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Essential roles of the Tap42-regulated protein phosphatase 2A (PP2A) family in wing imaginal disc development of Drosophila melanogaster.

Wang N, Leung HT, Mazalouskas MD, Watkins GR, Gomez RJ, Wadzinski BE - PLoS ONE (2012)

Bottom Line: 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.The Tap42(RNAi)-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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, Vanderbilt University Medical Center, Nashville, Tennessee, United States of America.

ABSTRACT
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 Tap42(RNAi)-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.

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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; +/+.
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pone-0038569-g001: 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; +/+.

Mentions: As revealed by EGFP expression, pnr-Gal4 activity is restricted to the notum area of the wing disc (Fig. 1-A1), which gives rise to the adult thorax [20], [21]. Suppression of Tap42 in the pnr domain resulted in the appearance of a marked thorax cleft (Fig. 1-B2), but the wings appeared normal (Fig. 1-C2). In comparison to pnr-Gal4 activity, ap-Gal4 activity extends from the stalk to the dorsal/ventral boundary and not only includes the notum, but hinge and wing compartments as well (Fig. 1-A2). As expected, given the broader activity of the ap-Gal4 driver in the wing discs, Drosophila lines expressing Tap42RNAi under the control of ap-Gal4 exhibited more complex phenotypes that included varying degrees of a cleft thorax (Fig. 1-B3) as well as significant wing deformities (Fig. 1-C3). Necrosis of the front leg joints was also observed in some of these flies (Fig. S2-B). These findings suggest that Tap42 is involved in wing imaginal disc morphogenesis and plays a crucial role in the patterning and differentiation of wing discs.


Essential roles of the Tap42-regulated protein phosphatase 2A (PP2A) family in wing imaginal disc development of Drosophila melanogaster.

Wang N, Leung HT, Mazalouskas MD, Watkins GR, Gomez RJ, Wadzinski BE - PLoS ONE (2012)

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; +/+.
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Related In: Results  -  Collection

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pone-0038569-g001: 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; +/+.
Mentions: As revealed by EGFP expression, pnr-Gal4 activity is restricted to the notum area of the wing disc (Fig. 1-A1), which gives rise to the adult thorax [20], [21]. Suppression of Tap42 in the pnr domain resulted in the appearance of a marked thorax cleft (Fig. 1-B2), but the wings appeared normal (Fig. 1-C2). In comparison to pnr-Gal4 activity, ap-Gal4 activity extends from the stalk to the dorsal/ventral boundary and not only includes the notum, but hinge and wing compartments as well (Fig. 1-A2). As expected, given the broader activity of the ap-Gal4 driver in the wing discs, Drosophila lines expressing Tap42RNAi under the control of ap-Gal4 exhibited more complex phenotypes that included varying degrees of a cleft thorax (Fig. 1-B3) as well as significant wing deformities (Fig. 1-C3). Necrosis of the front leg joints was also observed in some of these flies (Fig. S2-B). These findings suggest that Tap42 is involved in wing imaginal disc morphogenesis and plays a crucial role in the patterning and differentiation of wing discs.

Bottom Line: 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.The Tap42(RNAi)-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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, Vanderbilt University Medical Center, Nashville, Tennessee, United States of America.

ABSTRACT
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 Tap42(RNAi)-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.

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