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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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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/+.
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pone-0038569-g005: 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/+.

Mentions: Since PP2A family members have been implicated in the regulation of cell proliferation and mitosis [1], [9], we asked whether suppression of their common regulatory subunit, Tap42, in wing discs influences these cellular processes. Proliferating cells undergoing mitosis were visualized using a phospho-histone3 antibody, which is a marker of cells in late G2 and M phase [9]. As shown in Fig. 5-A2, cell proliferation was arrested within the notum region of wing discs harboring Tap42RNAi under the control of the ap driver, but no obvious changes in cell proliferation were observed in the wing compartment. TUNEL staining also revealed strong apoptosis around the wing blade in ap-Gal4>Tap42RNAi wing discs but only random apoptotic signals were found in the control discs (Fig. 5-A4). Although it remains to be determined whether defective cell cycle progression and apoptosis are direct consequences of Tap42 knockdown, alterations in these biological processes could provide an explanation for the morphological defects seen in the ap-Gal4>Tap42RNAi wing discs and the adults.


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)

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/+.
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pone-0038569-g005: 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/+.
Mentions: Since PP2A family members have been implicated in the regulation of cell proliferation and mitosis [1], [9], we asked whether suppression of their common regulatory subunit, Tap42, in wing discs influences these cellular processes. Proliferating cells undergoing mitosis were visualized using a phospho-histone3 antibody, which is a marker of cells in late G2 and M phase [9]. As shown in Fig. 5-A2, cell proliferation was arrested within the notum region of wing discs harboring Tap42RNAi under the control of the ap driver, but no obvious changes in cell proliferation were observed in the wing compartment. TUNEL staining also revealed strong apoptosis around the wing blade in ap-Gal4>Tap42RNAi wing discs but only random apoptotic signals were found in the control discs (Fig. 5-A4). Although it remains to be determined whether defective cell cycle progression and apoptosis are direct consequences of Tap42 knockdown, alterations in these biological processes could provide an explanation for the morphological defects seen in the ap-Gal4>Tap42RNAi wing discs and the adults.

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.

Show MeSH
Related in: MedlinePlus