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A rapid one-generation genetic screen in a Drosophila model to capture rhabdomyosarcoma effectors and therapeutic targets.

Galindo KA, Endicott TR, Avirneni-Vadlamudi U, Galindo RL - G3 (Bethesda) (2014)

Bottom Line: Here, we report a new approach to dissect RMS, exploiting a highly efficient Drosophila PAX7-FOXO1 model uniquely configured to uncover PAX-FOXO1 RMS genetic effectors in only one generation.Additionally, we reveal that mutation of mastermind, a gene encoding a MEF2 transcriptional coactivator, similarly suppresses PAX7-FOXO1, further pointing toward MEF2 transcriptional activity as a PAX-FOXO1 underpinning.These studies show the utility of the PAX-FOXO1 Drosophila system as a robust one-generation (F1) RMS gene discovery platform and demonstrate how Drosophila transgenic conditional expression models can be configured for the rapid dissection of human disease.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75390-9072.

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PAX7-FOXO1 drives myogenesis in Drosophila embryos. (A) Whole-mount wild-type and daughterless-Gal4;UAS-PAX7-FOXO1 (da>>PAX7-FOXO1) gastrulated embryos probed for expression of green fluorescent protein (GFP) from a Myosin Heavy Chain (MHC)-GFP reporter transgene. Because Drosophila embryos initiate native expression of MHC at embryonic stage 13, we focused on embryos at stage 12 or younger. Diffuse expression of MHC-GFP is only detected in the da>>PAX7-FOXO1 embryos. (B) Greater resolution images of embryo segments noted by the white bars in (A) MHC-GFP = GFP immunofluorescence from the MHC-GFP reporter; DAPI = 4′,6-diamidino-2-phenylindole nuclear staining.
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fig1: PAX7-FOXO1 drives myogenesis in Drosophila embryos. (A) Whole-mount wild-type and daughterless-Gal4;UAS-PAX7-FOXO1 (da>>PAX7-FOXO1) gastrulated embryos probed for expression of green fluorescent protein (GFP) from a Myosin Heavy Chain (MHC)-GFP reporter transgene. Because Drosophila embryos initiate native expression of MHC at embryonic stage 13, we focused on embryos at stage 12 or younger. Diffuse expression of MHC-GFP is only detected in the da>>PAX7-FOXO1 embryos. (B) Greater resolution images of embryo segments noted by the white bars in (A) MHC-GFP = GFP immunofluorescence from the MHC-GFP reporter; DAPI = 4′,6-diamidino-2-phenylindole nuclear staining.

Mentions: Toward validating the new fly PAX-FOXO1 system, we tested whether human PAX7-FOXO1 promotes myogenesis in Drosophila. We used the daughterless-Gal4 driver, which directs ubiquitous expression of UAS-transgenes, to express PAX7-FOXO1 during embryogenesis. We then probed for expression of a GFP-tagged Myosin Heavy Chain (MHC) reporter transgene, a marker specific for myogenesis and a reporter previously used in embryonic screens to successfully identify genes involved in Drosophila somatic muscle development and patterning (Chen and Olson 2001; Chen et al. 2003). Drosophila embryos initiate native expression of MHC at embryonic stage 13—thus, we focused on embryos at stage 12 or younger for ectopic MHC expression. We observed robust expression of MHC-GFP in cells of all three germ layers, including nonmyogenic cells within the ectoderm and endoderm primordia (Figure 1), findings similar to PAX3-FOXO1 misexpression in mouse embryonic primordial cells (Scuoppo et al. 2007). These results [as well as similar results described below (MEF2 as a PAX-FOXO gene target and putative RMS effector) (Figure 4C)] show that Drosophila precursors are vulnerable to the myogenic programming properties intrinsic to the PAX-FOXO1 chimera.


A rapid one-generation genetic screen in a Drosophila model to capture rhabdomyosarcoma effectors and therapeutic targets.

Galindo KA, Endicott TR, Avirneni-Vadlamudi U, Galindo RL - G3 (Bethesda) (2014)

PAX7-FOXO1 drives myogenesis in Drosophila embryos. (A) Whole-mount wild-type and daughterless-Gal4;UAS-PAX7-FOXO1 (da>>PAX7-FOXO1) gastrulated embryos probed for expression of green fluorescent protein (GFP) from a Myosin Heavy Chain (MHC)-GFP reporter transgene. Because Drosophila embryos initiate native expression of MHC at embryonic stage 13, we focused on embryos at stage 12 or younger. Diffuse expression of MHC-GFP is only detected in the da>>PAX7-FOXO1 embryos. (B) Greater resolution images of embryo segments noted by the white bars in (A) MHC-GFP = GFP immunofluorescence from the MHC-GFP reporter; DAPI = 4′,6-diamidino-2-phenylindole nuclear staining.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4321029&req=5

fig1: PAX7-FOXO1 drives myogenesis in Drosophila embryos. (A) Whole-mount wild-type and daughterless-Gal4;UAS-PAX7-FOXO1 (da>>PAX7-FOXO1) gastrulated embryos probed for expression of green fluorescent protein (GFP) from a Myosin Heavy Chain (MHC)-GFP reporter transgene. Because Drosophila embryos initiate native expression of MHC at embryonic stage 13, we focused on embryos at stage 12 or younger. Diffuse expression of MHC-GFP is only detected in the da>>PAX7-FOXO1 embryos. (B) Greater resolution images of embryo segments noted by the white bars in (A) MHC-GFP = GFP immunofluorescence from the MHC-GFP reporter; DAPI = 4′,6-diamidino-2-phenylindole nuclear staining.
Mentions: Toward validating the new fly PAX-FOXO1 system, we tested whether human PAX7-FOXO1 promotes myogenesis in Drosophila. We used the daughterless-Gal4 driver, which directs ubiquitous expression of UAS-transgenes, to express PAX7-FOXO1 during embryogenesis. We then probed for expression of a GFP-tagged Myosin Heavy Chain (MHC) reporter transgene, a marker specific for myogenesis and a reporter previously used in embryonic screens to successfully identify genes involved in Drosophila somatic muscle development and patterning (Chen and Olson 2001; Chen et al. 2003). Drosophila embryos initiate native expression of MHC at embryonic stage 13—thus, we focused on embryos at stage 12 or younger for ectopic MHC expression. We observed robust expression of MHC-GFP in cells of all three germ layers, including nonmyogenic cells within the ectoderm and endoderm primordia (Figure 1), findings similar to PAX3-FOXO1 misexpression in mouse embryonic primordial cells (Scuoppo et al. 2007). These results [as well as similar results described below (MEF2 as a PAX-FOXO gene target and putative RMS effector) (Figure 4C)] show that Drosophila precursors are vulnerable to the myogenic programming properties intrinsic to the PAX-FOXO1 chimera.

Bottom Line: Here, we report a new approach to dissect RMS, exploiting a highly efficient Drosophila PAX7-FOXO1 model uniquely configured to uncover PAX-FOXO1 RMS genetic effectors in only one generation.Additionally, we reveal that mutation of mastermind, a gene encoding a MEF2 transcriptional coactivator, similarly suppresses PAX7-FOXO1, further pointing toward MEF2 transcriptional activity as a PAX-FOXO1 underpinning.These studies show the utility of the PAX-FOXO1 Drosophila system as a robust one-generation (F1) RMS gene discovery platform and demonstrate how Drosophila transgenic conditional expression models can be configured for the rapid dissection of human disease.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75390-9072.

Show MeSH
Related in: MedlinePlus