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International Journal of Applied Earth Observation and Geoinformation

Volume 92, October 2020, 102173

Tree species classification using UAS-based digital aerial photogrammetry point clouds and multispectral imageries in subtropical natural forests

Author links open overlay panelZhongXua1XiangqianWua

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https://doi.org/10.1016/j.jag.2020.102173Get rights and content

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open access

Highlights

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Classifying dominant tree species using UAS multispectral and point cloud data.

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A structural from motion algorithm is used to derive 3D point cloud from imagery.

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Image- and point-based segmentation algorithms are compared and assessed.

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Multiresolution segmentation algorithm has a higher accuracy than the others (82.5 %).

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Combination of spectral and structural metrics has positive impacts on classification.

Abstract

Tree species composition of forest stand is an important indicator of forest inventory attributes for assessing ecosystem health, understanding successional processes, and digitally displaying forest biodiversity. In this study, we acquired high spatial resolution multispectral and RGB imagery over a subtropical natural forest in southwest China using a fixed-wing UAV system. Digital aerial photogrammetric (DAP) technique was used to generate multi-spectral and RGB derived point clouds, upon which individual tree crown (ITC) delineation algorithms and a machine learning classifier were used to identify dominant tree species. To do so, the structure-from-motion method was used to generate RGB imagery-based DAP point clouds. Then, three ITC delineation algorithms (i.e., point cloud segmentation (PCS), image-based multiresolution segmentation (IMRS), and advanced multiresolution segmentation (AMRS)) were used and assessed for ITC detection. Finally, tree-level metrics (i.e., multispectral, texture and point cloud metrics) were used as metrics in the random forest classifier used to classify eight dominant tree species. Results indicated that the accuracy of the AMRS ITC segmentation was highest (F1-score = 82.5 %), followed by the segmentation using PCS (F1-score = 79.6 %), the IMRS exhibited the lowest accuracy (F1-score = 78.6 %); forest types classification (coniferous and deciduous) had a higher accuracy than the classification of all eight tree species, and the combination of spectral, texture and structural metrics had the highest classification accuracy (overall accuracy = 80.20 %). In the classification of both eight tree species and two forest types, the classification accuracies were lowest when only using spectral metrics, indicated that the texture metrics and point cloud structural metrics had a positive impact on the classification (the overall accuracy and kappa accuracy increased by 1.49–4.46 % and 2.86–6.84 %, respectively).

Industrie 4.0 is the German description for the 4th industrial revolution. While in Germany "Industrie 4.0" aims at putting the strong German manufacturing industry in a position of future readiness through integrated digitization, for the ICT-dominant USA, "Smart Manufacturing" is ought to revive the country's re-industrialization. Fraunhofer, a major European Research and Technology Organization (RTO), has a strong focus on Industrie 4.0 technologies throughout the whole production value chain. Together with research partners from universities, Fraunhofer supports the German and European industry to benefit from the new possibilities enabled through Industrie 4.0 developments. With several thousand experts and researchers Fraunhofer works to realize the development of the smart factory, e.g. in areas like production planning, manufacturing technologies ranging from deep-drawing to laser applications, as well as Internet of Things (IoT) applications and services, supply chain management or efficient buildings. Key enablers for harnessing the benefits of digitization for Industrie 4.0 are widely accepted standards, extreme low latency in digital communication as well as safety and security for data analytics and data exchange. To foster a straightforward communication of the challenges and developments described under the term Industrie 4.0 and corresponding technologies, an Industrie 4.0 description model, the "Fraunhofer layer model", was developed. This paper describes the concept of Industrie 4.0, the technological challenges and the extensive "Fraunhofer layer model" together with its bottom-up genesis based on Fraunhofer technologies.

Positron emission tomography (PET) is a minimally invasive imaging procedure with a wide range of clinical and research applications. PET allows for the three-dimensional mapping of administered positron-emitting radiopharmaceuticals such as 18F-fluorodeoxyglucose (for imaging glucose metabolism). PET enables the study of biologic function in both health and disease, in contrast to magnetic resonance imaging (MRI) and computed tomography (CT), that are more suited to study a body's morphologic changes, although functional MRI can also be used to study certain brain functions by measuring blood flow changes during task performance. This chapter first provides an overview of the basic physics principles and instrumentation behind PET methodology, with an introduction to the merits of merging functional PET imaging with anatomic CT or MRI imaging. We then focus on clinical neurologic disorders, and reference research on relevant PET radiopharmaceuticals when applicable. We then provide an overview of PET scan interpretation and findings in several specific neurologic disorders such as dementias, epilepsy, movement disorders, infection, cerebrovascular disorders, and brain tumors.

Putamen

Related terms:

Positron Emission Tomography

Dopamine Agonist

Parkinson's Disease

Dopamine

Substantia nigra

Thalamus

Basal Ganglion

Globus pallidus

Caudate Nucleus

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Parkinson's Disease and Related Disorders, Part II

Federico Eduardo Micheli, María Graciela Cersósimo, in Handbook of Clinical Neurology, 2007

51.11 Positron emission tomography

Putamen 18F‐dopa uptake of PD patients is reduced by at least 35% at onset of symptoms; therefore, PET scans can be used to detect preclinical disease in clinically unaffected twins and relatives of patients with PD. PET scans can be used to detect underlying nigral pathology in patients with isolated tremor and patients taking dopamine receptor‐blocking agents who become rigid. Patients with familial essential tremor have normal, whereas those with isolated rest tremor have consistently low, putamen 18F‐dopa uptake. DIP is infrequently associated with underlying nigral pathology (Brooks, 1991).

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Imaging the Addicted Brain

C.A. Hanlon, ... J.L. Jones, in International Review of Neurobiology, 2016

2.2.3 Putamen

The putamen is also part of the dorsal striatum. It is generally involved in movement and learning (Balleine et al., 2007), and its volume has also been found to be larger among cocaine users (Ide et al., 2014; Jacobsen et al., 2001). The putamen has a role in cocaine craving, with increased BOLD activity during craving following cocaine administration (Breiter et al., 1997). During a PET study of cocaine cue-induced cravings, the putamen showed increased levels of dopamine receptor occupancy (Wong et al., 2006). Alterations of the activity of the putamen have also been associated with worse cognitive performance, showing decreased BOLD signal in cocaine users compared to nonusing controls in a working memory task (Moeller et al., 2010). Similarly, hypoactivation in the putamen during the Stroop test (a task of working memory) predicted shorter length of abstinence (Brewer et al., 2008).

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Spontaneous Intracerebral Hemorrhage

José Biller, Michael J. Schneck, in Stroke in Children and Young Adults (Second Edition), 2009

Putaminal Hemorrhage

The putamen is the most common site for hypertensive ICH.228 Hemorrhages may remain localized to the putamen; enlarge to involve the internal capsule, corona radiata, centrum semiovale, or temporal lobe; or rupture into the ventricular system (Fig. 14-14). The clinical picture is characterized by contralateral hemiparesis or hemiplegia, accompanied by conjugate preference to the side of the hematoma. There may be less severe contralateral hemisensory loss. Left putaminal hemorrhages result in aphasia; right putaminal hemorrhages produce apractagnosia, left visual field neglect, and constructional apraxia. Homonymous hemianopsia may be present.

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Brain Banking

Nicola Palomero-Gallagher, Karl Zilles, in Handbook of Clinical Neurology, 2018

Glutamate receptors

The caudate-putamen has higher AMPA, but lower kainate, NMDA or mGluR2/3 receptor densities than those found in most cortical regions (Fig. 24.5A–D; Dure et al., 1991; Zilles and Palomero-Gallagher, 2001; Zilles et al., 2002b; Palomero-Gallagher et al., 2006, 2009a; Zilles and Amunts, 2009; Amunts et al., 2010). Regional differences include:

AMPA, NMDA, kainate: accumbens = caudate = putamen > globus pallidus (Ball et al., 1994; Meador-Woodruff et al., 2001; Villares and Stavale, 2001);

AMPA, NMDA: matrix > striosomes (Dure et al., 1991, 1992);

kainate: matrix < striosomes (Dure et al., 1991, 1992).

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Normal Brain Histopathology

Daniel J. Brat MD, PhD, in Practical Surgical Neuropathology: A Diagnostic Approach (Second Edition), 2018

Basal Ganglia

The caudate, putamen, and nucleus accumbens (i.e., the neostriatum) are developmentally related and histologically similar (Fig. 2.10A). They contain a variety of small and large neuronal populations that have relatively uniform density. About 95% are small and midsize (10 to 18 µm) gamma-aminobutyric acid-ergic (GABAergic) spiny neurons that provide projections to the globus pallidus (i.e., the paleostriatum). These have extensive dendritic trees packed with spines for connection with the large array of input fibers from the cerebral cortex, thalamus, and brainstem. Other populations consist of large cholinergic neurons (approximately 2% of neurons) and smaller cells containing neuropeptide Y, somatostatin, or nitric oxide synthetase. Interspersed among the neurons and neuropil of the striatum are small, white matter bundles of the internal capsule that can only be seen microscopically. These "pencil fibers of Wilson" are specific for this region and serve as a guide to location when included in small biopsy specimens.

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Subpallial Structures

Loreta Medina, Antonio Abellán, in The Mouse Nervous System, 2012

Striatal Projection Neurons

The caudate-putamen and the nucleus accumbens of the basal ganglia (or striatum proper) contain GABAergic projection neurons, which in rodents represent about 90% of all neurons in the striatum (Gerfen, 1992; 2004; Medina, 2008b; Parent and Hazrati, 1995a; Reiner et al., 1998). As noted above, data in mouse indicate that most, if not all, projection neurons of the striatum originate in vLGE (Marín et al., 2000; Stenman et al., 2003a). Nevertheless, at present we cannot exclude the possibility that some neuronal subpopulations of the striatum originate in dLGE (for example, some patch neurons [Toresson and Campbell, 2001], see below). There are two major subtypes of projection neurons in the striatum (Fig. 7.7A): half of them co-contain GABA and the neuropeptides substance P/dynorphin (GABA/SP/DYN), whereas the other half co-contain GABA and the neuropeptide enkephalin (GABA/ENK). Nevertheless, some examples of projection neurons co-containing a combination of substance P, dynorphin and/or enkephalin are found (this happens less often in the caudate-putamen [only 4%] than in the nucleus accumbens; reviewed in Gerfen, 2004; Medina, 2008b; Reiner et al., 1998). These two types of projection neurons differ in their projections and functions (Fig. 7.7B), as explained in a separate section and reviewed elsewhere (Gerfen, 2004; Medina, 2008b; Parent et al., 1995a,b; Reiner et al., 1998; see also chapter on the functional aspects of the Basal Ganglia in this book). For example, SP-containing neurons of the caudate-putamen primarily project to the internal segment of the globus pallidus and the substantia nigra and are involved in promoting voluntary movements, while ENK-containing neurons project to the external segment of the globus pallidus and are involved in blocking involuntary movements (Fig. 7.7B). It is not known whether substance P- or enkephalin-containing projection striatal neurons originate in different LGE subdomains, or in the same subdomain(s) at different moments. Interestingly, the differentiation of these two types of projection neurons is regulated by different genetic cascades/networks, involving the transcription factor Ebf1 in the case of SP-containing neurons (Garel et al., 1999; Lobos et al., 2008), and Ikaros-1 in the case of ENK-containing neurons (Agoton et al., 2007; Martín-Ibañez et al., 2010). These two types of striatal projection neurons die at different moments in Huntington';s disease (ENK neurons die earlier than SP neurons), and this is correlated with distinct motor deficits at different stages of the disease (reviewed by Reiner et al., 1998). The study of the genetic regulatory programs involved in the differentiation of these two types of neurons may help to understand these differences.

FIGURE 7.7. (A) Scheme of a frontal telencephalic section at the level of the caudate-putamen (CPu) and globus pallidus, representing the neuron subpopulations of these nuclei, and their embryonic origin using a color code (explained in a separate list on the right). In the CPu, about 90% of the neurons are GABAergic projection neurons (which typically are medium-sized and with spiny dendrites), and these originate in LGE. About 10% of the remaining neurons include five different subtypes of interneurons, the majority of which originate in the pallidal (MGE) or the preoptic (POC) subdivisions. The globus pallidus contains two major subtypes of principal GABAergic neurons showing descending projections: about two-thirds of them (66%) contain parvalbumin (these cells originate in MGE); and one third of them contain calbindin and enkephalin, and have a descending axon with a collateral projecting back to the striatum (these cells appear to originate in LGE). In addition, the GP contains a subpopulation of cholinergic neurons that belong to the Ch4 corticopetal system (with ascending projections to the cortex/pallium), which appear to originate in the POC. (B) Lateral view of the brain (rostral is to the left, and dorsal to the top) showing the main projections of the SP+ or ENK+ projection neurons of the CPu, and the direct (green) and indirect (blue) pathways to influence thalamo-cortical neurons, involved in the control of motor behavior. Activation of the SP+ striatal neuron (direct pathway) produces disinhibition of the thalamocortical target and releases voluntary movement. On the contrary, activation of the ENK+ striatal neuron disinhibits the subthalamic nucleus, which by excitatory projections activates the inhibitory pallidal neurons that project to the thalamus (part of the direct pathway), thus blocking involuntary movements. See text for more details. For abbreviations see list.

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Normal Brain Histopathology

Daniel J. Brat, in Practical Surgical Neuropathology, 2010

Basal Ganglia

The caudate, putamen, and the nucleus accumbens (a.k.a. the neostriatum) are developmentally related and histologically similar (Fig. 2-10A). They contain a variety of small- and large-sized neuronal populations that have relatively uniform density. About 95% are small- and medium-sized (10–18 μm) γ-aminobutyric acid (GABA)-ergic spiny neurons that provide projections to the globus pallidus (a.k.a. the paleostriatum). These have extensive dendritic trees packed with spines for connection with the large array of input fibers from the cerebral cortex, thalamus, and brainstem. Other populations consist of large cholinergic neurons (approximately 2% of neurons) and smaller cells containing neuropeptide Y, somatostatin, or nitric oxide synthetase. Interspersed among the neurons and neuropil of the striatum are small white matter bundles of the internal capsule that can only be seen microscopically. These "pencil fibers of Wilson" are specific for this region and serve as a guide to location when included in small biopsy specimens.

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Deep Brain Stimulation for Movement Disorders

Ludvic Zrinzo, Jonathan A. Hyam, in Principles of Neurological Surgery (Fourth Edition), 2018

Globus Pallidus

Together with the putamen, the globus pallidus forms the so-called lentiform nucleus that is bounded laterally by the external capsule and overlying claustrum, extreme capsule, and insula and medially by the internal capsule.59 The pallidum, or "pale nucleus," owes its name to the multitude of myelinated fibers coursing through its substance. The pallidum is separated from the putamen by the external medullary laminae and is in turn divided into internal and external segments by the medial medullary lamina. An accessory lamina lies within the internal segment (Fig. 57.5). An inferior subcommissural portion is termed the ventral pallidum (GPv).60

The pallidum is populated by large neurons with characteristically disk-shaped dendritic arborization lying parallel to the lateral surface of the pallidum perpendicular to the incoming striatal axons.60

The ansa lenticularis and lenticular fasciculus form the major outflow pathways from GPi. The ansa lenticularis winds medially around the anterior and inferior aspect of the posterior limb of the internal capsule. The lenticular fasciculus traverses the posterior limb of the internal capsule to form the H2 field of Forel before merging with the ansa in the prerubral field (H of Forel) prior to reaching the thalamic fasciculus (H1).61

The sensorimotor pallidum is located in the inferior, posterior, and lateral portion of the pallidum.62 There is evidence from nonhuman primate studies that a somatotopic arrangement persists within the pallidum.63 The limbic areas of the GPe and GPi lie in the anterior inferior and medial regions.60

Pallidal afferents from the striatum are mainly GABAergic and inhibitory. Reciprocal GABAergic connections also exist between GPi and GPe. Glutamatergic excitatory inputs arrive from the STN. The pallidum also receives afferents from the thalamus (mainly CM-Pf), pedunculopontine nucleus, SNc, and VTA.

Pallidal efferents are GABAergic and inhibitory. GPe projects mainly to STN, GPi/SNr, and pedunculopontine nuclei. GPi efferents from GPi to the ventral anterior and ventral lateral thalamus constitute part of the information loop back to cortex. GPi efferents to CM-Pf thalamus and thereafter striatum form part of a subcortical feedback loop between basal ganglia output and input structures.

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Parkinson's Disease

Néstor Gálvez-Jiménez MD, MSc, MHSA, FACP, in Neurobiology of Disease, 2007

D. Histaminergic System

Histamine concentration in the putamen, SNc, and globus pallidus increases in patients with PD when compared with that seen with other parkinsonian syndromes such as multiple system atrophy [79]. The significance of these findings is unknown, but histamine may be implicated in the motor and behavioral alterations in PD. Histamine binds to four types of G-protein–coupled transmembrane receptors (H1–H4) with possible modulatory influences at the dopamine D2 family of receptors. Most of the histaminergic afferents originate in the tuberomammillary hypothalamic nuclei, composed of 64,000 neurons, projecting widely to the central nervous system. Of all histamine receptors, H3 has caught the attention of most researchers because of its unique function as a presynaptic autoreceptor with high expression in the striatum, cerebellum, limbic system, and thalamus, hence the interest as a new therapeutic target in PD [80]. Studies have shown high levels of H3 autoreceptors in medium-size interneurons, co-localizing with the medium spiny cholinergic striatal neurons resulting in modulation of the direct and indirect nigrostriatal pathways, and dopamine, acetylcholine, and glutamate release [81]. Using in situ hybridization, Anichtchik et al. [82] have shown an increase of H3 receptor expression in the SN, putamen, globus pallidus, and cortex of patients with PD. The authors hypothesized that the increase expression of H3 receptors results as a consequence of the modulatory effects of dopamine on the expression of messenger RNA (mRNA) in the striatum and SN. The authors further concluded that the modulation of the histaminergic system results in alterations of other neurotransmitter such as GABA, serotonin, acetylcholine, and dopamine. Histamine has been implicated in the regulation of hibernation, circadian rhythm, locomotion, movement, memory, and cognition. Also, Goodchild et al. [83] have shown the presence of H3 receptors in the striatonigral projection neurons to the direct and indirect pathways, further supporting the influence histamine may have over the activity of these motor circuits.

During aging, there is a decrease in histamine receptor mRNA levels, particularly H1; H2, and H3, especially in the cortex, hypothalamus, hippocampus, and medulla in 3-month-old rats. In addition, H2 levels are decreased in the cerebellum and pons [84]. We can hypothesize that the activation of the histaminergic system with its resultant inhibition of dopamine release may be partly responsible for the motor and behavioral alterations seen in PD. This will await additional study results.

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Caudate Nucleus

Enrique L. Labadie, in Encyclopedia of the Neurological Sciences, 2003

Neuronal Organization and Main Neurotransmitters

Neurons forming the caudate and putamen are distributed in two main groups; approximately 80% are medium-sized spiny neurons localized at the matrix, and 20% form patches called striatosomes. A significant number of both neuronal groups produce the inhibitory neurotransmitter γ-aminobutyric acid (GABA). Some caudate neurons also produce substance P and opioid peptides, and other large excitatory neurons contain acetylcholine.

The majority of matrix neurons have D2 dopamine receptors on their surface, whereas the striatosome neurons have D1 dopamine receptors. The D2 receptors are several times more sensitive to dopamine than are the D1 receptors. Whereas dopamine acts on D2 receptors to produce a net inhibitory influence on GABA release, dopamine is excitatory to D1 receptors.

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Polyglutamine

Polyglutamine expanded mutant Htt protein appears to have altered physical and functional properties leading to a dominant toxic gain of function, although a role for loss of normal Htt function in HD pathogenesis remains a possibility.

From: Animal and Translational Models for CNS Drug Discovery, 2008

Related terms:

Huntingtin

Protein

Pathogenesis

Toxicity

Nerve Degeneration

Spinocerebellar Degeneration

Huntington Chorea

Kennedy Disease

Androgen Receptor

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Cellular and Molecular Basis of Neurodegeneration in the CAG–Polyglutamine Repeat Diseases

Rabaab Zahra, ... George J. Siegel, in Basic Neurochemistry (Eighth Edition), 2012

RNA Toxicity in the Polyglutamine Repeat Diseases?

PolyQ disease pathology has been extensively studied, and there are considerable data supporting proteotoxicity as the primary cause of the pathogenesis in these diseases. A number of studies, including especially work done on SCA1 and SBMA, have precluded a role for RNA toxicity in polyQ disease, as mutant RNAs do not cause any neurodegeneration in mice, when the polyQ-expanded protein does not enter the nucleus (Katsuno et al., 2002; Klement et al., 1998). However, in a Drosophila screen for modifiers of polyQ-expanded ataxin-3 degeneration, muscleblind (mbl), a splicing regulator implicated in the RNA toxicity of CUG repeat expansion diseases, was found to modify ataxin-3 polyQ toxicity (Li et al., 2008). Although this is a provocative finding, the mechanism underlying the effect of muscleblind has remained elusive, leaving a possible role for RNA toxicity in polyQ diseases undefined.

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Intranuclear Amyloid – Local and Quantitative Analysis of Protein Fibrillation in the Cell Nucleus

Florian Arnhold, Anna von Mikecz, in Bio-nanoimaging, 2014

Abstract

Homopolymeric polyglutamine (polyQ) repeats are inherently functional and toxic at the same time. PolyQ motifs occur in proteins that are involved in gene expression and promote the formation of nuclear assemblies such as the transcription initiation complex. Transition of these functional complexes to insoluble protein aggregates is constitutively prevented by proteasomal proteolysis that occurs in locally separated nucleoplasmic microdomains/proteolytic centers. However, conditions that exhaust the ubiquitin–proteasome system, such as the extensive production of expanded polyQ proteins, aging, and xenobiotic stress, induce a congested state in which nuclear proteins, including those with polyQ stretches, fibrillate and form amyloid-like aggregates. Here, we describe imaging methods that enable quantitative monitoring of nuclear protein fibrillation. We show that amyloid indicators, such as Congo Red, bind to distinct nucleoplasmic microdomains that are describable by application of discrete mathematics on the image information. As the formation of Congo Red-binding nuclear microdomains correlates with increased amyloid formation, and decreased solubility of endogenous polyQ proteins, the idea is put forward that different protein fibrillation steps can be described intracellularly by graph theory-aided pattern recognition.

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Spinocerebellar Atrophy

M. Manto, P. Jissendi, in Encyclopedia of Neuroscience, 2009

Gain of Function of Proteins

Polyglutamine expansions are associated with transcriptional alterations. For several SCAs (SCA1, SCA2, SCA3, SCA6, SCA7, SCA8, and SCA17), an abnormally long polyglutamine tract has been reported as in Huntington's disease, hence the terminology of polyglutamine expansion disorders. Phenotypic differences among the diseases are determined not only by the repeat length but also by the instrinsic function of the disease-causing protein. Formation of unusual structures by repetitive DNA seems to be a core mechanism of repeat instability which leads to elongated polyglutamine tracts.

Proteins resulting from defective genes aggregate both in the cytoplasm and in the nucleus. They are cleaved before entering the nuclei. Transcriptional deregulation might be a key factor in the pathogenesis of most polyglutamine disorders, polyglutamine tracts interacting closely with well-defined transcriptional regulators such as cAMP responsive element-binding protein (CREB). For instance, the SCA7 gene product, ataxin 7, is part of a transcriptional coactivator complex, called STAGA, which has histone acetyltransferase activity. In SCA17, the CAG repeat expansion occurs in TATA-binding protein (TBP), a transcription factor. SBMA (X-linked) is caused by a polyglutamine expansion in the androgen receptor. Impaired protein conformation leads to protein accumulation and a cascade of aberrant protein–protein interactions, resulting finally in cell death. Chaperones and proteasomes are used to refold or dispose polyglutamine-containing fragments in order to prevent further aggregation. Accumulation of insoluble aggregates occurs in the nuclei for SCA1, SCA7, and SCA17; in the cytoplasm for SCA2 and SCA6; and in both the nuclei and cytoplasm for DRPLA, SBMA, and SCA3. Small aggregates sequester other proteins and protein complexes including proteasomes and transcription factors, leading to inclusion bodies. Formation of inclusions might be a protective mechanism against the toxicity of expanded polyglutamine proteins. A mechanism of impaired Ca2+ influx into neurons in SCA6, caused by mutations in the α-1A subunit of the voltage-gated neuronal Ca2+ channel, could contribute to the neurological deficits in this relatively pure cerebellar syndrome.

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Ataxias

Thomas Klockgether, in Textbook of Clinical Neurology (Third Edition), 2007

PATHOGENESIS AND PATHOPHYSIOLOGY

All polyglutamine disorders share important pathogenetic features that are due to deleterious actions mediated by the elongated polyglutamine tracts. Specifically, proteins containing elongated polyglutamine tracts have a strong tendency to aggregate and to undergo abnormal interactions with other cellular proteins. Aggregations of the disease proteins forming ubiquitinated neuronal intranuclear inclusions are found in affected brain areas of most polyglutamine disorders. However, research has shown that these inclusions are not required for neurodegeneration. Instead, it is assumed that the abnormal interaction of the elongated proteins with general transcription factors such as TATA‐binding protein (TBP) or TBP‐associated factor (TAF), which contain polyglutamine tracts, is a common and essential pathogenetic mechanism. As a consequence, there is a general suppression of gene transcription.41 Interestingly, SCA17 is due to a polyglutamine expansion in the TBP gene.42 In addition, there are pathogenetic mechanisms that are specific for each disorder and that are related to the specific properties of the involved proteins and their alterations through the polyglutamine expansion. This dual model explains both the overlapping and the disease‐specific phenotypical features of the polyglutamine disorders.

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Nanoparticle-Based Peptide Vaccines

Y. Fujita, H. Taguchi, in Micro and Nanotechnology in Vaccine Development, 2017

8.6.1 Self-assembling protein antigens

A polyglutamine (polyQ) domain is found in several types of aggregation-prone proteins associated with neurodegeneration (eg, Huntington's disease). Short polyQ domains (<35 Qs) are soluble, while longer polyQ domains (>36 Qs) tend to aggregate.96,97 Ilyinski et al. investigated a model fusion protein, in which a polyQ domain (longer than 100 glutamine residues) was attached to the weak immunogenic green fluorescent protein (GFP).98 When used as a plasmid DNA vaccine, polyQ-associated aggregation of expressed antigen strongly enhanced both antibody and cytotoxic responses against GFP in mice immunization, suggesting that the linkage of an elongated polyQ domain to antigens has the potency of an adjuvant. Peptides that are able to adopt a coiled-coil conformation tend to aggregate into fibers. Burkhard et al. studied self-assembling peptide-based nanoparticles, which consisted of a pentameric coiled-coil oligomerization domain derived from cartilage oligomeric matrix protein (COMP)99 and a de novo designed trimeric coiled-coil sequence.100 Subsequently, they applied them in the design of multiple antigen-presenting vaccines.101,102 The self-assembling peptide, conjugated with the C-terminal heptad repeat region (HRC1) epitope derived from the severe acute respiratory syndrome coronavirus (SARS-CoV) S protein as a B cell epitope, formed nanoparticles that were about 25 nm in size and had multiple copies of HRC1 epitopes on their surface. Immunological evaluation of the conjugate containing SARS epitope showed the production of specific antibodies and their neutralization activity against SARS-CoV infection.101 The same building block has been used for a self-assembling peptide-based vaccine against malaria. The conjugate with a B cell epitope (DPPPPNPN)2D derived from the circumsporozoite protein of the malaria parasite Plasmodium berghei self-assembled into nanoparticles (∼25 nm in diameter). The conjugate containing the malaria epitope elicited a high level of antibody production after immunization in mice. The immunized mice showed a long-lasting protection against the parasite for up to 6 months. This self-assembling peptide-based nanosystem has also been used for the development of an HIV vaccine103 and antitumor vaccine.104

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Triplicate Repeats: Huntington's disease

J.-H.J. Cha, K.B. Kegel, in Encyclopedia of Neuroscience, 2009

Polyglutamines and Aggregates

The polyglutamine moiety that is expanded in mutant forms of the huntingtin protein gives rise to the neuropathologic changes observed in HD. A transgenic mouse model expressing only exon 1 of the huntingtin gene (the portion containing the polyglutamine moiety) develops an abnormal neurologic phenotype. The fact that such a striking phenotype could be produced in an animal expressing only exon 1 of the HD gene added weight to the early 'toxic fragment' hypothesis. One of the striking findings which emerged out of the findings with transgenic animals is the observation of novel inclusions in neuronal nuclei, although huntingtin normally exists as a predominantly cytoplasmic protein. This remarkable finding prompted reexamination of human biopsy and necropsy material, and abnormal huntingtin- and ubiquitin-positive nuclear and cytoplasmic aggregates were also found. Although abnormal aggregates are a striking feature of human HD and transgenic mouse HD models, recent studies have raised controversy as to the importance of inclusions. In cell culture models, the presence of inclusions was not correlated with neuronal death. Other models have suggested that inclusions may actually serve as a protective function.

Several theories have developed concerning the gain-of-function which emerges when the length of the polyglutamine moiety extends into the pathologic range. One theory proposes that polyglutamine moieties serve as a substrate for the enzyme transglutaminase. Nuclear inclusions in HD are reminiscent of protein deposits that are pathologic hallmarks of other neurodegenerative diseases; an emerging theme is that neurodegenerative diseases may be disorders of altered protein folding. Polyglutamine moieties are found within other proteins, including transcription factors. Many studies suggest that polyglutamine aggregates may serve as sinks to sequester transcription factors or to deplete proteins involved in vesicle movement. Aggregates present in the cytoplasm may also physically block vesicles, preventing transport within axons and dendrites.

Huntingtin aggregates are dynamic. In in vitro systems, one can visualize the appearance and growth of aggregates. In a transgenic mouse model that expressed a portion of the huntingtin protein under the control of an inducible promoter, stopping expression of the mutant transgene led to regression of the size of intranuclear aggregates, attesting to the recuperative ability of neurons with aggregates.

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SK-Ca3 Small Conductance Calcium Activated Potassium Channel

Stephan Grissmer, in xPharm: The Comprehensive Pharmacology Reference, 2007

Disorders

Longer polyglutamine repeats are over-represented in schizophrenic individuals Chandy et al (1998), Cardno et al (1999) and in patients with anorexia nervosa Koronyo-Hamaoui et al (2002) and spinocerebellar ataxia Figueroa et al (2001). A four base deletion has been found in a patient with schizophrenia Bowen et al (2001) that truncates the protein just before the S1 segment and causes dominant-negative suppression of endogeneous SK channels Miller et al (2001). Protein and mRNA levels are increased in skeletal muscle after denervation Pribnow et al (1999), Neelands et al (2001) and in patients with myotonic muscular dystrophy Renaud et al (1986), Behrens et al (1994), Kimura et al (2000).

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SCA17

S. Tsuji, in Encyclopedia of Movement Disorders, 2010

Pathogenesis/Pathophysiology

Among the nine polyglutamine diseases, the physiological functions of the gene products have been known only for SCA6, SBMA, and SCA17. Thus, SCA17 is a good target for investigating the pathophysiologic mechanisms of neurodegeneration. TBP is an important general transcription initiation factor and is the DNA-binding subunit of RNA polymerase II transcription factor D (TFIID), the multisubunit complex crucial for the expression of most genes. Intranuclear accumulation of mutant proteins carrying expanded polyglutamine stretches and subsequent nuclear dysfunction through association of mutant proteins with various transcriptional factors have been considered to play essential roles in the pathogenesis of polyglutamine diseases. Intranuclear inclusions identified in autopsied brains of SCA17 cases support this hypothesis. In contrast to the 'gain-of-toxic function' hypothesis, interference with the physiological functions of TBP may also be involved in the pathophysiologic mechanisms.

Recent studies have demonstrated that expansion of polyglutamine stretches causes abnormal interaction of TBP with the general TFIIB and induces neurodegeneration in transgenic SCA17 mice. Furthermore, it has been shown that mutant TBP with expanded polyglutamine stretches with a deletion spanning part of the DNA-binding domain does not bind DNA in vitro but forms nuclear aggregates and inhibits TATA-dependent transcription activity in cultured cells. These findings suggest that the polyglutamine stretches affect the binding of TBP to promoter DNA and that mutant TBP with expanded polyglutamine stretches can induce neuronal toxicity independently of its interaction with DNA.

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Clinical and Pathological Features of Hereditary Ataxiasemsp;

TETSUO ASHIZAWA, S.H. SUBRAMONY, in Animal Models of Movement Disorders, 2005

2. SCA2

The polyglutamine repeat expansion in SCA2 is in the ataxin 2 protein encoded by the SCA2 gene on chromosome 2q23–24.1 (table 1) (Pulst et al., 1996; Sanpei et al., 1996; Imbert et al., 1996). The CAG repeat lengths inversely correlate with the age of onset, and tend to expand further during paternal transmission with anticipation (Riess et al., 1997).

Patients with SCA2 show gait ataxia, which is characteristically accompanied by slow saccades, kinetic or postural tremor, and decrease of muscle tone and tendon reflexes. SCA2 patients may also have Parkinsonian features, dystonia, chorea, supranuclear ophthalmoplegia, and dementia. MRI of the brain shows non-specific cerebellar or pontocerebellar atrophy. Nerve conduction studies often show evidence of axonal sensory-motor neuropathy.

Macroscopic changes of SCA2 brains are characterized by dramatic cerebellar and pontine atrophy, similar to sporadic olivo-ponto-cerebellar atrophy (OPCA) (Figure 4). MRI of the brain of SCA2 patients clearly shows these dramatic changes (Figure 5). The most conspicuous microscopic findings are severe loss of Purkinje cells and cells in the pontine and olivary nuclei. Cells in the dentate nuclei and the substantia nigra are preserved. In advanced stages, neuronal losses are more extensive, involving regions such as substantia nigra, striatum, pallidum, and later even the neocortex. The spinal cord may also show severe atrophy of the dorsal columns and reduction in the number of neurons in the motor pool and Clarke's nuclei (Pang et al., 2000). This widespread degeneration pattern is clearly more extensive than most other spinocerebellar ataxias, and involves regions known to degenerate in Huntington disease and multi-system atrophy (Estrada et al., 1999). SCA2 has an intriguing pathophysiological difference from other polyglutamine diseases; ataxin-2, the protein product of the SCA2 gene, is diffusely and densely distributed in the cytoplasm in cerebellar neurons of SCA2 patients (Figure 6). The ataxin-2 loaded neurons are particularly numerous in the pontine nuclei (Huynh et al., 2000), and have been shown to contain ubiquitin (Koyano et al., 1999). SCA2 patients show the most severe overall synaptic destruction in cerebellum and brain stem (Koeppen et al., 2002).

FIGURE 4. The midsagittal section of the brain from a patient with SCA2. Severe cerebellar and pontine atrophy, wide interfolial spaces, and dilatation of the fourth ventricle and aqueduct are shown. The uvula and nodulus of the vermis are not significantly atrophied.

FIGURE 5. T1-weighted brain MRI of three SCA2 patients. Mediosagittal plane (A) and axial images at the level of the pons (B) show atrophy of the cerebellum and the brain stem in a 21 year-old-patient with the disease duration 5 years and 51 CAGs. Similar axial images show mild brain stem and cerebellar atrophy in a 61-year-old patient with 4 year duration and 36 CAGs (C), compared with more severe atrophy in a 39-year-old patient with a 14 year disease duration and 43 CAGs (D).

FIGURE 6. Ataxin-2 in the cerebellum of SCA2 patients. Levels of ataxin-2 immunoreactivity in the cerebellum from a 49-year-old female with SCA2 (B, E and H), and her 41-year-old daughter with SCA2 (C, F and I) are more diffuse and intense than in the cerebellum of a 49-year-old female control (A, D and G). A-C are cerebellar cortex, D-F are Purkinje cells, G and I are dentate neurons, and H is cerebellar granule neurons. Nuclear immunoreactivity was seen in some granular neurons (H).

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Drosophila Models of Huntington Disease

LESLIE M. THOMPSON, J. LAWRENCE MARSH, in Animal Models of Movement Disorders, 2005

E. PolyQ Models

Investigators tested the issue of whether polyQ chains are toxic to neurons independent of any disease gene context by engineering transgenic flies expressing various forms of polyQ peptides [27,28]. These studies show that polyQ peptides alone are intrinsically cytotoxic and cause neuronal degeneration and early adult death and that the inclusion of other amino acids modified and generally reduced toxicity. The influence of protein context is further highlighted by the insertion of an expanded polyQ repeat within a cytosolic protein, Disheveled, that normally contains a polyQ repeat tract [27]. When this repeat was expanded, polyglutamine-mediated phenotypes were not observed and effects on protein activity were modest.

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Transgenic Drosophila

Transgenic Drosophila containing LacZ under the control of hsp70 promoter has extensively contributed to the understanding of stress response mechanism underlying heavy metal and/or pesticide toxicity (reviewed in Gupta et al., 2010).

From: Animal Biotechnology (Second Edition), 2020

Related terms:

Wild Type

Alpha-Synuclein

Nested Gene

Drosophila

Phenotype

Mutation

Lifespan

Fruit Fly Model

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Biology of Serpins

Thomas R. Jahn, ... Damian C. Crowther, in Methods in Enzymology, 2011

Abstract

Transgenic Drosophila melanogaster have been used to model both the physiological and pathological behavior of serpins. The ability to generate flies expressing serpins and to rapidly assess associated phenotypes contributes to the power of this paradigm. While providing a whole-organism model of serpinopathies the powerful toolkit of genetic interventions allows precise molecular dissection of important biological pathways. In this chapter, we summarize the contribution that flies have made to the serpin field and then describe some of the experimental methods that are employed in these studies. In particular, we will describe the generation of transgenic flies, the assessment of phenotypes, and the principles of how to perform a genetic screen.

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Drosophila Models of Huntington Disease

LESLIE M. THOMPSON, J. LAWRENCE MARSH, in Animal Models of Movement Disorders, 2005

B. Spinocerebellar Ataxia 3 (Machado-Joseph Disease)

Transgenic Drosophila expressing a truncated form of the human SCA3/MJD protein in neurons exhibited nuclear inclusions and late-onset cellular degeneration [24]. Cell death appeared to be apoptotic based on the observation that co-expression of a viral antiapoptotic gene, P35, mitigated the pathogenesis. This suppression was not observed in models of HD [20], suggesting that while many features are shared among the different Drosophila disease models, some aspects of biology will be unique. Even with high levels of expression and significant cellular toxicities, tissue specific effects were observed. Expression in the nervous system caused severe consequences whereas comparable targeted expression in epithelial cells generated less toxicity, confirming an in vivo system where some cells are more vulnerable to polyglutamine expression. Understanding the basis of this selective susceptibility is a major unresolved aspect of polyQ pathogenesis.

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The Microbiome in Health and Disease

Matthew Silbergleit, ... Ikuko Kato, in Progress in Molecular Biology and Translational Science, 2020

8.5 AvrA toxicity in animal models

In both transgenic Drosophila and murine models, AvrA was shown to potently inhibit c-Jun N-terminal kinase (JNK) and NF-κB signaling pathways and, in the mouse intestinal mucosa, infection with AvrA-positive Salmonella dampened the proapoptotic innate immune response to Salmonella, suggesting a survival strategy for intracellular pathogen, where the bacteria elicit transient inflammation but do not destroy epithelial cells.271 In a murine short-term infection model, epithelial cell proliferation of the large intestine was increased in mice infected with AvrA-positive Salmonella than those infected with AvrA-negative Salmonella.276,278 Further studies have revealed that infection with AvrA-expressing Salmonella increased the Wnt/β-catenin activity, intestinal stem cell population, and cell proliferation in the infected intestinal mucosa.280 In their murine chronic infection model, Lu et al. found that AvrA persistently regulated β-catenin post-translational modifications, including phosphorylation and acetylation. Moreover, the upstream regulator Akt, transcription factors, T cell factors, nuclear β-catenin, and β-catenin target genes were enhanced in mice infected with Salmonella-expressing AvrA, suggesting its functional role in promoting intestinal renewal.281 The subsequent studies using mouse chemical carcinogenesis models have demonstrated that colorectal tumor incidence indeed markedly increased (almost doubled) in the AvrA + Salmonella infected mice, compared with mice without bacterial gavage or mice infected with AvrA − Salmonella.282 While the Wnt1 protein level in the mouse intestine was decreased in the AvrA + infected mice compared with those infected with an AvrA − strain,279 Wint11 expression was enhanced in animals infected with AvrA + strains.277 In the same animal model colonized with Salmonella AvrA-sufficient or AvrA-deficient bacterial strains, AvrA-expressing bacteria activated the STAT3 pathway, which is predicted to enhance proliferation and promote tumorigenesis, providing new insights regarding a STAT3-dependent mechanism by which the specific bacterial product AvrA enhances the development of infection-associated colon cancer.283

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Homology Effects

Judith A. Kassis, in Advances in Genetics, 2002

I. Introduction

A commonly used vector for making transgenic Drosophila is the P-element-based vector CaSpeR (Pirrotta, 1988). CaSpeR includes a minigene for the selectable marker white; expression of the white minigene causes mutant white-eyed flies to have colored eyes. The eye color is sensitive to the dose of white, i.e., the higher the levels of white mRNA, the darker the eye color. At saturation, a red, wild-type color is observed. In addition, the white gene is cell autonomous. Thus, different cells in the eye can have different colors if white expression levels differ. Mini-white lacks most of the regulatory DNA present at the endogenous white locus. It contains only a minimal white promoter that is expressed at a low level in its ground state and produces yellow-eyed flies (Kellum and Schedl, 1991). However, the eye color of CaSpeR transgenic flies varies tremendously with the site of insertion, since flanking genomic enhancers and silencers influence its expression. These properties of the mini-white gene make it a sensitive marker of gene expression and led to the discovery of pairing-sensitive silencing and related phenomena.

In 1991 we reported that a particular fragment of cis-regulatory DNA from the segmentation gene engrailed had an unusual effect on the expression of the mini-white gene in CaSpeR (Kassis et al., 1991). Normally, flies homozygous for a given CaSpeR insertion have a darker eye color than heterozygotes. However, when a particular engrailed DNA fragment was included in that transposon, homozygotes often had a lighter eye color than heterozygotes. Thus, the engrailed DNA caused the mini-white gene to be repressed in homozygotes. Chromosomes are somatically paired in Drosophila (Metz, 1916; Lifschytz and Harevan, 1982), thus, in the homozygous state the two CaSpeR insertions would be near each other in the genome. Duplicating the transposon on the same chromosome also led to mini-white silencing. Silencing did not occur when two insertions were located on different chromosomes. I called this type of repression "pairing-sensitive suppression" (Kassis, 1994), since the repression of white was dependent on two copies of the transposon in close proximity in the genome—either in cis (on the same chromosome) or in trans (on homologous chromosomes). We originally proposed that "pairing-sensitive" (PS) sites might be involved in mediating interactions between regulatory DNAs located throughout the 70-kb engrailed locus; i.e., PS sites located near the engrailed promoter might allow it to interact with distant silencers and enhancers (Kassis et al., 1991; Kassis, 1994). Since our initial report, DNA fragments from many genes have been found to cause mini-white silencing in homozygotes and/or heterozygotes (Table 14.1). Many of these DNA fragments are "Polycomb group response elements" (PREs). PREs are regulatory DNA necessary for the action of the Polycomb group (PcG) of transcriptional repressors (Simon et al., 1993; Chan et al., 1994; reviewed in Pirrotta, 1997a, 1997b). Here I attempt a comprehensive review of the DNA fragments that cause silencing of mini-white in CaSpeR. I will describe the different types of silencing observed and explore the relationships between PREs and PS sites. The data suggest that not all PREs are PS sites, and vice versa. However, the data suggest that PS sites may potentiate the action of PREs and may be integral parts of these elements. I will also discuss another related phenomenon, transposon homing. Both pairing-sensitive silencing and transposon homing may be due to the formation of protein complexes that can bring together distant DNA sites.

Table 14.1. Regulatory DNA Reported to Cause Pairing-Sensitive Silencing

Regulatory DNAVariegatedaPatternedbPSScEye color sensitive to PcG mutationsdPRE in embryosePHO binding sitesfpho sensitivegengrailed 2.6-kbnoyesyesnoyesyesND Fragment 8hnoyesyesNDyesyesyes Fragment 5hnoyesyesNDNDyesNDpolyhomeoticyesyesyesyesNDyesNDescargotnonoyesNDNDyesNDeven-skippedyesyesyesNDNDyesNDSex combs reduced SSRN + 8.2-kb XbalyesnoyesyesNDeyesnog SSRN+ 5.5-kb Hind IIInonoyesnoNDyesND 10-kb XbaIyesnonoyesyesyesnogProbosipedia 0.58-kbyesyesyesnoNDyesND pbZRyesyesnonoNDyesND 2.1-kb enhancer + pbZRyesyesyesnoNDyesNDiab-2 (1.7) enhancerNRNRyesnonoyesyesbxd PRE 2212H6.5yesNRNRyesyesyesND 1.5-kb EcoRI-StyIyesNRyesNDyesyesND HS × 3iyesnoyeskNDNDnoND HH2 × 4iyesnoyeskNDNDnoND HA × 4iyesnoyeskNDNDnoND AB × 6inoyesyesyesnoyesND BP × 6iyesnoyesyesyesyesND PF × 4iyesnoyesyesnoyesNDMcp2.9-kbyesyesyesyesyesyesyes810-bp corejnononoNDyesyesNDcore + ftz DNAjnoyesyesyesNDyesyesiab-7 PREyesyesyesyesyesyesyesiab-8 PREyesNRyesNDyesyesND

NR, not reported; ND, not done.

aEye color variegated in heterozygotes. Pigmentation occurs in patches that are variable from one eye to the next.bPatterned eyes are those in which one portion of the eye is regularly and reproducibly more highly pigmented than others. This can occur in either homo- or heterozygotes. For engrailed DNA, this occurs in about 10% of insertion sites.cPSS-pairing-sensitive silencing. The eye color of homozygotes is lighter than heterozygotes. A fragment is said to cause pairing-sensitive silencing if mini-white is silenced in greater than 20% of chromosomal insertion sites.dHeterozygous mutations in PcG genes cause an increase in eye color of insertions at a high percentage of chromosomal insertion sites. Effect of pho mutations is listed separately.ePcG-dependent silencing of a reporter construct in embryos. Although the activity of the 8.2-kb Xbal fragment from Scr was tested in embryos, it was not tested in a vector designed to test for PRE function.fAt least one consensus binding site for PHO present in the DNA.gEye color is darker in a pho mutant. For the engrailed fragment, effects were not seen in pho/+ heterozygotes, but were observed in pho1/phocv flies. The Scr fragments were tested only in pho heterozygotes.hFragment 8 and Fragment 5 contain nonoverlapping subsets of the 2.6-kb engrailed fragment.iThese fragments contain subsets of the 1.5-kb EcoRI-StyI fragment. These fragments were tested as multimers (i.e., HS × 3 means that three copies of the HS fragment were present in CaSpeR).jThis is a subset of the 2.9-kb fragment. The Mcp core is required for the activity of the 2.9-kb fragment. Although the Mcp core has no silencing activity on its own, when combined with ftz (or yellow) regulatory sequences, silencing is observed.kHomozygotes have variegated eyes. Complete silencing is not observed.

References: engrailed: Kassis et al., 1991; Kassis, 1994; Brown et al., 1998; J. Americo, M. Fujioka, M. Whiteley, J. B. Jaynes, and J. A. Kassis, in preparation. polyhomeotic: Fauvarque and Dura, 1993. escargot: Kassis, 1994. even-skipped: Fujioka et al., 1999; M. Fujioka and J. Jaynes, personal communication. Sex combs reduced: Gindhart and Kaufman, 1995. Probosipedia: Kapoun and Kaufman, 1995. bxd PRE: Chan et al., 1994; Horard et al., 2000; Sigrist and Pirrotta, 1997; V. Pirrotta, personal communication. Mcp: Muller et al., 1999; M. Muller, personal communication; Bustaria et al., 1997. iab-7 PRE: Hagstrom et al., 1997; Cavalli and Paro 1999; Mishra et al., 2001. iab-8 PRE: Barges et al., 2000; Zhou et al., 1999.

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The Chromodomain of Polycomb

S. Qin, ... J. Min, in Polycomb Group Proteins, 2017

Polycomb Chromodomain Specifically Recognizes H3K27me3

On the basis of elegant studies using Drosophila transgenic cell lines as well as transient tissue culture cells, it was revealed that the chromodomain of Pc is absolutely required for the binding of Pc protein to chromatin, which is important for its functions [12]. Specifically, mutations of the Pc chromodomain, including deletion as well as point mutations, abolish the chromosomal binding capability of the Pc protein, whereas carboxy-terminal truncations of the Pc protein do not affect its chromosomal binding ability [12]. Interestingly, a similar phenomenon was also observed for HP1. The HP1 chromodomain, like that of the Pc protein, has chromosome-binding activities, but it binds at distinct chromosomal sites. Analogously, point mutations in the HP1 chromodomain effectively nullify the ability of HP1 to promote gene silencing [13]. Furthermore, in the fission yeast Schizosaccharomyces pombe, the correct localization of Swi6 (the HP1 equivalent) depends on Clr4, a homolog of histone methyltransferase SUV39H1 that specifically methylates H3K9me3 [14,15]. These results led scientists to propose the HP1 chromodomain as a reader of the H3K9me3 mark 10 years after the identification of the chromodomain [10,11]. A point mutation in the chromodomain, which disrupts the gene silencing activity of HP1 in Drosophila, also abolishes its methyl-lysine–binding activity [10]. Genetic and biochemical analyses in S. pombe showed that the methyltransferase activity of Clr4 is necessary for the targeted localization of Swi6 at centromeric heterochromatin and for gene silencing [10]. These results provide a stepwise model for the formation of transcriptionally silent heterochromatin: SUV39H1/Clr4 places a "methyl mark" on histone H3, which is then recognized by HP1/Swi6 through its chromodomain [10]. In particular, the association of HP1 with methylated mononucleosomes could be completely disrupted by the addition of excess H3K9me3 peptide, suggesting that HP1 recognizes H3K9me3 in the context of mononucleosome [10]. Functionally, the interaction of HP1 with H3K9me3 is essential for the epigenetic control of heterochromatin assembly in vivo [10,16]. Therefore, the finding that the HP1 chromodomain specifically binds to H3K9me3 is a major breakthrough in the field of chromatin biology. Soon after that, the Pc chromodomain was reported to bind H3K27me3, a mark of repressed homeotic genes generated by a multiprotein complex containing E(z) and Esc, or their human counterparts EZH2 and EED [6–8].

The amino acid sequences surrounding lysine 9 and lysine 27 in the H3 tail are very similar. In particular, they share a consensus sequence ARKS (Ala-Arg-Lys-Ser) (Fig. 3.1A). However, quantitative measurements showed a strong preference of the chromodomain of Drosophila Pc protein for H3K27me3, whereas the chromodomain of Drosophila HP1 protein binds preferentially to H3K9me3 (Table 3.1) [17]. Specifically, the Pc protein's dissociation constant (Kd) is about 5 μM for the H3K27me3 peptide, but becomes 25-fold weaker or about 125 μM for the H3K9me3 peptide. In striking contrast, the HP1 chromodomain bound to the H3K9me3 peptide with an affinity of about 4 μM, but bound 16-fold weaker to the H3K27me3 peptide with an affinity of about 64 μM [17]. Furthermore, the binding affinities of the Pc protein to H3K27me1/2 peptides were about five times weaker than its binding affinity to the H3K27me3 peptide, but were still much stronger than its binding affinity to the H3K9me3 peptide. On the other hand, the binding affinities of HP1 to H3K9me1/2 peptides decreased about 15-fold and 2-fold, respectively, when compared with its binding affinity to H3K9me3 peptide. Collectively, these results indicate that Pc prefers H3K27me3, whereas HP1 prefers both H3K9me3 and H3K9me2 [17].

Figure 3.1. Sequence alignments. (A) ARKS (Ala-Arg-Lys-Ser)-like motif-containing histone and nonhistone targets of chromodomain; the methyl-lysine site is highlighted in yellow and the ARKS-like motif is underlined. The residues of particular notice are shown in red. H3t, testis-specific histone H3 variant. (B) Chromodomains of Pc and HP1 proteins from Drosophila and human; the secondary structure elements of Pc are shown on top and the theoretical isoelectric points of each protein are shown on the right. The residues involved in the methyl-lysine interactions are highlighted in yellow. The "hydrophobic clasp" of Pc proteins and the "polar clasp" of HP1 proteins are highlighted in cyan and magenta, respectively.

Table 3.1. Dissociation Constants (μM) of Polycomb and HP1 Chromodomains to H3K9me and H3K27me Peptides [17,25,26]

K9me1K9me2K9me3K27me1K27me2K27me3Fruit flyPc>1000>1000125 ± 2820 ± 328 ± 45 ± 1MouseCbx2382 ± 29396 ± 3641 ± 6>500143 ± 444 ± 5Cbx4>500261 ± 2149 ± 9>500>500150 ± 20Cbx6>500>500>500>500>500330 ± 120Cbx7267 ± 4879 ± 1212 ± 3>500136 ± 2322 ± 5Cbx8>500>500>500>500>500165 ± 20HumanCBX2>500185 ± 20CBX470 ± 7205 ± 20CBX6>500>500CBX755 ± 5110 ± 17CBX8>500>500Fruit flyHP146 ± 97 ± 24 ± 164 ± 7MouseCbx51072 ± 1>500286204HumanCBX15 ± 2NBCBX315 ± 8NBCBX530 ± 5NB

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Calcium in Living Cells

Michael Whitaker, in Methods in Cell Biology, 2010

3 Camgaroos and Inverse Pericam

UAS/Gal 4 expression was used to create transgenic Drosophila that expressed camgaroos-1 and-2 in the mushroom bodies of adult brain (Yu et al., 2003). Dissected fly brains were used. Camgaroo-2 fluorescence in the mushroom bodies was much more intense than that of camgaroo-1, but the camgaroo-1 emission ratio signal on potassium depolarization was more than double that of camgaroo-2 (38% vs. 14% in the mushroom body lobe and 83% vs. 28% in the mushroom body itself ). It was shown that these increases were not due to changes in pH. Application of the putative mushroom body transmitter, acetylcholine, causes ratio changes of a few percent. In this setting, camgaroo-2, although brighter, showed substantially lower ratio changes than camgaroo-1; it also underwent significantly faster photobleaching.

Inverse pericam is an intensity-coded sensor that decreases its fluorescence as calcium increases. Addition of DsRed2 to the C-terminal of inverse pericam produces a ratiometric indicator whose 615/510 nm emission ratio increases as calcium increases. This indicator (DsRed2-referenced inverse pericam (DRIP)) requires dual excitation and dual emission optics (Shimozono et al., 2004). The DsRed2 fluorescence is a passive, calcium-independent signal that is proportional to the concentration of the sensor and helps control for alterations in overall fluorescence intensity due for example to movement artifacts. DRIP was expressed transgenically in worms under the control of the myo 2 promoter that is specific for pharyngeal muscle. Ratio changes of 30–40% were measured in worms undergoing fast pharyngeal pumping.

After screening six sensors (flash pericam, inverse pericam, G-CaMP, camgaroo-2, YC2.12, and YC3.12) for calcium sensitivity in stably transfected fibroblast cell lines, the two with the greatest dynamic range (inverse pericam: − 40% and camgaroo-2: + 170%), together with YC3.12 that gave inconclusive results in the fibroblast expression screen but is optimized for expression at 37 °C, were used to generate transgenic mice under the control of the TET expression system (Hasan et al., 2004); the TET system allows tissue-specific expression by crossing the TET mice with mice expressing the TET transactivator under tissue-specific control. TET sensor mice were crossed with a line expressing the transactivator under the control of the alpha-calmodulin/calcium dependent kinase II (αCamKII) promoter. All mice developed normally. Five highly expressing lines were obtained out of 36 transgenic lines: two YC3.12, two camgaroo-2, and one inverse pericam. Expression patterns in brain slices and excised retina were analyzed by two-photon microscopy. They appeared to be mosaic, not mapping directly to the known patterns of αCamKII expression. Neocortical expression could also be imaged through the thinned skull in anaesthetized mice. Two photon fluoresence recovery after photobleaching suggested that as much as half the fluorescence signal was immobile and this together with punctuate staining patterns suggested that this immobile sensor fraction might be due to interaction between the M13 and CaM moieties of the sensors and their normal cellular targets. Cellular and synaptic stimulation of pyramidal neurones in cortical slices using sharp and patch microelectroded gave 5–10% increases in 535 nm fluorescence using wide field imaging and around 20–100% for camgaroo-2 and − 30% for inverse pericam using two photon imaging. In the retina, a ganglion cell subset was strongly labeled in YC3.1-expressing mice, but no light-evoked responses were detected. In camgaroo-2 expressing lines, bleaching occurred in the retina too quickly for measurements to be made. In one inverse pericam-expressing mouse, 7 of 12 ganglion cells tested showed a transient decrease in fluorescence attributable to a calcium increase in response to light. Sensors were imaged in the olfactory bulb in vivo using wide field microscopy. Camgaroo-2 expressing mice showed a 1–3% increase in response to odors, while inverse pericam gave ~ 8% decrease. Each distinct odor evoked a unique pattern of activity, similar odors evoking similar patterns.

This thoughtful study established four main facts: around half of the transgenically expressed cameleon family sensor was immobile; this reduced sensitivity and made quantitation of the calcium signals impossible; nonetheless, it was possible to observe patterns of neuronal activity; YC3.12 was not an effective transgenic sensor. The study also reports unpublished experiments in which transgenic mice expressing YC3.0 under the control of a β-actin promoter gave only 1–2% ratio changes during wide filed imaging in cerebellar slices. The high proportion of immobile sensor in transgenic animals remains for the moment inexplicable—it was not seen in the stably transfected fibroblast lines.

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Membrane Proteins—Production and Functional Characterization

Yvonne Hackmann, ... Irmgard Sinning, in Methods in Enzymology, 2015

4 Case Study: Signal Peptide Peptidase

We applied the protocol outlined in this chapter to generate transgenic Drosophila expressing HsSPP and DmSPP in the photoreceptor cells. As shown in Fig. 4A–C, the GFP fusion construct is specifically expressed in the eye of transgenic flies. When we analyzed the exact localization of HsSPP in the photoreceptor cells, we noticed that it was predominantly retained by its KKXX motif in the enlarged ER and in contrast to rhodopsin did not localize to the rhabdomeres (compare Fig. 4D + E and F + G). We purified DmSPP from fly eye membranes and analyzed the elution by Western blot. The height of the observed band corresponds to DmSPP (43 kDa) appended with GFP (27 kDa) (Fig. 4H).

Figure 4. Expression of human and Drosophila SPP in photoreceptor cells. (A–C) Bright field (A) and fluorescent (B + C) image of a transgenic Drosophila expressing human (A + B) or Drosophila SPP (C). Inset in (B) and (C) show the specific expression in the facetted ommatidia. (D–G) Bright field and epifluorescence images of extracted rhabdomeres from a transgenic fly expressing rhodopsin–GFP (D + E) or HsSPP–GFP (F + G). (H) Western blot image showing a single band for DmSPP–GFP (~ 70 kDa) expressed in transgenic Drosophila and purified by nickel affinity chromatography. Detection was performed with an anti-GFP antibody.

This example shows the broad applicability of the Drosophila expression system in that even an ER-resident protein can be successfully expressed and purified. Interestingly though, expression of SPP in the photoreceptor cells using our standard strategy led to a dramatic increase in ER, which allowed us to purify a substantial amount of DmSPP from the eyes, comparable to the "canonical" expression in rhabdomeres. At the same time, the advantages of the fly eye system were maintained, such as the homogeneity of the sample.

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Polycomb Group Response Elements in Drosophila and Vertebrates

Judith A. Kassis, J. Lesley Brown, in Advances in Genetics, 2013

3.2 Homing and PcG target genes

P-element based vectors have been used to make transgenic Drosophila for about 30 years (Rubin & Spradling, 1982). Aside from some hotspots and a preference to insert near the 5′ end of transcription units, P-based vectors insert in the genome in a relatively nonselective manner and as such, have been used as a tool to generate thousands of transposon-induced mutations in Drosophila (Bellen et al., 2011). In 1990, Hama, Ali, and Kornberg reported that P-based constructs that contain en regulatory DNA (P[en]) insert near or into the en/inv domain at a very high frequency. They called this phenomenon "homing" (Hama, Ali, & Kornberg, 1990). Further studies on P[en] homing by en regulatory DNA showed that a 2-kb fragment, extending from − 2.4- to − 0.4-kb upstream of the en transcription start site was sufficient for homing, and that the en PREs contributed to the homing activity of this fragment (Cheng et al., 2012). Further, P[en] inserted near PcG targets at an increased frequency compared with a P-based vector used to generate insertions for the Drosophila gene disruption project (Cheng et al., 2012). These data suggest that either the chromatin structure or PcG proteins themselves bring P[en] to the region of PcG-regulated loci. This is consistent with the view that PcG targets colocalize in the nucleus in PcG bodies (Delest et al., 2012; Pirrotta & Li, 2012) and suggest that these bodies also occur in germ cells where homing occurs. Transgenes containing polyhomeotic PREs and BX-C PREs have also been reported to insert at a high frequency in or near PcG target genes (reviewed in Kassis, 2002).

Fragments of DNA from the BX-C and eve PcG targets have been shown to mediate homing (Bender & Hudson, 2000; Fujioka, Wu, & Jaynes, 2009). Like P[en], insertions mediated by the BX-C and eve homing fragments occurred over a large region, causing insertions throughout and near the parent locus. For these two cases, the fragments that mediate homing are most likely insulators, not PREs (Bender & Hudson, 2000; Fujioka et al., 2009). Anecdotally, 1/5 insertions of a P-based vector that contained one of the Psc/Su(z)2 PREs was inserted near the promoter of the Su(z)12 gene, strongly suggesting that this fragment mediates homing. Note that one reported case of homing, that of the linotte gene (now called drl; Taillenbourg & Dura, 1999), is not reported to be a PcG target gene in embryos, larvae, or S2 cells; however, it is possible it could be a PcG-regulated gene in germ cells where P-element homing occurs.

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Aging and Stress, Biology of*

M.A. Horan, ... G.J. Lithgow, in Encyclopedia of Stress (Second Edition), 2007

Drosophila melanogaster

D. melanogaster is an insect has been widely used in both stress and aging studies. Transgenic Drosophila lines containing additional copies of the Cu/ZnSOD and catalase genes have been generated, and most lines that have elevated antioxidant–enzyme levels are also long-lived. This demonstrates the importance of these two enzymes in determining life span. More recent experiments suggest that the expression of MnSOD in neurons alone is sufficient to extend life span.

The transgenic Drosophila experimental system has also been used to test the role of molecular chaperones in determining aging rates. In these experiments, it was shown that additional copies of heat shock protein (HSP)-70 could reduce age-specific mortality rates in Drosophila. Just how HSP-70 retards aging is unknown, but these findings are consistent with other experiments in both C. elegans and Drosophila in which animals given a mild thermal stress and allowed to recover go on to develop acquired thermotolerance and exhibit extended life span. In these experiments, HSPs are induced and may be the principal cause of slowed aging.

Drosophila has also been used for studying the effects of multiple genes on stress responses and aging. Selection has been undertaken for Drosophila lines that differ in their levels of stress tolerance, and at least one laboratory has demonstrated that selection for stress-resistance produces lines with increased longevity. In addition, some long-lived Drosophila lines, selected for late reproduction, are stress resistant.

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Animal Models of Spinocerebellar Ataxia Type 1

Puneet Opal, Harry T. Orr, in Movement Disorders (Second Edition), 2015

63.3 Concluding Remarks

Animal models of the polyglutamine diseases have been extremely useful in gaining insights into the pathogenic mechanisms of these disorders. In the case of SCA1, numerous transgenic mouse and Drosophila models have been used to uncover many important aspects of the disease. At this time, the data indicate that nuclear localization of the mutant protein is required for disease, nuclear inclusions are not necessary to initiate disease, and protein folding and clearance pathways are vital to the disease process. In fact, formation of inclusions may be a cellular defense mechanism against polyglutamine-mediated toxicity. Neurons that are the last to form inclusions are the most vulnerable to neurodegeneration. Hence, neuron-specific, rate-limiting components of protein folding and clearance pathways may be responsible for the cell-specific neurodegeneration observed in each of the polyglutamine disorders. It also appears that transcriptional dysregulation underlies some aspects of pathogenesis. Last, events outside the CAG repeat in cis can modulate pathogenesis. Mutation of serine 776 of ATXN1 to alanine dampened the pathogenicity of mutant ATXN1. Moreover, this mutation rendered mutant ATXN1 substantially more soluble, perhaps allowing for more prompt disposal of the mutant protein by the neuron.

An important and relevant conclusion has come out of the numerous SCA1 transgenic mouse studies; the disease pathway appears to be acting through ATXN1's normal cellular pathways (Table 63.1). Aspects involved in the normal cellular role of ATXN1 appear to require nuclear localization and phosphorylation of the protein at serine 776. Both wild-type and mutant ATXN1 are found primarily in the nucleus of neurons. Preventing the mutant protein from entering the nucleus by mutating the NLS signal prevents disease. Second, both wild-type and mutant ATXN1 are phosphorylated. Mutation of serine 776 in the mutant protein mitigates disease. Thus, defining the role of phosphorylated ATXN1 in the nucleus may provide further insights into SCA1 pathogenesis.

TABLE 63.1. Biological Properties of ATXN1 in SCA1 Transgenic Mice

TransgenicpQ LengthLocalizationInclusionsP-S7761DiseaseA0230QNuclear−+NoB0582QNuclear+++++YesK772T82QCytoplasmic−±NoΔSAD77QNuclear−+YesA77682QNuclear++NoATXN1[30Q]-d77630QNuclear−Phosphomimetic (but no neuronal loss)ATXN1[30Q]-d776ATXN1[82Q]-d77682QNuclear++++Phosphomimetic acceleratedATXN1[82Q]-d776Conditional SCA1[82Q] miceNuclearUsed to study reversibility of phenotype or to delay onset of mutant proteinExpressionConditional SCA1[82Q] miceATXN1[30Q]-d77630QNuclear−Phosphomimetic (but no neuronal loss)ATXN1[30Q]-d776ATXN1[82Q]-d77682QNuclear++++Phosphomimetic acceleratedATXN1[82Q]-d776

ATXN1, ataxin-1; SCA1, spinocerebellar ataxia type 1.

1Serine 776 phosphorylated ATXN1 determined by immunohistochemical analysis.

From Emamian et al. (2003).

Progress in understanding the disease process in SCA1 has uncovered numerous potential sites for therapeutic intervention, including upregulation of molecular chaperone or protein degradation pathways, inhibition of ATXN1 message by RNAi or microRNA strategies, regulation of subcellular localization, and altering the solubility of the mutant protein. In addition, modulation of ATXN1 phosphorylation, reversing transcriptional dysregulation with drugs or strategic gene products such as neurotrophic factors, and targeting mutant-specific protein–protein interactions are promising therapeutic avenues to further investigation.

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Drosophila

Drosophila develop ethanol tolerance after a single exposure, as evidenced by their increased resistance to sedation and delayed slowing of locomotor behavior upon a repeated exposure to ethanol vapor (Scholz et al., 2000).

From: Advances in Genetics, 2002

Related terms:

Nested Gene

Phenotype

Mutation

Yeast

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Insect

Mammal

Caenorhabditis Elegans

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Drosophila

K. Ravi Ram, D. Kar Chowdhuri, in Animal Biotechnology, 2014

Principle

Drosophila is a well-studied and highly tractable genetic model system to decipher the molecular mechanisms underlying various biological processes. The completion of genome sequencing and annotation discovered the high degree of conservation of fundamental biological processes between Drosophila and mammals. This has prompted biotechnologists to utilize Drosophila to understand the molecular basis of human diseases. The ease with which Drosophila transgenics can be created was also been instrumental in the success of this model for understanding human diseases. Using a plethora of molecular tools available for Drosophila, biotechnologists genetically manipulated Drosophila by either inserting human genes in the fly genome or by modifying the function of human disease orthologs in Drosophila and sensibly developed Drosophila-based models for human diseases.

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Fly Models of Human Diseases

M. Sonoshita, R.L. Cagan, in Current Topics in Developmental Biology, 2017

4 Conclusion

Drosophila has a long and proud history of solving complex problems with powerful genetics. In the developmental field, flies have proven an excellent discovery tool: genetic screens in particular identify new and surprising mechanisms, a "hypothesis-building" tool that is rapid and inexpensive. These same qualities make model systems such as Drosophila useful in translational work: surprising new mechanisms and therapeutics can be identified that address the complexity of diseases such as cancer. Similar to problems in development, the power of Drosophila lies in its ability to take a fresh, whole animal look at a disease that has joined heart disease as the major sources of mortality of Americans. Flies will not replace mammalian models, but similar to their role in development they provide a powerful and complementary tool.

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Genetics of Memory in Drosophila

E.D. Gonzales, J.C.P. Yin, in Encyclopedia of Behavioral Neuroscience, 2010

Drosophila neurogenetics has revolutionized learning and memory research using classical forward and reverse genetics to identify cellular and molecular pathways (including the cyclic AMP signaling cascade and translational regulation) integral to memory formation. Some principal genes in memory are discussed. Advances in genetic technology now permit temporal and spatial manipulation of transgene expression, as well as selective activation/inactivation of subsets of neurons. Novel tools are being used to map the olfactory memory circuitry and determine molecular components of distinct memory phases. Drosophila neurogenetics is transitioning from a discovery-driven discipline to one driven by defined hypotheses.

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Drosophila

Rami R. Ajjuri, ... Janis M. O'Donnell, in Movement Disorders (Second Edition), 2015

Abstract

The fruit fly, Drosophila melanogaster, has been the focus of genetics research for over a century. One consequence of this long history as a genetic model is a remarkably robust and diverse set of genetic tools, including loss of function mutations in most genes, a powerful transgenesis system, whole-genome RNA interference libraries, and expression systems with temporal and spatial specificity. Since approximately 75% of known human disease genes are conserved in Drosophila, these powerful genetic reagents available for Drosophila research are increasingly being applied to human disease research. Relative to the mammalian brain, the fruit fly brain is greatly simplified, yet retains significant complexity, with conserved neurotransmitter systems. These features, coupled with simple, quantitative mobility assays, make the fly a valuable model for movement disorders research. Applications include the investigation of pathogenic mechanisms using either human disease transgenes or Drosophila homologs, genetic modifier screens to detect functionally related genes, and therapeutic drug screens.

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Axonal Ensheathment and Intercellular Barrier Formation in Drosophila

Kevin Blauth, ... Manzoor A. Bhat, in International Review of Cell and Molecular Biology, 2010

6 Concluding Remarks

Drosophila ensheathment research continues to be a field which yields interesting and relevant findings that provide insights into nervous system function, and are often highly translational to studies in vertebrates. One area of Drosophila research which remains to be explored in depth is axonal ensheathment by glia in the brain. While CNS glial subtypes have been classified based on morphology, location, and molecular profile, much work regarding the diverse functions of brain glia remains to be accomplished. One interesting finding was the discovery that swiss cheese (sws) mutants exhibit an excess of axonal wrapping by glia in the pupal and adult Drosophila brain, suggesting a yet-to-be defined role for sws in axonal ensheathment (Kretzschmar et al., 1997). Ultimately sws mutants experience neurodegeneration and shortened lifespan. This may be relevant to human disorders such as Charcot-Marie-Tooth neuropathy, a heterogeneous group of demyelinating diseases (Kretzschmar et al., 1997). In contrast to sws mutants, drop-dead mutants exhibit incomplete wrapping of axons in the adult brain, and like sws mutants also ultimately leads to neurodegeneration (Buchanan and Benzer, 1993). These results point to an interesting potential field of inquiry in Drosophila: does improper ensheathment lead to neurodegeneration? Answering this question could have ramifications for vertebrate neurobiology, as the most common human demyelinating disorder, MS, becomes a largely neurodegenerative disorder in its later stages (Bjartmar and Trapp, 2003).

Several aspects of Drosophila axonal ensheathment resemble vertebrate myelination, and ensheathment research has already provided critical insights into vertebrate neuron–glial interactions. One aspect of Drosophila PNS ensheathment in particular that is highly relevant to vertebrate myelination is the formation of neuron–glial SJs between peripheral glia, which exhibits a high degree of molecular homology to vertebrate paranodal SJs. As mechanisms of ensheathment are in many ways highly homologous to mammalian myelination, the study of ensheathment is particularly pertinent to our understanding of human demyelinating disorders, and in the years ahead this research should yield results which will help us better understand and treat human nervous system disorders.

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Circadian Rhythms and Biological Clocks, Part A

Sofia Axelrod, ... Michael W. Young, in Methods in Enzymology, 2015

1 Introduction: Studying Circadian Behavior in the Fruit Fly, Drosophila melanogaster

Drosophila exhibits a multitude of innate and adaptive behaviors that allow researchers to study complex behaviors in a genetically tractable organism. Fruit flies, like all animals, need to correctly interpret and respond to their environment.

All life on earth is subject to the changes in light and temperature due to the earth's rotation. Many animals and plants exhibit diurnal or nocturnal behavior depending on their habitat and lifestyle. French scientist Jean-Jaques d'Ortous de Mairan discovered in 1729 that the daily opening and closing of plant leaves persisted in a dark room, indicating that this circadian behavior was not merely a reaction to light, but was effected by internal processes (de Mairan, 1729). It was not until over 200 years later that Konopka and Benzer analyzed the role of endogenous forces—genes—on the daily eclosion rhythm of the fruit fly Drosophila melanogaster (Konopka & Benzer, 1971). Since then, studies in Drosophila have played a prominent role in elucidating the genes and molecular mechanisms driving circadian behavior (Blau et al., 2007; Stanewsky, 2003). Analogous studies in mammals have revealed that these genes and mechanisms are largely conserved through evolution, indicating that these mechanisms are fundamental and underlie the conservation of animal behavior across evolution (Wager-Smith & Kay, 2000). Insights from Drosophila continue to have a broad impact on our understanding of circadian biology in vertebrates, including mechanisms of human circadian dysfunction that alter core clock components homologous to those characterized in Drosophila (Toh, Jones, He, Eide, & Hinz, 2001; Xu, Padiath, Shapiro, Jones, & Wu, 2005).

More recently, Drosophila has been used to study sleep, a behavior that is functionally linked to the circadian clock. Like other invertebrates that have been carefully examined (Campbell & Tobler, 1984), Drosophila displays the key behavioral attributes of sleep (Hendricks, Finn, Panckeri, & Chavkin, 2000; Shaw, Cirelli, Greenspan, & Tononi, 2000). These attributes include postural changes specific to sleep, immobility correlated with an increased arousal threshold, a homeostatic rebound in sleep duration and intensity after sleep deprivation, changes in brain electrical activity during sleep (Nitz, van Swinderen, Tononi, & Greenspan, 2002), and alterations in sleep by stimulants and hypnotics that parallel their effects in mammals (Hendricks et al., 2000; Shaw et al., 2000). Recently, it has been suggested that sleep in fruit flies, like that of humans, has different stages of depth during the sleep cycle (van Alphen, Yap, Kirszenblat, Kottler, & van Swinderen, 2013).

Although the adoption of Drosophila as a model organism to study sleep is relatively recent, considerable enthusiasm exists for its potential impact on our understanding of the molecular underpinnings of sleep regulation and function. Despite intensive studies over the past several decades, many aspects of sleep have remained elusive.

How sleep is regulated by circadian inputs and in a homeostatic manner (Borbély, 1982) is one focus of investigation. A second focus concerns the essential functions of sleep, as well as how sleep or lack thereof affects other physiological and behavioral processes. Theories for the functions of sleep invoke memory consolidation, synaptic downscaling, cell repair, metabolic and immune augmentation, and removal of toxins from the brain (Crocker & Sehgal, 2010; Xie et al., 2013). How sleep might function within the brain and somatic tissues to achieve these functions is still unclear, particularly at a molecular and cellular level, and these questions are the subject of several studies in Drosophila.

The impact of Drosophila in studies of circadian rhythms and sleep, as in other areas of biology, stems from the ability to perform large-scale and unbiased forward genetic screens and from powerful genetic tools that enable the fruits of these screens to be exploited (St Johnston, 2002). This chapter reviews recent genetic screens to gain further insight into the molecular basis circadian rhythm and sleep. We touch briefly on prior screens for rhythm and sleep mutants and proceed to the genetic screens for circadian rhythm and sleep that have been performed in recent years with an emphasis on transgenic mutagenesis in comparison with classical methods of genomic mutagenesis.

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Learning and Memory in Invertebrates: Drosophila

D.-B.G. Akalal, R.L. Davis, in Encyclopedia of Neuroscience, 2009

Drosophila are able to learn many different things. They can form associations between olfactory cues and aversive or appetitive reinforcers in operant or classical paradigms. They can form associations between geometric or color visual cues and the aversive stimulus of heat. Male Drosophila can also learn from unsuccessful courtship bouts with fertilized females and respond by limiting subsequent courtship. Successful performance in many of these learning paradigms requires the normal function of genes encoding components of cAMP signaling, cell adhesion and signaling, and ribonucleoprotein transport and local translation. Learning based completely or partly on olfactory stimuli requires the normal function of the mushroom bodies. Learning about visual cues is generally independent of the mushroom bodies but requires the function of neurons in the central complex.

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Recent advances in the use of Drosophila in neurobiology and neurodegeneration

Zhenming Yu, Nancy M. Bonini, in International Review of Neurobiology, 2011

III Conclusion

Drosophila is a powerful system for studying human trinucleotide repeat diseases, including polyglutamine diseases and RNA-based toxicity diseases. Genetic modifiers identified using forward genetic screens or candidate gene approaches have provided valuable insights into pathogenic mechanisms. With the rapid advance of fly molecular genetic tools, it is likely that additional screens will result in additional modifiers, leading to greater understanding of pathogenic mechanisms. This understanding should provide the foundation for therapeutic targets. In addition, Drosophila disease models can be used for chemical compound screens to identify possible therapeutic drugs (Chang et al., 2008; Garcia-Lopez et al., 2008). Thus, application of the fly system to human diseases, including the trinucleotide repeat diseases, will continue to prove a fruitful approach.

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Nucleic Acid Sensing and Immunity - Part B

Chaitali Khan, ... B.J. Rao, in International Review of Cell and Molecular Biology, 2019

5 Perspectives: Possible Role of DNA Repair Players in Patterning Tissue and Organism

Drosophila and C. elegans have been widely used as model organisms by many developmental biologists across the world. However, they are relatively under-studied in relation to DDR. It is only our work coupled with a few others, which have attempted to define the roles of various DDR players in functions other than DNA damage in Drosophila, with a special emphasis on developmental themes. A separate piece of work conducted by us also posits that Drosophila rad51 has functions which can regulate tissue patterning (Khan et al., 2017a,b). There is emerging evidence from work done in C. elegans, which shows that Notch can modulate DDR by binding to ATM and inactivates it. This mode of regulation of DDR by Notch appears to be independent of its transcriptional activity, and appears to be conserved in humans (Vermezovic et al., 2015). This is an example of how work done in nematodes has paved the way for further explorations in other organisms including humans. Interestingly, this study also shows how Notch appears to impair DDR signaling in the gonad cells of the nematode. Additionally, it also highlights the importance of using model organisms, to uncover tissue-specific relationships shared between complex signaling networks. This fact is especially important owing to different susceptibilities of various cells and tissues to DDR, which might be linked to their distinct cell cycle status during development (discussed in Section 2.4).

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Glycobiology

Shoko Nishihara, in Methods in Enzymology, 2010

1.1 Glycosaminoglycan structures

Drosophila GAGs comprise two major types, heparan sulfate (HS) and chondroitin sulfate (CS) (Fig. 15.1). Each of these types contains unique disaccharide repeats, (GlcNAcα1-4GlcAβ1-4)n in HS and (GalNAcβ1-4GlcAβ1-3)n in CS, but they share a so-called linkage region, which is a common tetrasaccharide structure, GlcAβ1-3Galβ1-3Galβ1-4Xylβ1-O-Ser. Structural analyses of GAGs from Drosophila melanogaster have been performed (Lawrence et al., 2008; Toyoda et al., 2000a,b; Yamada et al., 2002). Drosophila HS contains N-, 2-O-, and 6-O-sulfated disaccharide structures, and sulfation can give rise to mono-, di-, and tri-sulfated forms (Fig. 15.1). The relative amount of each type of sulfation is regulated developmentally, probably by the expression of sulfotransferases (Toyoda et al., 2000b). Drosophila CS contains nonsulfated and 4-O-sulfated disaccharide structures. The nonsulfated structure is the more common type and corresponds to approximately 80–90% of CS in adult flies (Fig. 15.1). However, the ratio of the two types is also regulated developmentally (Toyoda et al., 2000b). In addition to the sulfation described above, the xylosyl (Xyl) residue in the linkage region can be phosphorylated in both HS and CS. In adult flies, approximately 70% of Xyl residues in HS are unphosphorylated, whereas 100% of Xyl residues in CS are phosphorylated at the C2-position (Yamada et al., 2002).

Figure 15.1. Pattern diagrams of Drosophila glycosaminoglycan structures. Heparan sulfate (HS) and chondroitin sulfate (CS) share a common tetrasaccharide structure (the so-called linkage region), GlcAβ1-3Galβ1-3Galβ1-4Xylβ1-O-Ser, but they also contain unique disaccharide repeats, (GlcNAcα1-4GlcAβ1-4)n and (GalNAcβ1-4GlcAβ1-3)n in HS and CS, respectively. This pattern diagram is based on published information. *, only 30% of the Xyl residues of HS are phosphorylated at the C2-position in adult flies. **, the relative amount of each disaccharide structure in the unique disaccharide repeats of HS and CS. The abbreviations indicate the following disaccharide structures: ΔUA-GlcNAc, ΔHexUAα1-4GlcNAc; ΔUA-GlcNS, ΔHexUAα1-4GlcN(2-N-sulfate); ΔUA-GlcNAc6S, ΔHexUAα1-4GlcNAc(6-O-sulfate); ΔUA-GlcNS6S, ΔHexUAα1-4GlcN(2-N-,6-O-disulfate); ΔUA2S-GlcNS, ΔHexUA(2-O-sulfate)α1-4GlcN(2-N-sulfate); ΔUA2S-GlcN6S, ΔHexUA(2-O-sulfate) α1-4GlcN(2-N-,6-O-disulfate); ΔUA-GalNAc, ΔHexUAβ1-3GalNAc; ΔUA-GalNAc4S, ΔHexUAβ1-3GalNAc(4-O-sulfate); 2P, 2-O-phosphate.

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A novel Drosophila receptor-like protein tyrosine phosphatase gene, DPTP4E, was isolated and characterized. DPTP4E, located at cytological position 4E1-2, is comprised of 10 exons; its RNA products are widely expressed during embryonic development, including the developing central nervous system. DPTP4E produces three major developmentally regulated transcripts of 6.5, 7.0, and 7.5 kilobases. The two major embryonic transcripts arise as the result of the alternative splicing of exon IX; as a consequence, two proteins (200 and 183 kDa) are produced which differ in their carboxyl-terminal sequences. The deduced extracellular domain, which lies between two putative hydrophobic transmembrane segments, contains 11 fibronectin type III-like repeats and 25 putative N-glycosylation sites. A single conserved protein tyrosine phosphatase (PTPase) catalytic domain, which shows a high level of amino acid identity to the Drosophila PTPase DPTP10D and human HPTP beta, is found in the predicted intracellular domain; this PTPase domain, when expressed as a fusion protein in Escherichia coli, exhibits PTPase activity. The possible implications of these findings are discussed.A simple, inexpensive, environmentally friendly and efficient route for Michael addition of indoles to α,β-unsaturated ketones using pentafluorophenylammonium triflate (PFPAT) as a catalyst is described. Various indole derivatives were synthesized in good to excellent yields. The preparation of PFPAT catalyst from simple and readily available starting materials makes this method more affordable.

To be continued

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