The splicing factor hnRNPL demonstrates conserved myocardial regulation across species and is altered in heart failure.

Heart failure (HF) is highly prevalent. Mechanisms underlying HF remain incompletely understood. Splicing factors (SF), which control pre-mRNA alternative splicing, regulate cardiac structure and function. This study investigated regulation of the splicing factor heterogeneous nuclear ribonucleoprotein-L (hnRNPL) in the failing heart. hnRNPL protein increased in left ventricular tissue from mice with transaortic constriction-induced HF and from HF patients. In left ventricular tissue, hnRNPL was detected predominantly in nuclei. Knockdown of the hnRNPL homolog Smooth in Drosophila induced cardiomyopathy. Computational analysis of predicted mouse and human hnRNPL binding sites suggested hnRNPL-mediated alternative splicing of tropomyosin, which was confirmed in C2C12 myoblasts. These findings identify hnRNPL as a sensor of cardiac dysfunction and suggest that disturbances of hnRNPL affect alternative splicing in HF.

Dominant stop-loss HNRNPA1 variants in juvenile-onset myopathy.

INTRODUCTION/AIMS: Heterogeneous nuclear ribonucleoprotein A1 is involved in nucleic acid homeostatic functions. The encoding gene HNRNPA1 has been associated with several neuromuscular disorders including an amyotrophic lateral sclerosis-like phenotype, distal hereditary motor neuropathy, multisystem proteinopathy, and various myopathies. We report two unrelated individuals with monoallelic stop loss variants affecting the same codon of HNRNPA1. METHODS: Two individuals with unsolved juvenile-onset myopathy were enrolled under approved institutional protocols. Phenotype data were collected and genetic analyses were performed, including whole-exome sequencing (WES). RESULTS: The two probands (MNOT002-01 and K1440-01) showed a similar onset of slowly progressive extremity and facial weakness in early adolescence. K1440-01 presented with facial weakness, winged scapula, elevated serum creatine kinase (CK) levels, and mild neck weakness. MNOT002-01 also exhibited elevated CK levels along with facial weakness, cardiomyopathy, respiratory dysfunction, pectus excavatum, a mildly rigid spine, and loss of ambulation. On quadriceps muscle biopsy, K1440-01 displayed rounded myofibers, mild variation in fiber diameter, and type 2 fiber hypertrophy, while MNOT002-01 displayed rimmed vacuoles. Monoallelic stop-loss variants in HNRNPA1 were identified for both probands: c.1119A>C p.*373Tyrext*6 (K1440-01) and c.1118A>C p.*373Serext*6 (MNOT002-01) affect the same codon and are both predicted to lead to the addition of six amino acids before termination at an alternative stop codon. DISCUSSION: Both stop-loss variants in our probands are likely pathogenic. Our findings contribute to the disease characterization of pathogenic variants in HNRNPA1. This gene should be screened in clinical diagnostic testing of unsolved cases of sporadic or dominant juvenile-onset myopathy.

Effects of HMGCR deficiency on skeletal muscle development.

Pathogenic variants in HMGCR were recently linked to a limb-girdle muscular dystrophy (LGMD) phenotype. The protein product HMG CoA reductase (HMGCR) catalyzes a key component of the cholesterol synthesis pathway. The two other muscle diseases associated with HMGCR, statin-associated myopathy (SAM) and autoimmune anti-HMGCR myopathy, are not inherited in a Mendelian pattern. The mechanism linking pathogenic variants in HMGCR with skeletal muscle dysfunction is unclear. We knocked down Hmgcr in mouse skeletal myoblasts, knocked down hmgcr in Drosophila, and expressed three pathogenic HMGCR variants (c.1327C>T, p.Arg443Trp; c.1522_1524delTCT, p.Ser508del; and c.1621G>A, p.Ala541Thr) in Hmgcr knockdown mouse myoblasts. Hmgcr deficiency was associated with decreased proliferation, increased apoptosis, and impaired myotube fusion. Transcriptome sequencing of Hmgcr knockdown versus control myoblasts revealed differential expression involving mitochondrial function, with corresponding differences in cellular oxygen consumption rates. Both ubiquitous and muscle-specific knockdown of hmgcr in Drosophila led to lethality. Overexpression of reference HMGCR cDNA rescued myotube fusion in knockdown cells, whereas overexpression of the pathogenic variants of HMGCR cDNA did not. These results suggest that the three HMGCR-related muscle diseases share disease mechanisms related to skeletal muscle development.

Drosophila noktochor regulates night sleep via a local mushroom body circuit.

We show that a sleep-regulating, Ig-domain protein (NKT) is secreted from Drosophila mushroom body (MB) α'/ß' neurons to act locally on other MB cell types. Pan-neuronal or broad MB expression of membrane-tethered NKT (tNkt) protein reduced sleep, like that of an NKT null mutant, suggesting blockade of a receptor mediating endogenous NKT action. In contrast, expression in neurons requiring NKT (the MB α'/ß' cells), or non-MB sleep-regulating centers, did not reduce night sleep, indicating the presence of a local MB sleep-regulating circuit consisting of communicating neural subtypes. We suggest that the leucocyte-antigen-related like (Lar) transmembrane receptor may mediate NKT action. Knockdown or overexpression of Lar in the MB increased or decreased sleep, respectively, indicating the receptor promotes wakefulness. Surprisingly, selective expression of tNkt or knockdown of Lar in MB wake-promoting cells increased rather than decreased sleep, suggesting that NKT acts on wake- as well as sleep-promoting cell types to regulate sleep.

Cryptic splicing of stathmin-2 and UNC13A mRNAs is a pathological hallmark of TDP-43-associated Alzheimer's disease.

Nuclear clearance and cytoplasmic accumulations of the RNA-binding protein TDP-43 are pathological hallmarks in almost all patients with amyotrophic lateral sclerosis (ALS) and up to 50% of patients with frontotemporal dementia (FTD) and Alzheimer's disease. In Alzheimer's disease, TDP-43 pathology is predominantly observed in the limbic system and correlates with cognitive decline and reduced hippocampal volume. Disruption of nuclear TDP-43 function leads to abnormal RNA splicing and incorporation of erroneous cryptic exons in numerous transcripts including Stathmin-2 (STMN2, also known as SCG10) and UNC13A, recently reported in tissues from patients with ALS and FTD. Here, we identify both STMN2 and UNC13A cryptic exons in Alzheimer's disease patients, that correlate with TDP-43 pathology burden, but not with amyloid-ß or tau deposits. We also demonstrate that processing of the STMN2 pre-mRNA is more sensitive to TDP-43 loss of function than UNC13A. In addition, full-length RNAs encoding STMN2 and UNC13A are suppressed in large RNA-seq datasets generated from Alzheimer's disease post-mortem brain tissue. Collectively, these results open exciting new avenues to use STMN2 and UNC13A as potential therapeutic targets in a broad range of neurodegenerative conditions with TDP-43 proteinopathy including Alzheimer's disease.

hnRNPL expression dynamics in the embryo and placenta.

Heterogeneous nuclear ribonucleoprotein L (hnRNPL) is a conserved RNA binding protein (RBP) that plays an important role in the alternative splicing of gene transcripts, and thus in the generation of specific protein isoforms. Global deficiency in hnRNPL in mice results in preimplantation embryonic lethality at embryonic day (E) 3.5. To begin to understand the contribution of hnRNPL-regulated pathways in the normal development of the embryo and placenta, we determined hnRNPL expression profile and subcellular localization throughout development. Proteome and Western blot analyses were employed to determine hnRNPL abundance between E3.5 and E17.5. Histological analyses supported that the embryo and implantation site display distinct hnRNPL localization patterns. In the fully developed mouse placenta, nuclear hnRNPL was observed broadly in trophoblasts, whereas within the implantation site a discrete subset of cells showed hnRNPL outside the nucleus. In the first-trimester human placenta, hnRNPL was detected in the undifferentiated cytotrophoblasts, suggesting a role for this factor in trophoblast progenitors. Parallel in vitro studies utilizing Htr8 and Jeg3 cell lines confirmed expression of hnRNPL in cellular models of human trophoblasts. These studies [support] coordinated regulation of hnRNPL during the normal developmental program in the mammalian embryo and placenta.

Variants in DTNA cause a mild, dominantly inherited muscular dystrophy.

DTNA encodes α-dystrobrevin, a component of the macromolecular dystrophin-glycoprotein complex (DGC) that binds to dystrophin/utrophin and α-syntrophin. Mice lacking α-dystrobrevin have a muscular dystrophy phenotype, but variants in DTNA have not previously been associated with human skeletal muscle disease. We present 12 individuals from four unrelated families with two different monoallelic DTNA variants affecting the coiled-coil domain of α-dystrobrevin. The five affected individuals from family A harbor a c.1585G > A; p.Glu529Lys variant, while the recurrent c.1567_1587del; p.Gln523_Glu529del DTNA variant was identified in the other three families (family B: four affected individuals, family C: one affected individual, and family D: two affected individuals). Myalgia and exercise intolerance, with variable ages of onset, were reported in 10 of 12 affected individuals. Proximal lower limb weakness with onset in the first decade of life was noted in three individuals. Persistent elevations of serum creatine kinase (CK) levels were detected in 11 of 12 affected individuals, 1 of whom had an episode of rhabdomyolysis at 20 years of age. Autism spectrum disorder or learning disabilities were reported in four individuals with the c.1567_1587 deletion. Muscle biopsies in eight affected individuals showed mixed myopathic and dystrophic findings, characterized by fiber size variability, internalized nuclei, and slightly increased extracellular connective tissue and inflammation. Immunofluorescence analysis of biopsies from five affected individuals showed reduced α-dystrobrevin immunoreactivity and variably reduced immunoreactivity of other DGC proteins: dystrophin, α, ß, δ and γ-sarcoglycans, and α and ß-dystroglycans. The DTNA deletion disrupted an interaction between α-dystrobrevin and syntrophin. Specific variants in the coiled-coil domain of DTNA cause skeletal muscle disease with variable penetrance. Affected individuals show a spectrum of clinical manifestations, with severity ranging from hyperCKemia, myalgias, and exercise intolerance to childhood-onset proximal muscle weakness. Our findings expand the molecular etiologies of both muscular dystrophy and paucisymptomatic hyperCKemia, to now include monoallelic DTNA variants as a novel cause of skeletal muscle disease in humans.

The Notch signaling pathway in skeletal muscle health and disease.

The Notch signaling pathway is a key regulator of skeletal muscle development and regeneration. Over the past decade, the discoveries of three new muscle disease genes have added a new dimension to the relationship between the Notch signaling pathway and skeletal muscle: MEGF10, POGLUT1, and JAG2. We review the clinical syndromes associated with pathogenic variants in each of these genes, known molecular and cellular functions of their protein products with a particular focus on the Notch signaling pathway, and potential novel therapeutic targets that may emerge from further investigations of these diseases. The phenotypes associated with two of these genes, POGLUT1 and JAG2, clearly fall within the realm of muscular dystrophy, whereas the third, MEGF10, is associated with a congenital myopathy/muscular dystrophy overlap syndrome classically known as early-onset myopathy, areflexia, respiratory distress, and dysphagia. JAG2 is a canonical Notch ligand, POGLUT1 glycosylates the extracellular domain of Notch receptors, and MEGF10 interacts with the intracellular domain of NOTCH1. Additional genes and their encoded proteins relevant to muscle function and disease with links to the Notch signaling pathway include TRIM32, ATP2A1 (SERCA1), JAG1, PAX7, and NOTCH2NLC. There is enormous potential to identify convergent mechanisms of skeletal muscle disease and new therapeutic targets through further investigations of the Notch signaling pathway in the context of skeletal muscle development, maintenance, and disease.

hnRNP L is essential for myogenic differentiation and modulates myotonic dystrophy pathologies.

INTRODUCTION: RNA-binding proteins (RBPs) play an important role in skeletal muscle development and disease by regulating RNA splicing. In myotonic dystrophy type 1 (DM1), the RBP MBNL1 (muscleblind-like) is sequestered by toxic CUG repeats, leading to missplicing of MBNL1 targets. Mounting evidence from the literature has implicated other factors in the pathogenesis of DM1. Herein we sought to evaluate the functional role of the splicing factor hnRNP L in normal and DM1 muscle cells. METHODS: Co-immunoprecipitation assays using hnRNPL and MBNL1 expression constructs and splicing profiling in normal and DM1 muscle cell lines were performed. Zebrafish morpholinos targeting hnrpl and hnrnpl2 were injected into one-cell zebrafish for developmental and muscle analysis. In human myoblasts downregulation of hnRNP L was achieved with shRNAi. Ascochlorin administration to DM1 myoblasts was performed and expression of the CUG repeats, DM1 splicing biomarkers, and hnRNP L expression levels were evaluated. RESULTS: Using DM1 patient myoblast cell lines we observed the formation of abnormal hnRNP L nuclear foci within and outside the expanded CUG repeats, suggesting a role for this factor in DM1 pathology. We showed that the antiviral and antitumorigenic isoprenoid compound ascochlorin increased MBNL1 and hnRNP L expression levels. Drug treatment of DM1 muscle cells with ascochlorin partially rescued missplicing of established early biomarkers of DM1 and improved the defective myotube formation displayed by DM1 muscle cells. DISCUSSION: Together, these studies revealed that hnRNP L can modulate DM1 pathologies and is a potential therapeutic target.

Megf10 deficiency impairs skeletal muscle stem cell migration and muscle regeneration.

Biallelic loss-of-function MEGF10 mutations lead to MEGF10 myopathy, also known as early onset myopathy with areflexia, respiratory distress, and dysphagia (EMARDD). MEGF10 is expressed in muscle satellite cells, but the contribution of satellite cell dysfunction to MEGF10 myopathy is unclear. Myofibers and satellite cells were isolated and examined from Megf10-/- and wild-type mice. A separate set of mice underwent repeated intramuscular barium chloride injections. Megf10-/- muscle satellite cells showed reduced proliferation and migration, while Megf10-/- mouse skeletal muscles showed impaired regeneration. Megf10 deficiency is associated with impaired muscle regeneration, due in part to defects in satellite cell function. Efforts to rescue Megf10 deficiency will have therapeutic implications for MEGF10 myopathy and other inherited muscle diseases involving impaired muscle regeneration.

Selective serotonin reuptake inhibitors ameliorate MEGF10 myopathy.

MEGF10 myopathy is a rare inherited muscle disease that is named after the causative gene, MEGF10. The classic phenotype, early onset myopathy, areflexia, respiratory distress and dysphagia, is severe and immediately life-threatening. There are no disease-modifying therapies. We performed a small molecule screen and follow-up studies to seek a novel therapy. A primary in vitro drug screen assessed cellular proliferation patterns in Megf10-deficient myoblasts. Secondary evaluations were performed on primary screen hits using myoblasts derived from Megf10-/- mice, induced pluripotent stem cell-derived myoblasts from MEGF10 myopathy patients, mutant Drosophila that are deficient in the homologue of MEGF10 (Drpr) and megf10 mutant zebrafish. The screen yielded two promising candidates that are both selective serotonin reuptake inhibitors (SSRIs), sertraline and escitalopram. In depth follow-up analyses demonstrated that sertraline was highly effective in alleviating abnormalities across multiple models of the disease including mouse myoblast, human myoblast, Drosophila and zebrafish models. Sertraline also restored deficiencies of Notch1 in disease models. We conclude that SSRIs show promise as potential therapeutic compounds for MEGF10 myopathy, especially sertraline. The mechanism of action may involve the Notch pathway.

The impact of Megf10/Drpr gain-of-function on muscle development in Drosophila.

Recessive mutations in multiple epidermal growth factor-like domains 10 (MEGF10) underlie a rare congenital muscle disease known as MEGF10 myopathy. MEGF10 and its Drosophila homolog Draper (Drpr) are transmembrane receptors expressed in muscle and glia. Drpr deficiency is known to result in muscle abnormalities in flies. In the current study, flies that ubiquitously overexpress Drpr, or mouse Megf10, display developmental arrest. The phenotype is reproduced with overexpression in muscle, but not in other tissues, and with overexpression during intermediate stages of myogenesis, but not in myoblasts. We find that tubular muscle subtypes are particularly sensitive to Megf10/Drpr overexpression. Complementary genetic analyses show that Megf10/Drpr and Notch may interact to regulate myogenesis. Our findings provide a basis for investigating MEGF10 in muscle development using Drosophila.

Identification of a pathogenic mutation in ATP2A1 via in silico analysis of exome data for cryptic aberrant splice sites.

BACKGROUND: Pathogenic mutations causing aberrant splicing are often difficult to detect. Standard variant analysis of next-generation sequence (NGS) data focuses on canonical splice sites. Noncanonical splice sites are more difficult to ascertain. METHODS: We developed a bioinformatics pipeline that screens existing NGS data for potentially aberrant novel essential splice sites (PANESS) and performed a pilot study on a family with a myotonic disorder. Further analyses were performed via qRT-PCR, immunoblotting, and immunohistochemistry. RNAi knockdown studies were performed in Drosophila to model the gene deficiency. RESULTS: The PANESS pipeline identified a homozygous ATP2A1 variant (NC_000016.9:g.28905928G>A; NM_004320.4:c.1287G>A:p.(Glu429=)) that was predicted to cause the omission of exon 11. Aberrant splicing of ATP2A1 was confirmed via qRT-PCR, and abnormal expression of the protein product sarcoplasmic/endoplasmic reticulum Ca++ ATPase 1 (SERCA1) was demonstrated in quadriceps femoris tissue from the proband. Ubiquitous knockdown of SERCA led to lethality in Drosophila, as did knockdown targeting differentiating or fusing myoblasts. CONCLUSIONS: This study confirms the potential of novel in silico algorithms to detect cryptic mutations in existing NGS data; expands the phenotypic spectrum of ATP2A1 mutations beyond classic Brody myopathy; and suggests that genetic testing of ATP2A1 should be considered in patients with clinical myotonia.

Impact of PYROXD1 deficiency on cellular respiration and correlations with genetic analyses of limb-girdle muscular dystrophy in Saudi Arabia and Sudan.

Next-generation sequencing is commonly used to screen for pathogenic mutations in families with Mendelian disorders, but due to the pace of discoveries, gaps have widened for some diseases between genetic and pathophysiological knowledge. We recruited and analyzed 16 families with limb-girdle muscular dystrophy (LGMD) of Arab descent from Saudi Arabia and Sudan who did not have confirmed genetic diagnoses. The analysis included both traditional and next-generation sequencing approaches. Cellular and metabolic studies were performed on Pyroxd1 siRNA C2C12 myoblasts and controls. Pathogenic mutations were identified in eight of the 16 families. One Sudanese family of Arab descent residing in Saudi Arabia harbored a homozygous c.464A>G, p.Asn155Ser mutation in PYROXD1, a gene recently reported in association with myofibrillar myopathy and whose protein product reduces thiol residues. Pyroxd1 deficiency in murine C2C12 myoblasts yielded evidence for impairments of cellular proliferation, migration, and differentiation, while CG10721 (Pyroxd1 fly homolog) knockdown in Drosophila yielded a lethal phenotype. Further investigations indicated that Pyroxd1 does not localize to mitochondria, yet Pyroxd1 deficiency is associated with decreased cellular respiration. This study identified pathogenic mutations in half of the LGMD families from the cohort, including one in PYROXD1. Developmental impairments were demonstrated in vitro for Pyroxd1 deficiency and in vivo for CG10721 deficiency, with reduced metabolic activity in vitro for Pyroxd1 deficiency.

RNA helicase, DDX27 regulates skeletal muscle growth and regeneration by modulation of translational processes.

Gene expression in a tissue-specific context depends on the combined efforts of epigenetic, transcriptional and post-transcriptional processes that lead to the production of specific proteins that are important determinants of cellular identity. Ribosomes are a central component of the protein biosynthesis machinery in cells; however, their regulatory roles in the translational control of gene expression in skeletal muscle remain to be defined. In a genetic screen to identify critical regulators of myogenesis, we identified a DEAD-Box RNA helicase, DDX27, that is required for skeletal muscle growth and regeneration. We demonstrate that DDX27 regulates ribosomal RNA (rRNA) maturation, and thereby the ribosome biogenesis and the translation of specific transcripts during myogenesis. These findings provide insight into the translational regulation of gene expression in myogenesis and suggest novel functions for ribosomes in regulating gene expression in skeletal muscles.

Consequences of MEGF10 deficiency on myoblast function and Notch1 interactions.

Mutations in MEGF10 cause early onset myopathy, areflexia, respiratory distress, and dysphagia (EMARDD), a rare congenital muscle disease, but the pathogenic mechanisms remain largely unknown. We demonstrate that short hairpin RNA (shRNA)-mediated knockdown of Megf10, as well as overexpression of the pathogenic human p.C774R mutation, leads to impaired proliferation and migration of C2C12 cells. Myoblasts from Megf10-/- mice and Megf10-/-/mdx double knockout (dko) mice also show impaired proliferation and migration compared to myoblasts from wild type and mdx mice, whereas the dko mice show histological abnormalities that are not observed in either single mutant mouse. Cell proliferation and migration are known to be regulated by the Notch receptor, which plays an essential role in myogenesis. Reciprocal co-immunoprecipitation studies show that Megf10 and Notch1 interact via their respective intracellular domains. These interactions are impaired by the pathogenic p.C774R mutation. Megf10 regulation of myoblast function appears to be mediated at least in part via interactions with key components of the Notch signaling pathway, and defects in these interactions may contribute to the pathogenesis of EMARDD.

A critical role for the Drosophila dopamine D1-like receptor Dop1R2 at the onset of metamorphosis.

BACKGROUND: Insect metamorphosis relies on temporal and spatial cues that are precisely controlled. Previous studies in Drosophila have shown that untimely activation of genes that are essential to metamorphosis results in growth defects, developmental delay and death. Multiple factors exist that safeguard these genes against dysregulated expression. The list of identified negative regulators that play such a role in Drosophila development continues to expand. RESULTS: By using RNAi transgene-induced gene silencing coupled to spatio/temporal assessment, we have unraveled an important role for the Drosophila dopamine 1-like receptor, Dop1R2, in development. We show that Dop1R2 knockdown leads to pre-adult lethality. In adults that escape death, abnormal wing expansion and/or melanization defects occur. Furthermore we show that salivary gland expression of this GPCR during the late larval/prepupal stage is essential for the flies to survive through adulthood. In addition to RNAi-induced effects, treatment of larvae with the high affinity D1-like receptor antagonist flupenthixol, also results in developmental arrest, and in morphological defects comparable to those seen in Dop1R2 RNAi flies. To examine the basis for pupal lethality in Dop1R2 RNAi flies, we carried out transcriptome analysis. These studies revealed up-regulation of genes that respond to ecdysone, regulate morphogenesis and/or modulate defense/immunity. CONCLUSION: Taken together our findings suggest a role for Dop1R2 in the repression of genes that coordinate metamorphosis. Premature release of this inhibition is not tolerated by the developing fly.

Targeted inactivation of the rickets receptor in muscle compromises Drosophila viability.

Bursicon is a hormone that modulates wing expansion, cuticle hardening and melanization in Drosophila melanogaster. Bursicon activity is mediated through its cognate G protein-coupled receptor (GPCR), rickets. We have developed a membrane-tethered bursicon construct that enables spatial modulation of rickets-mediated physiology in transgenic flies. Ubiquitous expression of tethered bursicon throughout development results in arrest at the pupal stage. The few organisms that eclose fail to undergo wing expansion. These phenotypes suggest that expression of tethered bursicon inhibits rickets-mediated function. Consistent with this hypothesis, we show in vitro that sustained stimulation of rickets by tethered bursicon leads to receptor desensitization. Furthermore, tissue-specific expression of the tethered bursicon inhibitor unraveled a critical role for rickets in a subset of adult muscles. Taken together, our findings highlight the utility of membrane-tethered inhibitors as important genetic/pharmacological tools to dissect the tissue-specific roles of GPCRs in vivo.

Silencing of drpr leads to muscle and brain degeneration in adult Drosophila.

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.