Figure 1.
Silencing of Tap42 in wing discs leads to pleiotrophic defects that include deformed thorax and wings.
pnr-Gal4 and ap-Gal4 imaginal disc drivers were used to drive expression of EGFP or Tap42RNAi in Drosophila. Wing discs obtained from 3rd instar larvae expressing EGFP (green) reveal the pnr-Gal4 (A1) and ap-Gal4 (A2) expression domain in wing discs. Control flies harboring the UAS-Tap42RNAi construct lacked any noticeable defect in the adult thorax (B1, with head left) or wing (C1, with wing margin to left). Tap42RNAi expression using the pnr-Gal4 driver caused a marked cleft phenotype on the adult thorax (B2, red arrow) with no notable defects in fly wing (C2). Silencing the Tap42 gene with the ap-Gal4 driver resulted in a thorax cleft phenotype ranging in severity from mild (B3, red arrow) to severe (Fig. 6-B1) as well as drastically shriveled wings (C3). Genotypes: (A1) UAS-EGFP/+; pnr-Gal4/+. (A2) ap-Gal4/UAS-EGFP. (B1 & C1) UAS-Tap42RNAi/+ as control. (B2 & B3) UAS-Tap42RNAi/+; pnr-Gal4/+. (C2 & C3) ap-Gal4/UAS-Tap42RNAi; +/+.
Table 1.
The effects of mtsXE2258, Tap42WT, and Tap42ED on the viability of Tap42RNAi flie a, b.
Figure 2.
Tap42 is expressed in imaginal discs and primarily localized in the peripodial epithelium (PE) region.
Panel A: Wing (A1–A3), haltere/3rd leg (A4–A6), 2nd leg (A7–A9), and eye imaginal discs (A10–A12) isolated from 3rd instar larvae were immunostained for Tap42 protein expression (green) and counter-stained with the nucleic acid dye TO-PRO3 (purple). UAS-Tap42RNAi control flies exhibited abundant expression of Tap42 in the PE region of these imaginal discs (A1, A4, A7, & A10). Tap42RNAi expression with the pnr (A2, A5, A8, & A11) and ap (A3, A6, A9, & A12) drivers dramatically reduced Tap42 expression to nearly undetectable levels. Of note, ap-Gal4-mediated silencing of Tap42 also disrupted the morphological patterning of the wing disc, as revealed by TO-PRO3 staining (A3). Panel B: The localization of Tap42 in the PE region was confirmed by immunofluorescence histochemistry. Immunostaining of wing discs obtained from wild type flies revealed an overlap of Ubx (red) and Tap42 (green) expression (B1). An amplified view of the merged image highlights strong Tap42 expression around the presumptive medial edge (ME) cells of the PE, which localizes near the boundary of the PE and DP (B2). Some Tap42 expression was visualized in the disc proper (DP) cells. Wing discs were counter-stained with the nucleic acid dye TO-PRO3 (blue). Genotypes: (A1, A4, A7, & A10) UAS-Tap42RNAi/+ as control. (A2, A5, A8, & A11) UAS-Tap42RNAi/+; pnr-Gal4/+. (A3, A6, A9, & A12) ap-Gal4/UAS-Tap42RNAi; +/+. (B1 & B2) wild type w1118.
Figure 3.
JNK and DPP signaling are altered in wing imaginal discs following depletion of Tap42.
The activity and expression of BSK was monitored in wing imaginal discs using antibodies recognizing phospho-JNK or total JNK. The pattern of active JNK/BSK (green, A1-3) was not different between control UAS-Tap42RNAi flies (A1) and flies co-expressing the pnr driver (A2). However, hyperphosphorylation of JNK/BSK was observed in the wing disc dorsal compartment (red arrows) along with hypophosphorylation of JNK/BSK in the ventral wing compartment when Tap42RNAi was driven by ap-Gal4 (A3). Total levels of JNK/BSK (green, B1-B3) did not change as a result of Tap42 knockdown. Dpp gene expression (purple, C1-C3), as monitored by X-GAL staining of dpp-LaZ, in the scutellum and along the anterior/posterior boundary of the wing blade was similar in both control (C1) and pnr-Gal4 driven Tap42RNAi flies (C2). ap-Gal4 driven Tap42RNAi flies demonstrated decreased DPP signal in the scutellum (red arrow, C3) and expanded staining in the wing blade compartment (red dashed line, C3). Genotypes: (A1, B1, & C1) UAS-Tap42RNAi/+ as control. (A2, B2, & C2) UAS-Tap42RNAi/+; pnr-Gal4/+. (A3, B3, & C3) ap-Gal4/UAS-Tap42RNAi; +/+.
Figure 4.
Suppression of Tap42 expression in wing imaginal discs interrupts HH signaling, hampers mitosis, and triggers apoptosis.
Panel A: Isolated wing imaginal discs were immunostained with antibodies recognizing Tap42 (green) and multiple components in the HH signaling pathway, including Ptc, Smo, and Ci (red). Control wing discs displayed strong Tap42 (A1) expression and the expected expression pattern for Ptc (B1), Smo (C1), and Ci (D1). Suppression of Tap42 with the pnr-Gal4 or ap-Gal4 driver effectively reduced Tap42 levels in wing discs (A2 & A3). While the levels of the HH receptor Ptc were unaffected by Tap42 silencing (B3), the expression of other downstream components of HH signaling, Smo (C3) and Ci (D3), were abrogated. Suppression of Tap42 with the pnr-Gal4 driver did not alter the expression pattern of HH signaling as shown in B2 (Ptc), C2 (Smo) and D2 (Ci). Genotypes: (A1, B1, C1, & D1) UAS-Tap42RNAi/+ as control. (A2, B2, C2, & D2) UAS-Tap42RNAi/+; pnr-Gal4/+. (A3, B3, C3, & D3) ap-Gal4/UAS-Tap42RNAi; +/+.
Figure 5.
The mtsXE2258 allele partially rescues Tap42RNAi-induced thorax and wing phenotypes.
Panel A: Mitosis and apoptosis in wing discs were monitored using a phospho-Histone H3 (p-H3, green) antibody and TUNEL staining (red), respectively. Control wing discs exhibited phospho-Histone expression throughout the wing disc (A1) with sporadic apoptotic signals (A3). Tap42RNAi under the control of the ap driver arrested mitosis in the notum area (red dashed line, A2) and triggered massive apoptosis, especially within the wing compartment (red dashed line, A4). Genotypes: (A1 & A3) UAS-Tap42RNAi/+ as control. (A2 & A4) ap-Gal4/UAS-Tap42RNAi; +/+. Panel B: Adult control flies (UAS-Tap42RNAi; mtsXE2258), as well as flies harboring the mtsXE2258 allele alone, did not exhibit any noticeable defect in the thorax (B1 & B3) or wings (B4 & B6). Introduction of the mtsXE2258 allele into the Tap42RNAi background resulted in a milder thorax cleft phenotype as compared to flies lacking the mtsXE2258 allele (compare B2 with Figs. 1-B3 & 6-B1). Furthermore, the presence of the mtsXE2258 allele resulted in a more developed wing (compare B5 with Fig. 1-C3). Genotypes: (B1 & B4) UAS-Tap42RNAi, mtsXE2258/CyO. (B2 & B5) ap-Gal4/UAS-Tap42RNAi, mtsXE2258. (B3 & B6) mtsXE2258/+.
Figure 6.
Tap42 interacts with all three PP2A members and is required for normal wing disc development.
Panel A: FLAG immunoprecipitations (FLAG IPs) were performed from extracts of Drosophila S2 cells expressing HA3-Mts, HA3-PP4, or HA3-PPV alone or together with wildtype (FLAG3-Tap42WT) or mutant Tap42 (FLAG3-Tap42ED). The FLAG immune complexes and corresponding cell extracts (lysates) were analyzed by Western blotting using the indicated epitope tag antibodies. Panel B: Adult flies expressing Tap42RNAi in the ap domain displayed a marked thorax cleft (red arrow, B1) and shriveled wings (B5). Expression of Tap42WT in this background completely rescued both thorax (B2) and wing defects (B6). However, introduction of the Tap42ED mutant in this background failed to rescue the defects and the flies lacked the scutum (B3) and formed blistered wings (B7). Expression of Tap42ED alone resulted in a mild defect around the scutum (B4) and the formation of a forked wing vein (B8). Genotypes: (B1 & B5) ap-Gal4/UAS-Tap42RNAi; +/+. (B2 & B6) ap-Gal4/UAS-Tap42RNAi; +/UAS-Tap42WT. (B3 & B7) ap-Gal4/UAS-Tap42RNAi; +/UAS-Tap42ED. (A4 & B4) ap-Gal4/+; +/UAS-Tap42ED.