How Has The Use Of Model Organisms Advanced Our Knowledge Of The Genes That Control Human Diseases?

Using Drosophila melanogaster To Discover Human Illness Genes: An Educational Primer for Use with "Amyotrophic Lateral Sclerosis Modifiers in Drosophila Reveal the Phospholipase D Pathway as a Potential Therapeutic Target"

Surya Banerjee,

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Abstract

Since the dawn of the 20th century, the fruit fly Drosophila melanogaster has been used as a model organism to understand the nature of genes and how they control development, behavior, and physiology. Ane of the most powerful experimental approaches employed in Drosophila is the forward genetic screen. In the 21st century, genome-wide screens have become popular tools for identifying evolutionarily conserved genes involved in complex human diseases. In the accompanying article "Amyotrophic Lateral Sclerosis Modifiers in Drosophila Reveal the Phospholipase D Pathway every bit a Potential Therapeutic Target," Kankel and colleagues depict a forward genetic modifier screen to discover factors that contribute to the astringent neurodegenerative disease amyotrophic lateral sclerosis (ALS). This primer briefly traces the history of genetic screens in Drosophila and introduces students to ALS. Nosotros then provide a set of guided reading questions to aid students piece of work through the information presented in the research article. Finally, several ideas for literature-based research projects are offered as opportunities for students to aggrandize their appreciation of the potential scope of genetic screens. The primer is intended to aid students and instructors thoroughly examine a current study that uses forward genetics in Drosophila to place human disease genes.

One of the most powerful approaches for discovering the functions of genes is the forward genetic screen. Screens in model organisms like Drosophila melanogaster can identify evolutionarily conserved genes involved in complex human diseases. As an instance, in the accompanying article "Amyotrophic Lateral Sclerosis Modifiers in Drosophila Reveal the Phospholipase D Pathway as a Potential Therapeutic Target," Kankel and colleagues describe a forward genetic screen to discover factors that contribute to the severe neurodegenerative disease amyotrophic lateral sclerosis (ALS). This primer guides students through the data in the article and offers ideas for literature-based inquiry projects to expand students' appreciation of genetic screens.

Model Organisms

It is impossible to imagine the successes of modernistic medicine without the contributions from so-called "model organisms." Because fundamental genetic and cell biological processes are mutual to all life on Globe, profound insight into human biology tin exist gained by studying species simpler than our own. Research on organisms as distant from us as bacteria and their phages have helped provide the thorough and all-encompassing understanding we have today of the genetic fabric, the lawmaking past which it stores data, and the process by which this information is decoded by cells. That foundational knowledge, congenital in model organisms, laid the groundwork for the molecular biology revolution of the mid-20th century, which in turn underpins the modern biomedical enterprise. Likewise, beast studies in rats, mice, chicks, frogs, fish, and invertebrates accept been providing insight into the evolutionarily conserved mechanisms of our own development, physiology, and behavior, and related pathologies, for well over a century. In their contempo article, Kankel and coauthors written report on a genetic study using the fruit fly, D. melanoga st er, equally a model organism ( Kankel et al. 2020).

Drosophila melanogaster

D. melanoga st er has a particularly long history every bit a model organism, having first accomplished fame in the early 20th century for its role in establishing the chromosome theory of inheritance. Because of its long history, its resurgence in popularity starting in the mid-20th century, and its "convenient" attributes, including short life-wheel, pocket-size size, loftier fecundity, and relatively compact genome, Drosophila has get a research platform with a plethora of experimental tools ( Hales et al. 2015). These tools, coupled with the fact that more than half of Drosophila genes have orthologs in humans, take fabricated the fruit fly a leading model arrangement for studying the mechanisms of human biology and disease (Bellen and Yamamoto 2015). Additionally, its complex brain and behavioral repertoire make it a specially useful model for understanding neurobiology ( Bellen et al. 2010; McGurk et al. 2015).

Genetic Screens

Later on early on experiments in Drosophila helped elucidate the rules of inheritance and the nature of genes, studies in this organism turned to the question of how genes direct biology. Past examining what happens when genes are mutated, scientists have been able to tease autonomously how unmutated wild-type alleles contribute to the normal functioning of an organism. Since the 1960s, forward genetic screening has been used to identify novel genes involved in myriad biological processes (St Johnston 2013). Forrad genetic screens begin by creating de novo mutations randomly throughout the genome, so that any factor has a gamble of being altered ("striking") and the researcher is unbiased toward whatever specific gene or grade of genes (St Johnston 2013; Hales et al. 2015). Mutants then are surveyed for singled-out phenotypic alterations every bit compared to the wild type. When practical on a large calibration, this powerful method has the potential to capture any, and theoretically all, genes involved in a particular process (Nüsslein-Volhard and Wieschaus 1980; St Johnston 2002). In contrast to "reverse" genetics approaches, in which a specific factor or group of genes is targeted a priori, forward genetic screens are uniquely poised to detect pathways previously unknown to be involved in the process of interest. Some of the earliest screens centered on stereotyped behaviors, thereby demonstrating that even biological functions as complex as neurobiology and beliefs are regulated by the activity of specific genes (due east.g., Benzer 1967; Hotta and Benzer 1969; Pak et al. 1969; January and January 2008).

Dominant Modifiers

I of the most obvious physical features of the D. melanoga st er wing is its middle—a large, complex, and beautiful neurological organ. Since the very first described Drosophila mutant with white middle color instead of crimson, some of the near notable frontward genetic screens take looked for mutations that perturb the center (Morgan 1910; St Johnston 2002). A number of these screens have advantage of the fact that genes in the same pathway have a tendency to be sensitive to each other's dosage (e.chiliad., Rogge et al. 1991; Simon et al. 1991). Because changes to the sequence of nucleotide bases in a cistron oft reduce the functionality of the encoded factor product (protein or RNA), many mutant alleles are loss of function. In diploid organisms, including Drosophila and humans, a single wild-blazon allele typically produces enough factor product to maintain normal activity; hence, most loss of role mutant alleles are recessive and must exist homozygous to bear upon phenotype. However, if another component of the same pathway is altered in its genetic dosage, loss of function mutant alleles tin can exacerbate (heighten) or ameliorate (suppress) the phenotype as heterozygotes, and thus appear ascendant. In this way, forward genetic modifier screens can be performed by irresolute the dosage of i pathway component, thereby creating a sensitized background, generating novel mutations randomly throughout the genome, and then screening for those second-site alleles that are dominant modifiers of the starting phenotype. This enables identification of genes that act in the same pathway as the original component, with the added advantage that the screen tin be performed with only one generation of flies, as the dominant modifiers do not need to be homozygous to manifest the contradistinct phenotype. This strategy is especially valuable considering mutant alleles that are otherwise homozygous lethal can be identified as heterozygotes, with survival of the organism permitted. Furthermore, the center, beingness a nonessential organ, is an ideal context in which to study development and physiology without impacting survival or fertility ( Bakery et al. 2014). In their study, Kankel et al. (2020) perform a genetic screen for dominant modifiers of a mutant eye phenotype.

Expressing Human Genes in Drosophila

The experimental range of Drosophila was expanded vastly past transgenic technology. Once the tools of molecular biology made it possible to isolate individual genes from the genome and engineer them in bacterial plasmids, methods were developed to deliver whatsoever factor of interest into the genome of another organism, of the same or different species (Rubin and Spradling 1982; Spradling and Rubin 1982). With this engineering science in hand, human genes now could exist introduced into Drosophila (east.g., Jowett et al. 1991; Luo et al. 1992). In the decades since, this procedure has been used to demonstrate time and once again the strong evolutionary conservation between flies and humans, with man proteins capable of rescuing mutant phenotypes conferred past loss of their wing counterparts. Furthermore, expressing human being disease alleles in flies often mimics disease pathology, again highlighting the robust underlying conservation of genetic pathways and cellular networks (e.g., Jackson et al. 1998; Warrick et al. 1998). Kankel et al. (2020) apply this arroyo of expressing human disease alleles in the fly eye, which creates a visible mutant phenotype.

GAL4-UAS

In order for an exogenous transgene to be expressed in the host organism, information technology must include regulatory sequences for transcription and translation. Early transgenes were engineered with specific Drosophila regulatory elements, just the GAL4-UAS system introduced a great technological comeback by separating the gene of interest from its regulation (Make and Perrimon 1993). In this system, Drosophila transgenes are engineered downstream of an upstream activation sequence (UAS) from the Saccharomyces cerevisiae genome that binds the GAL4 transcription factor. Because the Drosophila genome does non endogenously encode GAL4, UAS-driven transgenes are effectively non expressed, thereby protecting the transgenic organisms from expression of deleterious transgenes. A vast array of divide transgenic Drosophila stocks has been generated and shared inside the scientific customs, each conveying the yeast GAL4 transcription factor expressed in a tissue- or prison cell-specific design nether the command of an endogenous regulatory chemical element ( Hales et al. 2015). Thus, any UAS-driven transgene now tin be expressed in almost whatsoever tissue or cell type by simply mating to a GAL4 stock. Kankel et al. (2020) use the GMR-GAL4 driver, which produces GAL4 protein in cells of the eye, to induce expression of UAS-controlled human disease alleles.

Genome-Wide Mutant Collections

Past the last decade of the 20th century, the fly enquiry community was inspired to generate new collections of mutants that could exist used for rapid forward genetic screening and subsequent mapping (Cooley et al. 1988; Spradling et al. 1995). Collections aiming to represent every gene in the genome made utilize of mobile transposable elements that could integrate throughout the genome at random, creating loss of role mutations by disrupting local regulatory and/or coding sequences (Artavanis-Tsakonas 2004; Thibault et al. 2004; Bellen et al. 2011). In this study, Kankel et al. (2020) screen one of these collections of Drosophila insertion mutations, the Exelixis collection. These insertions tin be mapped immediately by isolation of the inserted element along with its neighboring genomic Dna, greatly reducing labor- and time-intensive procedures for mapping hits from screens. The utility of collections similar these has been augmented further by whole genome sequencing, which revolutionized the study of biology at the plough of the 21st century ( Adams et al. 2000). Today, scientists have at their fingertips about the entire Drosophila genome sequence, along with libraries of mutants disrupting a substantial proportion of poly peptide-coding loci, making possible rapid genetic screening, identification, and functional analysis. The availability of the homo genome sequence, together with the revelation that 60%–70% of human genes have orthologs in Drosophila, and that this pct is even higher (∼75%) for illness genes, has prompted the scientific customs to use the powerful tools of Drosophila genetics to understand human disease ( Lander et al. 2001; Venter et al. 2001; McGurk et al. 2015; Wangler et al. 2017; Johnston 2020).

Organizing Screen Hits

A successful large-calibration genome-wide genetic screen tin identify hundreds of hits, genes whose mutation leads to interesting phenotypic alterations. To organize these hits, scientists frequently use publicly available gene ontology (GO) classification, which annotates each gene with molecular functions, biological processes, and cellular sites of activity (http://geneontology.org/). Annotations are based on data from many dissimilar model systems, also as on computer-based analyses, and are curated by a large global consortium of scientists ( Gaudet et al. 2017). This diversity of inputs, along with a controlled vocabulary that is clearly defined and adheres to a consistent logical framework, makes the GO classification structure universally applicable (Hastings 2017). Become nomenclature can help grouping screen hits according to role and/or cellular localization, thereby illuminating biological pathways of import for the process under investigation.

Amyotrophic Lateral Sclerosis

Human life expectancy has increased dramatically over the course of the last century due largely to improved diet, public health, and medical care, merely with increased lifespan comes greater risk of age-associated diseases, including neurodegenerative affliction (ND, https://world wide web.nia.nih.gov/research/dbsr/global-aging). ND encompasses a range of conditions that cause the death of neurons and loss of neurological function, including Alzheimer's affliction, Lewy body diseases like Parkinson'due south, polyglutamine diseases similar Huntington's disease, and amyotrophic lateral sclerosis (ALS). Despite much investigation, a comprehensive understanding of ND has been confounded by the complex contribution of a variety of genetic and environmental factors. ALS, commonly known as Lou Gehrig'south illness, is a severe ND in which motor neurons dice and their target muscles cloudburst (Brown and Al-Chalabi 2017). Although a modest proportion of ALS cases are conspicuously inherited (familial ALS, fALS), ∼80%–90% of ALS cases are sporadic (sALS), with only ∼20% of desultory cases linked to specific genes ( Vucic et al. 2014; Brown and Al-Chalabi 2017; Martin et al. 2017). This, together with the limited treatment options, creates a pressing need to empathise better the genetic and cellular pathways underlying this serious disease. Considering fALS and sALS acquit high clinical and pathological similarity, and because the identified genetic loci are common to both, it is presumed that fALS and sALS share molecular and cellular etiologies (Chocolate-brown and Al-Chalabi 2017; Martin et al. 2017). Numerous genes have been identified in fALS (https://alsod.air conditioning.united kingdom of great britain and northern ireland/), with four loci accounting for the majority of cases, namely FUS, TARDBP/TDP-43, C9orf72, and SOD1 (Brown and Al-Chalabi 2017; Hardiman et al. 2017). Interestingly, FUS and TDP-43 are both RNA-bounden proteins, and abnormal RNA processing may contribute to affliction progression ( Ranganathan et al. 2020; Yerbury et al. 2020). Nonetheless, ALS-associated mutations in FUS or TDP-43 increase the tendency of the encoded proteins to misfold and aggregate abnormally, a property likewise observed for ALS-associated SOD1 mutants, suggesting that dysregulation of protein homeostasis (proteostasis) and resultant proteotoxicity is the cause of neurodegeneration ( Martin et al. 2017; Yerbury et al. 2020). In back up of this idea, TDP-43 aggregates are establish in upward to 97% of ALS patients, representing both sporadic and familial cases, including in patients with wild-type TDP-43 alleles ( Hardiman et al. 2017). Toxic aggregates are also a feature of ALS linked to mutations in c9orf72. The protein encoded by c9orf72 is implicated in autophagy, an important cellular process for regulating proteostasis, merely ALS-causing mutations derive from expansion of a hexanucleotide GGGGCC repeat in a noncoding intron of the gene. These repeated sequences are transcribed into RNAs that aggregate and sequester RNA binding proteins, and the repeat containing RNAs can be translated via a noncanonical protein translation pathway into dipeptide repeats that form toxic protein aggregates ( Ranganathan et al. 2020). Much is still unknown about how ALS develops, including whether and how dysregulation of RNA processing and proteostasis lead to neurodegeneration, whether ALS-associated mutations crusade disease by loss of their wild-type functions or gain of toxicity, and whether these four genes, as well as others, human activity in the same genetic and biochemical pathway. Moreover, despite the prevalence of RNA processing defects and proteotoxic aggregates in ALS, many other cellular activities are affected as well ( Hardiman et al. 2017). The goal of this report is to link previously unassociated genes to ALS, in society to better understand how the disease progresses and to reveal potential therapeutic targets. Prior studies have demonstrated that expressing human disease variants of FUS or TDP-43 with the GAL4-UAS system in photoreceptor neurons of the Drosophila center induces degeneration, mimicking the cellular pathology of the disease ( Ritson et al. 2010; Lanson et al. 2011). The authors screen the Exelixis collection of Drosophila insertion mutations for dominant modifiers of this eye degeneration phenotype, to elucidate the molecular pathways leading from the human disease alleles to ALS pathology (Figure ane).

Figure 1

To comport their screen, the authors nerveless flies from (A) a Drosophila strain carrying an ALS-associated allele, for instance hFUS^R521C , nether control of a UAS regulatory element along with the GMR-GAL4 driver. Expression of UAS-hFUS^R521C with GMR-GAL4 causes a degenerative rough eye phenotype. These flies were crossed to (B) flies from the Exelixis collection, each carrying an individual mutation caused by random insertion of a transposable element in the genome (light-green triangle). F1 progeny (C) were examined for enhancement or suppression of the rough eye phenotype.

Unpacking the Work

In order to conduct genome-wide screens for ALS genes, the authors develop a strategy to cross transgenic Drosophila strains expressing ALS-associated human alleles to insertion mutations from the Exelixis collection and to examine the F1 progeny (Effigy 1). Females were collected from Drosophila strains carrying either of 2 ALS-associated alleles, hFUS^R521C or hTDP-43^M337V , each controlled by a UAS regulatory element. Each strain also carries the GMR-GAL4 driver, which produces GAL4 protein in cells of the eye, thereby causing expression of the human ALS-associated proteins there. These females were mated to males from the Exelixis collection, each strain of which carries an individual insertion mutation.

All starting strains also carry balancer chromosomes [meet ( Hales et al. 2015) for a detailed description of balancer chromosomes], which permit stocks to be maintained as heterozygotes by bearing recessive lethal mutations and suppressing meiotic recombination with homologous chromosomes. The balancers are marked with the dominant wing phenotype Curly (Cy, curly wings instead of straight, on the balancer CyO), or the dominant larval phenotype Tubby (Tb, larvae are shorter and fatter than wild type, on the balancer TM6B). The 2d chromosome balancers in the strains expressing the human ALS alleles additionally carry the yeast gene GAL80 expressed in all cells. GAL80 poly peptide inhibits GAL4 activity, preventing expression of the ALS alleles in the starting strains (St Johnston 2013).

Technical Glossary

This glossary provides some technical data to assistance with agreement the experiments in the research commodity.

RNAi (RNA interference) uses a small-scale double-stranded RNA molecule to target an endogenous mRNA for post-transcriptional silencing. To employ RNAi in vivo, a transgene is generated encoding an RNAi transcript under command of a UAS regulatory element, allowing directed expression past the GAL4-UAS system. The transgene is designed with sequence complementarity to an endogenous target mRNA and an inverted repeat structure, which facilitates folding of the RNAi transcript into a double-stranded RNA. The double-stranded RNA promotes destruction of the target mRNA with matching sequence, thereby inhibiting production of functional protein from the target gene. This process of postal service-transcriptional knockdown frequently produces similar phenotypes as loss of function alleles and circumvents the time and labor needed to create genomic mutations. Furthermore, the Drosophila research community has produced libraries of RNAi transgenes targeting every gene in the genome, which are readily available (Mohr and Perrimon 2012).
Imaginal discs are epithelial sacs of primordial cells that requite rising to the external anatomical structures of the adult fly, too chosen the imago (Beira and Paro 2016). For instance, the eye imaginal discs are the precursors of the adult eyes. Imaginal discs are specified during embryogenesis, are patterned and abound during larval development, and course their singled-out morphologies during metamorphosis.
The neuromuscular junction (NMJ) is the site of contact between motor neurons and musculus tissue. In the Drosophila larva, motor neuron axon terminals at the NMJ class synaptic boutons, round structures that contain the synaptic active zones. Each Drosophila motor neuron creates a stereotypical number of boutons, providing a quantitative measurement of the fidelity of NMJ development and maintenance ( Menon et al. 2013). In ALS, motor neuron degeneration is accompanied by disassembly of the NMJ and atrophy of muscle tissue (Cappello and Francolini 2017).
A ascendant negative allele is one that interferes with the activity of the wild-type allele. Ascendant negative alleles are functionally dominant, i.due east., they generate a mutant phenotype every bit heterozygotes, but because their mutant phenotypes effect from reduced activity of the wild-type allele, they are loss of function.

Guided Reading Questions

The guided reading questions below are intended to assistance students work through the results of Kankel et al. (2020). Students can be expected to spend ∼xv–30 min on each figure, on boilerplate. Questions can be assigned as homework in preparation for class discussions or every bit in-grade grouping work. The overall logic of the experiments presented in the commodity is diagrammed in Effigy 2. Answers to the guided reading questions are bachelor as Supplemental Material online.

Effigy 2

$Workflow diagram illustrating the logic of the screening process. Genetic screens begin with a hypothesis, i.e., that genetic screens in Drosophila can be used to identify novel genes associated with ALS. Because human ALS-associated alleles hFUSR521C or hTDP-43M337V cause a visible degenerative eye phenotype in adult Drosophila, libraries of mutants like the Exelixis collection can be screened for dominant modifiers of this phenotype. Once the screens have been planned, proof-of-principle experiments are conducted to demonstrate that the screen has the potential to be successful, using selected Drosophila mutants of other ALS gene orthologs as positive controls. The screens are then performed, and a list of hits is generated. The list can be organized by which ALS allele is affected by each modifier, whether modifiers are enhancers or suppressors, and according to GO classification for \biological function and/or cellular localization. Screen hits also are validated experimentally by assaying whether they modify other ALS model systems, including c9orf72(G4C2)30-mediated degeneration of the adult Drosophila eye, dTDP-43mNLS aggregation in the larval eye imaginal disc, and dTDP-43N493D perturbation of the larval NMJ. Candidates that show activity in the secondary assays may be chosen for further study, which includes examination in an ALS model mouse and in human ALS patient data.$

Workflow diagram illustrating the logic of the screening procedure. Genetic screens begin with a hypothesis, i.eastward., that genetic screens in Drosophila can exist used to identify novel genes associated with ALS. Considering human ALS-associated alleles hFUS^R521C or hTDP-43^M337V cause a visible degenerative middle phenotype in adult Drosophila, libraries of mutants like the Exelixis collection can exist screened for dominant modifiers of this phenotype. Once the screens have been planned, proof-of-principle experiments are conducted to demonstrate that the screen has the potential to be successful, using selected Drosophila mutants of other ALS gene orthologs equally positive controls. The screens are so performed, and a list of hits is generated. The listing can exist organized by which ALS allele is affected by each modifier, whether modifiers are enhancers or suppressors, and according to GO classification for \biological office and/or cellular localization. Screen hits also are validated experimentally by assaying whether they change other ALS model systems, including c9orf72(G4C2)₃₀ -mediated degeneration of the adult Drosophila centre, dTDP-43^mNLS assemblage in the larval heart imaginal disc, and dTDP-43^N493D perturbation of the larval NMJ. Candidates that evidence activity in the secondary assays may be chosen for further written report, which includes examination in an ALS model mouse and in homo ALS patient information.

Figure 1. A genome-wide screening strategy for dominant modifiers of human ALS-associated allele induced centre degeneration in Drosophila .

What is the purpose of the screens described in this article? How were the screens conducted?
Explain each of the starting Drosophila strains used for the screens, shown in Figure 1A of Kankel et al. (2020). What genetic elements does each starting strain comport?
Depict Punnett squares representing the screen crosses. Which are the desired progeny and how will they exist selected?
Which photomicrograph console shows a normal fly eye? How exercise Figure one, C and H compare to normal?
Describe what is shown in Figure i, D–G and I–L in your own words. For each of the four genes shown, dSETX, dco, Hsc70Cb, and Ask, explain how it was tested and whether it is a suppressor or enhancer of hFUS^R521C and hTDP-43^M337V .

Figure ii. Ascendant modifiers of homo ALS-associated allele induced eye degeneration are identified in the screens.

How many insertion mutations were screened? How many crosses were established in the screens?
How former were the F1 flies when they were screened?
How many hits were recovered from the screens? What percentage of the full do the hits stand for? Show your calculations. Is this percentage higher, lower, or on par with expectations?
How many hits bear upon both hFUS^R521C and hTDP-43^M337V transgenes? Do those common hits always touch on the two transgenes in the same way? Give an example and propose a hypothesis to explain these results.

Figure 3. Validating screen hits using another ALS genetic model .

Explicate the experiment shown in Figure 3. How does it differ from the original screens? What are the advantages and disadvantages of this experiment compared to the original screens?
What does the graph in Figure 3I show? How is each percentage calculated?
What percentage of genes tested shows furnishings in this experiment?

Effigy 4. Validating screen hits using some other ALS-associated phenotype .

How do the transgenes in the experiment shown in Figure 4 differ from those in the experiments shown in Figure 1?
Compare and dissimilarity the proteins expressed from these transgenes with the original constructs. What additional disease-relevant belongings do these proteins show?
Is this boosted property modified past the hits from the screens? What does that suggest almost how this holding relates to degeneration?

Figure v. Validating screen hits in another cellular context .

In Figure five, the authors switch their focus abroad from the eye. What anatomical construction is the focus of this figure? In which cells is OK371-GAL4 expressed? Why do the authors choose to examine this structure?
The authors use the OK371-GAL4 driver to express iii different dTDP-43 variants. Amidst these three variants, which one has the most astringent mutant phenotype and how do yous know? How does this chronicle to the anatomical structure shown in the photomicrographs? What does this suggest well-nigh this variant?
The authors exam 3 genes, SF2, lilli, and klp98A, for their ability to modify the phenotypes caused by OK371-GAL4 expression of dTDP-43. How were these iii genes chosen?
Which of these three genes has a significant effect on the OK371-dTDP-43 phenotype? Does any of the three fail to touch on the OK371-dTDP-43 phenotype? Propose a hypothesis to explain these results.

Figure 6. Phospholipase D is an important player in ALS .

From their screens, the authors discover the Phospholipase D (PLD) pathway (schematized in Figure 9). How practice they exam the importance of PLD in ALS in Figure half dozen? Describe 5 results shown in this figure that corroborate the importance of PLD.
What effect would you await if Drosophila RalA expression were reduced instead of PLD in the same type of experiments?

Figure seven. The Phospholipase D pathway is of import for ALS progression.

Is your expectation above (Figure half dozen, question two) confirmed? Explain why or why not.
How do the results in Figure seven further bolster the importance of the PLD pathway in ALS?
Based on the experiments presented in Figures half-dozen and seven, are PLD pathway effectors likely to function upstream or downstream of FUS, TDP-43, and c9orf72 in ALS disease evolution?

Figure eight. Validating Phospholipase D in another model organism .

What are the advantages of performing genetic screens in Drosophila melanogaster as opposed to mice? What are the advantages of testing genetic interactions in mice as opposed to Drosophila?
Compare the grip strength of wild-type mice, SOD1^G93A-expressing mice, and SOD1^G93A-expressing mice with mutations in PLD1, PLD2, or PLD1 and 2. How does mutating PLD modify the effects of SOD1^G93A expression?
Are the results shown in Figure 8 consistent with those shown in Figure 6? Why or why not?

Putting It All Together

Other contempo research articles accept found that PLD pathway genes are upregulated in sALS patients with early-onset disease ( Rabin et al. 2010; Kaplan et al. 2014). Is this finding consistent with the data presented in Figures 6–eight? Explicate.
Do you think the PLD pathway is a worthwhile therapeutic target for ALS? Why or why non?
Advise an experiment to follow upward on the idea of PLD as a therapeutic target for ALS.

Student Projects

After reading the article and working through the guided reading questions, instructors may choose to give their students the opportunity to develop a project of their own on a related topic. Below are two different options for student-directed projects. We recommend giving students several weeks to consummate the assignment and submit in midsemester or at the end of the term. Projects can be formatted equally a written paper or as an oral presentation, with students working as individuals or in groups.

Cull a affliction besides ALS that interests you. Does this disease accept a genetic component? Has this disease been modeled in Drosophila or another model organism? Pattern a genetic screen to identify novel factors in this disease.
Using PubMed (https://pubmed.ncbi.nlm.nih.gov/), find another research article that describes a screen for ALS components. Compare and dissimilarity with this article, using the questions beneath to help you.
- What model organism or model organization was used in your commodity?
- How was ALS imitation in your article? What phenotypic aspects of ALS were examined?
- What are the advantages and disadvantages of the model system in your article compared to the one used in Kankel et al. (2020)?
- How was the screen conducted?
- What were the outcomes of the screen?
- Are there whatever screen hits in common betwixt your article and Kankel et al. (2020)? Why might this be? What does this advise?

Acknowledgments

We thank Elizabeth De Stasio, Molly Gallop, Rebecca Delventhal, Mark Kankel, Anindya Sen, and Spyros Artavanis-Tsakonas for helpful comments on the manuscript. The Steinhauer laboratory is supported by National Institutes of Health (NIH) grant R15-HD080511.

Communicating editor: E. De Stasio

Literature Cited

Adams

Grand D

S E

Celniker

R A

Holt

C A

Evans

J D

Gocayne

et al. ,

2000

The genome sequence of Drosophila melanogaster.

Science

287

2185

–

2195

Artavanis-Tsakonas

2004

Accessing the Exelixis drove.

Nat. Genet.

207

Baker

Due north Eastward

Quiquand

Ruggiero

, and

50 H

Wang

2014

Eye evolution.

Methods

252

–

259

Beira

J V

, and

Paro

2016

The legacy of Drosophila imaginal discs.

Chromosoma

125

573

–

592

Bellen

H J

, and

South

Yamamoto

2015

Morgan's legacy: fruit flies and the functional annotation of conserved genes.

Cell

163

–

(erratum: Cell 163: 772).

Bellen

H J

Tong

, and

Tsuda

2010

100 years of Drosophila research and its impact on vertebrate neuroscience: a history lesson for the time to come.

Nat. Rev. Neurosci.

514

–

522

Bellen

H J

R W

Levis

J Westward

Carlson

Evans-Holm

et al. ,

2011

The Drosophila factor disruption project: progress using transposons with distinctive site specificities.

Genetics

188

731

–

743

Benzer

1967

Behavioral mutants of Drosophila isolated by countercurrent distribution.

Proc. Natl. Acad. Sci. USA

1112

–

1119

Make

A H

, and

Perrimon

1993

Targeted gene expression as a means of altering cell fates and generating dominant phenotypes.

Development

118

401

–

415

Chocolate-brown

R H

, and

Al-Chalabi

2017

Amyotrophic lateral sclerosis.

N. Engl. J. Med.

377

162

–

172

Cappello

, and

One thousand

Francolini

2017

Neuromuscular junction dismantling in amyotrophic lateral sclerosis.

Int. J. Mol. Sci.

2092

Cooley

Kelley

, and

Spradling

1988

Insertional mutagenesis of the Drosophila genome with single P elements.

Science

239

1121

–

1128

Gaudet

Škunca

J C

, and

Dessimoz

2017

Primer on the gene ontology.

Methods Mol. Biol.

1446

–

Hales

K G

C A

Korey

A Thou

Larracuente

, and

D M

Roberts

2015

Genetics on the fly: a primer on the Drosophila model organisation.

Genetics

201

815

–

842

Hardiman

Al-Chalabi

Chio

E Yard

Corr

Thou

Logroscino

et al. ,

2017

Amyotrophic lateral sclerosis.

Nat. Rev. Dis. Primers

17071

(erratum: Nat. Rev. Dis. Primers 3: 17085).

Hastings

2017

Primer on ontologies.

Methods Mol. Biol.

1446

–

Hotta

, and

Southward

Benzer

1969

Abnormal electroretinograms in visual mutants of Drosophila.

Nature

222

354

–

356

Jackson

G R

Salecker

Dong

Yao

Due north

Arnheim

et al. ,

1998

Polyglutamine-expanded man Huntingtin transgenes induce degeneration of Drosophila photoreceptor neurons.

Neuron

633

–

642

Jan

Y North

, and

Fifty

Jan

2008

Retrospective: Seymour Benzer (1921-2007).

Scientific discipline

319

Editorial,

2020

Model organisms: nature'south souvenir to disease research.

Genetics

214

233

–

234

Jowett

Chiliad F

Wajidi

East

Oxtoby

, and

C R

Wolf

1991

Mammalian genes expressed in Drosophila: a transgenic model for the report of mechanisms of chemical mutagenesis and metabolism.

EMBO J.

1075

–

1081

Kankel

G W

Sen

Chiliad

Theodorou

D North

Dimlich

et al. ,

2020

Amyotrophic lateral sclerosis modifiers in Drosophila reveal the phospholipase D pathway as a potential therapeutic target.

Genetics

215

747

–

766

Kaplan

Grand J

Spiller

Towne

Grand C

Kanning

G T

Choe

et al. ,

2014

Neuronal matrix metalloproteinase-9 is a determinant of selective neurodegeneration.

Neuron

333

–

348

Lander

E S

L M

Linton

Birren

Nusbaum

Thou C

Zody

et al. ;

International Human Genome Sequencing Consortium

2001

Initial sequencing and analysis of the human genome.

Nature

409

860

–

921

(erratum: Nature 412: 565).

Lanson

N A

Maltare

Male monarch

Smith

J H

Kim

et al. ,

2011

A Drosophila model of FUS-related neurodegeneration reveals genetic interaction betwixt FUS and TDP-43.

Hum. Mol. Genet.

2510

–

2523

Luo

Tully

, and

White

1992

Human amyloid precursor protein ameliorates behavioral deficit of flies deleted for Appl gene.

Neuron

nine

595

–

605

Martin

Al Khleifat

, and

Al-Chalabi

2017

What causes amyotrophic lateral sclerosis?

F1000Res.

371

McGurk

Berson

, and

Due north M

Bonini

2015

Drosophila equally an in vivo model for human neurodegenerative disease.

Genetics

201

377

–

402

Menon

K P

R A

Carrillo

, and

Thou

Zinn

2013

Evolution and plasticity of the Drosophila larval neuromuscular junction.

Wiley Interdiscip. Rev. Dev. Biol.

647

–

670

Mohr

S E

, and

Northward

Perrimon

2012

RNAi screening: new approaches, understandings, and organisms.

Wiley Interdiscip. Rev. RNA

iii

145

–

158

Morgan

T H

1910

Sex limited inheritance in Drosophila.

Science

120

–

122

Nüsslein-Volhard

, and

Wieschaus

1980

Mutations affecting segment number and polarity in Drosophila.

Nature

287

795

–

801

Pak

Westward Fifty

Grossfield

, and

North V

White

1969

Nonphototactic mutants in a study of vision of Drosophila.

Nature

222

351

–

354

Rabin

Southward J

J M

Kim

Thou

Baughn

R T

Libby

Y J

Kim

et al. ,

2010

Sporadic ALS has compartment-specific abnormal exon splicing and altered cell-matrix adhesion biology.

Hum. Mol. Genet.

nineteen

313

–

328

Ranganathan

Haque

Coley

Shepheard

Cooper-Knock

et al. ,

2020

Multifaceted genes in amyotrophic lateral sclerosis-frontotemporal dementia.

Front. Neurosci.

xiv

684

Ritson

K P

S K

Custer

B D

Freibaum

J B

Guinto

Geffel

et al. ,

2010

TDP-43 mediates degeneration in a novel Drosophila model of disease caused past mutations in VCP/p97.

J. Neurosci.

7729

–

7739

Rogge

R D

C A

Karlovich

, and

Banerjee

1991

Genetic autopsy of a neurodevelopmental pathway: son of sevenless functions downstream of the sevenless and EGF receptor tyrosine kinases.

Cell

–

Rubin

Chiliad M

, and

A C

Spradling

1982

Genetic transformation of Drosophila with transposable element vectors.

Science

218

348

–

353

Simon

One thousand A

D D

Bowtell

Thousand South

Dodson

T R

Laverty

, and

Chiliad M

Rubin

1991

Ras1 and a putative guanine nucleotide exchange factor perform crucial steps in signaling by the sevenless protein tyrosine kinase.

Cell

701

–

716

Spradling

A C

, and

G M

Rubin

1982

Transposition of cloned P elements into Drosophila germ line chromosomes.

Scientific discipline

218

341

–

347

Spradling

A C

D M

Stern

Kiss

Roote

Laverty

et al. ,

1995

Gene disruptions using P transposable elements: an integral component of the Drosophila genome projection.

Proc. Natl. Acad. Sci. USA

10824

–

10830

St Johnston

2002

The art and design of genetic screens: Drosophila melanogaster.

Nat. Rev. Genet.

176

–

188

St Johnston

2013

Using mutants, knockdowns, and transgenesis to investigate gene function in Drosophila.

Wiley Interdiscip. Rev. Dev. Biol.

587

–

613

Thibault

Southward T

K A

Vocaliser

Westward Y

Miyazaki

Milash

N A

Dompe

et al. ,

2004

A complementary transposon tool kit for Drosophila melanogaster using P and piggyBac.

Nat. Genet.

283

–

287

Venter

J C

M D

Adams

E Westward

Myers

P Westward

R J

Mural

et al. ,

2001

The sequence of the human genome.

Science

291

1304

–

1351

(erratum: Scientific discipline 292: 1838).

Vucic

J D

Rothstein

, and

M C

Kiernan

2014

Advances in treating amyotrophic lateral sclerosis: insights from pathophysiological studies.

Trends Neurosci.

433

–

442

Wangler

M F

Yamamoto

H T

Chao

J E

Posey

Chiliad

Westerfield

et al. ,

2017

Model organisms facilitate rare disease diagnosis and therapeutic research.

Genetics

207

nine

–

Warrick

J M

H Fifty

Paulson

1000 L

Gray-Board

Q T

Bui

K H

Fischbeck

et al. ,

1998

Expanded polyglutamine protein forms nuclear inclusions and causes neural degeneration in Drosophila.

Cell

939

–

949

Yerbury

J J

Due north Due east

Farrawell

, and

McAlary

2020

Proteome homeostasis dysfunction: a unifying principle in ALS pathogenesis.

Trends Neurosci.

274

–

284