Figure 1—figure supplement 1F-F'' | Hsp70 > cre; UAS-dBrainbow; byn-Gal4 papillae dissected at 62 (D), 69 (D’), or 80 (D’’) hours post-puparium formation (HPPF) at 25°C. Hindguts were stained with Rabbit anti-GFP (Thermo-Fisher, A11122, 1:1000), Rat anti-HA (Sigma, 3F10, 1:100), and DAPI at 5 μg/ml. |
Figure 1G | Hsp70 > cre; UAS-dBrainbow; byn-Gal4 papillae dissected at various HPPF at 25°C. The area labeled by mKO2 was divided by total papillar area. |
Figure 1H | Hsp70 > cre; UAS-dBrainbow; byn-Gal4 papillae live-imaged at 69HPPF at 25°C. |
Figure 1H' | Fluorescence intensity measured in neighboring cells during sharing onset (1H). |
Figure 1I-I' | byn-Gal4/UAS-GFPPA, live-imaged during adulthood. Single secondary and principal cells were photoactivated and imaged every 3 s. |
Figure 2A | UAS-RNAis and dominant-negative versions of 77 genes representing a wide range of cellular roles were screened (Hsp70 > cre; UAS-dBrainbow; byn-Gal4) for sharing defects. Animals expressing both UAS-dBrainbow and an UAS-driven RNAi or mutant gene were raised at 25°C and shifted to 29°C at L3. If a given RNAi or DN line was lethal when expressed with the byn-Gal4 driver, a Gal80ts was crossed in and the animals raised at 18°C with a shift to 29°C at pupation. Given the robustness of cytoplasmic sharing in WT animals, gene knockdowns or mutants with even single cell defects in sharing were considered ‘hits’. |
Figure 2B | Secondary screen of 36 genes representing various categories of membrane trafficking (Hsp70 > cre; UAS-dBrainbow; byn-Gal4) for sharing defects. Animals expressing both UAS-dBrainbow and an UAS-driven RNAi were raised at 25°C and shifted to 29°C at L3. If a given RNAi line was lethal when expressed with the byn-Gal4 driver, a Gal80ts was crossed in and the animals raised at 18°C with a shift to 29°C at pupation. Given the robustness of cytoplasmic sharing in WT animals, gene knockdowns with even single cell defects in sharing were considered ‘hits’. |
Figure 2C | Secondary screen (Hsp70 > cre; UAS-dBrainbow; byn-Gal4) of dominant-negative and constitutively-active variants of the Drosophila Rab GTPases. UAS-Rab11DN and UAS-Rab14DN required a Gal80ts repressor and temperature shifts from 18 to 29°C at pupation. UAS-Rab1DN and UAS-Rab5DN required papillar-specific expression using an alternative Gal4 driver (60 H12-Gal4), Gal80ts repressor, and temperature shifts from 18 to 29°C at pupation. |
Figure 2D | Hsp70 > cre; UAS-dBrainbow; byn-Gal4, Gal80ts animals dissected pre-sharing (48 HPPF at 29°C). |
Figure 2D' | Hsp70 > cre; UAS-dBrainbow; byn-Gal4, Gal80ts animals raised at 18°C and shifted to 29°C at pupation and dissected post-sharing (young adult). |
Figure 2E | Young adult animals expressing UAS-shi RNAi #1 in a Hsp70 > cre; UAS-dBrainbow; byn-Gal4, Gal80ts background. Animals were shifted from 18 to 29°C at pupation to maximize RNAi and minimize animal lethality. |
Figure 2F | Young adult animals expressing UAS-Rab5 RNAi #1 in a Hsp70 > cre; UAS-dBrainbow; byn-Gal4, Gal80ts background. Animals were shifted from 18 to 29°C at 1–2 days PPF to maximize RNAi and minimize animal lethality. |
Figure 2G | Young adult animals expressing UAS-Rab11 RNAi #2 in a Hsp70 > cre; UAS-dBrainbow; byn-Gal4, Gal80ts background. Animals were shifted from 18 to 29°C at 1–2 days PPF to maximize RNAi and minimize animal lethality. |
Figure 2H | Animals were shifted and dissected as in 2D-G. Additionally, Hsp70 > cre; UAS-dBrainbow; byn-Gal4, Gal80ts animals expressing UAS-shi RNAi #2 were raised at 18°C and shifted to 29°C at pupation, animals expressing UAS-Rab5 RNAi #2 were raised at 18°C and shifted to 29°C at L3, and animals expressing UAS-Rab11 RNAi #1 were raised at 18°C and shifted to 29°C at 1–2 days PPF. |
Figure 3A-A' | Pupae expressing the early and late endosome marker UAS-GFP-myc-2x-FYVE were dissected pre (A, 48HPPF at 29°C) and post (A’, 72HPPF at 29°C) sharing onset. |
Figure 3B | Pupae expressing UAS-GFP-myc-2x-FYVE in a UAS-shi RNAi #1 background at a post-sharing time point (24HPPF at 18°C + 72 hr at 29°C). |
Figure 3C | Aggregated line profiles of UAS-GFP-myc-2x-FYVE intensity across papilla. |
Figure 3D-D' | Pupae expressing UAS-shi-Venus were dissected pre (D, 48HPPF at 29°C) and post (D’, 72HPPF at 29°C) sharing onset. |
Figure 3E | Aggregated line profiles of Shi-Venus intensity from the basal (0% distance) to the apical (100% distance) edges of the papilla. See 3C. |
Figure 3F-F'' | Transmission electron micrographs of the microvillar-like structures of pupal papillae pre (F, 60HPPF at 25°C), mid (F’, 66HPPF at 25°C), and post (F’’, 69HPPF at 25°C) cytoplasm sharing onset. |
Figure 3G-G'' | Electron micrographs of mitochondria and surrounding membrane material pre (G, 60HPPF at 25°C), mid (G’, 66HPPF at 25°C), and post (G’’, 69HPPF at 25°C) |
Figure 3H | Electron micrograph of microvillar-like structures of WT (w1118) young adult papillar cells. |
Figure 3I | Electron micrograph of microvillar-like structures of young adult byn-Gal4, Gal80ts, UAS-shi RNAi #2 (raised at 18°C, shifted at pupation to 29°C). |
Figure 3J | Electron micrograph of microvillar-like structures of young adult byn-Gal4, Gal80ts, UAS-Rab5 RNAi #1 animals (raised at 18°C, shifted at 1–2 days PPF to 29°C). |
Figure 3K | Electron micrograph of mitochondria and surrounding membrane material of WT (w1118) young adult papillar cells. |
Figure 3L | Electron micrograph of mitochondria and surrounding membrane material of young adult byn-Gal4, Gal80ts, UAS-shi RNAi #2 (raised at 18°C, shifted at pupation to 29°C). |
Figure 3M | Electron micrograph of mitochondria and surrounding membrane material of young adult byn-Gal4, Gal80ts, UAS-Rab5 RNAi #1 animals (raised at 18°C, shifted at 1–2 days PPF to 29°C). |
Figure 3N | Electron micrograph of post-sharing WT (TM3/UAS-shi RNAi #1) pupa (24HPPF at 18°C, shifted to 29°C for 50 hr, then dissected) |
Figure 3O | Electron micrograph of post-sharing byn-Gal4, Gal80ts,UAS-shi RNAi #1 pupa (24HPPF at 18°C, shifted to 29°C for 50 hr, then dissected) |
Figure 3P | Gap junction length / (gap junction length + septate junction length) measured in WT and UAS-shi RNAi #1 pupae (see 3N-3O). Each point represents an image of a junction. |
Figure 4A-A'' | Electron micrographs of apical junctions (adherens, septate, and gap) pre (A, 60HPPF at 25°C), mid (A’, 66HPPF at 25°C), and post (A’’, 69HPPF at 25°C) |
Figure 4B | Gap junction length / (gap junction length + septate junction length) measured in pupae pre (60HPPF at 25°C), mid (66HPPF at 25°C), and post (69HPPF at 25°C) sharing onset. Each point represents an image of a junction. |
Figure 4C | Relative innexin transcript abundance (innexin X transcripts/total innexin transcripts) using data from Fly Atlas 2 (Leader et al., 2018) and RNA-seq of adult w1118 rectums performed in the Fox Lab. |
Figure 4D-D' | Pupae with endogenously GFP-tagged NrxIV (NrxIV-GFP) dissected pre (D, 48HPPF) and post (D', 72HPPF) sharing onset. |
Figure 4E-E' | Pupae stained with Inx3 antibody (gift from Reinhard Bauer, rabbit, 1:75) pre (E, 48HPPF) and post (E', 58HPPF, papillae do not stain well at later timepoints) sharing onset. |
Figure 4F | Young adult animals expressing no transgene (WT), UAS-ogreDN, UAS-ogre RNAi, or containing a deficiency covering ogre, Inx2, and Inx7 in a Hsp70 > cre; UAS-dBrainbow; byn-Gal4, Gal80ts background. Animals were raised at 25°C until L3 and then shifted to 29°C until dissection at young adulthood. |
Figure 4G | See Figure 4F. |
Figure 4H | 60 H12-Gal4, Gal80ts driving UAS-shiDN and WT siblings were shifted from 18 to 29°C at pupation. byn-Gal4, Gal80ts driving UAS-ogreDN animals and WT siblings were raised at 25°C and shifted to 29°C at L3. Animals 1–3 days post-eclosion were sorted into sex-matched groups and fed a control diet or a high salt (2% NaCl) diet. Survival was assessed once per day for 10 days. |
Figure 1—figure supplement 1A | Hsp70 > cre; UAS-dBrainbow; tubulin-Gal4 animals raised at 29°C. Tissues dissected at adulthood. |
Figure 1—figure supplement 1D | byn-Gal4/UAS-Gapdh2-GFPPA raised at 29°C and live-imaged during adulthood. Principal cells were photoactivated and imaged every 15 s. |
Figure 1—figure supplement 1E | Hsp70 > cre; UAS-dBrainbow; byn-Gal4 animals were shifted from 25 to 29°C during L3 and dissected at adulthood. |
Figure 1—figure supplement 1F | Hsp70 > cre; UAS-dBrainbow/UAS-fzr RNAi; byn-Gal4 animals were shifted from 25 to 29°C during L2 to maximize fzr knock down during endocycling. Animals were dissected at adulthood. |
Figure 1—figure supplement 1G | Hsp70 > cre; UAS-dBrainbow; byn-Gal4/UAS-NDN animals were shifted from 25 to 29°C during L3 to ensure maximum UAS-NDN expression during mitoses. Animals were dissected at adulthood. |
Figure 2—figure supplement 1A | Hsp70 > cre; UAS-dBrainbow; byn-Gal4, Gal80ts animals expressing various previously published myoblast fusion RNAis raised at 25°C and shifted to 29°C at L3 and dissected post-sharing (young adult). |
Figure 2—figure supplement 1B | Hsp70 > cre; UAS-dBrainbow; byn-Gal4, Gal80ts animals expressing various previously published UAS-dominant-negative active regulators raised at 18°C and shifted to 29°C at L3 and dissected post-sharing (young adult). |
Figure 2—figure supplement 1C | Papillar cells were identified using byn-Gal4, Gal80ts, driving UAS-GFPNLS expression. Cells were counted in one, z-sectioned half of the papillae and multiplied by two to give an approximate cell count. |
Figure 2—figure supplement 1D | Hsp70 > cre; UAS-dBrainbow; byn-Gal4, Gal80ts animals were raised at 18°C until 3–4 days PPF and shifted to 29°C and dissected at young adulthood. |
Figure 2—figure supplement 1E | Hsp70 > cre; UAS-dBrainbow; byn-Gal4, Gal80ts animals expressing UAS-shi RNAi #1 were raised at 18°C until 3–4 days PPF and shifted to 29°C and dissected at young adulthood. |
Figure 3—figure supplement 1A | See Figure 3A-C. Basal and apical membrane defined as 10–20% and 90–100% total distance of papillae, respectively. |
Figure 3—figure supplement 1B-B' | byn-Gal4 > UAS-Rab5-YFP animals dissected pre (48HPPF, 29°C) and post (72HPPF, 29°C) sharing onset. |
Figure 3—figure supplement 1B'' | See Figure 3—figure supplement 1B-B' and Figure 3C. |
Figure 3—figure supplement 1C-C'' | Electron micrographs of apical junctions (adherens, septate, and gap) pre (D, 60HPPF at 25°C), mid (D’, 66HPPF at 25°C), and post (D’’, 69HPPF at 25°C) |
Figure 3—figure supplement 1D | Electron micrograph of apical junctions (adherens, septate, and gap) of WT (w1118) young adult papillar cells. |
Figure 3—figure supplement 1E | Electron micrograph of apical junctions (adherens, septate, and gap) of young adult byn-Gal4, Gal80tsts, UAS-shi RNAi #2 (raised at 18°C, shifted at pupation to 29°C). |
Figure 3—figure supplement 1F | Electron micrograph of apical junctions (adherens, septate, and gap) of young adult byn-Gal4, Gal80tsts, UAS-Rab5 RNAi #1 animals (raised at 18°C, shifted at 1–2 days PPF to 29°C). |
Figure 3—figure supplement 1G | See Figure 3N-O. Junction width was measured throughout and averaged per image. Each point represents one image of a junction. |
Figure 3—figure supplement 1G' | See Figure 3N-O. Junction width was measured throughout and averaged per image. Each point represents one image of a junction. |
Figure 3—figure supplement 1G'' | See Figure 3N-O. Raw lengths shown were used to calculate ‘fraction gap junction’ in 3P. Each point represent one image of a junction. |
Figure 3—figure supplement 2A | TEM of young adult (w1118) papilla. |
Figure 4—figure supplement 1A | See Figure 4A-B. Junction width was measured throughout and averaged per image. Each point represents one image of a junction. |
Figure 4—figure supplement 1A' | See Figure 4A-B. Junction width was measured throughout and averaged per image. Each point represents one image of a junction. |
Figure 4—figure supplement 1A'' | See Figure 4A-B. Raw lengths shown were used to calculate ‘fraction gap junction’ in 3P. Each point represent one image of a junction. |
Figure 4—figure supplement 1B-B' | Pupae expressing byn-Gal4, Gal80tsts, UAS-ogreDN (UAS-GFP-ogre) dissected pre (B, 48HPPF, 29°C) and post (B', 72HPPF, 29°C) sharing onset. |
Figure 4—figure supplement 1C | byn-Gal4, Gal80ts pupae raised at 18°C until 0HPPF and then shifted to 29°C until dissection at 58HPPF. Pupal rectums were stained with Inx3 antibody (gift from Reinhard Bauer, rabbit, 1:75). |
Figure 4—figure supplement 1C' | byn-Gal4, Gal80tsts, UAS-shi RNAi #2 pupae raised at 18°C until 0HPPF and then shifted to 29°C until dissection at 58HPPF. Pupal rectums were stained with Inx3 antibody (gift from Reinhard Bauer, rabbit, 1:75). |
Figure 4—figure supplement 1D | byn-Gal4 > UAS-GFPNLS dissected pre (48HPPF, 29°C) sharing onset. |
Figure 4—figure supplement 1D' | 60H12-Gal4 > UAS-GFPNLS dissected pre (48HPPF, 29°C) sharing onset. The pan-hindgut driver used in previous experiments, brachyenteron (byn-Gal4), causes animal lethality with shi, Rab5, and Rab11 knockdown within a few days. We therefore screened for and identified an alternative, papillae-specific driver (60H12-Gal4), derived from regulatory sequences of the hormone receptor gene Proctolin Receptor. 60H12-Gal4 > shiDN animals are viable on a control diet allowing us to test papillar function on a high-salt diet. |
Figure 4—figure supplement 1E | Hsp70 > cre; UAS-dBrainbow; 60H12-Gal4 animals raised at 18°C and shifted to 29°C at pupation and dissected as young adults. |
Figure 4—figure supplement 1E' | Hsp70 > cre; UAS-dBrainbow; 60H12-Gal4 / UAS-shiDN animals raised at 18°C and shifted to 29°C at pupation and dissected as young adults. |
Figure 4—figure supplement 1E'' | See Figure 4—figure supplement 1E-E'. |