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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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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.
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pone-0038569-g002: 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.

Mentions: To begin to explore the mechanism underlying Tap42 regulation of wing disc development, we examined the expression pattern of Tap42 in wing discs using immunofluorescence histochemistry and a Tap42-specific rabbit polyclonal antibody. Tap42 is highly expressed in the wing disc stalk and squamous peripodial epithelium (PE) cells but weakly expressed in the columnar disc proper (DP) cells (Fig. 2-A1 & Fig. S1). Silencing of the Tap42 gene using the pnr- or ap-Gal4 drivers almost completely eliminated the Tap42 signal (Fig. 2-A2 & A3), thus verifying the specificity of the Tap42 antibody and demonstrating the high efficacy of the Tap42-targeted RNAi. Although pnr-Gal4 activity was found in a more restricted compartment of the wing disc as compared to ap-Gal4 activity (Fig. 1-A1 & A2), both drivers effectively eliminated Tap42 expression in wing disc. Interestingly, we also observed that the morphological structures and patterns of the ap-Gal4>Tap42RNAi wing disc (as revealed using the nucleus stain TO-PRO3) were disrupted in the DP cells (Fig. 2-A3), which eventually gives rise to the thorax and wings [20], [21]. However, no obvious alterations of the wing disc morphological structures and patterns were found in the pnr-Gal4>UAS-Tap42RNAi flies (Fig. 2-A2).


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)

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.
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pone-0038569-g002: 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.
Mentions: To begin to explore the mechanism underlying Tap42 regulation of wing disc development, we examined the expression pattern of Tap42 in wing discs using immunofluorescence histochemistry and a Tap42-specific rabbit polyclonal antibody. Tap42 is highly expressed in the wing disc stalk and squamous peripodial epithelium (PE) cells but weakly expressed in the columnar disc proper (DP) cells (Fig. 2-A1 & Fig. S1). Silencing of the Tap42 gene using the pnr- or ap-Gal4 drivers almost completely eliminated the Tap42 signal (Fig. 2-A2 & A3), thus verifying the specificity of the Tap42 antibody and demonstrating the high efficacy of the Tap42-targeted RNAi. Although pnr-Gal4 activity was found in a more restricted compartment of the wing disc as compared to ap-Gal4 activity (Fig. 1-A1 & A2), both drivers effectively eliminated Tap42 expression in wing disc. Interestingly, we also observed that the morphological structures and patterns of the ap-Gal4>Tap42RNAi wing disc (as revealed using the nucleus stain TO-PRO3) were disrupted in the DP cells (Fig. 2-A3), which eventually gives rise to the thorax and wings [20], [21]. However, no obvious alterations of the wing disc morphological structures and patterns were found in the pnr-Gal4>UAS-Tap42RNAi flies (Fig. 2-A2).

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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