We thank Jose Salazar, Julia Wang, and Dr. Karen Schulze for critical reading of the manuscript. We acknowledge Drs. Ning Liu and Xi Luo for the functional characterization of the TBX2 variants discussed here. Undiagnosed Diseases Network Model Organisms Screening Center was supported through the National Institutes of Health (NIH) Common Fund (U54 NS093793). H. T. C. was further supported by the NIH[CNCDP-K12 and NINDS (1K12 NS098482)], American Academy of Neurology (Neuroscience Research grant), Burroughs Wellcome Fund (Career Award for Medical Scientists), Child Neurology Society and Child Neurology Foundation (PERF Elterman grant), and the NIH Director&#8217;s Early Independence Award (DP5 OD026426). M. F. W. was further supported by Simons Foundation (SFARI Award: 368479). S. Y. was further supported by the NIH (R01 DC014932), the Simons Foundation (SFARI Award: 368479), the Alzheimer&#8217;s Association (New Investigator Research Grant: 15-364099), Naman Family Fund for Basic Research, and Caroline Wiess Law Fund for Research in Molecular Medicine. Confocal microscopy at BCM is supported in part by NIH Grant U54HD083092 to the Intellectual and Developmental Disabilities Research Center (IDDRC) Neurovisualization Core.

The authors have nothing to disclose.

Experimental studies using Drosophila melanogaster provide a robust assay system to assess the consequences of disease-associated human variants. This is due to the large body of knowledge and diverse genetic tools that have been generated by many researchers in the fly field over the past century89. Just like any other experimental system, however, it is important to acknowledge the caveats and limitations that exist.
Caveats Associated with Data Mining 
Although the first step in this protocol is to mine databases for information pertaining to a gene of interest, it is important to use it only as a starting point. For example, although in silico prediction of variant function provides valuable insights, these data should always be interpreted with caution. There are some instances in which all major algorithms predict that a human variant is benign, yet functional studies in Drosophila clearly demonstrated the damaging nature of such variant24. Similarly, although protein-protein interactions, co-expression, and structural modeling data are all insightful pieces of information, there may be pseudo-positive and pseudo-negative information present in these large -omics data sets. For example, some of the previously identified or predicted protein-protein interactions may be artificial or only seen in certain cell and tissue types.
In addition, there may be many false negative interactions not captured in these data sets, since certain protein-protein interactions are transient (e.g., enzyme-substrate interactions). Experimental validation is critical to demonstrating that certain genes or proteins genetically or physically interact in vivo in the biological context of interest. Similarly, structures predicted based on homology modeling should be treated as a model rather than solved structure. Although this information may be useful if it is found that an amino acid of interest is present in a structurally important part of the protein, negative data does not rule out the possibility that the variant may be damaging. Finally, some of the previously reported genotype-phenotype information should also be treated with caution, since some information archived in public databases may not be accurate. For example, some information in MO databases are based on experiments that have been well-controlled and performed rigorously, whereas others may come from a large screen paper with no further follow-up studies and stringent controls.
&#34;Humanization&#34; Experiments using T2A-GAL4 Strategy Not Always Successful 
While rescue- and overexpression-based functional studies using human cDNAs allow assessment of variants in the context of the human protein, this approach is not always successful. If a reference human cDNA cannot rescue the fly mutant phenotype, there are two probable explanations. The first possibility is that the human protein is nonfunctional or has significantly reduced activity in the context of a fly cell. This may be due to 1) reduced protein expression, stability, activity and/or localization or 2) a lack of compatibility with fly proteins that work in a multi-protein complex. Since the UAS/GAL4 system is temperature sensitive, the flies can be raised at a relatively high temperature (e.g., 29 &#176;C) to see the possibility of a rescue in this condition. In addition, a UAS-fly cDNA construct and transgene as a positive control can be generated. If the variant of interest affects a conserved amino acid, the analogous variant can be introduced into the fly cDNA for functional study of the variant in the context of the fly ortholog. Although this is not necessary, it greatly helps the study in cases that using human cDNA transgenic lines give negative or inconclusive results (Figure 3).
The second possibility is that expression of the human protein causes some cellular- or organism-level toxicity. This may be due to antimorphic (e.g., acting as a dominant negative protein), hypermorphic (e.g., too much activity), or neomorphic (e.g., gain of a novel toxic function such as protein aggregation that is not always related to the endogenous function of the gene of interest) effects. In this case, keeping the flies in a low temperature (e.g., 18 &#176;C) may alleviate some of these problems. Finally, there are some scenarios in which overexpression of a fly cDNA may not rescue the fly T2A-GAL4 line as seen in the TBX2 example, likely due to trict dosage dependence of the gene product. To avoid overexpression of a protein of interest, the fly gene of interest can be modified directly via CRISPR, a genomic rescue construct can be engineered that contains the variant of interest, or rescue experiments can be performed using a LOF allele21. For small genes, &#34;humanizing&#34;&#160;the fly genomic rescue construct can be considered to test human variants that affect non-conserved amino acids24. In summary, alternate strategies should be considered when the humanization experiment does not allow for functional assessment of the variant of interest.
Interpreting Negative and Positive Results 
If 1) both the reference and variant human cDNAs rescue the fly mutant phenotypes to a similar degree and 2) there is no difference observed in all conditions tested, then it can be assumed that the variant is functionally indistinguishable in Drosophila in vivo. It is important to note, however, that this information is not sufficient to rule out that the variant of interest is non-pathogenic, since the Drosophila assay may not be sensitive enough or capture all potential functions of the gene/protein of interest relevant to humans. Positive data, on the other hand, is a strong indication that the variant has damaging consequences on protein function, but this data alone is still not sufficient to claim pathogenicity. The American College of Medical Genetics and Genomics (ACMG) has published a set of standards and guidelines to classify variants in human disease associated genes into &#34;benign&#34;, &#34;likely benign&#34;, &#34;variant of unknown significance (VUS)&#34;, &#34;likely pathogenic&#34;, and &#34;pathogenic&#34;90. Although this classification only applies to established disease-associated genes and is not directly applicable to variants in &#34;genes of uncertain significance&#34;&#160;(GUS), all individuals involved in human variant functional studies are strongly encouraged to adhere to this guideline when reporting variant function.
Extracting Useful Biological Information when MO Phenotypes Do Not Model A Human Disease Condition 
It is important to keep in mind that overexpression-based functional assays have limitations, especially since some of the phenotypes being scored may have little relevance to the disease condition of interest. Similarly, phenotypes that are being assessed through rescue experiments may not have any direct relevance to the disease of interest. Since these experiments are conducted outside the endogenous contexts in an invertebrate system, they should not be considered disease models but rather as a gene function test using Drosophila as a &#34;living test tube&#34;.
Even if the model organism does not mimic a human disease condition, scorable phenotypes used in rescue experiments can often provide useful biological insights into disease conditions. The concept of &#34;phenologs (non-obvious homologous phenotypes)&#34;91 can be used to further determine underlying molecular connections between Drosophila and human phenotypes. For example, morphological phenotypes in the fly wing, thorax, legs, and eyes are excellent phenotypic readouts for defects in Notch signaling pathway, an evolutionarily conserved pathway linked to many congenital disorders, including cardiovascular defects in humans62. By understanding the molecular logic behind certain phenotypes in Drosophila, it is possible to identify hidden biological links between genes and phenotypes in humans that are not yet understood.
Continuous Communication with Clinical Collaborators 
When working with clinicians to study the function of a rare variant found in patient, it is important to establish a strong collaborative relationship. Although clinical and basic biomedical researchers may share interests in the same genes/genetic pathways, there is a large cultural and linguistic (e.g., medical jargon, model organism-specific nomenclature) gap between the clinical and scientific fields. A strong, trust-based relationship between the two parties can be built through extensive communication. Furthermore, bidirectional communication is critical to establishing and maintaining this relationship. For example, in the two cases described in the representative results section, identification of additional patients with similar genotypes and phenotypes, as well as subsequent functional study, were critical to prove pathogenicity of the variants of interest. Even with strong functional data, researchers and clinicians often have difficulties convincing human geneticists that a variant identified in &#34;n = 1&#34;&#160;cases is the true cause of disease.
Once the MO researcher identifies that a variant of interest is damaging, it is critical to communicate back to clinical collaborators as soon as possible so they can actively try to identify matching cases by networking with other clinicians and human geneticists. Tools such as Geno2MP [Genotypes to Mendelian Phenotypes: a de-identified database of 9,650 individuals enrolled in the University of Washington&#39;s Center for Mendelian Genomics Study41; includes patients and family members suspected of having genetic disorders] can be searched to assess individuals that may have the same disorder. Then, the lead clinician can be contacted using a messaging feature.
Alternatively, GeneMatcher can be used, which is a matchmaking website for clinicians, basic researchers, and patients who share interests in the same genes to identify additional patients that carry rare variants. Since GeneMatcher is part of a larger integrative network of matchmaking websites called Matchmaker Exchange42, additional databases around the world can be searched, including the Australian Genomics Health Alliance Patient Archive, Broad Matchbox, DECIPHER, MyGene2, and PhenomeCentral in a single GeneMatcher gene submission. Although participation in GeneMatcher is possible as a &#34;researcher&#34;, it is recommended that basic scientists utilize this website with their clinical collaborators, since communication with other clinicians after a match requires certain levels of medical expertise.

Patients with rare diseases often undergo an arduous journey referred to as the &#34;diagnostic odyssey&#34;&#160;to obtain an accurate diagnosis1. Most rare diseases are thought to have a strong genetic origin, making genetic/genomic analyses critical elements of the clinical workup. In addition to candidate gene panel sequencing and copy number variation analysis based on chromosomal microarrays, whole-exome (WES) and whole-genome sequencing (WGS) technologies have become increasingly valuable tools over the past decade2,3. Currently, the diagnostic rate for identifying a known pathogenic variant in WES and WGS is ~25% (higher in pediatric cases)4,5. For most cases that remain undiagnosed after clinical WES/WGS, a common issue is that there are many candidate genes and variants. Next-generation sequencing often identifies novel or ultra-rare variants in many genes, and interpreting whether these variants contribute to disease phenotypes is challenging. For example, although most nonsense or frameshift mutations in genes are thought to be loss-of-function (LOF) alleles due to nonsense-mediated decay of the encoded transcript, truncating mutations found in the last exons escape this process and may function as benign or gain-of-function (GOF) alleles6.
Moreover, predicting the effects of a missense allele is a daunting task, since it can result in a number of different genetic scenarios as first described by Herman Muller in the 1930s (i.e., amorph, hypomorph, hypermorph, antimorph, neomorph, or isomorph)7. Numerous in silico programs and methodologies have been developed to predict the pathogenicity of missense variants based on evolutionary conservation, type of amino acid change, position within a functional domain, allele frequency in the general population, and other parameters8. However, these programs are not a comprehensive solution to solving the complicated problem of variant interpretation. Interestingly, a recent study demonstrated that five broadly used variant pathogenicity prediction algorithms (Polyphen9, SIFT10, CADD11, PROVEAN12, Mutation Taster) agree on pathogenicity ~80% of the time8. Notably, even when all algorithms agree, they return an incorrect prediction of pathogenicity up to 11% of the time. This not only leads to flawed clinical interpretation but also may dissuade researchers from following up on new variants by falsely listing them as benign. One way to complement the current limitation of in silico modeling is to provide experimental data that demonstrates the effect of variant function in vitro, ex vivo (e.g., cultured cells, organoids), or in vivo.
In vivo functional studies of rare disease associated variants in MO have unique strengths13 and have been adopted by many rare disease research initiatives around the world, including the Undiagnosed Diseases Network (UDN) in the United States and Rare Diseases Models &#38; Mechanisms (RDMM) Networks in Canada, Japan, Europe, and Australia14. In addition to these coordinated efforts to integrate MO researchers into the workflow of rare disease diagnosis and mechanistic studies at a national scale, a number of individual collaborative studies between clinical and MO researchers have led to the discovery and characterization of many new human disease-causing genes and variants82,83,84.
In the UDN, a centralized Model Organisms Screening Center (MOSC) receives submissions of candidate genes and variants with a description of the patient&#8217;s condition and assesses whether the variant is likely to be pathogenic using informatics tools and in vivo experiments. In Phase I (2015-2018) of the UDN, the MOSC comprised of a Drosophila Core [Baylor College of Medicine (BCM)] and Zebrafish Core (University of Oregon) that worked collaboratively to assess cases. Using informatics analysis and a number of different experimental strategies in Drosophila and zebrafish, the MOSC has so far contributed to the diagnosis of 132 patients, identification of 31 new syndromes55, discovery of several new human disease genes (e.g., EBF315, ATP5F1D16, TBX217, IRF2BPL18, COG419, WDR3720) and phenotypic expansion of known disease genes (e.g., CACNA1A21, ACOX122).
In addition to projects within the UDN, MOSC Drosophila Core researchers have contributed to new disease gene discoveries in collaboration with the Centers for Mendelian Genomics and other initiatives (e.g., ANKLE223, TM2D324, NRD125, OGDHL25, ATAD3A26, ARIH127, MARK328, DNMBP29) using the same set of informatics and genetic strategies developed for the UDN. Given the significance of MO studies on rare disease diagnosis, the MOSC was expanded to include a C. elegans Core and second Zebrafish core (both at Washington University at St. Louis) for Phase II (2018-2022) of the UDN.
This manuscript describes an in vivo functional study protocol that is actively used in the UDN MOSC Drosophila Core to determine if missense variants have functional consequences on the protein of interest using transgenic flies that express human proteins. The goal of this protocol is to help MO researchers work collaboratively with clinical research groups to provide experimental evidence that a candidate variant in a gene of interest has functional consequences, thus facilitating clinical diagnosis. This protocol is most useful in a scenario in which a Drosophila researcher is approached by a clinical investigator who has a rare disease patient with a specific candidate variant in a gene of interest.
This protocol can be broken down into three elements: (1) gathering information to assess the likelihood of the variant of interest being responsible for the patient phenotype and the feasibility of a functional study in Drosophila, (2) gathering existing genetic tools and establishing new ones, and (3) performing functional studies in vivo. The third element can further be subdivided into two sub-elements based on how the function of a variant of interest can be assessed (rescue experiment or overexpression-based strategies). It is important to note that this protocol can be adapted and optimized to many scenarios outside of rare monogenic disease research (e.g., common diseases, gene-environment interactions, and pharmacological/genetic screens to identify therapeutic targets). The ability to determine the functionality and pathogenicity of variants will not only benefit the patient of interest by providing accurate molecular diagnosis but will also have broader impacts on both translational and basic scientific research.

<table><tbody><tr><td>&lt;strong&gt;&lt;em&gt;Drosophila&lt;/em&gt; Stocks for UAS-human cDNA transgenesis&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Injection strains for transgenesis (&lt;em&gt;D. melanogaster&lt;/em&gt;)</td><td>BDSC</td><td>#24871</td><td>Specific Reagent: VK33 (3rd chromosome) Injection line</td></tr><tr><td>Injection strains for transgenesis (&lt;em&gt;D. melanogaster&lt;/em&gt;)</td><td>BDSC</td><td>#24872</td><td>Specific Reagent: VK37 (2nd chromosome) Injection line</td></tr><tr><td>&lt;strong&gt;Plasmid DNA&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Cloning vector</td><td>Thermo Fisher</td><td>#12536-017</td><td>Specific Reagent: pDONR221</td></tr><tr><td>&lt;em&gt;Drosophila&lt;/em&gt; transgenesis vector</td><td>Gift from Drs. Johannes Bischof and Konrad Basler (Bischof et al., 2013 PNAS)</td><td></td><td>Specific Reagent: pGW-HA.attB</td></tr><tr><td>&lt;strong&gt;Molecular biology kits and reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Agarose</td><td>Sigma-Aldrich</td><td>#A2790</td><td>Specific Reagent: Agarose (molecular biology grade)</td></tr><tr><td>Chemically Competent Cells (&lt;em&gt;E. coli&lt;/em&gt;)</td><td>Thermo Fisher</td><td>#18265017</td><td>Specific Reagent: DH5&amp;alpha;</td></tr><tr><td>DNA Gel Extraction kit</td><td>Thermo Fisher</td><td>#K210012</td><td>Specific Reagent: PureLink Gel Extraction Kit</td></tr><tr><td>DNA Isolation and purification kit</td><td>Qiagen</td><td>#27104</td><td>Specific Reagent: QIAprep Spin Miniprep Kit</td></tr><tr><td>High Fidelity Polymerase</td><td>NEB</td><td>#M0491</td><td>Specific Reagent: Q5 Polymerase kit</td></tr><tr><td>Recombinase mediated cloning system</td><td>Thermo Fisher</td><td>#11789020</td><td>Specific Reagent: Gateway BP Clonase kit</td></tr><tr><td>Recombinase mediated cloning system</td><td>Thermo Fisher</td><td>#11791100</td><td>Specific Reagent: Gateway LR Clonase II Enzyme kit</td></tr><tr><td>Site Directed Mutagenesis kit</td><td>Agilent</td><td>#200523</td><td>Specific Reagent: Quick Change II Mutagenesis kit</td></tr><tr><td>&lt;strong&gt;Electroretinogram Rig related equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>ERG Analysis</td><td>Molecular Devices</td><td>N/A</td><td>Specific Reagent: Axon pCLAMP 10 Data Software Package</td></tr><tr><td>ERG Data Collection</td><td>LabX</td><td>#R150358</td><td>Specific Reagent: ISO-DAM Isolated Biologic Amplifier</td></tr><tr><td>ERG Stimulator</td><td>Astro-Med</td><td>#S48</td><td>Specific Reagent: Square Pulse Stimulator</td></tr></tbody></table>

in vivo functional study of disease-associated rare human variants using drosophila

Advances in sequencing technology have made whole-genome and whole-exome datasets more accessible for both clinical diagnosis and cutting-edge human genetics research. Although a number of in silico algorithms have been developed to predict the pathogenicity of variants identified in these datasets, functional studies are critical to determining how specific genomic variants affect protein function, especially for missense variants. In the Undiagnosed Diseases Network (UDN) and other rare disease research consortia, model organisms (MO) including Drosophila, C. elegans, zebrafish, and mice are actively used to assess the function of putative human disease-causing variants. This protocol describes a method for the functional assessment of rare human variants used in the Model Organisms Screening Center Drosophila Core of the UDN. The workflow begins with gathering human and MO information from multiple public databases, using the MARRVEL web resource to assess whether the variant is likely to contribute to a patient&#39;s condition as well as design effective experiments based on available knowledge and resources. Next, genetic tools (e.g., T2A-GAL4 and UAS-human cDNA lines) are generated to assess the functions of variants of interest in Drosophila. Upon development of these reagents, two-pronged functional assays based on rescue and overexpression experiments can be performed to assess variant function. In the rescue branch, the endogenous fly genes are &#34;humanized&#34;&#160;by replacing the orthologous Drosophila gene with reference or variant human transgenes. In the overexpression branch, the reference and variant human proteins are exogenously driven in a variety of tissues. In both cases, any scorable phenotype (e.g., lethality, eye morphology, electrophysiology) can be used as a read-out, irrespective of the disease of interest. Differences observed between reference and variant alleles suggest a variant-specific effect, and thus likely pathogenicity. This protocol allows rapid, in vivo assessments of putative human disease-causing variants of genes with known and unknown functions.

Watch this Scientific Journal Video about In Vivo Functional Study of Disease-associated Rare Human Variants Using Drosophila at JoVE.com

In Vivo Functional Study of Disease-associated Rare Human Variants Using Drosophila

The goal of this protocol is to outline the design and performance of in vivo experiments in Drosophila melanogaster to assess the functional consequences of rare gene variants associated with human diseases.

In Vivo Functional Study of Disease-associated Rare Human Variants Using Drosophila

Advances in sequencing technology have made whole-genome and whole-exome datasets more accessible for both clinical diagnosis and cutting-edge human ...

in-vivo-functional-study-disease-associated-rare-human-variants-using

Nanyang Technological University

Research

JoVE Journal

Genetics

13.5K Views.  Baylor College of Medicine. The goal of this protocol is to outline the design and performance of in vivo experiments in Drosophila melanogaster to assess the functional consequences of rare gene variants associated with human diseases.

Video: In Vivo Functional Study of Disease-associated Rare Human Variants Using Drosophila

In vivo Visualization of Synaptic Vesicles Within Drosophila Larval Segmental Axons

Elucidating the mechanisms of axonal transport has shown to be very important in determining how defects in long distance transport affect different neurological diseases. Defects in this essential process can have detrimental effects on neuronal functioning and development. We have developed a dissection protocol that is designed to expose the Drosophila larval segmental nerves to view axonal transport in real time. We have adapted this protocol for live imaging from the one published by Hurd and Saxton (1996) used for immunolocalizatin of larval segmental nerves. Careful dissection and proper buffer conditions are critical for maximizing the lifespan of the dissected larvae. When properly done, dissected larvae have shown robust vesicle transport for 2-3 hours under physiological conditions. We use the UAS-GAL4 method 1 to express GFP-tagged APP or synaptotagmin vesicles within a single axon or many axons in larval segmental nerves by using different neuronal GAL4 drivers. Other fluorescently tagged markers, for example mitochrondria (MitoTracker) or lysosomes (LysoTracker), can be also applied to the larvae before viewing. GFP-vesicle movement and particle movement can be viewed simultaneously using separate wavelengths.

In vivo Visualization of Synaptic Vesicles Within Drosophila Larval Segmental Axons

This protocol discusses the live dissection of Drosophila larvae for the purpose of imaging the movement of GFP tagged axonal vesicles on microtubule tracks.

Elucidating the mechanisms of axonal transport has shown to be very important in determining how defects in long distance transport affect different ...

Neuroscience

Navigating MARRVEL, a Web-Based Tool that Integrates Human Genomics and Model Organism Genetics Information

Through whole-exome/genome sequencing, human geneticists identify rare variants that segregate with disease phenotypes. To assess if a specific variant is pathogenic, one must query many databases to determine whether the gene of interest is linked to a genetic disease, whether the specific variant has been reported before, and what functional data is available in model organism databases that may provide clues about the gene&#8217;s function in human. MARRVEL (Model organism Aggregated Resources for Rare Variant ExpLoration) is a one-stop data collection tool for human genes and variants and their orthologous genes in seven model organisms including in mouse, rat, zebrafish, fruit fly, nematode worm, fission yeast, and budding yeast. In this Protocol, we provide an overview of what MARRVEL can be used for and discuss how different datasets can be used to assess whether a variant of unknown significance (VUS) in a known disease-causing gene or a variant in a gene of uncertain significance (GUS) may be pathogenic. This protocol will guide a user through searching multiple human databases simultaneously starting with a human gene with or without a variant of interest. We also discuss how to utilize data from OMIM, ExAC/gnomAD, ClinVar, Geno2MP, DGV and DECHIPHER. Moreover, we illustrate how to interpret a list of ortholog candidate genes, expression patterns, and GO terms in model organisms associated with each human gene. Furthermore, we discuss the value protein structural domain annotations provided and explain how to use the multiple species protein alignment feature to assess whether a variant of interest affects an evolutionarily conserved domain or amino acid. Finally, we will discuss three different use-cases of this website. MARRVEL is an easily accessible open access website designed for both clinical and basic researchers and serves as a starting point to design experiments for functional studies.

Here, we present a protocol to access and analyze many human and model organism databases efficiently. This protocol demonstrates the use of MARRVEL to analyze candidate disease-causing variants identified from next-generation sequencing efforts.

Through whole-exome/genome sequencing, human geneticists identify rare variants that segregate with disease phenotypes. To assess if a specific variant is ...

In Vivo Forward Genetic Screen to Identify Novel Neuroprotective Genes in Drosophila melanogaster

There is much to understand about the onset and progression of neurodegenerative diseases, including the underlying genes responsible. Forward genetic screening using chemical mutagens is a useful strategy for mapping mutant phenotypes to genes among Drosophila and other model organisms that share conserved cellular pathways with humans. If the mutated gene of interest is not lethal in early developmental stages of flies, a climbing assay can be conducted to screen for phenotypic indicators of decreased brain functioning, such as low climbing rates. Subsequently, secondary histological analysis of brain tissue can be performed in order to verify the neuroprotective function of the gene by scoring neurodegeneration phenotypes. Gene mapping strategies include meiotic and deficiency mapping that rely on these same assays can be followed by DNA sequencing to identify possible nucleotide changes in the gene of interest.

In Vivo Forward Genetic Screen to Identify Novel Neuroprotective Genes in Drosophila melanogaster

We present a protocol using a forward genetic approach to screen for mutants exhibiting neurodegeneration in Drosophila melanogaster. It incorporates a climbing assay, histology analysis, gene mapping and DNA sequencing to ultimately identify novel genes related to the process of neuroprotection.

There is much to understand about the onset and progression of neurodegenerative diseases, including the underlying genes responsible. Forward genetic ...

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the proteins involved in these pathways are evolutionarily conserved between mammals and other eukaryotes, such as the fruit fly Drosophila melanogaster, suggesting that similar organizing principles exist during the development of these organisms. Importantly, Drosophila has been used extensively to identify cellular and molecular mechanisms regulating processes that are required in mammals including neurogenesis, differentiation, axonal guidance, and synaptogenesis. Flies have also been used successfully to model a variety of human neurodevelopmental diseases. Here we describe a protocol for the step-by-step microdissection, fixation, and immunofluorescent localization of proteins within the adult Drosophila brain. This protocol focuses on two example neuronal populations, mushroom body neurons and retinal photoreceptors, and includes optional steps to trace individual mushroom body neurons using Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. Example data from both wild-type and mutant brains are shown along with a brief description of a scoring criteria for axonal guidance defects. While this protocol highlights two well-established antibodies for investigating the morphology of mushroom body and photoreceptor neurons, other Drosophila brain regions and the localization of proteins within other brain regions can also be investigated using this protocol.

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

This protocol describes the dissection and immunostaining of adult Drosophila melanogaster brain tissues. Specifically, this protocol highlights the use of Drosophila mushroom body and photoreceptor neurons as example neuronal subsets that can be accurately used to uncover general principles underlying many aspects of neuronal development.

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the ...

Developmental Biology

A Simple Technique to Assay Locomotor Activity in Drosophila

Drosophila melanogaster is an ideal model organism for studying various diseases due to its abundance of advanced genetic manipulation techniques and diverse behavioral features. Identifying behavioral deficiency in animal models is a crucial measure of disease severity, for example, in neurodegenerative diseases where patients often experience impairments in motor function. However, with the availability of various systems to track and assess motor deficits in fly models, such as drug-treated or transgenic individuals, an economical and user-friendly system for precise evaluation from multiple angles is still lacking. A method based on the AnimalTracker application programming interface (API) is developed here, which is compatible with the Fiji image processing program, to systematically evaluate the movement activities of both adult and larval individuals from recorded video, thus allowing for the analysis of their tracking behavior. This method requires only a high-definition camera and a computer peripheral hardware integration to record and analyze behavior, making it an affordable and effective approach for screening fly models with transgenic or environmental behavioral deficiencies. Examples of behavioral tests using pharmacologically treated flies are given to show how the techniques can detect behavioral changes in both adult flies and larvae in a highly repeatable manner.

A Simple Technique to Assay Locomotor Activity in Drosophila

The present protocol assesses the locomotor activity of Drosophila by tracking and analyzing the movement of flies in a hand-made arena using open-source software Fiji, compatible with plugins to segment pixels of each frame based on high-definition video recording to calculate parameters of speed, distance, etc.

Drosophila melanogaster is an ideal model organism for studying various diseases due to its abundance of advanced genetic manipulation techniques and ...

Behavior

Quantitative Analysis of Climbing Defects in a Drosophila Model of Neurodegenerative Disorders

Locomotive defects resulting from neurodegenerative disorders can be a late onset symptom of disease, following years of subclinical degeneration, and thus current therapeutic treatment strategies are not curative. Through the use of whole exome sequencing, an increasing number of genes have been identified to play a role in human locomotion. Despite identifying these genes, it is not known how these genes are crucial to normal locomotive functioning. Therefore, a reliable assay, which utilizes model organisms to elucidate the role of these genes in order to identify novel targets of therapeutic interest, is needed more than ever. We have designed a sensitized version of the negative geotaxis assay that allows for the detection of milder defects earlier and has the ability to evaluate these defects over time. The assay is performed in a glass graduated cylinder, which is sealed with a wax barrier film. By increasing the threshold distance to be climbed to 17.5 cm and increasing the experiment duration to 2 min&#160;we have observed a greater sensitivity in detecting mild mobility dysfunctions. The assay is cost effective and does not require extensive training to obtain highly reproducible results. This makes it an excellent technique for screening candidate drugs in Drosophila mutants with locomotion defects.

We present an optimized inexpensive and reliable negative geotaxis assay in Drosophila melanogaster as a model for neurodegenerative disorders. Being more sensitive to mild locomotor defects, this assay will help screen for potential genetic interactions and drug targets.

Locomotive defects resulting from neurodegenerative disorders can be a late onset symptom of disease, following years of subclinical degeneration, and ...

Mass Histology to Quantify Neurodegeneration in Drosophila

Progressive neurodegenerative diseases like Alzheimer&#39;s disease (AD) or Parkinson&#39;s disease (PD) are an increasing threat to human health worldwide. Although mammalian models have provided important insights into the underlying mechanisms of pathogenicity, the complexity of mammalian systems together with their high costs are limiting their use. Therefore, the simple but well-established Drosophila model-system&#160;provides an alternative for investigating the molecular pathways that are affected in these diseases. Besides behavioral deficits, neurodegenerative diseases are characterized by histological phenotypes such as neuronal death and axonopathy. To quantify neuronal degeneration and to determine how it is affected by genetic and environmental factors, we use a histological approach that is based on measuring the vacuoles in adult fly brains. To minimize the effects of systematic error and to directly compare sections from control and experimental flies in one preparation, we use the &#39;collar&#39; method for paraffin sections. Neurodegeneration is then assessed by measuring the size and/or number of vacuoles that have developed in the fly brain. This can either be done by focusing on a specific region of interest or by analyzing the entire brain by obtaining serial sections that span the complete head. Therefore, this method allows one to measure not only severe degeneration but also relatively mild phenotypes that are only detectable in a few sections, as occurs during normal aging.

Mass Histology to Quantify Neurodegeneration in Drosophila

Drosophila is widely used as a model system to study neurodegeneration. This protocol describes a method by which degeneration, as determined by vacuole formation in the brain, can be quantified. It also minimizes effects due to the experimental procedure by processing and sectioning control and experimental flies as one sample.

Progressive neurodegenerative diseases like Alzheimer&#39;s disease (AD) or Parkinson&#39;s disease (PD) are an increasing threat to human health ...

Unbiased Quantification of Ommatidia in Drosophila Eye Models

Quantifying Drosophila melanogaster Eye Phenotypes: A Computational Approach Integrating ilastik and Flynotyper

The Drosophila melanogaster compound eye is a well-structured and comprehensive array of around 800 ommatidia, exhibiting a symmetrical and hexagonal pattern. This regularity and ease of observation make the Drosophila eye system a powerful tool to model various human neurodegenerative diseases. However, ways of quantifying abnormal phenotypes, such as manual ranking of eye severity scores, have limitations, especially when ranking weak alterations in eye morphology. To overcome these limitations, computational approaches have been developed such as Flynotyper. The use of a ring light allows for better qualitative images accessing the intactness of individual ommatidia. However, these images cannot be analyzed by Flynotyper directly due to shadows on ommatidia introduced by the ring light. Here, we describe an unbiased way to quantify rough eye phenotypes observed in Drosophila disease models by combining two software, ilastik and Flynotyper. By preprocessing the images with ilastik, successful quantification of the rough eye phenotype can be achieved with Flynotyper.

Author Spotlight: Quantifying Rough Eye Phenotypes in Drosophila Models of Amyotrophic Lateral Sclerosis with Frontotemporal Dementia

The Drosophila eye system is a useful tool for studying various biological processes, particularly human neurodegenerative diseases. However, manual quantification of rough eye phenotypes can be biased and unreliable. Here we describe a method by which ilastik and Flynotyper are used to quantify eye phenotype in an unbiased way.

The Drosophila melanogaster compound eye is a well-structured and comprehensive array of around 800 ommatidia, exhibiting a symmetrical and hexagonal ...

Purification of Transcripts and Metabolites from Drosophila Heads

For the last decade, we have tried to understand the molecular and cellular mechanisms of neuronal degeneration using Drosophila as a model organism. Although fruit flies provide obvious experimental advantages, research on neurodegenerative diseases has mostly relied on traditional techniques, including genetic interaction, histology, immunofluorescence, and protein biochemistry. These techniques are effective for mechanistic, hypothesis-driven studies, which lead to a detailed understanding of the role of single genes in well-defined biological problems. However, neurodegenerative diseases are highly complex and affect multiple cellular organelles and processes over time. The advent of new technologies and the omics age provides a unique opportunity to understand the global cellular perturbations underlying complex diseases. Flexible model organisms such as Drosophila are ideal for adapting these new technologies because of their strong annotation and high tractability. One challenge with these small animals, though, is the purification of enough informational molecules (DNA, mRNA, protein, metabolites) from highly relevant tissues such as fly brains. Other challenges consist of collecting large numbers of flies for experimental replicates (critical for statistical robustness) and developing consistent procedures for the purification of high-quality biological material. Here, we describe the procedures for collecting thousands of fly heads and the extraction of transcripts and metabolites to understand how global changes in gene expression and metabolism contribute to neurodegenerative diseases. These procedures are easily scalable and can be applied to the study of proteomic and epigenomic contributions to disease.

Purification of Transcripts and Metabolites from Drosophila Heads

We describe here the procedures for the extraction and purification of mRNA and metabolites from Drosophila heads. We are applying these techniques to better understand the cellular perturbations underlying neuronal degeneration. These methodologies can be easily scaled and adapted for other "omic" projects.

For the last decade, we have tried to understand the molecular and cellular mechanisms of neuronal degeneration using Drosophila as a model organism. ...

Biology

In Vivo Modeling of the Morbid Human Genome using Danio rerio

Here, we present methods for the development of assays to query potentially clinically significant nonsynonymous changes using in vivo complementation in zebrafish. Zebrafish (Danio rerio) are a useful animal system due to their experimental tractability; embryos are transparent to enable facile viewing, undergo rapid development ex vivo, and can be genetically manipulated.1 These aspects have allowed for significant advances in the analysis of embryogenesis, molecular processes, and morphogenetic signaling. Taken together, the advantages of this vertebrate model make zebrafish highly amenable to modeling the developmental defects in pediatric disease, and in some cases, adult-onset disorders. Because the zebrafish genome is highly conserved with that of humans (~70% orthologous), it is possible to recapitulate human disease states in zebrafish. This is accomplished either through the injection of mutant human mRNA to induce dominant negative or gain of function alleles, or utilization of morpholino (MO) antisense oligonucleotides to suppress genes to mimic loss of function variants. Through complementation of MO-induced phenotypes with capped human mRNA, our approach enables the interpretation of the deleterious effect of mutations on human protein sequence based on the ability of mutant mRNA to rescue a measurable, physiologically relevant phenotype. Modeling of the human disease alleles occurs through microinjection of zebrafish embryos with MO and/or human mRNA at the 1-4 cell stage, and phenotyping up to seven days post fertilization (dpf). This general strategy can be extended to a wide range of disease phenotypes, as demonstrated in the following protocol. We present our established models for morphogenetic signaling, craniofacial, cardiac, vascular integrity, renal function, and skeletal muscle disorder phenotypes, as well as others.

In Vivo Modeling of the Morbid Human Genome using Danio rerio

Here, we present a systematic approach for developing physiologically relevant, sensitive and specific in vivo assays for interpreting variation in human pathology. Transient genetic manipulation via microinjection of WT and mutant human mRNA and morpholino (MO) antisense oligonucleotides harness the tractability of the developing zebrafish embryo to rapidly assay pathogenic mutations, especially, but not exclusively, in the context of human developmental disorders.

Here,  we present methods for the development of assays to query potentially clinically  significant nonsynonymous changes using in  vivo complementation ...

In Vivo Functional Study of Disease-associated Rare Human Variants Using Drosophila

Summary

Explore More Videos

Chapters in this video

In vivo Visualization of Synaptic Vesicles Within Drosophila Larval Segmental Axons

Navigating MARRVEL, a Web-Based Tool that Integrates Human Genomics and Model Organism Genetics Information

In Vivo Forward Genetic Screen to Identify Novel Neuroprotective Genes in Drosophila melanogaster

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

A Simple Technique to Assay Locomotor Activity in Drosophila

Quantitative Analysis of Climbing Defects in a Drosophila Model of Neurodegenerative Disorders

Mass Histology to Quantify Neurodegeneration in Drosophila

Quantifying Drosophila melanogaster Eye Phenotypes: A Computational Approach Integrating ilastik and Flynotyper

Purification of Transcripts and Metabolites from Drosophila Heads

In Vivo Modeling of the Morbid Human Genome using Danio rerio