Essential Roles of the Tap42-Regulated Protein Phosphatase 2A (PP2A) Family in Wing Imaginal Disc Development of Drosophila melanogaster

Protein ser/thr phosphatase 2A family members (PP2A, PP4, and PP6) are implicated in the control of numerous biological processes, but our understanding of the in vivo function and regulation of these enzymes is limited. In this study, we investigated the role of Tap42, a common regulatory subunit for all three PP2A family members, in the development of Drosophila melanogaster wing imaginal discs. RNAi-mediated silencing of Tap42 using the binary Gal4/UAS system and two disc drivers, pnr- and ap-Gal4, not only decreased survival rates but also hampered the development of wing discs, resulting in a remarkable thorax cleft and defective wings in adults. Silencing of Tap42 also altered multiple signaling pathways (HH, JNK and DPP) and triggered apoptosis in wing imaginal discs. The Tap42RNAi-induced defects were the direct result of loss of regulation of Drosophila PP2A family members (MTS, PP4, and PPV), as enforced expression of wild type Tap42, but not a phosphatase binding defective Tap42 mutant, rescued fly survivorship and defects. The experimental platform described herein identifies crucial roles for Tap42•phosphatase complexes in governing imaginal disc and fly development

The effects of <i>mts^XE2258</i>, <i>Tap42^WT</i>, and <i>Tap42^ED</i> on the viability of <i>Tap42^RNAi</i> flie ^{a, b}.

<p>a. The actual surviving ratios of F1 progeny were quantified from the following crosses:</p><p>Cross 1: +/+; pnr-Gal4/TM3, Ser ♀ x UAS-Tap42^RNAi/CyO; +/+ ♂.</p><p>Cross 2: ap-Gal4/CyO ♀ x UAS-Tap42^RNAi/CyO ♂.</p><p>Cross 3: ap-Gal4/CyO ♀ x UAS-Tap42^RNAi, mts^XE2258/CyO ♂.</p><p>Cross 4: +/+; pnr-Gal4/TM3, Ser ♀ x UAS-Tap42^RNAi/CyO; UAS-Tap42^WT/MKRS ♂.</p><p>Cross 5: +/+; pnr-Gal4/TM3, Ser ♀ x UAS-Tap42^RNAi/CyO; UAS-Tap42^ED/MKRS ♂.</p><p>Cross 6: +/+ ♀ x mts^XE2258/CyO ♂.</p><p>b. Crosses were repeated at least three times and flies that enter eclosion were counted as survivors.</p

Silencing of <i>Tap42</i> in wing discs leads to pleiotrophic defects that include deformed thorax and wings.

<p><i>pnr-Gal4</i> and <i>ap-Gal4</i> imaginal disc drivers were used to drive expression of <i>EGFP</i> or <i>Tap42^RNAi</i> in <i>Drosophila</i>. Wing discs obtained from 3^rd instar larvae expressing EGFP (green) reveal the <i>pnr-Gal4</i> (A1) and <i>ap-Gal4</i> (A2) expression domain in wing discs. Control flies harboring the <i>UAS-Tap42^RNAi</i> construct lacked any noticeable defect in the adult thorax (B1, with head left) or wing (C1, with wing margin to left). <i>Tap42^RNAi</i> expression using the <i>pnr-Gal4</i> driver caused a marked cleft phenotype on the adult thorax (B2, red arrow) with no notable defects in fly wing (C2). Silencing the <i>Tap42</i> gene with the <i>ap-Gal4</i> 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) <i>UAS-EGFP/+; pnr-Gal4/+</i>. (A2) <i>ap-Gal4/UAS-EGFP</i>. (B1 & C1) <i>UAS-Tap42^RNAi/+</i> as control. (B2 & B3) <i>UAS-Tap42^RNAi/+; pnr-Gal4/+</i>. (C2 & C3) <i>ap-Gal4/UAS-Tap42^RNAi; +/+</i>.</p

Tap42 is expressed in imaginal discs and primarily localized in the peripodial epithelium (PE) region.

<p>Panel A: Wing (A1–A3), haltere/3^rd leg (A4–A6), 2^nd leg (A7–A9), and eye imaginal discs (A10–A12) isolated from 3^rd instar larvae were immunostained for Tap42 protein expression (green) and counter-stained with the nucleic acid dye TO-PRO3 (purple). <i>UAS-Tap42^RNAi</i> control flies exhibited abundant expression of <i>Tap42</i> in the PE region of these imaginal discs (A1, A4, A7, & A10). <i>Tap42^RNAi</i> expression with the <i>pnr</i> (A2, A5, A8, & A11) and <i>ap</i> (A3, A6, A9, & A12) drivers dramatically reduced <i>Tap42</i> expression to nearly undetectable levels. Of note, <i>ap-Gal4</i>-mediated silencing of <i>Tap42</i> 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) <i>UAS-Tap42^RNAi/+</i> as control. (A2, A5, A8, & A11) <i>UAS-Tap42^RNAi/+; pnr-Gal4/+</i>. (A3, A6, A9, & A12) <i>ap-Gal4/UAS-Tap42^RNAi; +/+</i>. (B1 & B2) wild type <i>w¹¹¹⁸</i>.</p

Tap42 interacts with all three PP2A members and is required for normal wing disc development.

<p>Panel A: FLAG immunoprecipitations (FLAG IPs) were performed from extracts of <i>Drosophila</i> S2 cells expressing HA₃-Mts, HA₃-PP4, or HA₃-PPV alone or together with wildtype (FLAG₃-Tap42^WT) or mutant Tap42 (FLAG₃-Tap42^ED). 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 <i>Tap42^RNAi</i> in the <i>ap</i> domain displayed a marked thorax cleft (red arrow, B1) and shriveled wings (B5). Expression of <i>Tap42^WT</i> in this background completely rescued both thorax (B2) and wing defects (B6). However, introduction of the <i>Tap42^ED</i> mutant in this background failed to rescue the defects and the flies lacked the scutum (B3) and formed blistered wings (B7). Expression of <i>Tap42^ED</i> alone resulted in a mild defect around the scutum (B4) and the formation of a forked wing vein (B8). Genotypes: (B1 & B5) <i>ap-Gal4/UAS-Tap42^RNAi; +/+</i>. (B2 & B6) <i>ap-Gal4/UAS-Tap42^RNAi; +/UAS-Tap42^WT</i>. (B3 & B7) <i>ap-Gal4/UAS-Tap42^RNAi; +/UAS-Tap42^ED.</i> (A4 & B4) <i>ap-Gal4/+; +/UAS-Tap42^ED</i>.</p

Suppression of Tap42 expression in wing imaginal discs interrupts HH signaling, hampers mitosis, and triggers apoptosis.

<p>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 <i>Tap42</i> with the <i>pnr-Gal4</i> or ap<i>-Gal4</i> 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 <i>pnr-Gal4</i> 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) <i>UAS-Tap42^RNAi/+</i> as control. (A2, B2, C2, & D2) <i>UAS-Tap42^RNAi/+; pnr-Gal4/+</i>. (A3, B3, C3, & D3) <i>ap-Gal4/UAS-Tap42^RNAi; +/+</i>.</p

JNK and DPP signaling are altered in wing imaginal discs following depletion of Tap42.

<p>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 <i>UAS-Tap42^RNAi</i> flies (A1) and flies co-expressing the <i>pnr</i> 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 <i>Tap42^RNAi</i> was driven by <i>ap-Gal4</i> (A3). Total levels of JNK/BSK (green, B1-B3) did not change as a result of <i>Tap42</i> knockdown. <i>Dpp</i> gene expression (purple, C1-C3), as monitored by X-GAL staining of <i>dpp-LaZ</i>, in the scutellum and along the anterior/posterior boundary of the wing blade was similar in both control (C1) and <i>pnr</i>-<i>Gal4</i> driven <i>Tap42^RNAi</i> flies (C2). <i>ap-Gal4</i> driven <i>Tap42^RNAi</i> 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) <i>UAS-Tap42^RNAi/+</i> as control. (A2, B2, & C2) <i>UAS-Tap42^RNAi/+; pnr-Gal4/+</i>. (A3, B3, & C3) <i>ap-Gal4/UAS-Tap42^RNAi; +/+</i>.</p