The main goal of this protocol is to use the power of forward genetics to identify genes which when dysregulated lead to neurodegeneration. The main advantage of this technique is that it provides an unbiased and straightforward approach for the identification of neuroprotective genes which might be more difficult to perform in mammalian models. This technique can be used to identify novel genes implicated in the process of neurodegeneration which is associated with many disease conditions such as Alzheimer's and Parkinson's disease.
In addition to identifying neuroprotective genes, the forward genetic approach can be used to discover genes involved in other biological processes such as neuronal function and transmission. To begin, collect homozygous flies from lines belonging to the ENU mutagenized collection of Drosophila mutants mapped to the second chromosome. Flip the flies aged 10 to 12 days into a testing chamber without anesthesia one line at a time and allow them to recover for five minutes.
With the five centimeter line mark side down, forcibly tap the chamber three times on a mouse pad placed on a solid surface so that all flies begin the assay at the bottom. Observe fly locomotion over a period of 10 seconds. Record the number of flies that reach or pass the five centimeter line within this time as well as the total number of flies tested.
Wait one minute before starting a second trial. Repeat a minimum of three trial replicates per line. After anesthetizing the flies on a carbon dioxide pad, sever fly heads with a surgical blade and use a paintbrush to gently place them in one milliliter of Carnoy's fixative in a 1.5 milliliter microcentrifuge tube.
Then store the tube at four degrees Celsius overnight. The following day, make sure the heads are sunk in the fixative solution. Under a fume hood, use a P1000 pipette to replace the fixative solution with one milliliter of 70%ethanol and maintain the samples at four degrees Celsius for future analysis.
Using a Pasteur pipette, transfer the fixed heads into microbiopsy cassettes and close the cassettes. Send the cassettes to a histology facility for processing or place them in an automated tissue processing machine if available on site. Run the program to dehydrate, clear and infiltrate the heads with paraffin.
Then transfer the cassettes into a paraffin embedding station with paraffin heated at 60 degrees Celsius and place metal base molds that fit the cassettes on the heated station. Use fine forceps to orient the heads such that they face the top in the base mold. Proper head orientation is critical for the histology analysis.
After hardening, trim each paraffin block to minimize the surface that will be sectioned. Cut five micrometer sections of each block on the microtome. Next, place the sectioned paraffin ribbons in a heated water bath at 35 degrees Celsius for a maximum of five minutes.
Immerse a polylysine-coated slide in the heated water bath and use a wood applicator to place the sectioned ribbons on the slide. Let the slides air dry overnight at room temperature. The next day, use a slide warmer to heat the slides for 15 minutes at 63 degrees Celsius.
Under a fume hood, place the slides on a slide staining rack and place the rack in HistoChoice for five minutes twice followed by several ethanol and distilled water bath treatments according to the manuscript. Transfer the rack into a container filled with Harris Hematoxylin for two to five minutes to stain. Then take the rack out of the Hematoxylin solution and place it under running tap water for 10 minutes before the following staining steps.
After staining, using forceps, take the slides out of the HistoChoice or Xylene solution. Drip a thin layer of permanent mounting medium on the sample and gently place a coverslip on the top. Avoid formation of bubbles.
After the mounting medium is hardened, place the slide under a light microscope to obtain images of representative brain sections at approximately midbrain. First, prepare a food containing vial for generating heterozygous female flies. After carbon dioxide anesthesia, use a paintbrush to transfer five males of the mutant line and 10 virgin females of the sternopleural jammed lobe and pin over Curly O line into the vial to make a cross.
Place the vial at 25 degrees Celsius. After 10 to 12 days, collect at least 15 virgin female progeny carrying the mutated chromosome and the marker chromosome. Cross them to five males from a balanced line that carries another visible dominant marker Curly O over scutoid or equivalent.
After 10 to 12 days, collect the heterozygous male progeny carrying either scutoid or Curly O and the potentially recombined chromosome from the cross. Individually mate them to virgin females from the stock carrying the mutated chromosome. Ten to 12 days later, collect the progeny from the last cross into new food containing vials and age the flies at 29 degrees Celsius to 10 to 14 days.
Perform histology on fly heads and repeat for progeny of each individual cross. With this information, perform deficiency mapping on narrowed region of the chromosome to refine the location of the recessive mutation. Each deficiency line has a deletion of different region of the chromosomes allowing them to be used for complementation testing.
Next, cross lines from the Drosophila deficiency kit for the second chromosome and look for non-complementation of the phenotype of the interest. To obtain reference for DNA sequence analysis, download a FASTA format file containing the genomic consensus sequence of the BRAT gene from FlyBase or use a file containing results for BRAT sequence of a genetic background control, for example the originally mutagenized Drosophila line. Open sequencing files in the A Plasmid Editor.
Go to Edit and click Align Sequences. Using the computer's mouse, select all sequences to compare. In the drop menu, indicate the reference sequence for this alignment.
Inspect the sequenced region for nucleotide changes in comparison with the reference sequence. If desired, save the alignment on a computer in RTF format by clicking on Text then Save. Perform the rescue experiment by collecting F1 progeny from each cross.
Carefully select away marked balancers. Age the flies on Drosophila food medium at 29 degrees Celsius and perform histology analysis on brains to look for neurodegeneration as previously. To perform age-dependent analysis of neurodegeneration in 867 mutants, age the flies to five, 15 and 25 days and perform subsequent histology analysis.
This protocol presents a forward genetic approach to screen through a collection of chemically mutagenized flies using a climbing assay. Among 235 homozygous lines, about 37%of the tested lines exhibited a climbing pass rate below 50%when tested at the age of 10 to 12 days. 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 neurodegeneration.
The neurodegeneration phenotype in 867 flies is recessive because the brains of heterozygous 867 flies that have been out-crossed to a wild type strain were comparable to those of controls. The deficiency line Df24116 does not complement the 867 mutant phenotype based on the climbing assay. Histological verification shows the deficiency line Df24116 does not complement the 867 neurodegeneration phenotype whereas Df9296 does.
Examining the brain phenotype of 867 mutants crossed to additional deficiency lines confirmed that the mutation is contained in the region of the genome that includes the gene BRAT. It's important to remember during this procedure to work as rigorously and precisely as possible as this protocol combines several steps which if executed improperly can affect the identification of mutants with neurodegeneration in candidate genes. Depending on the genes identified, subsequent analysis can include testing additional ovules of the gene of the interest or interactions with genes that play a role in known pathways of neurodegeneration.
During the histology procedure, remember to handle the Carnoy's fixative solution with caution since it contains chloroform. Wear gloves and use with adequate ventilation ideally under the fume hood. Similarly, perform all histology staining under the fume hood.
