using hh::gal4 label distinct vesicle pools that are often found adjacent to each other (examples marked by arrows) but rarely overlap (colocalization 2.9±0.3%, n=224). These steady stade colocalization values differ from those observed in the hub, indicating underlying differences in intracellular trafficking rates between these tissues. Scale bars 5 µm. -11-

Supplementary Figure S12: Colocalization of Rab11 and GFP-Dpp in Sec5 clones in the wing disc. Colocalization at local maxima of GFP-Dpp and Rab11 immunoflorescence following image segmentation. a, control tissue outside clone. b, homozygous Sec5 mutant cells within clone. Channels were thresholded independently, position of Sec5 clone outlined by dashed line. Output of particle tracking after segmentation shown in lower panels. Scale bars 5 µm. -12-

Supplementary Figure S13: Colocalization of Rab11 and DE-Cadherin in Sec5 clones in the wing disc. Colocalization at local maxima of DE-Cadherin and Rab11 immunoflorescence following image segmentation. a, control tissue outside clone. b, homozygous Sec5 mutant cells within clone. Channels were thresholded independently, output of particle tracking after segmentation shown in lower panels. Scale bars 5 µm. -13-

Supplementary Table S1: Rescue of tkv mutants by ubiquitous TIPF A) ubi::tipf transgene on the 2nd chromosome, two copies Prediction Prediction w / Y; p{w+; ubi::tipf 4-1}, tkv 8 / CyO for no for full x rescue rescue w / w; p{w+; ubi::tipf 4-1}, tkv A12 / CyO Class Phenotype expected expected observed observed frequency Rescued tkv mutants heterozygous progeny Cy + w + 0 % 33.3 % 99 33.1 % Cy - w + 100 % 66.7 % 200 66.9 % B) ubi::tipf transgene on the 2nd chromosome, one copy Rescued tkv mutants heterozygous progeny Phenotype Prediction for no rescue expected frequency Prediction for full rescue expected frequency w / Y; p{w+; ubi::tipf} 4-1, tkv 8 / CyO x w / w;, tkv A12 / CyO observed observed frequency incidence Cy + w + 0 % 33.3 % 14 18.9 % Cy - 100 % 66.7 % 60 81.1 % C) ubi::tipf transgene on X chromosome Prediction for no rescue Class Phenotype expected frequency Rescued tkv mutants (females) Non-rescued tkv mutants (males) tkv 8 heterozygous siblings tkv A12 heterozygous siblings (excluded) Prediction for full rescue expected frequency w, p{w+; ubi::tipf} 2 / Y; tkv A12 / Kr If x w/w; tkv 8 / CyO observed observed frequency incidence Cy + Kr If + 0 % 33 % 19 27.9 % Cy + Kr If + 0 % 0 % 0 0 % Cy + Kr If - 100 % 67 % 49 72.1 % Cy - n.d. n.d. n.d. n.d. -14-

Supplementary Table S2: TIPF reporter activity in zebrafish embryos Injection of TIPF mrna, 50 pg / embryo TIPF fluorescence at shield ventral only ventral side, laterally TIPF fluorescence stage expanded on dorsal side Incidence 30 12 4 frequency 65.2% 26.1% 8.7% -15-

Supplementary Table S3: Rescue of Sec15 mutants by ubiquitous expression of Sec15-Cherry shows that Sec15-Cherry is functional Prediction for no rescue Prediction for full rescue w; p{w+; Ubi::Sec15-Cherry} / +; Sec15 1 / TM3, Sb x w / w ;; Sec15 2 / TM3, Sb Class genotype expected frequency expected frequency observed incidence observed frequency heterozygous progeny, Sb - w; p{w+; Ubi::Sec15-Cherry} / + ; Sec15 1/2 / TM3, Sb or w; + / + ; Sec15 1/2 / TM3, Sb 100% 80 % 190 78.2% rescued homozyg. mutant progeny (w + Sb + ) homozyg. mutant progeny (w - Sb + ) w; p{w+; Ubi::Sec15-Cherry} 0% 20 % 53 21.8% / + ; Sec15 1 / Sec15 2 w; + / + ; Sec15 1 / Sec15 2 0 % 0 % 0 0% -16-

Supplementary Table S4: Rescue of GFP-Dpp overexpression pupal lethality by reducing Sec15 gene dosage A) Lethality of GFP-Dpp overexpression in the endogenous domain w, tub::gal80 / Y ;; dpp::gal4, UAS::GFP-Dpp / TM6B, Tb x w / w Class genotype 40 larvae sorted for Tb + phenotype females w, tub::gal80 / w ;; dpp::gal4, UAS::GFP-Dpp / + males w / Y ;; dpp::gal4, UAS::GFP-Dpp / + 19 hatching 0 hatching B) Suppression by Sec15 1 w, tub::gal80 / Y ;; dpp::gal4, UAS::GFP-Dpp / TM6B, Tb x w / w ;; Sec15 1 / TM6B, Tb Class genotype 40 larvae sorted for Tb + phenotype females w, tub::gal80 / w ;; dpp::gal4, UAS::GFP-Dpp / Sec15 1 males w / Y ;; dpp::gal4, UAS::GFP-Dpp / Sec15 1 18 hatching 0 hatching, 3 pharate adult males dissected from pupal case C) Suppression by Sec15 2 w, tub::gal80 / Y ;; dpp::gal4, UAS::GFP-Dpp / TM6B, Tb x w / w ;; Sec15 2 / TM3, Sb Class genotype 50 larvae sorted for Tb + phenotype females w, tub::gal80 / w ;; dpp::gal4, UAS::GFP-Dpp / TM3, Sb males w / Y ;; dpp::gal4, UAS::GFP-Dpp / TM3, Sb females w, tub::gal80 / w ;; dpp::gal4, UAS::GFP-Dpp / Sec15 2 males w / Y ;; dpp::gal4, UAS::GFP-Dpp / Sec15 2 excluded from analysis 0 hatching, 0 found among pharate adults 25 hatching 2 hatching, 3 Sb + pharate adult males dissected from pupal case -17-

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