Phenotypes In Drosophila Research Articles

Inhibition of Hsp70 by Methylene Blue Affects Signaling Protein Function and Ubiquitination and Modulates Polyglutamine Protein Degradation

The Hsp90/Hsp70-based chaperone machinery regulates the activity and degradation of many signaling proteins. Cycling with Hsp90 stabilizes client proteins, whereas Hsp70 interacts with chaperone-dependent E3 ubiquitin ligases to promote protein degradation. To probe these actions, small molecule inhibitors of Hsp70 would be extremely useful; however, few have been identified. Here we test the effects of methylene blue, a recently described inhibitor of Hsp70 ATPase activity, in three well established systems of increasing complexity. First, we demonstrate that methylene blue inhibits the ability of the purified Hsp90/Hsp70-based chaperone machinery to enable ligand binding by the glucocorticoid receptor and show that this effect is due to specific inhibition of Hsp70. Next, we establish that ubiquitination of neuronal nitric-oxide synthase by the native ubiquitinating system of reticulocyte lysate is dependent upon both Hsp70 and the E3 ubiquitin ligase CHIP and is blocked by methylene blue. Finally, we demonstrate that methylene blue impairs degradation of the polyglutamine expanded androgen receptor, an Hsp90 client mutated in spinal and bulbar muscular atrophy. In contrast, degradation of an amino-terminal fragment of the receptor, which lacks the ligand binding domain and, therefore, is not a client of the Hsp90/Hsp70-based chaperone machinery, is enhanced through homeostatic induction of autophagy that occurs when Hsp70-dependent proteasomal degradation is inhibited by methylene blue. Our data demonstrate the utility of methylene blue in defining Hsp70-dependent functions and reveal divergent effects on polyglutamine protein degradation depending on whether the substrate is an Hsp90 client.

In Vivo RNAi Rescue in Drosophila melanogaster with Genomic Transgenes from Drosophila pseudoobscura

BackgroundSystematic, large-scale RNA interference (RNAi) approaches are very valuable to systematically investigate biological processes in cell culture or in tissues of organisms such as Drosophila. A notorious pitfall of all RNAi technologies are potential false positives caused by unspecific knock-down of genes other than the intended target gene. The ultimate proof for RNAi specificity is a rescue by a construct immune to RNAi, typically originating from a related species.Methodology/Principal FindingsWe show that primary sequence divergence in areas targeted by Drosophila melanogaster RNAi hairpins in five non-melanogaster species is sufficient to identify orthologs for 81% of the genes that are predicted to be RNAi refractory. We use clones from a genomic fosmid library of Drosophila pseudoobscura to demonstrate the rescue of RNAi phenotypes in Drosophila melanogaster muscles. Four out of five fosmid clones we tested harbour cross-species functionality for the gene assayed, and three out of the four rescue a RNAi phenotype in Drosophila melanogaster.Conclusions/SignificanceThe Drosophila pseudoobscura fosmid library is designed for seamless cross-species transgenesis and can be readily used to demonstrate specificity of RNAi phenotypes in a systematic manner.

A New Chamber for Studying the Behavior of Drosophila

Methods available for quickly and objectively quantifying the behavioral phenotypes of the fruit fly, Drosophila melanogaster, lag behind in sophistication the tools developed for manipulating their genotypes. We have developed a simple, easy-to-replicate, general-purpose experimental chamber for studying the ground-based behaviors of fruit flies. The major innovative feature of our design is that it restricts flies to a shallow volume of space, forcing all behavioral interactions to take place within a monolayer of individuals. The design lessens the frequency that flies occlude or obscure each other, limits the variability in their appearance, and promotes a greater number of flies to move throughout the center of the chamber, thereby increasing the frequency of their interactions. The new chamber design improves the quality of data collected by digital video and was conceived and designed to complement automated machine vision methodologies for studying behavior. Novel and improved methodologies for better quantifying the complex behavioral phenotypes of Drosophila will facilitate studies related to human disease and fundamental questions of behavioral neuroscience.

Drosophila morgue influences cell numbers and positions in the embryonic nervous system

Morgue is a unique multi-domain protein that contains a zinc finger motif, an F box, and a variant E2 conjugase domain. The presence of these domains suggests potentially complex and novel functions for Morgue in ubiquitination pathways. Morgue was originally identified via its gain-of-function enhancement of eye cell death phenotypes in Drosophila and ectopic expression of Morgue also influences circadian rhythms. However, there is as yet little known about Morgues normal developmental or physiological functions. To address this issue, we generated several morgue loss-of-function mutants via P element excision mutagenesis and analyzed the mutant phenotypes during the fly life cycle. These studies revealed that morgue null mutants are viable, though approximately 10% of the mutants exhibit defects in pupal spiracle eversion and malformations in the adult abdominal cuticle. In addition, a similar subset of morgue mutant embryos exhibited alterations in the normal number, position, or morphology of specific neurons and glia. Analysis of Morgue protein localization was addressed through generation of a transgenic fly strain that expresses a GFP::Morgue fusion protein. Use of this strain revealed Morgue protein localization in multiple cellular compartments, including nuclei, cytoplasm and membranes. Taken together, these diverse phenotypes and distribution patterns suggest pleiotropic functions for Morgue.

Exploring the functional interaction between POSH and ALIX and the relevance to HIV-1 release

The ALG2-interacting protein X (ALIX)/AIP1 is an adaptor protein with multiple functions in intracellular protein trafficking that plays a central role in the biogenesis of enveloped viruses. The ubiquitin E3-ligase POSH (plenty of SH3) augments HIV-1 egress by facilitating the transport of Gag to the cell membrane. Recently, it was reported, that POSH interacts with ALIX and thereby enhances ALIX mediated phenotypes in Drosophila. In this study we identified ALIX as a POSH ubiquitination substrate in human cells: POSH induces the ubiquitination of ALIX that is modified on several lysine residues in vivo and in vitro. This ubiquitination does not destabilize ALIX, suggesting a regulatory function. As it is well established that ALIX rescues virus release of L-domain mutant HIV-1, HIV-1ΔPTAP, we demonstrated that wild type POSH, but not an ubiquitination inactive RING finger mutant (POSHV14A), substantially enhances ALIX-mediated release of infectious virions derived from HIV-1ΔPTAP L-domain mutant (YPXnL-dependent HIV-1). In further agreement with the idea of a cooperative function of POSH and ALIX, mutating the YPXnL-ALIX binding site in Gag completely abrogated augmentation of virus release by overexpression of POSH. However, the effect of the POSH-mediated ubiquitination appears to be auxiliary, but not necessary, as silencing of POSH by RNAi does not disturb ALIX-augmentation of virus release. Thus, the cumulative results identified ALIX as an ubiquitination substrate of POSH and indicate that POSH and ALIX cooperate to facilitate efficient virus release. However, while ALIX is obligatory for the release of YPXnL-dependent HIV-1, POSH, albeit rate-limiting, may be functionally interchangeable.

Exploring the functional interaction between POSH and ALIX and the relevance to HIV-1 release

BackgroundThe ALG2-interacting protein X (ALIX)/AIP1 is an adaptor protein with multiple functions in intracellular protein trafficking that plays a central role in the biogenesis of enveloped viruses. The ubiquitin E3-ligase POSH (plenty of SH3) augments HIV-1 egress by facilitating the transport of Gag to the cell membrane. Recently, it was reported, that POSH interacts with ALIX and thereby enhances ALIX mediated phenotypes in Drosophila.ResultsIn this study we identified ALIX as a POSH ubiquitination substrate in human cells: POSH induces the ubiquitination of ALIX that is modified on several lysine residues in vivo and in vitro. This ubiquitination does not destabilize ALIX, suggesting a regulatory function. As it is well established that ALIX rescues virus release of L-domain mutant HIV-1, HIV-1ΔPTAP, we demonstrated that wild type POSH, but not an ubiquitination inactive RING finger mutant (POSHV14A), substantially enhances ALIX-mediated release of infectious virions derived from HIV-1ΔPTAP L-domain mutant (YPXnL-dependent HIV-1). In further agreement with the idea of a cooperative function of POSH and ALIX, mutating the YPXnL-ALIX binding site in Gag completely abrogated augmentation of virus release by overexpression of POSH. However, the effect of the POSH-mediated ubiquitination appears to be auxiliary, but not necessary, as silencing of POSH by RNAi does not disturb ALIX-augmentation of virus release.ConclusionThus, the cumulative results identified ALIX as an ubiquitination substrate of POSH and indicate that POSH and ALIX cooperate to facilitate efficient virus release. However, while ALIX is obligatory for the release of YPXnL-dependent HIV-1, POSH, albeit rate-limiting, may be functionally interchangeable.

Evidence of a tick RNAi pathway by comparative genomics and reverse genetics screen of targets with known loss-of-function phenotypes in Drosophila

BackgroundThe Arthropods are a diverse group of organisms including Chelicerata (ticks, mites, spiders), Crustacea (crabs, shrimps), and Insecta (flies, mosquitoes, beetles, silkworm). The cattle tick, Rhipicephalus (Boophilus) microplus, is an economically significant ectoparasite of cattle affecting cattle industries world wide. With the availability of sequence reads from the first Chelicerate genome project (the Ixodes scapularis tick) and extensive R. microplus ESTs, we investigated evidence for putative RNAi proteins and studied RNA interference in tick cell cultures and adult female ticks targeting Drosophila homologues with known cell viability phenotype.ResultsWe screened 13,643 R. microplus ESTs and I. scapularis genome reads to identify RNAi related proteins in ticks. Our analysis identified 31 RNAi proteins including a putative tick Dicer, RISC associated (Ago-2 and FMRp), RNA dependent RNA polymerase (EGO-1) and 23 homologues implicated in dsRNA uptake and processing. We selected 10 R. microplus ESTs with >80% similarity to D. melanogaster proteins associated with cell viability for RNAi functional screens in both BME26 R. microplus embryonic cells and female ticks in vivo. Only genes associated with proteasomes had an effect on cell viability in vitro. In vivo RNAi showed that 9 genes had significant effects either causing lethality or impairing egg laying.ConclusionWe have identified key RNAi-related proteins in ticks and along with our loss-of-function studies support a functional RNAi pathway in R. microplus. Our preliminary studies indicate that tick RNAi pathways may differ from that of other Arthropods such as insects.

Pink1 suppresses α-synuclein-induced phenotypes in a Drosophila model of Parkinson’s disease

Parkinson's disease (PD) is the most prevalent human neurodegenerative movement disorder and is characterized by a selective and progressive loss of the dopaminergic neurons. Mutations in the genes parkin and PTEN-induced putative kinase 1 (PINK1) result in autosomal recessive forms of PD. It has been suggested that parkin and Pink1 function in the same pathway in Drosophila, with Pink1 acting upstream of parkin. Previous work in our laboratory has shown the ability of parkin to rescue an alpha-synuclein-induced PD-like phenotype in Drosophila. To investigate the ability of Pink1 to protect against alpha-synuclein-induced toxicity, we have performed longevity, mobility, and histological studies to determine whether Drosophila Pink1 can rescue the alpha-synuclein phenotypes. We have found that overexpression of Pink1 results in the rescue of the alpha-synuclein-induced phenotype of premature loss of climbing ability, suppression of degeneration of the ommatidial array, and the suppression of alpha-synuclein-induced developmental defects in the Drosophila eye. These results mark the first demonstration of Pink1 counteracting PD phenotypes in a protein toxicity animal model, and they show that Pink1 is able to impart protection against potentially harmful proteins such as alpha-synuclein that would otherwise result in cellular stress.

Ribosomal mutations cause p53-mediated dark skin and pleiotropic effects

Mutations in genes encoding ribosomal proteins cause the Minute phenotype in Drosophila and mice, and Diamond-Blackfan syndrome in humans. Here we report two mouse dark skin (Dsk) loci caused by mutations in Rps19 (ribosomal protein S19) and Rps20 (ribosomal protein S20). We identify a common pathophysiologic program in which p53 stabilization stimulates Kit ligand expression, and, consequently, epidermal melanocytosis via a paracrine mechanism. Accumulation of p53 also causes reduced body size and erythrocyte count. These results provide a mechanistic explanation for the diverse collection of phenotypes that accompany reduced dosage of genes encoding ribosomal proteins, and have implications for understanding normal human variation and human disease.

A Genetic Screen for Sleep and Circadian Mutants Reveals Mechanisms Underlying Regulation of Sleep in Drosophila

In order to characterize the genetic mechanisms underlying sleep, we have carried out a large-scale screen in Drosophila to identify short-sleeping mutants. The objectives of this study were as follows: (1) characterize the phenotypes of the shortest-sleeping mutants; (2) examine whether changes in arousal threshold or sleep homeostasis underlie short-sleeping phenotypes; (3) clone a gene affected in one of the shortest sleepers; and (4) investigate whether circadian mutants can be identified using light:dark (L:D) locomotor data obtained for studying sleep behavior. Locomotor activity was measured using the Drosophila Activity Monitoring System in a 12:12 L:D cycle. Drosophila research laboratory. Adult flies from the 2nd chromosome Zuker collection, which contain mutations in most of the nonessential genes on the Drosophila 2nd chromosome. Our analysis of sleep characteristics suggests that daily activity (but not waking activity) correlates with daily sleep time and that defects in sleep maintenance are more common than defects in sleep initiation. Our shortest sleepers have intact or increased sleep rebound following sleep deprivation but show reduced thresholds for arousal. Molecular analysis of one of the short-sleeping lines indicates that it is a novel allele of a dopamine transporter (DAT). Finally, we describe a novel approach for identifying circadian mutants using L:D data. Our data suggest that most short-sleeping mutant phenotypes in Drosophila are characterized by an inability to stay asleep, most likely because of a reduced arousal threshold.

Identification of small molecules rescuing fragile X syndrome phenotypes in Drosophila

Fragile X syndrome is caused by the functional loss of the fragile X mental retardation 1 (FMR1) gene. Deletion of the FMR1 ortholog in Drosophila melanogaster (Fmr1) recapitulates many phenotypes associated with fragile X syndrome. We have discovered that Fmr1 mutant Drosophila die during development when reared on food containing increased levels of glutamate, which is consistent with the theory that FMR1 loss results in excess glutamate signaling. Using this lethal phenotype, we screened a chemical library of 2,000 compounds and identified nine molecules that rescued the lethality, including three that implicate the GABAergic inhibitory pathway. Indeed, GABA treatment rescued several known Fmr1 mutant phenotypes in flies, including mushroom bodies defects, excess Futsch translation and abnormal male courtship behavior. These data are consistent with GABAergic inhibition of the enhanced excitatory pathway in fragile X syndrome. In addition, our screen reveals that the muscarinic cholinergic receptors may have a role in fragile X syndrome in parallel to the GABAergic pathway. These results point to potential therapeutic approaches for treating fragile X syndrome.

The Role of Carcinine in Signaling at the Drosophila Photoreceptor Synapse

The Drosophila melanogaster photoreceptor cell has long served as a model system for researchers focusing on how animal sensory neurons receive information from their surroundings and translate this information into chemical and electrical messages. Electroretinograph (ERG) analysis of Drosophila mutants has helped to elucidate some of the genes involved in the visual transduction pathway downstream of the photoreceptor cell, and it is now clear that photoreceptor cell signaling is dependent upon the proper release and recycling of the neurotransmitter histamine. While the neurotransmitter transporters responsible for clearing histamine, and its metabolite carcinine, from the synaptic cleft have remained unknown, a strong candidate for a transporter of either substrate is the uncharacterized inebriated protein. The inebriated gene (ine) encodes a putative neurotransmitter transporter that has been localized to photoreceptor cells in Drosophila and mutations in ine result in an abnormal ERG phenotype in Drosophila. Loss-of-function mutations in ebony, a gene required for the synthesis of carcinine in Drosophila, suppress components of the mutant ine ERG phenotype, while loss-of-function mutations in tan, a gene necessary for the hydrolysis of carcinine in Drosophila, have no effect on the ERG phenotype in ine mutants. We also show that by feeding wild-type flies carcinine, we can duplicate components of mutant ine ERGs. Finally, we demonstrate that treatment with H3 receptor agonists or inverse agonists rescue several components of the mutant ine ERG phenotype. Here, we provide pharmacological and genetic epistatic evidence that ine encodes a carcinine neurotransmitter transporter. We also speculate that the oscillations observed in mutant ine ERG traces are the result of the aberrant activity of a putative H3 receptor.

Characterization of lamin Mutation Phenotypes in Drosophila and Comparison to Human Laminopathies

Lamins are intermediate filament proteins that make up the nuclear lamina, a matrix underlying the nuclear membrane in all metazoan cells that is important for nuclear form and function. Vertebrate A-type lamins are expressed in differentiating cells, while B-type lamins are expressed ubiquitously. Drosophila has two lamin genes that are expressed in A- and B-type patterns, and it is assumed that similarly expressed lamins perform similar functions. However, Drosophila and vertebrate lamins are not orthologous, and their expression patterns evolved independently. It is therefore of interest to examine the effects of mutations in lamin genes. Mutations in the mammalian lamin A/C gene cause a range of diseases, collectively called laminopathies, that include muscular dystrophies and premature aging disorders. We compared the sequences of lamin genes from different species, and we have characterized larval and adult phenotypes in Drosophila bearing mutations in the lam gene that is expressed in the B-type pattern. Larvae move less and show subtle muscle defects, and surviving lam adults are flightless and walk like aged wild-type flies, suggesting that lam phenotypes might result from neuromuscular defects, premature aging, or both. The resemblance of Drosophila lam phenotypes to human laminopathies suggests that some lamin functions may be performed by differently expressed genes in flies and mammals. Such still-unknown functions thus would not be dependent on lamin gene expression pattern, suggesting the presence of other lamin functions that are expression dependent. Our results illustrate a complex interplay between lamin gene expression and function through evolution.

Design and Isolation of Temperature-sensitive Mutants of Gal4 in Yeast and Drosophila

Little is known about mechanisms responsible for the temperature-sensitive (ts) phenotype, or of the transferability of ts mutants of a specific gene between organisms. Using a structure-based approach, nine ts mutants of Gal4 were generated in yeast by mutating four DNA binding residues. Two of these nine yeast ts mutants were cloned into P element vectors under control of the Elav and GMR promoters and transgenic Drosophila lines were generated. These were crossed to UAS reporter lines and progeny were characterized for reporter gene expression as a function of temperature. Both of these yeast ts mutants show a ts phenotype in Drosophila and result in rapid induction of reporter gene expression upon shifting to the permissive temperature. Exposed, functional residues involved in protein–ligand or protein–protein interactions appear to be attractive candidate sites for generating ts mutants that are transferable between organisms.

RNAi knockdown of Nopp140 induces Minute-like phenotypes in Drosophila.

Nopp140 associates with small nucleolar RNPs to chaperone pre-rRNA processing and ribosome assembly. Alternative splicing yields two isoforms in Drosophila: Nopp140-True is homologous to vertebrate Nopp140 particularly in its carboxy terminus, whereas Nopp140-RGG contains a glycine and arginine-rich (RGG) carboxy terminus typically found in vertebrate nucleolin. Loss of ribosome function or production at critical points in development leads to Minute phenotypes in Drosophila or the Treacher Collins syndrome (TCS) in humans. To ascertain the functional significance of Nopp140 in Drosophila development, we expressed interfering RNA using the GAL4/UAS system. Reverse transcription-PCR showed variable losses of Nopp140 mRNA in larvae from separate RNAi-expressing transgenic lines, whereas immunofluorescence microscopy with isoform-specific antibodies showed losses of Nopp140 in imaginal and polyploid tissues. Phenotypic expression correlated with the percent loss of Nopp140 transcripts: a >or=50% loss correlated with larval and pupal lethality, disrupted nuclear structures, and in some cases melanotic tumors, whereas a 30% loss correlated with adult wing, leg, and tergite deformities. We consider these adult phenotypes to be Minute-like and reminiscent of human craniofacial malformations associated with TCS. Similarly, overexpression of either isoform caused embryonic and larval lethality, thus indicating proper expression of Nopp140 is critical for normal development.

The Microtubule Plus-End Tracking Protein EB1 Is Required for Kv1 Voltage-Gated K + Channel Axonal Targeting

Axonal Kv1 channels regulate action potential propagation-an evolutionarily conserved function important for the control of motor behavior as evidenced from the linkage of human Kv1 channel mutations to myokymia/episodic ataxia type 1 (EA1) and the Shaker mutant phenotype in Drosophila. To search for the machinery that mediates axonal targeting of Kv1 channels composed of both alpha and beta subunits, we first demonstrate that Kvbeta2 is responsible for targeting Kv1 channels to the axon. Next, we show that Kvbeta2 axonal targeting depends on its ability to associate with the microtubule (MT) plus-end tracking protein (+TIP) EB1. Not only do Kvbeta2 and EB1 move in unison down the axon, Brefeldin A-sensitive Kv1-containing vesicles can also be found at microtubule ends near the cell membrane. In addition, we found that Kvbeta2 associates with KIF3/kinesin II as well. Indeed, Kv1 channels rely on both KIF3/kinesin II and EB1 for their axonal targeting.

P1-006: Distinct accumulation properties of Aβ42 in the brain are associated with specific pathological phenotypes in Drosophila

P1-006: Distinct accumulation properties of Aβ42 in the brain are associated with specific pathological phenotypes in Drosophila

Two Distinct Pathways for O-Fucosylation of Epidermal Growth Factor-like or Thrombospondin Type 1 Repeats

Epidermal growth factor-like (EGF) repeats and thrombospondin type 1 repeats (TSRs) are both small cysteine-knot motifs known to be O-fucosylated. The enzyme responsible for the addition of O-fucose to EGF repeats, protein O-fucosyltransferase 1 (POFUT1), has been identified and shown to be essential in Notch signaling. Fringe, an O-fucose beta1,3-N-acetylglucosaminyltransferase, elongates O-fucose on specific EGF repeats from Notch to form a disaccharide that can be further elongated to a tetrasaccharide. TSRs are found in many extracellular matrix proteins and are involved in protein-protein interactions. The O-fucose moiety on TSRs can be further elongated with glucose to form a disaccharide. The discovery of O-fucose on TSRs raised the question of whether POFUT1, or a different enzyme, adds O-fucose to TSRs. Here we demonstrate the existence of a TSR-specific O-fucosyltransferase distinct from POFUT1. Similar to POFUT1, the novel TSR-specific O-fucosyltransferase is a soluble enzyme that requires a properly folded TSR as an acceptor substrate. In addition, we found that a previously identified fucose-specific beta1,3-glucosyltransferase adds glucose to O-fucose on TSRs, but it does not modify O-fucose on an EGF repeat. Similarly, Lunatic fringe, Manic fringe, and Radical fringe are all capable of modifying O-fucose on an EGF repeat, but not on a TSR. Taken together, these results suggest that two distinct O-fucosylation pathways exist in cells, one specific for EGF repeat and the other for TSRs.

Disease-related phenotypes in a Drosophila model of hereditary spastic paraplegia are ameliorated by treatment with vinblastine

Hereditary spastic paraplegias (HSPs) are a group of neurodegenerative diseases characterized by progressive weakness and spasticity of the lower limbs. Dominant mutations in the human SPG4 gene, encoding spastin, are responsible for the most frequent form of HSP. Spastin is an ATPase that binds microtubules and localizes to the spindle pole and distal axon in mammalian cell lines. Furthermore, its Drosophila homolog, Drosophila spastin (Dspastin), has been recently shown to regulate microtubule stability and synaptic function at the Drosophila larval neuromuscular junction. Here we report the generation of a spastin-linked HSP animal model and show that in Drosophila, neural knockdown of Dspastin and, conversely, neural overexpression of Dspastin containing a conserved pathogenic mutation both recapitulate some phenotypic aspects of the human disease, including adult onset, locomotor impairment, and neurodegeneration. At the subcellular level, neuronal expression of both Dspastin RNA interference and mutant Dspastin cause an excessive stabilization of microtubules in the neuromuscular junction synapse. In addition, we provide evidence that administration of the microtubule targeting drug vinblastine significantly attenuates these phenotypes in vivo. Our findings demonstrate that loss of spastin function elicits HSP-like phenotypes in Drosophila, provide novel insights into the molecular mechanism of spastin mutations, and raise the possibility that therapy with Vinca alkaloids may be efficacious in spastin-associated HSP and other disorders related to microtubule dysfunction.

Tuning of RNA editing by ADAR is required in Drosophila

RNA editing increases during development in more than 20 transcripts encoding proteins involved in rapid synaptic neurotransmission in Drosophila central nervous system and muscle. Adar (adenosine deaminase acting on RNA) mutant flies expressing only genome-encoded, unedited isoforms of ion-channel subunits are viable but show severe locomotion defects. The Adar transcript itself is edited in adult wild-type flies to generate an isoform with a serine to glycine substitution close to the ADAR active site. We show that editing restricts ADAR function since the edited isoform of ADAR is less active in vitro and in vivo than the genome-encoded, unedited isoform. Ubiquitous expression in embryos and larvae of an Adar transcript that is resistant to editing is lethal. Expression of this transcript in embryonic muscle is also lethal, with above-normal, adult-like levels of editing at sites in a transcript encoding a muscle voltage-gated calcium channel.