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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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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; +/+.
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pone-0038569-g003: 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; +/+.

Mentions: To explore the molecular mechanism underlying the thorax and wing phenotypes of Tap42RNAi flies, we examined a number of signaling pathways that are known to be involved in the control of wing disc development. We initially monitored JNK and DPP signaling as these pathways play important roles in the epithelium sheet migration and fusion, and their disruption can lead to a remarkable thorax cleft phenotype [22], [28]. The activity of Drosophila JNK (BSK) was assessed by immunostaining the discs with a phospho-specific antibody recognizing the active form of JNK. Suppression of the Tap42 gene in the pnr gene domain did not have a significant effect on the p-JNK signal in the scutellum area of the dorsal compartment (Fig. 3-A2), which develops into the adult notum. However, silencing of Tap42 in the ap gene domain had a profound effect on the JNK activity pattern in the wing discs, especially along the ventral/dorsal boundary, as evident by hyperphosphorylation of JNK in the dorsal side and almost complete loss of p-JNK in the ventral part (Fig. 3-A3 & Fig. S1). Overexpression of a dominant-negative BSK in the ap domain failed to rescue the Tap42RNAi thorax cleft phenotype (Fig. S3-A & B). Together, these findings indicate that alterations in JNK signaling contribute very little, if any, to formation of the thorax cleft in Tap42RNAi flies.


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)

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; +/+.
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pone-0038569-g003: 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; +/+.
Mentions: To explore the molecular mechanism underlying the thorax and wing phenotypes of Tap42RNAi flies, we examined a number of signaling pathways that are known to be involved in the control of wing disc development. We initially monitored JNK and DPP signaling as these pathways play important roles in the epithelium sheet migration and fusion, and their disruption can lead to a remarkable thorax cleft phenotype [22], [28]. The activity of Drosophila JNK (BSK) was assessed by immunostaining the discs with a phospho-specific antibody recognizing the active form of JNK. Suppression of the Tap42 gene in the pnr gene domain did not have a significant effect on the p-JNK signal in the scutellum area of the dorsal compartment (Fig. 3-A2), which develops into the adult notum. However, silencing of Tap42 in the ap gene domain had a profound effect on the JNK activity pattern in the wing discs, especially along the ventral/dorsal boundary, as evident by hyperphosphorylation of JNK in the dorsal side and almost complete loss of p-JNK in the ventral part (Fig. 3-A3 & Fig. S1). Overexpression of a dominant-negative BSK in the ap domain failed to rescue the Tap42RNAi thorax cleft phenotype (Fig. S3-A & B). Together, these findings indicate that alterations in JNK signaling contribute very little, if any, to formation of the thorax cleft in Tap42RNAi flies.

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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