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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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Isolation of the myogenesis benchmark gene D-Mef2 as a PAX7-FOXO1 suppressor and gene target. (A) Smaller, overlapping chromosomal deletions reduce the PAX7-FOXO1 deletion suppressor Df(2R)X1 to chromosomal segments 46C1-46C7, which includes D-Mef2, the master regulator of Drosophila myogenesis. (B) D-Mef2 loss-of-function mutation dominantly suppresses PAX7-FOXO1 lethality. PAX7-FOXO1-expression is semilethal. In the presence of Df(2R)X1, which deletes D-Mef2, the population of PAX7-FOXO1−positive adults is increased 2.4-fold and is a PAX7-FOXO1 suppressor. Two smaller overlapping deletions, Df(2R)BSC152 and Df(2R)BSC298, also delete D-Mef2 and suppress PAX7-FOXO1, whereas Df(2R)eve neither deletes D-Mef2 nor acts as a PAX7-FOXO1 suppressor. The D-Mef222-21  allele (n = 193 F1 adults scored) is a strong suppressor of PAX7-FOXO1 lethality (P = 0.0018), confirming that D-Mef2 genetically interacts with PAX7-FOXO1. Of note—although the Df(2R)BSC298 deletion showed a fold change of slightly less than 1.9, the increase in PAX7-FOXO1 adults (1.8-fold) was highly significant (P = 0.0004), and in this test we considered a suppressor. (C) PAX7-FOXO1 drives D-Mef2 expression. Whole-mount wild-type and daughterless-Gal4;UAS-PAX7-FOXO1 (da>>PAX7-FOXO1) gastrulated embryos (dorsal surface upper right corner, posterior surface, lower right corner) probed for expression of yellow fluorescent protein (YFP) from a D-Mef2-YFP embryonic reporter transgene. In wild-type embryos, D-Mef2 expression is limited to differentiating myoblasts within the mesoderm. In da>>PAX7-FOXO1 embryos, D-Mef2-YFP reporter expression is seen throughout the embryo, including ectodermal and endodermal derivatives. D-Mef2 is also detectably overexpressed in myoblasts, visible in a segmentally repeating pattern. The black lines note the posterior aspect of both embryos shown in the right-most, greater resolution images. Mef2-YFP = YFP immunofluorescence from the D-Mef2-YFP reporter; DAPI = 4′,6-diamidino-2-phenylindole nuclear staining. *P < 0.05, **P < 0.01, ***P < 0.001 vs. control.
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fig4: Isolation of the myogenesis benchmark gene D-Mef2 as a PAX7-FOXO1 suppressor and gene target. (A) Smaller, overlapping chromosomal deletions reduce the PAX7-FOXO1 deletion suppressor Df(2R)X1 to chromosomal segments 46C1-46C7, which includes D-Mef2, the master regulator of Drosophila myogenesis. (B) D-Mef2 loss-of-function mutation dominantly suppresses PAX7-FOXO1 lethality. PAX7-FOXO1-expression is semilethal. In the presence of Df(2R)X1, which deletes D-Mef2, the population of PAX7-FOXO1−positive adults is increased 2.4-fold and is a PAX7-FOXO1 suppressor. Two smaller overlapping deletions, Df(2R)BSC152 and Df(2R)BSC298, also delete D-Mef2 and suppress PAX7-FOXO1, whereas Df(2R)eve neither deletes D-Mef2 nor acts as a PAX7-FOXO1 suppressor. The D-Mef222-21 allele (n = 193 F1 adults scored) is a strong suppressor of PAX7-FOXO1 lethality (P = 0.0018), confirming that D-Mef2 genetically interacts with PAX7-FOXO1. Of note—although the Df(2R)BSC298 deletion showed a fold change of slightly less than 1.9, the increase in PAX7-FOXO1 adults (1.8-fold) was highly significant (P = 0.0004), and in this test we considered a suppressor. (C) PAX7-FOXO1 drives D-Mef2 expression. Whole-mount wild-type and daughterless-Gal4;UAS-PAX7-FOXO1 (da>>PAX7-FOXO1) gastrulated embryos (dorsal surface upper right corner, posterior surface, lower right corner) probed for expression of yellow fluorescent protein (YFP) from a D-Mef2-YFP embryonic reporter transgene. In wild-type embryos, D-Mef2 expression is limited to differentiating myoblasts within the mesoderm. In da>>PAX7-FOXO1 embryos, D-Mef2-YFP reporter expression is seen throughout the embryo, including ectodermal and endodermal derivatives. D-Mef2 is also detectably overexpressed in myoblasts, visible in a segmentally repeating pattern. The black lines note the posterior aspect of both embryos shown in the right-most, greater resolution images. Mef2-YFP = YFP immunofluorescence from the D-Mef2-YFP reporter; DAPI = 4′,6-diamidino-2-phenylindole nuclear staining. *P < 0.05, **P < 0.01, ***P < 0.001 vs. control.

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)

Isolation of the myogenesis benchmark gene D-Mef2 as a PAX7-FOXO1 suppressor and gene target. (A) Smaller, overlapping chromosomal deletions reduce the PAX7-FOXO1 deletion suppressor Df(2R)X1 to chromosomal segments 46C1-46C7, which includes D-Mef2, the master regulator of Drosophila myogenesis. (B) D-Mef2 loss-of-function mutation dominantly suppresses PAX7-FOXO1 lethality. PAX7-FOXO1-expression is semilethal. In the presence of Df(2R)X1, which deletes D-Mef2, the population of PAX7-FOXO1−positive adults is increased 2.4-fold and is a PAX7-FOXO1 suppressor. Two smaller overlapping deletions, Df(2R)BSC152 and Df(2R)BSC298, also delete D-Mef2 and suppress PAX7-FOXO1, whereas Df(2R)eve neither deletes D-Mef2 nor acts as a PAX7-FOXO1 suppressor. The D-Mef222-21  allele (n = 193 F1 adults scored) is a strong suppressor of PAX7-FOXO1 lethality (P = 0.0018), confirming that D-Mef2 genetically interacts with PAX7-FOXO1. Of note—although the Df(2R)BSC298 deletion showed a fold change of slightly less than 1.9, the increase in PAX7-FOXO1 adults (1.8-fold) was highly significant (P = 0.0004), and in this test we considered a suppressor. (C) PAX7-FOXO1 drives D-Mef2 expression. Whole-mount wild-type and daughterless-Gal4;UAS-PAX7-FOXO1 (da>>PAX7-FOXO1) gastrulated embryos (dorsal surface upper right corner, posterior surface, lower right corner) probed for expression of yellow fluorescent protein (YFP) from a D-Mef2-YFP embryonic reporter transgene. In wild-type embryos, D-Mef2 expression is limited to differentiating myoblasts within the mesoderm. In da>>PAX7-FOXO1 embryos, D-Mef2-YFP reporter expression is seen throughout the embryo, including ectodermal and endodermal derivatives. D-Mef2 is also detectably overexpressed in myoblasts, visible in a segmentally repeating pattern. The black lines note the posterior aspect of both embryos shown in the right-most, greater resolution images. Mef2-YFP = YFP immunofluorescence from the D-Mef2-YFP reporter; DAPI = 4′,6-diamidino-2-phenylindole nuclear staining. *P < 0.05, **P < 0.01, ***P < 0.001 vs. control.
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fig4: Isolation of the myogenesis benchmark gene D-Mef2 as a PAX7-FOXO1 suppressor and gene target. (A) Smaller, overlapping chromosomal deletions reduce the PAX7-FOXO1 deletion suppressor Df(2R)X1 to chromosomal segments 46C1-46C7, which includes D-Mef2, the master regulator of Drosophila myogenesis. (B) D-Mef2 loss-of-function mutation dominantly suppresses PAX7-FOXO1 lethality. PAX7-FOXO1-expression is semilethal. In the presence of Df(2R)X1, which deletes D-Mef2, the population of PAX7-FOXO1−positive adults is increased 2.4-fold and is a PAX7-FOXO1 suppressor. Two smaller overlapping deletions, Df(2R)BSC152 and Df(2R)BSC298, also delete D-Mef2 and suppress PAX7-FOXO1, whereas Df(2R)eve neither deletes D-Mef2 nor acts as a PAX7-FOXO1 suppressor. The D-Mef222-21 allele (n = 193 F1 adults scored) is a strong suppressor of PAX7-FOXO1 lethality (P = 0.0018), confirming that D-Mef2 genetically interacts with PAX7-FOXO1. Of note—although the Df(2R)BSC298 deletion showed a fold change of slightly less than 1.9, the increase in PAX7-FOXO1 adults (1.8-fold) was highly significant (P = 0.0004), and in this test we considered a suppressor. (C) PAX7-FOXO1 drives D-Mef2 expression. Whole-mount wild-type and daughterless-Gal4;UAS-PAX7-FOXO1 (da>>PAX7-FOXO1) gastrulated embryos (dorsal surface upper right corner, posterior surface, lower right corner) probed for expression of yellow fluorescent protein (YFP) from a D-Mef2-YFP embryonic reporter transgene. In wild-type embryos, D-Mef2 expression is limited to differentiating myoblasts within the mesoderm. In da>>PAX7-FOXO1 embryos, D-Mef2-YFP reporter expression is seen throughout the embryo, including ectodermal and endodermal derivatives. D-Mef2 is also detectably overexpressed in myoblasts, visible in a segmentally repeating pattern. The black lines note the posterior aspect of both embryos shown in the right-most, greater resolution images. Mef2-YFP = YFP immunofluorescence from the D-Mef2-YFP reporter; DAPI = 4′,6-diamidino-2-phenylindole nuclear staining. *P < 0.05, **P < 0.01, ***P < 0.001 vs. control.
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