Drosophila sensory neurons require Dscam for dendritic self-avoidance and proper dendritic field organization
A neuron's dendrites typically do not cross one another. This intrinsic self-avoidance mechanism ensures unambiguous processing of sensory or synaptic inputs. Moreover, some neurons respect the territory of others of the same type, a phenomenon known as tiling. Different types of neurons, however, often have overlapping dendritic fields. We found that Down's syndrome Cell Adhesion Molecule (Dscam) is required for dendritic self-avoidance of all four classes of Drosophila dendritic arborization (da) neurons. However, neighboring mutant class IV da neurons still exhibited tiling, suggesting that self-avoidance and tiling differ in their recognition and repulsion mechanisms. Introducing 1 of the 38,016 Dscam isoforms to da neurons in Dscam mutants was sufficient to significantly restore self-avoidance. Remarkably, expression of a common Dscam isoform in da neurons of different classes prevented their dendrites from sharing the same territory, suggesting that coexistence of dendritic fields of different neuronal classes requires divergent expression of Dscam isoforms.
The type II clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system has emerged recently as a powerful method to manipulate the genomes of various organisms. Here, we report a toolbox for high-efficiency genome engineering of Drosophila melanogaster consisting of transgenic Cas9 lines and versatile guide RNA (gRNA) expression plasmids. Systematic evaluation reveals Cas9 lines with ubiquitous or germ-line-restricted patterns of activity. We also demonstrate differential activity of the same gRNA expressed from different U6 snRNA promoters, with the previously untested U6:3 promoter giving the most potent effect. An appropriate combination of Cas9 and gRNA allows targeting of essential and nonessential genes with transmission rates ranging from 25-100%. We also demonstrate that our optimized CRISPR/Cas tools can be used for offset nicking-based mutagenesis. Furthermore, in combination with oligonucleotide or long double-stranded donor templates, our reagents allow precise genome editing by homology-directed repair with rates that make selection markers unnecessary. Last, we demonstrate a novel application of CRISPR/Cas-mediated technology in revealing loss-of-function phenotypes in somatic cells following efficient biallelic targeting by Cas9 expressed in a ubiquitous or tissue-restricted manner. Our CRISPR/Cas tools will facilitate the rapid evaluation of mutant phenotypes of specific genes and the precise modification of the genome with single-nucleotide precision. Our results also pave the way for high-throughput genetic screening with CRISPR/Cas.
Topographic projection of afferent terminals into 2D maps in the CNS is a general strategy used by the nervous system to encode the locations of sensory stimuli. In vertebrates, it is known that although guidance cues are critical for establishing a coarse topographic map, neural activity directs fine-scale topography between adjacent afferent terminals [1-4]. However, the molecular mechanism underlying activity-dependent regulation of fine-scale topography is poorly understood. Molecular analysis of the spatial relationship between adjacent afferent terminals requires reliable localization of the presynaptic terminals of single neurons as well as genetic manipulations with single-cell resolution in vivo. Although both requirements can potentially be met in Drosophila melanogaster [5, 6], no activity-dependent topographic system has been identified in flies . Here we report a topographic system that is shaped by neuronal activity in Drosophila. With this system, we found that topographic separation of the presynaptic terminals of adjacent nociceptive neurons requires different levels of Trim9, an evolutionarily conserved signaling molecule [8-11]. Neural activity regulates Trim9 protein levels to direct fine-scale topography of sensory afferents. This study offers both a novel mechanism by which neural activity directs fine-scale topography of axon terminals and a new system to study this process at single-neuron resolution.
The putative Na(+)/Cl(-)-dependent neurotransmitter/osmolyte transporter inebriated in the Drosophila hindgut is essential for the maintenance of systemic water homeostasis
Most organisms are able to maintain systemic water homeostasis over a wide range of external or dietary osmolarities. The excretory system, composed of the kidneys in mammals and the Malpighian tubules and hindgut in insects, can increase water conservation and absorption to maintain systemic water homeostasis, which enables organisms to tolerate external hypertonicity or desiccation. However, the mechanisms underlying the maintenance of systemic water homeostasis by the excretory system have not been fully characterized. In the present study, we found that the putative Na(+)/Cl(-)-dependent neurotransmitter/osmolyte transporter inebriated (ine) is expressed in the basolateral membrane of anterior hindgut epithelial cells. This was confirmed by comparison with a known basolateral localized protein, the alpha subunit of Na(+)-K(+) ATPase (ATPalpha). Under external hypertonicity, loss of ine in the hindgut epithelium results in severe dehydration without damage to the hindgut epithelial cells, implicating a physiological failure of water conservation/absorption. We also found that hindgut expression of ine is required for water conservation under desiccating conditions. Importantly, specific expression of ine in the hindgut epithelium can completely restore disrupted systemic water homeostasis in ine mutants under both conditions. Therefore, ine in the Drosophila hindgut is essential for the maintenance of systemic water homeostasis.
Astrocytes engage unique molecular programs to engulf pruned neuronal debris from distinct subsets of neurons
Precise neural circuit assembly is achieved by initial overproduction of neurons and synapses, followed by refinement through elimination of exuberant neurons and synapses. Glial cells are the primary cells responsible for clearing neuronal debris, but the cellular and molecular basis of glial pruning is poorly defined. Here we show that Drosophila larval astrocytes transform into phagocytes through activation of a cell-autonomous, steroid-dependent program at the initiation of metamorphosis and are the primary phagocytic cell type in the pupal neuropil. We examined the developmental elimination of two neuron populations-mushroom body (MB) gamma neurons and vCrz(+) neurons (expressing Corazonin [Crz] neuropeptide in the ventral nerve cord [VNC])-where only neurites are pruned or entire cells are eliminated, respectively. We found that MB gamma axons are engulfed by astrocytes using the Draper and Crk/Mbc/dCed-12 signaling pathways in a partially redundant manner. In contrast, while elimination of vCrz(+) cell bodies requires Draper, elimination of vCrz(+) neurites is mediated by Crk/Mbc/dCed-12 but not Draper. Intriguingly, we also found that elimination of Draper delayed vCrz(+) neurite degeneration, suggesting that glia promote neurite destruction through engulfment signaling. This study identifies a novel role for astrocytes in the clearance of synaptic and neuronal debris and for Crk/Mbc/dCed-12 as a new glial pathway mediating pruning and reveals, unexpectedly, that the engulfment signaling pathways engaged by glia depend on whether neuronal debris was generated through cell death or local pruning.
Neurite degeneration is a hallmark feature of nearly all neurodegenerative diseases, occurs after most brain trauma, and is thought to be the underlying cause of functional loss in patients. Understanding the genetic basis of neurite degeneration represents a major challenge in the neuroscience field. If it is possible to define key signaling pathways that promote neurite destruction, their blockade represents an exciting new potential therapeutic approach to suppressing neurological loss in patients. This review highlights recently developed models that can be used to study fundamental aspects of neuronal injury using the fruit fly Drosophila. The speed, precision, and powerful molecular-genetic tools available in the fruit fly make for an attractive system in which to dissect neuronal signaling after injury. Their use has led to the identification of some of the first molecules whose endogenous function includes promoting axonal degeneration after axotomy, and these signaling pathways appear functionally well conserved in mammals.
Axons damaged by acute injury, toxic insults, or neurodegenerative diseases execute a poorly defined autodestruction signaling pathway leading to widespread fragmentation and functional loss. Here, we describe an approach to study Wallerian degeneration in the Drosophila L1 wing vein that allows for analysis of axon degenerative phenotypes with single-axon resolution in vivo. This method allows for the axotomy of specific subsets of axons followed by examination of progressive axonal degeneration and debris clearance alongside uninjured control axons. We developed new Flippase (FLP) reagents using proneural gene promoters to drive FLP expression very early in neural lineages. These tools allow for the production of mosaic clone populations with high efficiency in sensory neurons in the wing. We describe a collection of lines optimized for forward genetic mosaic screens using MARCM (mosaic analysis with a repressible cell marker; i.e., GFP-labeled, homozygous mutant) on all major autosomal arms ( approximately 95% of the fly genome). Finally, as a proof of principle we screened the X chromosome and identified a collection eight recessive and two dominant alleles of highwire, a ubiquitin E3 ligase required for axon degeneration. Similar unbiased forward genetic screens should help rapidly delineate axon death genes, thereby providing novel potential drug targets for therapeutic intervention to prevent axonal and synaptic loss.
Neuron-glia interactions through the Heartless FGF receptor signaling pathway mediate morphogenesis of Drosophila astrocytes
Astrocytes are critically important for neuronal circuit assembly and function. Mammalian protoplasmic astrocytes develop a dense ramified meshwork of cellular processes to form intimate contacts with neuronal cell bodies, neurites, and synapses. This close neuron-glia morphological relationship is essential for astrocyte function, but it remains unclear how astrocytes establish their intricate morphology, organize spatial domains, and associate with neurons and synapses in vivo. Here we characterize a Drosophila glial subtype that shows striking morphological and functional similarities to mammalian astrocytes. We demonstrate that the Fibroblast growth factor (FGF) receptor Heartless autonomously controls astrocyte membrane growth, and the FGFs Pyramus and Thisbe direct astrocyte processes to ramify specifically in CNS synaptic regions. We further show that the shape and size of individual astrocytes are dynamically sculpted through inhibitory or competitive astrocyte-astrocyte interactions and Heartless FGF signaling. Our data identify FGF signaling through Heartless as a key regulator of astrocyte morphological elaboration in vivo.
Comment on Transcellular degradation of axonal mitochondria. [Proc Natl Acad Sci U S A. 2014]
The Huntingtin (Htt) protein is essential for a wealth of intracellular signaling cascades and when mutated, causes multifactorial dysregulation of basic cellular processes. Understanding the contribution to each of these intracellular pathways is essential for the elucidation of mechanisms that drive pathophysiology. Using appropriate models of Huntington's disease (HD) is key to finding the molecular mechanisms that contribute to neurodegeneration. While mouse models and cell lines expressing mutant Htt have been instrumental to HD research, there has been a significant contribution to our understating of the disease from studies utilizing Drosophila melanogaster. Flies have an Htt protein, so the endogenous pathways with which it interacts are likely conserved. Transgenic flies engineered to overexpress the human mutant HTT gene display protein aggregation, neurodegeneration, behavioral deficits and a reduced lifespan. The short life span of flies, low cost of maintaining stocks and genetic tools available for in vivo manipulation make them ideal for the discovery of new genes that are involved in HD pathology. It is possible to do rapid genome wide screens for enhancers or suppressors of the mutant Htt-mediated phenotype, expressed in specific tissues or neuronal subtypes. However, there likely remain many yet unknown genes that modify disease progression, which could be found through additional screening approaches using the fly. Importantly, there have been instances where genes discovered in Drosophila have been translated to HD mouse models.
A tale of two receptors: Dual roles for ionotropic acetylcholine receptors in regulating motor neuron excitation and inhibition
Nicotinic or ionotropic acetylcholine receptors (iAChRs) mediate excitatory signaling throughout the nervous system, and the heterogeneity of these receptors contributes to their multifaceted roles. Our recent work has characterized a single iAChR subunit, ACR-12, which contributes to two distinct iAChR subtypes within the C. elegans motor circuit. These two receptor subtypes regulate the coordinated activity of excitatory (cholinergic) and inhibitory (GABAergic) motor neurons. We have shown that the iAChR subunit ACR-12 is differentially expressed in both cholinergic and GABAergic motor neurons within the motor circuit. In cholinergic motor neurons, ACR-12 is incorporated into the previously characterized ACR-2 heteromeric receptor, which shows non-synaptic localization patterns and plays a modulatory role in controlling circuit function.(1) In contrast, a second population of ACR-12-containing receptors in GABAergic motor neurons, ACR-12GABA, shows synaptic expression and regulates inhibitory signaling.(2) Here, we discuss the two ACR-12-containing receptor subtypes, their distinct expression patterns, and functional roles in the C. elegans motor circuit. We anticipate our continuing studies of iAChRs in the C. elegans motor circuit will lead to novel insights into iAChR function in the nervous system as well as mechanisms for their regulation.
Transcriptional Control of Synaptic Remodeling through Regulated Expression of an Immunoglobulin Superfamily Protein
Neural circuits are actively remodeled during brain development, but the molecular mechanisms that trigger circuit refinement are poorly understood. Here, we describe a transcriptional program in C. elegans that regulates expression of an Ig domain protein, OIG-1, to control the timing of synaptic remodeling. DD GABAergic neurons reverse polarity during larval development by exchanging the locations of pre- and postsynaptic components. In newly born larvae, DDs receive cholinergic inputs in the dorsal nerve cord. These inputs are switched to the ventral side by the end of the first larval (L1) stage. VD class GABAergic neurons are generated in the late L1 and are postsynaptic to cholinergic neurons in the dorsal nerve cord but do not remodel. We investigated remodeling of the postsynaptic apparatus in DD and VD neurons using targeted expression of the acetylcholine receptor (AChR) subunit, ACR-12::GFP. We determined that OIG-1 antagonizes the relocation of ACR-12 from the dorsal side in L1 DD neurons. During the L1/L2 transition, OIG-1 is downregulated in DD neurons by the transcription factor IRX-1/Iroquois, allowing the repositioning of synaptic inputs to the ventral side. In VD class neurons, which normally do not remodel, the transcription factor UNC-55/COUP-TF turns off IRX-1, thus maintaining high levels of OIG-1 to block the removal of dorsally located ACR-12 receptors. OIG-1 is secreted from GABA neurons, but its anti-plasticity function is cell autonomous and may not require secretion. Our study provides a novel mechanism by which synaptic remodeling is set in motion through regulated expression of an Ig domain protein.
Light is a crucial input for circadian clocks. In Drosophila, short light exposure can robustly shift the phase of circadian behavior. The model for this resetting posits that circadian photoreception is cell autonomous: CRYPTOCHROME senses light, binds to TIMELESS (TIM), and promotes its degradation, which is mediated by JETLAG (JET). However, it was recently proposed that interactions between circadian neurons are also required for phase resetting. We identify two groups of neurons critical for circadian photoreception: the morning (M) and the evening (E) oscillators. These neurons work synergistically to reset rhythmic behavior. JET promotes acute TIM degradation cell autonomously in M and E oscillators but also nonautonomously in E oscillators when expressed in M oscillators. Thus, upon light exposure, the M oscillators communicate with the E oscillators. Because the M oscillators drive circadian behavior, they must also receive inputs from the E oscillators. Hence, although photic TIM degradation is largely cell autonomous, neural cooperation between M and E oscillators is critical for circadian behavioral photoresponses.
Circadian clocks integrate light and temperature input to remain synchronized with the day/night cycle. Although light input to the clock is well studied, the molecular mechanisms by which circadian clocks respond to temperature remain poorly understood. We found that temperature phase shifts Drosophila circadian clocks through degradation of the pacemaker protein TIM. This degradation is mechanistically distinct from photic CRY-dependent TIM degradation. Thermal TIM degradation is triggered by cytosolic calcium increase and CALMODULIN binding to TIM and is mediated by the atypical calpain protease SOL. This thermal input pathway and CRY-dependent light input thus converge on TIM, providing a molecular mechanism for the integration of circadian light and temperature inputs. Mammals use body temperature cycles to keep peripheral clocks synchronized with their brain pacemaker. Interestingly, downregulating the mammalian SOL homolog SOLH blocks thermal mPER2 degradation and phase shifts. Thus, we propose that circadian thermosensation in insects and mammals share common principles.
This article describes the dissection of early Drosophila embryos to generate flat or fillet preparations. For the procedure, a modified chamber or well is designed using glass slides and sealant. Embryos are dissected by removing the chorionic (outer) membrane (performed outside the chamber) and then removing the vitelline (inner) membrane (performed inside the chamber and under saline).
Wnt signaling plays critical roles during synaptic development and plasticity. However, the mechanisms by which Wnts are released and travel to target cells are unresolved. During synaptic development, the secretion of Drosophila Wnt1, Wingless, requires the function of Evenness Interrupted (Evi)/Wls, a Wingless-binding protein that is secreted along with Wingless at the neuromuscular junction. Given that Evi is a transmembrane protein, these studies suggested the presence of a novel vesicular mechanism of trans-synaptic communication, potentially in the form of exosomes. To establish the mechanisms for the release of Evi vesicles, we used a dsRNA assay in cultured cells to screen for genes that when down-regulated prevent the release of Evi vesicles. We identified two proteins, Rab11 and Syntaxin 1A (Syx1A), that were required for Evi vesicle release. To determine whether the same mechanisms were used in vivo at the neuromuscular junction, we altered the activity of Rab11 and Syx1A in motoneurons and determined the impact on Evi release. We found that Syx1A, Rab11, and its effector Myosin5 were required for proper Evi vesicle release. Furthermore, ultrastructural analysis of synaptic boutons demonstrated the presence of multivesicular bodies, organelles involved in the production and release of exosomes, and these multivesicular bodies contained Evi. We also used mass spectrometry, electron microscopy, and biochemical techniques to characterize the exosome fraction from cultured cells. Our studies revealed that secreted Evi vesicles show remarkable conservation with exosomes in other systems. In summary, our observations unravel some of the in vivo mechanisms required for Evi vesicle release.
Book chapter discussing assessment of pain and physical function following total hip replacement surgery.
Web-based Comparative Patient-reported Outcome Feedback to Support Quality Improvement and Comparative Effectiveness Research in Total Joint Replacement
INTRODUCTION: Patient-reported outcomes (PROs) are rarely included in quality monitoring systems, surgeon comparative feedback reports, or registries. We present the design and implementation of a secure website in a federally funded research program-Function and Outcomes Research for Comparative Effectiveness in Total Joint Replacement (FORCE-TJR)-to return comparative PRO reports to participating surgeons, in addition to including traditional quality measures, in order to monitor and improve quality and health outcomes.
METHODS: The surgeon-specific comparative PRO reports were designed and structured based on user input for content, data elements, integration, and display. Three questions are addressed regarding the knee and hip joint symptom profiles of patients before TJR, as well as outcomes of surgery. The website is organized with a hierarchical structure to display data at national, practice, and individual surgeon levels, and provides a comprehensive site-level executive summary and surgeon-level data reports that can be downloaded.
EARLY RESULTS: As of September 2014, over 22,000 patients were enrolled from more than 130 surgeons in 22 states. The reporting website was launched in September 2012 and has been updated quarterly for all surgeons to review their site- and individual-specific outcomes data compared to national benchmarks.
DISCUSSION: In this novel system, quarterly comparative surgeon feedback extends beyond traditional measures of complication rates to include PROs of pain relief and functional gain. We anticipate that this enhanced data will facilitate patient-centered quality improvement (QI) and outcomes research from the registry. As the Centers for Medicare and Medicaid Services (CMS) and other insurers consider future implementation of PROs, surgeons will increasingly need comparative data by which to self-monitor their practice outcomes.
Patient report improves posthospital discharge event capture in total joint replacement: a novel approach to capturing all posthospital event data
INTRODUCTION: Current approaches to quantifying total posthospital complications and readmissions following surgical procedures are limited because the United States does not have a single health care payer. Patients seek posthospital care in varied locations, yet hospitals can only quantify those returning to the same facility. Seeking information directly from patients about health care utilization following hospital discharge holds promise to provide data that is missing for surgeons and health care systems.
BACKGROUND: Because total joint replacement (TJR) is the most common and costly elective surgical hospitalization, we examined the concordance between patients' self-report of potential short-term complications and their readmissions and our review of medical records in the initial hospital and surrounding facilities.
METHODS: Patients undergoing primary total hip or knee replacement from July 1, 2011, through December 3, 2012, at a large site participating in a national cohort of TJR patients were identified. Patients completed a six-month postoperative survey regarding emergency department (ED), day surgery (DS), or inpatient care for possible medical or mechanical post-TJR complications. We reviewed inpatient and outpatient medical records from all regional facilities and examined the sensitivity, specificity, and positive- and negative predictive values for patient self-report and medical records.
FINDINGS: There were 413 patients who had 431 surgeries and completed the six-month questionnaire. Patients reported 40 medical encounters (9 percent) including ED, DS or inpatient care, of which 20 percent occurred at hospitals different from the initial surgery. Review of medical records revealed 9 additional medical encounters that patients had not mentioned including five hospitalizations following surgery and four ED visits. Overall patient self-report of ED, DS, and inpatient care for possible complications was both sensitive (82 percent) and specific (100 percent). The positive predictive value was 100 percent and negative predictive value 98 percent.
DISCUSSION: Patient self-report of posthospital events was accurate. Substantial numbers of patients required care at outlying hospitals (not where the TJR occurred).
CONCLUSION: Methods that directly engage patients can augment current posthospital utilization surveillance to assure complete data.
Reply to the letter to the editor: preoperative pain and function profiles reflect consistent TKA patient selection among US surgeons
Comment on: Letter to the editor: preoperative pain and function profiles reflect consistent TKA patient selection among US surgeons. [Clin Orthop Relat Res. 2015] and Preoperative pain and function profiles reflect consistent TKA patient selection among US surgeons. [Clin Orthop Relat Res. 2015]