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 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.
Neurons are for the most part post-mitotic and incapable of dividing1,2. In most animals, neuroprotective mechanisms exist to maintain these cells throughout the organism's lifespan, especially at old age when neurons are most vulnerable to damage. Genes underlying these mechanisms can be identified in mutants exhibiting neurodegeneration, a phenotypic indicator for the loss of neuroprotection, using a forward genetic protocol. Forward genetic screens using chemical mutagens such as ethyl methanesulfonate (EMS) or N-ethyl-N-nitrosourea (ENU) are particularly useful due to the random point mutations they induce, resulting in an inherently unbiased approach that has shed light on numerous gene functions in eukaryotic model organisms3,4,5 (in contrast, X-ray mutagenesis creates DNA breaks and can result in rearrangement rather than point mutations6).
The common fruit fly Drosophila melanogaster is an ideal subject for these screens due to its high quality, well annotated genome sequence, its long history as a model organism with highly developed genetic tools, and most significantly, its shared evolutionary history with humans7,8. A limiting factor in the applicability of this protocol is early lethality caused by the mutated genes, which would prevent testing at old age9. However, for non-lethal mutations, a climbing assay, which takes advantage of negative geotaxis, is a simple, although extensive, method of quantifying impaired motor functioning10. To exhibit sufficient locomotor reactivity, flies depend on neural functions to determine direction, sense its position, and coordinate movement. The inability of flies to sufficiently climb in response to stimuli can therefore indicate neurological defects11. Once a particular defective climbing phenotype is identified, further testing using a secondary screen such as histological analysis of brain tissue, can be used to identify neurodegeneration in climbing-defective flies. Subsequent gene mapping can then be used to reveal the genomic region on the chromosome carrying the defective neuroprotective gene of interest. To narrow down the chromosomal region of interest, meiotic mapping using mutant fly lines carrying dominant marker genes with known locations on the chromosome can be performed. The marker genes serve as a reference point for the mutation as the frequency of recombination between two loci provides a measurable distance that can be used to map the approximate location of a gene. Finally, crossing the mutant lines with lines carrying balanced deficiencies on the meiotically mapped chromosomal region of interest creates a complementation test in which the gene of interest can be verified if its known phenotype is expressed5. Polymorphic nucleotide sequences in the identified gene, possibly resulting in an altered amino acid sequences, can be evaluated by sequencing the gene and comparing it to the Drosophila genome sequence. Subsequent characterization of the gene of interest can include testing of additional mutant alleles, mutation rescue experiments and examination of additional phenotypes.
1. Preparation and Aging of Flies
2. Protocol 1: Climbing Assay (Adapted from Ali et al.10)
3. Protocol 2: Selection of Drosophila Strains for Further Analysis
4. Protocol 3: Histology Analysis
5. Protocol 4: Gene Mapping of Recessive Mmutant Phenotype
6. Protocol 5: DNA Sequencing and Analysis
NOTE: Keep in mind that ENU mutagenesis introduces point mutations11.
7. Protocol 6: Further Analysis of Candidate Gene Function
In this aerticle, we present the steps used to identify the gene brain tumor (brat) as playing a role in maintenance of neuronal integrity (e.g., neuroprotection) in adult flies17; a methodology that can be used to identify genes involved in neuroprotection. We used a forward genetic approach (the strategy is outlined in Figure 1A) to screen through a collection of chemically mutagenized flies using a climbing assay (the apparatus used for this assay is shown in Figure 1B). Among 235 homozygous lines, about 37% of tested lines exhibited a climbing pass rate below 50% when tested at the age of 10-12 days (Figure 1C). 58% of tested lines exhibited significantly different climbing behavior when their percent climbing pass rate (CPR) was compared to the mean climbing percent value of all tested lines using one-way ANOVA (Figure 1D). Subsequent histological screen on 51 of the lines exhibiting the lowest climbing pass rate revealed that 29 of these lines showed visible appearance of holes in the brain neuropil (ranging from mild to severe) indicative of neurodegeneration20 (Figure 2). Among the lines showing defective climbing behavior and severe neurodegeneration, we choose to map the mutation underlying the phenotype in line 867 (0% CPR and P value= 1.36e-14 based on one-way ANOVA). The neurodegeneration phenotype in 867 flies is recessive because the brains of heterozygous 867 flies that have been outcrossed to a wild type strain are comparable to these of controls (Figure 3A). Using histology, meiotic mapping situated the mutation in the 31-51 cytological locations, between the phenotypic markers J (Jammed) and L (Lobe) on the second Drosophila chromosome. Using the climbing assay, deficiency mapping between cytological locations 31 and 51 with lines from the Drosophila Deficiency Kit for chromosome 2L (DK2L)16 mapped the mutation in a region encompassing 118 genes (Figure 3B; where 867/+ serves as control for the statistical analysis). Examining the brain phenotype of 867 mutants crossed to additional deficiency lines (Figure 3C) confirmed that the mutation is contained in a region of the genome that includes the gene brat. A complete list of all additional genes contained within these deletions can be found in Flybase by clicking on the link associated with the deleted segment (e.g., for Df(2L)ED1272 the deleted segment is 37C5-38A2). Based on previous observations that brat mutants have supernumerary cells in their brains21, a phenotype also observed in 867 mutants, brat was selected for DNA sequencing analysis. Sanger sequencing of the brat gene containing the exon-coding region identified G/A nucleotide change at position 37,739 of the gene (Figure 4A), leading to glycine to glutamic acid (G/E) change at position 470 of the Brat protein17 (Figure 4B). Rescue experiments confirm that Brat plays a neuroprotective role, as the overexpression of the functional gene in the 867 line suppresses the neurodegeneration phenotype (Figure 5A). Moreover, the phenotype in 867 mutants worsens over time, suggesting that brat plays an age-dependent role in neuroprotection17 (Figure 5B).
Figure 1: Forward genetic screen to identify novel neuroprotective genes. (A) Screen strategy. (B) Climbing assay testing apparatus. (C) Climbing assay results for males from 235 homozygous ENU-mutagenized lines. Pie chart represents the proportions of tested fly lines that fall into 4 groups of climbing pass rates: 0% , 1-20% , 21-50% , and 51-100% . (D) Results from 1 way ANOVA statistical analysis in which mutant lines were compared to the mean climbing percent value of all tested lines. Represented is the number for two P value categories: P < 0.05 (significant change) and P > 0.05 (not significant change).
Figure 2: Secondary histology screen. H&E-stained mid-brain sections illustrating, from left to right, no neurodegeneration, mild neurodegeneration, and confirmed neurodegeneration. A total of 51 homozygous lines were retested by histology after identification of candidates using the climbing assay. Arrows indicate the holes in the brain indicative of neurodegeneration. The region circled by the dotted lines shows the severe neurodegenration observed in line 867.
Figure 3: Identification of the neuroprotective gene brat following deficiency mapping using the climbing assay. (A) Shown are representative images of H&E-stained mid-brain sections of 18-20 days old w1118 (Control), heterozygous 867 (867/+) and homozygous 867 mutant flies. (B) The deficiency line Df(2L)ED1272 (BL_24116) does not complement the 867 mutant phenotype (black histogram) based on the climbing assay. Histological verification shows that Df(2L)ED1272 line doesn't complement the 867 neurodegeneration phenotype, whereas Df(2L)ED1315 (BL_9269) does. Graph represents mean and standard deviation of 5 climbing trials for each line. Asterisks indicate significance based on one-way ANOVA, in which the mean climbing percent value of each line was compared to the mean climbing percent value of line 867/+ (green histogram). * P < 0.05, ** P < 0.01 and ***P < 0.001. (C) Schematic representation of additional deficiency lines showing non-complementation of the 867 phenotype by histology. This figure has been modified from Loewen et al.17; an article published under the Creative Commons Attribution 4.0 International License.
Figure 4: Point mutation in the brat gene leads to an amino acid change in the Coiled-coil domain of the Brat protein. (A) brat gene model and primers used for the amplification and sequencing of the coding region. (B) DNA sequencing of the brat locus identifies a nucleotide change that leads to an amino acid change in the Coiled Coil domain of the Brat protein. This figure has been modified from Loewen et al.17; an article published under the Creative Commons Attribution 4.0 International License.
Figure 5: Further analysis of brat mutants confirms its neuroprotective role in Drosophila. (A) Using the Gal-4>UAS system22 , neuroblast-specific expression of the functional brat gene rescues the neurodegeneration phenotype in 867 mutants. (B) Age-dependent neurodegeneration is observed in 867 mutants carrying a reporter gene pcna-GFP. bratchs corresponds to the name of the brat allele in line 867, which was named cheesehead (chs). Shown are representative H&E-stained midbrain sections of yw (control) and homozygous bratchs; pcna-GFP flies of the indicated ages. This figure has been modified from Loewen et al.17; an article published under the Creative Commons Attribution 4.0 International License.
Station | Time | Temperature | Vacuum/Pressure | Solution |
1 | NO DELAY-QUICK START | |||
2 | OFF | OFF | OFF | |
3 | : 15 | 37 °C | ON/ON | 80%EtOH |
4 | : 15 | 37 °C | ON/ON | 95% EtOH |
5 | : 15 | 37 °C | ON/ON | 100% EtOH |
6 | : 15 | 37 °C | ON/ON | 100% EtOH |
7 | : 15 | 37 °C | ON/ON | 100% EtOH |
8 | : 15 | 37 °C | ON/ON | Xylenes |
9 | : 15 | 37 °C | ON/ON | Xylenes |
10 | : 15 | 37 °C | ON/ON | Xylenes |
11 | : 30 | 58 °C | ON/ON | Paraffin |
12 | : 15 | 58 °C | ON/ON | Paraffin |
13 | OFF | 58 °C | OFF | Paraffin |
14 | : 15 | 58 °C | ON/ON | Paraffin |
Table 1: Recommended program settings for automated tissue processor. The steps in the order listed here can be used with an automated tissue processor for fixed heads.
Reagent | Volume (μL) |
10x ExTaq Buffer (Mg2+ plus) (20 mM) | 2.5 |
Forward primer (10 μM) | 2.5 |
Reverse primer (10 μM) | 2.5 |
2.5 mM dNTPs | 2 |
Template DNA | 2 |
TaKaRa Ex Tq (5 U/μL) | 0.25 |
Nuclease-free water | 13.25 |
Total volume | 25 |
Table 2: Reagents used for PCR reaction. Reagents and respective volumes used in the PCR reaction to amplify the brat-coding region.
Step | Temperature | Time |
35 cycles | 95 °C | 15 s |
55 °C | 45 s | |
72 °C | 3 min 10 s | |
Hold | 4 °C | ∞ |
Table 3: Thermal cycling conditions used for PCR. The steps in the order listed here can be used to program a thermal cycler using the reaction mix shown in Table2.
Forward genetic screens in Drosophila have been an effective approach to identify genes involved in different biological processes, including age-dependent neuroprotection5,23,24,25. Using this strategy, we were successful in identifying brat as a novel neuroprotective gene17.
One critical step in this protocol involves the proper orientation of heads (as described in Section 4.4.3.) for histology analysis. Additionally, markers of the line used for the meiotic mapping should not interfere with the phenotype of the new mutation (in this study: neurodegeneration). We recommended an initial test to confirm that the chosen markers do not cause neurodegeneration of their own. For instance, Jammed (J) causes wing blisters that could possibly interfere with climbing as is the case for amyloid precursor protein (APP)-overexpressing flies that also have blistered wings26. Lobe (L) leads to reduction in eye size; however, we do not observe central brain degeneration and this marker can be used to map central brain neurodegeneration. Pin and Sternoplural (Sp) are also appropriate to use, as brains of flies carrying these markers are comparable to brains of wild type controls.
One disadvantage of the forward genetic methodology in flies is the length of time required to narrow regions of DNA to the gene of interest3,5. The use of improved mapping strategies27 along with the availability of next generation sequencing technologies 28 should allow even easier identification of mutations associated with phenotypes of interest. Forward genetic screens can be labor-intensive. For instance, histology confirmation, which assays directly for neurodegeneration in the brain, alone requires a considerable amount of time. However, this approach will be much more difficult and expensive to perform in mammalian models, not necessarily allowing extensive genetic studies. Chemical mutagenesis screens are advantageous because the identification of point mutations could provide critical information about the function of affected genes' products. The identification of a point mutation causing an amino acid change in a conserved domain of the Brat protein (Figure 4B) illustrates the importance of the coiled-coil domain of this TRIM-NHL protein in neuroprotection18. The climbing assay, which measures locomotor behavior, is certainly advantageous by allowing rapid testing of large numbers of flies for defects in mobility, also often seen in human neurodegeneration25. This assay could also be performed in another screen setting such as genome-wide in vivo RNA interference (RNAi) screen that could be used to identify neuroprotective genes. Although we decreased the distance between the bottom of the test chamber and the passing line from 8 cm10 to 5 cm to set a stricter passing rate in the present study, low climbing rates did not mirror neurodegeneration for large number of lines. Neurodegeneration may become apparent after the onset of behavioral defects, meaning that flies may need to be aged for longer before vacuoles appear. Another possibility is that the locomotor defects we observe in certain lines are not due to defective neurological functioning but rather to muscle weakness or more general unfitness of the flies. Additionally, it is possible that we are missing potential candidates, in which climbing defects do not manifest at younger age (e.g., 10 days old) but rather become prominent later in life. This screening strategy could certainly be applied to identify age-dependent genes, thus eliminating genes associated with potential defects of neural development. The protocol presented here can be adjusted depending on the desired probability of candidate lines to contain the mutated gene involved in neuroprotection. Lowering the passing rate threshold directly is another means of achieving the same result. These candidates would theoretically exhibit greater neurodegeneration, which could save time and resources during confirmation and mapping.
In general, the protocol describes a straightforward way to screen for neurodegeneration and subsequently identify neuroprotective gene candidates. This genetic approach is not limited to the behavior and histological assays described here, and may also be used to screen for other phenotypes like temperature-sensitive (ts) paralysis, egg laying or fertility phenotypes, just to cite a few. For instance, Drosophila ts-paralytic mutants are known to be enriched for neurodegeneration29 and could represent an additional source for the identification of neuroprotective genes.
The authors have nothing to disclose.
We are particularly grateful to Dr. Barry Ganetzky, in who's lab the genetic screen was performed, allowing the identification and characterization of brat as a neuroprotective gene. We thank Dr. Steven Robinow for kindly providing the collection of ENU-mutagenized flies used in the genetic screen presented in this article. We thank the members of Ganetzky lab, Drs. Grace Boekhoff-Falk and David Wassarman for helpful discussions throughout the duration of this project, Ling Ling Ho and Bob Kreber for technical assistance, Dr. Aki Ikeda for the use of his microtome facility at the University of Wisconsin and Dr. Kim Lackey and the Optical Analysis Facility at the University of Alabama.
Major equipment | |||
Fume hood for histology | |||
Light Microscope | Nikon | Eclipe E100 | Preferred objective for imaging is X20 |
Imaging software | Nikon | ||
Microscope Camera | Nikon | ||
Thermal cycler | Eppendorf | ||
Fly pushing and climbing assay | |||
VWR® Drosophila Vial, Narrow | VWR | 75813-160 | |
VWR® General-Purpose Laboratory Labeling Tape | VWR | 89097-912 | |
Standard mouse pad | |||
Stereoscope | Motic | Model SMZ-168 | |
CO2 anesthesia station (Blowgun, foot valve, Ultimate Flypad) | Genesee Scientific | 54-104, 59-121, 59-172 | Doesn’t iinclude CO2 tank |
Fine-Tip Brushes | SOLO HORTON BRUSHES, INC. | ||
Drosophila Incubator | VWR | 89510-750 | |
Gene mapping | |||
CantonS | Bloomington Drosophila Stock Center | 9517 | |
w1118 | Bloomington Drosophila Stock Center | 5905 | |
yw | Bloomington Drosophila Stock Center | 6599 | |
Drosophila line used for recombination mapping | Bloomington Drosophila Stock Center | 3227 | Genotype: wg[Sp-1] J[1] L[2] Pin[1]/CyO, P{ry[+t7.2]=ftz/lacB}E3 |
CyO/sno[Sco] | Bloomington Drosophila Stock Center | 2555 | Drosophila balancer line used for recombination mapping |
Deficiency Kit for chromosome 2L | Bloomington Drosophila Stock Center | DK2L | Cook et al., 2012 |
Histology analysis | |||
Ethanol, (100%) | Thermo Fischer Scientific | A4094 | |
Chloroform | Thermo Fischer Scientific | C298-500 | |
Glacial Acetic Acid | Thermo Fischer Scientific | A38-500 | |
Fisherbrand™ Premium Microcentrifuge Tubes: 1.5mL | Thermo Fischer Scientific | 05-408-129 | |
Histochoice clearing agent 1X | VWR Life Sciences | 97060-934 | |
Harris Hematoxylin | VWR | 95057-858 | |
Eosin | VWR | 95057-848 | |
Thermo Scientific™ Richard-Allan Scientific™ Mounting Medium | Thermo Scientific™ 4112 | 22-110-610 | CyO/sna[Sco] |
Unifrost Poly-L-Lysine microscope slides, 75x25x1mm, EverMark Select Plus | Azer Scientific | ||
Fisherbrand™ Cover Glasses: Rectangles | Fisherbrand | 12-545M | Dimensions: 24×60 mm |
Traceable timer | VWR | ||
Slide Warmer | Barnstead International | model no. 26025 | |
Slide tray and racks | DWK Life Sciences | Rack to hold 20 slides | |
Fisherbrand™ General-Purpose Extra-Long Forceps | Fisherbrand | 10-316A | |
Kimwipes™ | Kimberly-Clark™ Professional | ||
6 inch Puritan applicators | Hardwood Products Company, Guilford, Maine | 807-12 | |
VWR® Razor Blades | VWR | 55411-050 | |
Tupperware or glass containers for histology liquids | 16 + 1 for running water | ||
High Profile Coated Microtome Blades | VWR | 95057-834 | |
Corning™ Round Ice Bucket with Lid, 4L | Corning™ | ||
Beaker | Or other container for ice water and cassettes | ||
Tissue Bath | Precision Scientific Company | 66630 | |
Microtome | Leica Biosystems | ||
Molecular analysis | |||
Wizard® SV Gel and PCR Clean-Up System | Promega | A9282 | |
Ex Taq DNA polymerase | TaKaRa | 5 U/μl | |
Invitrogen™ SYBR™ Safe™ DNA Gel Stain | Invitrogen™ | ||
UltraPure™ Agarose | Invitrogen™ | ||
1 Kb Plus DNA Ladder | Invitrogen™ | ||
ApE-A plasmid Editor software | Available for free download | ||
Statistical analysis | |||
R software package | |||
Further analysis | |||
y[1] w[*]; wg[Sp-1]/CyO; Dr[1]/TM3, Sb[1] | Bloomington Drosophila Stock Center | 59967 |