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Silencing of drpr Leads to Muscle and Brain Degeneration in Adult Drosophila

2014; Elsevier BV; Volume: 184; Issue: 10 Linguagem: Inglês

10.1016/j.ajpath.2014.06.018

ISSN

1525-2191

Autores

Isabelle Draper, Lane J. Mahoney, Satomi Mitsuhashi, Christina A. Pacak, Robert Salomon, Peter B. Kang,

Tópico(s)

Ubiquitin and proteasome pathways

Resumo

Mutations in the gene encoding the single transmembrane receptor multiple epidermal growth factor-like domain 10 (MEGF10) cause an autosomal recessive congenital muscle disease in humans. Although mammalian MEGF10 is expressed in the central nervous system as well as in skeletal muscle, patients carrying mutations in MEGF10 do not show symptoms of central nervous system dysfunction. drpr is the sole Drosophila homolog of the human genes MEGF10, MEGF11, and MEGF12 (JEDI, PEAR). The functional domains of MEGF10 and drpr bear striking similarities, and residues affected by MEGF10 mutations in humans are conserved in drpr. Our analysis of drpr mutant flies revealed muscle degeneration with fiber size variability and vacuolization, as well as reduced motor performance, features that have been observed in human MEGF10 myopathy. Vacuolization was also seen in the brain. Tissue-specific RNAi experiments demonstrated that drpr deficiency in muscle, but not in the brain, leads to locomotor defects. The histological and behavioral abnormalities seen in the affected flies set the stage for further studies examining the signaling pathway modulated by MEGF10/Drpr in muscle, as well as assessing the effects of genetic and/or pharmacological manipulations on the observed muscle defects. In addition, the absence of functional redundancy for Drpr in Drosophila may help elucidate whether paralogs of MEGF10 in humans (eg, MEGF11) contribute to maintaining wild-type function in the human brain. Mutations in the gene encoding the single transmembrane receptor multiple epidermal growth factor-like domain 10 (MEGF10) cause an autosomal recessive congenital muscle disease in humans. Although mammalian MEGF10 is expressed in the central nervous system as well as in skeletal muscle, patients carrying mutations in MEGF10 do not show symptoms of central nervous system dysfunction. drpr is the sole Drosophila homolog of the human genes MEGF10, MEGF11, and MEGF12 (JEDI, PEAR). The functional domains of MEGF10 and drpr bear striking similarities, and residues affected by MEGF10 mutations in humans are conserved in drpr. Our analysis of drpr mutant flies revealed muscle degeneration with fiber size variability and vacuolization, as well as reduced motor performance, features that have been observed in human MEGF10 myopathy. Vacuolization was also seen in the brain. Tissue-specific RNAi experiments demonstrated that drpr deficiency in muscle, but not in the brain, leads to locomotor defects. The histological and behavioral abnormalities seen in the affected flies set the stage for further studies examining the signaling pathway modulated by MEGF10/Drpr in muscle, as well as assessing the effects of genetic and/or pharmacological manipulations on the observed muscle defects. In addition, the absence of functional redundancy for Drpr in Drosophila may help elucidate whether paralogs of MEGF10 in humans (eg, MEGF11) contribute to maintaining wild-type function in the human brain. Muscular dystrophy is a heterogeneous group of inherited muscle diseases characterized by persistent muscle degeneration and regeneration leading to muscle wasting and loss. Recently, mutations in the gene encoding multiple epidermal growth factor–like domain protein 10 (MEGF10) were found to cause a novel autosomal recessive congenital muscle disease in humans.1Boyden S.E. Mahoney L.J. Kawahara G. Myers J.A. Mitsuhashi S. Estrella E.A. Duncan A.R. Dey F. DeChene E.T. Blasko-Goehringer J.M. Bönnemann C.G. Darras B.T. Mendell J.R. Lidov H.G. Nishino I. Beggs A.H. Kunkel L.M. Kang P.B. Mutations in the satellite cell gene MEGF10 cause a recessive congenital myopathy with minicores.Neurogenetics. 2012; 13: 115-124Crossref PubMed Scopus (61) Google Scholar, 2Logan C.V. Lucke B. Pottinger C. Abdelhamed Z.A. Parry D.A. Szymanska K. Diggle C.P. van Riesen A. Morgan J.E. Markham G. Ellis I. Manzur A.Y. Markham A.F. Shires M. Helliwell T. Scoto M. Hubner C. Bonthron D.T. Taylor G.R. Sheridan E. Muntoni F. Carr I.M. Schuelke M. Johnson C.A. Mutations in megf10, a regulator of satellite cell myogenesis, cause early onset myopathy, areflexia, respiratory distress and dysphagia (EMARDD).Nat Genet. 2011; 43: 1189-1192Crossref PubMed Scopus (81) Google Scholar, 3Pierson T.M. Markello T. Accardi J. Wolfe L. Adams D. Sincan M. Tarazi N.M. Fajardo K.F. Cherukuri P.F. Bajraktari I. Meilleur K.G. Donkervoort S. Jain M. Hu Y. Lehky T.J. Cruz P. Mullikin J.C. Bonnemann C. Gahl W.A. Boerkoel C.F. Tifft C.J. Novel SNP array analysis and exome sequencing detect a homozygous exon 7 deletion of MEGF10 causing early onset myopathy, areflexia, respiratory distress and dysphagia (EMARDD).Neuromuscul Disord. 2013; 23: 483-488Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar Patients show progressive muscle weakness with features of muscular dystrophy and congenital myopathy.1Boyden S.E. Mahoney L.J. Kawahara G. Myers J.A. Mitsuhashi S. Estrella E.A. Duncan A.R. Dey F. DeChene E.T. Blasko-Goehringer J.M. Bönnemann C.G. Darras B.T. Mendell J.R. Lidov H.G. Nishino I. Beggs A.H. Kunkel L.M. Kang P.B. Mutations in the satellite cell gene MEGF10 cause a recessive congenital myopathy with minicores.Neurogenetics. 2012; 13: 115-124Crossref PubMed Scopus (61) Google Scholar, 2Logan C.V. Lucke B. Pottinger C. Abdelhamed Z.A. Parry D.A. Szymanska K. Diggle C.P. van Riesen A. Morgan J.E. Markham G. Ellis I. Manzur A.Y. Markham A.F. Shires M. Helliwell T. Scoto M. Hubner C. Bonthron D.T. Taylor G.R. Sheridan E. Muntoni F. Carr I.M. Schuelke M. Johnson C.A. Mutations in megf10, a regulator of satellite cell myogenesis, cause early onset myopathy, areflexia, respiratory distress and dysphagia (EMARDD).Nat Genet. 2011; 43: 1189-1192Crossref PubMed Scopus (81) Google Scholar, 3Pierson T.M. Markello T. Accardi J. Wolfe L. Adams D. Sincan M. Tarazi N.M. Fajardo K.F. Cherukuri P.F. Bajraktari I. Meilleur K.G. Donkervoort S. Jain M. Hu Y. Lehky T.J. Cruz P. Mullikin J.C. Bonnemann C. Gahl W.A. Boerkoel C.F. Tifft C.J. Novel SNP array analysis and exome sequencing detect a homozygous exon 7 deletion of MEGF10 causing early onset myopathy, areflexia, respiratory distress and dysphagia (EMARDD).Neuromuscul Disord. 2013; 23: 483-488Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar, 4Hartley L. Kinali M. Knight R. Mercuri E. Hubner C. Bertini E. Manzur A.Y. Jimenez-Mallebrera C. Sewry C.A. Muntoni F. A congenital myopathy with diaphragmatic weakness not linked to the SMARD1 locus.Neuromuscul Disord. 2007; 17: 174-179Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar MEGF10 is expressed in the central nervous system (CNS), retina, and skeletal muscle.2Logan C.V. Lucke B. Pottinger C. Abdelhamed Z.A. Parry D.A. Szymanska K. Diggle C.P. van Riesen A. Morgan J.E. Markham G. Ellis I. Manzur A.Y. Markham A.F. Shires M. Helliwell T. Scoto M. Hubner C. Bonthron D.T. Taylor G.R. Sheridan E. Muntoni F. Carr I.M. Schuelke M. Johnson C.A. Mutations in megf10, a regulator of satellite cell myogenesis, cause early onset myopathy, areflexia, respiratory distress and dysphagia (EMARDD).Nat Genet. 2011; 43: 1189-1192Crossref PubMed Scopus (81) Google Scholar, 5Kay J.N. Chu M.W. Sanes J.R. MEGF10 AND MEGF11 mediate homotypic interactions required for mosaic spacing of retinal neurons.Nature. 2012; 483: 465-469Crossref PubMed Scopus (138) Google Scholar In the brain, where it is abundantly expressed, MEGF10 is enriched in astrocytes and myelinating oligodendrocytes.6Cahoy J.D. Emery B. Kaushal A. Foo L.C. Zamanian J.L. Christopherson K.S. Xing Y. Lubischer J.L. Krieg P.A. Krupenko S.A. Thompson W.J. Barres B.A. A transcriptome database for astrocytes, neurons, and oligodendrocytes: a new resource for understanding brain development and function.J Neurosci. 2008; 28: 264-278Crossref PubMed Scopus (2346) Google Scholar This protein has been shown to mediate engulfment of apoptotic neurons7Scheib J.L. Sullivan C.S. Carter B.D. Jedi-1 and MEGF10 signal engulfment of apoptotic neurons through the tyrosine kinase Syk.J Neurosci. 2012; 32: 13022-13031Crossref PubMed Scopus (66) Google Scholar as well as synapse pruning in the developing and adult CNS8Chung W.S. Clarke L.E. Wang G.X. Stafford B.K. Sher A. Chakraborty C. Joung J. Foo L.C. Thompson A. Chen C. Smith S.J. Barres B.A. Astrocytes mediate synapse elimination through MEGF10 and MERTK pathways.Nature. 2013; 504: 394-400Crossref PubMed Scopus (830) Google Scholar and to participate in the uptake of amyloid-β peptide.9Singh T.D. Park S.Y. Bae J.S. Yun Y. Bae Y.C. Park R.W. Kim I.S. MEGF10 functions as a receptor for the uptake of amyloid-β.FEBS Lett. 2010; 584: 3936-3942Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar In addition, elegant studies in rodents have shown that MEGF10, together with MEGF11, regulates the arrangement of retinal mosaic.5Kay J.N. Chu M.W. Sanes J.R. MEGF10 AND MEGF11 mediate homotypic interactions required for mosaic spacing of retinal neurons.Nature. 2012; 483: 465-469Crossref PubMed Scopus (138) Google Scholar In resting muscle, MEGF10 expression is observed in myoblasts and quiescent satellite cells, where it suppresses the differentiation program.10Holterman C.E. Le Grand F. Kuang S. Seale P. Rudnicki M.A. Megf10 regulates the progression of the satellite cell myogenic program.J Cell Biol. 2007; 179: 911-922Crossref PubMed Scopus (71) Google Scholar Many gaps remain, however, in our understanding of the signaling pathway/physiological function mediated by this receptor in muscle, as well as of the molecular consequences of the pathogenic mutations that underlie the muscle disease in humans. MEGF10 encodes a single-pass membrane protein with an N-terminus EMI domain followed by multiple extracellular EGF-like domains. These structural features, together with the intracellular noncanonical immunoreceptor tyrosine-based activation motif (ITAM) signaling motifs, have been conserved from invertebrates to humans.5Kay J.N. Chu M.W. Sanes J.R. MEGF10 AND MEGF11 mediate homotypic interactions required for mosaic spacing of retinal neurons.Nature. 2012; 483: 465-469Crossref PubMed Scopus (138) Google Scholar, 7Scheib J.L. Sullivan C.S. Carter B.D. Jedi-1 and MEGF10 signal engulfment of apoptotic neurons through the tyrosine kinase Syk.J Neurosci. 2012; 32: 13022-13031Crossref PubMed Scopus (66) Google Scholar Previous structure–function analyses have highlighted similarities between human MEGF10 and its Drosophila (fruit fly) homolog Drpr (the corresponding gene is draper, drpr, CG2086),7Scheib J.L. Sullivan C.S. Carter B.D. Jedi-1 and MEGF10 signal engulfment of apoptotic neurons through the tyrosine kinase Syk.J Neurosci. 2012; 32: 13022-13031Crossref PubMed Scopus (66) Google Scholar, 11Ziegenfuss J.S. Biswas R. Avery M.A. Hong K. Sheehan A.E. Yeung Y.G. Stanley E.R. Freeman M.R. Draper-dependent glial phagocytic activity is mediated by Src and Syk family kinase signalling.Nature. 2008; 453: 935-939Crossref PubMed Scopus (153) Google Scholar raising the possibility that these proteins mediate parallel functions. In addition to its well-characterized role in cell corpse engulfment12Freeman M.R. Delrow J. Kim J. Johnson E. Doe C.Q. Unwrapping glial biology: Gcm target genes regulating glial development, diversification, and function.Neuron. 2003; 38: 567-580Abstract Full Text Full Text PDF PubMed Scopus (312) Google Scholar and glial response to degenerating axons,13MacDonald J.M. Beach M.G. Porpiglia E. Sheehan A.E. Watts R.J. Freeman M.R. The Drosophila cell corpse engulfment receptor Draper mediates glial clearance of severed axons.Neuron. 2006; 50: 869-881Abstract Full Text Full Text PDF PubMed Scopus (373) Google Scholar Drpr mediates the remodeling of the neuromuscular junction, where it is expressed both in glia that wrap around the motor neurons as well as postsynaptically.14Fuentes-Medel Y. Logan M.A. Ashley J. Ataman B. Budnik V. Freeman M.R. Glia and muscle sculpt neuromuscular arbors by engulfing destabilized synaptic boutons and shed presynaptic debris.PLoS Biol. 2009; 7: e1000184Crossref PubMed Scopus (116) Google Scholar Despite the phylogenetic distance between Drosophila and humans, fly models of human disease have shown potential for facilitating a better understanding of the development of the pathophysiological processes that underlie neuromuscular disorders. These include fly models of spinal muscular dystrophy, lamin-associated myopathies, actin myopathies, dystrophinopathies, and dystroglycanopathies.15Chang H.C. Dimlich D.N. Yokokura T. Mukherjee A. Kankel M.W. Sen A. Sridhar V. Fulga T.A. Hart A.C. Van Vactor D. Artavanis-Tsakonas S. Modeling spinal muscular atrophy in Drosophila.PLoS One. 2008; 3: e3209Crossref PubMed Scopus (143) Google Scholar, 16Dialynas G. Flannery K.M. Zirbel L.N. Nagy P.L. Mathews K.D. Moore S.A. Wallrath L.L. LMNA variants cause cytoplasmic distribution of nuclear pore proteins in Drosophila and human muscle.Hum Mol Genet. 2012; 21: 1544-1556Crossref PubMed Scopus (32) Google Scholar, 17Goldstein J.A. Kelly S.M. LoPresti P.P. Heydemann A. Earley J.U. Ferguson E.L. Wolf M.J. McNally E.M. SMAD signaling drives heart and muscle dysfunction in a Drosophila model of muscular dystrophy.Hum Mol Genet. 2011; 20: 894-904Crossref PubMed Scopus (33) Google Scholar, 18Rajendra T.K. Gonsalvez G.B. Walker M.P. Shpargel K.B. Salz H.K. Matera A.G. A Drosophila melanogaster model of spinal muscular atrophy reveals a function for SMN in striated muscle.J Cell Biol. 2007; 176: 831-841Crossref PubMed Scopus (133) Google Scholar, 19Sevdali M. Kumar V. Peckham M. Sparrow J. Human congenital myopathy actin mutants cause myopathy and alter Z-disc structure in Drosophila flight muscle.Neuromuscul Disord. 2013; 23: 243-255Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar, 20Shcherbata H.R. Yatsenko A.S. Patterson L. Sood V.D. Nudel U. Yaffe D. Baker D. Ruohola-Baker H. Dissecting muscle and neuronal disorders in a Drosophila model of muscular dystrophy.EMBO J. 2007; 26: 481-493Crossref PubMed Scopus (105) Google Scholar, 21Taghli-Lamallem O. Akasaka T. Hogg G. Nudel U. Yaffe D. Chamberlain J.S. Ocorr K. Bodmer R. Dystrophin deficiency in Drosophila reduces lifespan and causes a dilated cardiomyopathy phenotype.Aging Cell. 2008; 7: 237-249Crossref PubMed Scopus (90) Google Scholar, 22Wairkar Y.P. Fradkin L.G. Noordermeer J.N. DiAntonio A. Synaptic defects in a Drosophila model of congenital muscular dystrophy.J Neurosci. 2008; 28: 3781-3789Crossref PubMed Scopus (53) Google Scholar The identification of adult muscle precursor cells in Drosophila that share features with vertebrate satellite cells23Figeac N. Jagla T. Aradhya R. Da Ponte J.P. Jagla K. Drosophila adult muscle precursors form a network of interconnected cells and are specified by the rhomboid-triggered EGF pathway.Development. 2010; 137: 1965-1973Crossref PubMed Scopus (38) Google Scholar, 24Maqbool T. Jagla K. Genetic control of muscle development: learning from Drosophila.J Muscle Res Cell Motil. 2007; 28: 397-407Crossref PubMed Scopus (33) Google Scholar provides further rationale for using this model organism in muscle biology investigations. This study was designed to assess the extent to which drpr mutant Drosophila may be used to model skeletal muscle phenotypes with relevance to human MEGF10 myopathy. Of note, Megf10−/− mice5Kay J.N. Chu M.W. Sanes J.R. MEGF10 AND MEGF11 mediate homotypic interactions required for mosaic spacing of retinal neurons.Nature. 2012; 483: 465-469Crossref PubMed Scopus (138) Google Scholar do not have an obvious muscle phenotype (Peter Kang, personal communication). The characterization of drpr mutant and RNAi flies establishes a baseline on which to probe the signaling pathway modulated by MEGF10/Drpr in muscle, as well as to assess the effects of genetic and/or pharmacological manipulations on the observed muscle defects. The drprΔ5 mutant fly line [genotype: w–; sp/CyOact::GFP; drprΔ5rec8 (9)/TM6, sb, Tb, e; where w–; sp/CyOact::GFP; drprΔ5rec8 (9)/drprΔ5rec8 (9) null are adult viable] and UAS-ds-drpr RNAi mutant fly line (genotype: yw; UAS-drprRNAi#7b/CyO;+/TM6, sb, Tb, e) were gifts from Marc R. Freeman (University of Massachusetts Medical School, Boston, MA).12Freeman M.R. Delrow J. Kim J. Johnson E. Doe C.Q. Unwrapping glial biology: Gcm target genes regulating glial development, diversification, and function.Neuron. 2003; 38: 567-580Abstract Full Text Full Text PDF PubMed Scopus (312) Google Scholar The UAS-ds-drpr RNAi stock transformant ID 27086 (genotype: w1118; P{GD14423}v27086, FBst0456744) was purchased from the Vienna Drosophila RNAi Center (Vienna, Austria). The genetic background strain w1118 (FBal0018186) was purchased from the Bloomington Drosophila Stock Center, as were the following Gal4 driver lines: Actin5C-Gal4 (y1 w*; P{Act5C-Gal4}25FO1/CyO, y+; FBst0004414: ubiquitous expression), how-Gal4 (w∗; P{GawB}how24B; FBst0001767: expression in mesoderm), Elav-Gal4 (P{GawB}elavC155; FBst0000458: pan-neuronal expression), and twist-Gal4 (P{GAL4-twi.G}108.4, w1; FBst0000914: expression in adult muscle precursors). The repo-Gal4 driver line (expression in glia) was a generous gift from Mary Roberts (F. Rob Jackson laboratory, Tufts University School of Medicine, Boston, MA). All strains were raised at 25°C in a 12-hour light/12-hour dark cycle on standard Drosophila media. To generate transgenic drpr RNAi flies, UAS-ds-drpr flies were crossed at 29°C with flies carrying the Gal4 transgene. RT-PCR was used to assess transcription levels of the drpr gene. Total RNA was isolated from pooled adult male Drosophila by RNA STAT-60 (Tel-Test, Inc., Friendswood, TX) following the manufacturer's recommendations. Complementary cDNA was generated using MuLV Reverse Transcriptase (Applied Biosystems Inc/Life Technologies Corp., Grand Island, NY). Alternatively, genomic DNA was extracted from the corresponding flies and used. PCR was performed according to standard protocols. The sequences of the drpr locus–specific primers used for the PCR reaction were as follows: drpr_2, forward, 5′-AGGACCTGGAATCCACTGC-3′; CG18171, reverse, 5′-GCGGCAAGTAATCTGAGTCC-3′; drpr_5, reverse, 5′-GCCTGAAAAGGGCTCACATA-3′; drpr_5, forward, 5′-CGGTATGTGAGCCCTTTTCA-3′; drpr_6, reverse, 5′-GCAGGTCATGCTGCAGTT-3′; CG12035_1, forward, 5′-GCTGCTTAATATCCCCAGAGG-3′; and CG12035_1, reverse, 5′-GTCGTTACTCTTGGCAATGG-3′. Protein extraction and western blot analysis were performed as previously reported.25McPhee C.K. Logan M.A. Freeman M.R. Baehrecke E.H. Activation of autophagy during cell death requires the engulfment receptor Draper.Nature. 2010; 465: 1093-1096Crossref PubMed Scopus (108) Google Scholar Primary antibodies were used at the following dilutions: rabbit anti-Draper, 1:5000 (generated in the Freeman Laboratory12Freeman M.R. Delrow J. Kim J. Johnson E. Doe C.Q. Unwrapping glial biology: Gcm target genes regulating glial development, diversification, and function.Neuron. 2003; 38: 567-580Abstract Full Text Full Text PDF PubMed Scopus (312) Google Scholar); and mouse anti-Discs-Large, 1:500 (clone 4F3, Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA). To assess lifespan, 1-day-old male adults were collected and then regularly transferred to fresh medium every 7 days. The number of dead flies was recorded at the time of each transfer. For the drpr heterozygous versus null lifespan study, raw percentage survival data were collected weekly on three populations in each group. The total numbers of flies analyzed were 83 and 70 in the heterozygous and null groups, respectively. Statistical analysis on the lifespan study was performed using a t-test in SigmaPlot version 11 software (Systat Software Inc., Chicago, IL). Data are presented as means ± SEM. As an index of motor function, age-matched male adults in the control and drpr mutant groups (n = 10 to 12 flies per group) were examined using a negative geotaxis climbing assay. One-day-old flies were collected and maintained for 3 to 21 days in fresh vials before testing. Corresponding flies were transferred (without CO2 anesthesia) into a clear vial. The vial was gently tapped four times to collect flies at the bottom, thus prompting a negative geotaxis response. The number of flies crossing a 5-cm threshold during a 6-second interval was recorded. Each genotype was assessed three times consecutively. Independent groups of flies/genotype were evaluated on different days.26Draper I. Tabaka M.E. Jackson F.R. Salomon R.N. Kopin A.S. The evolutionarily conserved RNA binding protein SMOOTH is essential for maintaining normal muscle function.Fly (Austin). 2009; 3: 235-246Crossref PubMed Scopus (11) Google Scholar Histological analyses were performed in the Department of Pathology, Tufts Medical Center. Aged-matched (30 to 40 days old) control and drpr mutant Drosophila were immersed in Telly's fixative for 7 days, embedded in paraffin, and sectioned at 5 μm using standard techniques. All sections were stained with hematoxylin and eosin, then examined using standard bright-field light microscopy. Drpr is the Drosophila homolog of human MEGF10. Sequence alignment (CLUSTAL 2.1) of the longest Drpr isoform, Drpr-PE (1042 aa, http://www.ncbi.nlm.nsih.gov/protein, accession number NP_001261276.1), and human MEGF10 protein variant a (1140 aa, http://www.ncbi.nlm.nih.gov/protein, accession number EAW62406.1) revealed a high degree of similarity within the EGF-like domains. The fly protein comprises 15 EGF-like domains12Freeman M.R. Delrow J. Kim J. Johnson E. Doe C.Q. Unwrapping glial biology: Gcm target genes regulating glial development, diversification, and function.Neuron. 2003; 38: 567-580Abstract Full Text Full Text PDF PubMed Scopus (312) Google Scholar versus 17 in the human.27Nagase T. Kikuno R. Ohara O. Prediction of the coding sequences of unidentified human genes. XXII. The complete sequences of 50 new cDNA clones which code for large proteins.DNA Res. 2001; 8: 319-327Crossref PubMed Scopus (49) Google Scholar Notably, within these domains, two cysteines that are targeted by known pathogenic human gene mutations (ie, C326R and C774R)1Boyden S.E. Mahoney L.J. Kawahara G. Myers J.A. Mitsuhashi S. Estrella E.A. Duncan A.R. Dey F. DeChene E.T. Blasko-Goehringer J.M. Bönnemann C.G. Darras B.T. Mendell J.R. Lidov H.G. Nishino I. Beggs A.H. Kunkel L.M. Kang P.B. Mutations in the satellite cell gene MEGF10 cause a recessive congenital myopathy with minicores.Neurogenetics. 2012; 13: 115-124Crossref PubMed Scopus (61) Google Scholar are conserved in Drosophila. In contrast, the elastin microfibril interface located protein (EMILIN) domain–nested arginine, which is mutated in another patient (R71W),1Boyden S.E. Mahoney L.J. Kawahara G. Myers J.A. Mitsuhashi S. Estrella E.A. Duncan A.R. Dey F. DeChene E.T. Blasko-Goehringer J.M. Bönnemann C.G. Darras B.T. Mendell J.R. Lidov H.G. Nishino I. Beggs A.H. Kunkel L.M. Kang P.B. Mutations in the satellite cell gene MEGF10 cause a recessive congenital myopathy with minicores.Neurogenetics. 2012; 13: 115-124Crossref PubMed Scopus (61) Google Scholar was not conserved in Drosophila (an arginine is found in close proximity) (Figure 1). In addition to MEGF10, Drpr also shows homology to human MEGF11 and MEGF12 [alias platelet endothelial aggregation receptor 1 (PEAR1)/Jedi], with approximately 40% identity found in the region encompassing the EGF-like domains. The Drosophila drpr/CG2086 gene is localized on chromosome 3 (cytolocation 3L:1715595.. 731107). Recent annotation updates of the Drosophila genome have revealed that within the drpr locus two additional genes of unknown function are found: CG18171 in cis- (ie, same strand) and CG12035 in trans- (ie, opposite strand) (Figure 2A). The Δ5 drpr mutant allele is a deletion that was generated by imprecise excision of the EP(3)522 P-element inserted in the 5′ untranslated region of the drpr gene.12Freeman M.R. Delrow J. Kim J. Johnson E. Doe C.Q. Unwrapping glial biology: Gcm target genes regulating glial development, diversification, and function.Neuron. 2003; 38: 567-580Abstract Full Text Full Text PDF PubMed Scopus (312) Google Scholar Corresponding mutant flies are commonly used to study the function of drpr. To assess whether the molecular Δ5 lesion affects the expression of the two other genes nested in the drpr locus, PCR primers were designed to amplify selected exons within this region. PCR analysis of genomic DNA as well as cDNA revealed that the drprΔ5 lesion disrupts transcription of the drpr gene but did not affect the other two genes in the locus (Figure 2B). Western blot analysis confirmed that Δ5 homozygotes did not produce Drpr, using a polyclonal antibody directed against the intracellular domain of the protein, common to all Drpr isoforms, as previously reported12Freeman M.R. Delrow J. Kim J. Johnson E. Doe C.Q. Unwrapping glial biology: Gcm target genes regulating glial development, diversification, and function.Neuron. 2003; 38: 567-580Abstract Full Text Full Text PDF PubMed Scopus (312) Google Scholar, 25McPhee C.K. Logan M.A. Freeman M.R. Baehrecke E.H. Activation of autophagy during cell death requires the engulfment receptor Draper.Nature. 2010; 465: 1093-1096Crossref PubMed Scopus (108) Google Scholar (Figure 2C). drprΔ5 homozygous null flies were viable into adulthood. However, lifespan analyses demonstrated that these mutants displayed a significantly reduced median lifespan versus corresponding heterozygotes (drprΔ5 homozygous mutants versus drprΔ5 heterozygotes, approximately 6 weeks versus 8 weeks; P = 0.014) (Figure 3). To investigate whether reduced Drpr levels affect motor function, drprΔ5 null mutant flies were assessed using an established negative geotaxis assay.26Draper I. Tabaka M.E. Jackson F.R. Salomon R.N. Kopin A.S. The evolutionarily conserved RNA binding protein SMOOTH is essential for maintaining normal muscle function.Fly (Austin). 2009; 3: 235-246Crossref PubMed Scopus (11) Google Scholar Using this approach, a significant decrease in performance of drprΔ5 mutants was demonstrated versus that in corresponding control flies (Figure 3). The impaired motor function was readily noticeable in drpr null mutants, which often display abnormal position of the legs and showed a rapid age-dependent decline in locomotor activity from 3 days to 7 to 10 days of life compared with age-matched controls (Figure 3 and Supplemental Figure S1). To dissect the cellular requirement that underlies the observed motor phenotype, RNA-interference flies were generated in which drpr gene expression is down-regulated in a tissue-specific manner (using the well-characterized Gal4/UAS bipartite system28Brand A.H. Perrimon N. Targeted gene expression as a means of altering cell fates and generating dominant phenotypes.Development. 1993; 118: 401-415Crossref PubMed Google Scholar). RNAi flies that down-regulate drpr in the entire organism (genotype Actin5C-Gal4; UAS-ds drpr, using the ubiquitous cytosolic actin5C-Gal4 driver line) also displayed altered locomotor function (25-day-old flies versus age-matched controls) (data not shown). The locomotor phenotype was recapitulated when drpr RNA interference was either induced in the mesoderm (how-Gal4 driver), or in the adult muscle precursor cells, which express the twist transcription factor (twist-Gal4 driver) (Figure 4). Confirming a muscle-specific effect, the locomotor phenotype was not recapitulated in flies that down-regulate drpr in neurons (pan-neuronal effect using the Elav-Gal4 driver, Elav encodes a neuronal RNA-binding protein), or in glia (using repo-Gal4 driver, repo encodes a glial-specific homeodomain protein) (Figure 4). Assessment of the locomotor activity of the flies was complemented by histological examination of drprΔ5 adult male Drosophila. Flies that were 30 to 40 days old were compared to age-matched controls. Histological analyses of drpr null mutants revealed pathological changes in the skeletal muscles of the thorax. The defect, which includes hyalinization with loss of striation, variability in fiber size, as well as vacuolization, primarily affected the tergal depressor of the trochanter (jump muscle, composed of skeletal tubular fibers). In contrast, the flight muscles (which are composed of skeletal fibrillar fibers) appeared normal (Figure 5). The muscle phenotype was recapitulated in drpr RNAi flies that down-regulate drpr ubiquitously (genotype Actin5C-Gal4; UAS-ds drpr). Penetrance of the muscle defect was low to moderate in both drprΔ5 homozygotes and drpr RNAi flies (approximately 10%–20%). Parallel abnormalities were not observed in age-matched corresponding control flies (drprΔ5 heterozygotes, and w1118; Actin5C-Gal4, respectively). In addition, marked degeneration was readily observed in the nervous systems of adult drprΔ5 mutant flies as well as in drpr RNAi flies, both within the brain and in the thoracic ventral ganglion (Figure 5 and Supplemental Figure S2). Many vacuoles were seen, which clustered on the ventral part of the nervous system. Degeneration of the visual center was also evident in drpr mutant flies in which alterations were seen in the retina and within the optic ganglia (ie, lamina, medulla) (Figure 5). The brain/retinal phenotype was highly penetrant (100% adult flies were affected, with individual variability in the degree of severity). The clinical and pathological features associated with the rare MEGF10 myopathy vary broadly, depending on the specific mutation. Missense mutations have been shown to result in a mild neuromuscular phenotype with prolonged preservation of ambulation,1Boyden S.E. Mahoney L.J. Kawahara G. Myers J.A. Mitsuhashi S. Estrella E.A. Duncan A.R. Dey F. DeChene E.T. Blasko-Goehringer J.M. Bönnemann C.G. Darras B.T. Mendell J.R. Lidov H.G. Nishino I. Beggs A.H. Kunkel L.M. Kang P.B. Mutations in the s

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