SUMMER INTERN REPORT

SOUTH CAMPUS, UNIVERSITY OF DELHI

Ishita Chaudhry| Integrated BS-MS’27 IISERM | 4th June 2024

 Introduction
Neurodegenerative diseases are caused by the loss of structure and functioning of neurons, in the process known as
neurodegeneration, which involves cell death. Neurodegenerative diseases include amyotrophic lateral sclerosis,
multiple sclerosis, Parkinson's disease, Alzheimer's disease, polyglutamine diseases, multiple system atrophy,
tauopathies, and prion diseases.

The brain and spinal cord are complex, therefore we need to study simpler, analogous systems to address scientific
questions effectively. The fruit fly, Drosophila melanogaster, is a model organism in neuroscientific disease research,
enabling significant discoveries. Its genetic tools allow precise genome manipulation, and its short lifespan facilitates
rapid investigation of brain function, compared to models like mice. Drosophila's features are used as a model for
human neurodegenerative diseases, particularly in polyglutamine disease and amyotrophic lateral sclerosis (ALS)
models, L. McGurk et al (2015).

Human polyglutamine poly(Q) disorders are a group of neurodegenerative disorders caused by the atypical expansion of
tandemly arranged cytosine-adenine-guanine (CAG) trinucleotide repeats within the coding region of the causative
gene, resulting in an extended stretch of glutamine in the mutated protein. There are nine human poly(Q) disorders,
namely, Huntington's disease (HD), Spinal and bulbar muscular atrophy (SBMA or Kennedy's disease), six of the
Spinocerebellar ataxias: SCA1, SCA2, SCA3/Machado-Joseph Disease (MJD), SCA6, SCA7 and SCA17, and Dentatorubral-
pallidoluysian atrophy (DRPLA or Haw River syndrome). Notably, all poly(Q) diseases share some salient features, such
as: i) they are autosomal dominant, except SBMA which is X-linked recessive; ii) they predominantly affect the central
nervous system (CNS); iii) they are generally adult onset and progressive in nature; iv) they display genetic anticipation;
v) they show complete or high penetrance; vi) they show neuronal accumulation of the toxic protein aggregates; vii)
they culminate in death and have no cure till date. Besides, the expanded poly(Q) stretch, the causative proteins of all
poly(Q) disorders do not share any sequence homology or functional similarities. Therefore, due to fundamental
differences in the different causative proteins result in some disease-specific manifestations. The length of CAG repeat
plays a decisive role in determining the age of onset, the severity of the disease and the rate of progression; the longer
the poly(Q) repeat length, the earlier the disease onset with more severe symptoms and accelerated progression.
Poly(Q) diseases are also characterized by the phenomenon of anticipation, where the age of onset tends to decrease
in successive generations. This is because CAG repeats increase in size with repetition of cycles, hence, in the following
generations. (S. Tandon et al. 2024)

Objectives
▪ To learn the basic techniques used in a Drosophila-based laboratory.
▪ To set up genetic crosses, virgin collection and food preparation.
▪ To learn about Drosophila morphology and genetics, and the genetic tools associated with Drosophila.
▪ Learn about Poly(Q) disorders and cellular pathways involved in neurodegenerative disorders.

Why is Drosophila used as a model organism?

▪ According to the central dogma, metabolic and genetic pathways across all life forms are conserved, therefore
there are similarities between Drosophila and human genome and genetics.
▪ Drosophila has a short life cycle of about two weeks.
▪ The number of offspring produced is high (several hundred eggs per fly).
▪ Sexual dimorphism is present, making it easy to distinguish between male and female flies.
▪ Do not cause harm to the environment if the flies escape.
▪ Cost-effective, requires simple food, easy to culture, requires less space.
▪ 75% of disease-associated genes in humans have orthologs in Drosophila.

Drosophila life cycle

1 Embryogenesis(24 hours after fertilization): small, white coloured eggs.
1. Larval stage (3 instars – 4 days)
2. Pupal stage (4 days): The larva undergoes metamorphosis inside a protective casing called a puparium.
3. Adult life ( Lifespan 30 days; varies with temperature).
 Composition and method of preparation of Drosophila food

COMPONENTS AMOUNT IN GRAMS

1 2 3 6
UNIT UNITS UNITS UNITS

4 08 12 24
AGAR
MAIZE 34 68 102 204
POWDER

SUGAR 30 60 90 180
YEAST
EXTRACT 12 24 36 72

NAPAGIN 2 4 12
6
PROPIONIC
ACID(ml) 2 4 6 12

WATER(RO)(ml) 720 1440 2160 4320

Drosophila Food Preparation (FOR 1 UNIT)

❖ Collect 720 ml H2O. Split this volume into two:
▪ 120 ml: Add yeast, maize powder, sugar, nepagin
▪ 600 ml: Subdivide it into two parts:
• 500 ml: Near boil or 60C̊ +Agar with constant stirring
• 100 ml: For rising maize powder sticking to the walls of other utensils.
▪ Prevent agar to stick to the bottom and get charred.
▪ Pour a batch of maize powder paste into the agar-H2O mix.
▪ Stir well till sticky.
▪ When not steamy hot, add propionic acid.
▪ Pour food into a vial.
▪ Prevent overboiling while dissolving agar.
▪ Use the extra 100 ml of H2O to rinse the vessel with maize powder paste and add to food.
▪ Prevent leftovers which might change the composition of food.
▪ Prevent the formation of lumps.
▪ Post pouring, cover the tray with a muslin cloth to prevent contamination. Place tight cotton plugs on the mouth of
the vials once the food properly solidifies.
▪ The quantity of food needed varies depending on the specific culture.

How to Distinguish between Adult Male and Female Drosophila?

1) Abdominal Shape : Female Drosophila have a larger, more pointed abdomen, while in males, the abdomens are
smaller with rounded ends and curl inwards.
2) Pigmentation in the abdominal area : Males have darker pigmentation on their abdomens, whereas females
have a pale abdomen
3) Sex combs: Only males have sex combs, which are specialized bristles located on the
forelegs. These are used during mating. Sex combs are absent in females.
4) Size: Males may be smaller than females.
 Phenotype identification and Distinguishing between Male and Female Adult Drosophila

3-vials were given, labelled A, B and C. The number of males and females, and phenotypic characteristics of
Drosophila in each vial had to be noted.

VIAL A

Number of males Number of females

19 14

OBSERVATIONS:

▪ Eye colour: Brick red

▪ Body colour: Yellow-brown
▪ Wing shape: Full, rounded wings
▪ Specifications: Transverse black rings across the abdomen

CONCLUSION: Wild-type Drosophila melanogaster

VIAL B:

Number of males Number of females

8 0

▪ Eye colour: Less pigmented

▪ Body colour: Yellow-brown
▪ Wing shape: Full, rounded wings
▪ Specifications: Transverse black rings across the abdomen

CONCLUSION: 78(Q) SCA3 diseased Drosophila.

Since this disease affects the eyes, we observe the diseased white-eye phenotype. SCA3 Ataxin-3 Suppresses
Polyglutamine stands for Spinocerebellar Ataxia type 3 or Machado-Joseph Disease(MJD1).Neurodegeneration in
Drosophila 78(Q)S means that there are 78 repeats of the glutamine sequence, where S stands for severe.

VIAL C:
Number of males Number of females
02 13

OBSERVATIONS:
▪ Eye colour: Brick red
▪ Body colour: Yellow-brown
▪ Wing shape: Crooked, thin wings
▪ Specifications: Transverse black rings across the abdomen
CONCLUSION: Vestigial wing phenotype
Vg locus is present on chromosome 2. In the absence of a vg+ gene, extensive cell death occurs in the third instar,
resulting in a complete loss of adult wing margin structures.

The Need for Collecting Virgin Females?

Females can store sperm in their reproductive tract; therefore, collecting virgin females becomes necessary.
The reasons why we need to collect virgin females:
▪ This is done to ensure that the offspring that we get is of the desired parentage.
▪ Since females can store the sperm of the undesired male, there might be contamination in the experiment.
▪ To avoid uncertain parentage of offspring.
▪ Avoiding sperm competition; if a female mates with different partners, there will be sperm competition in the
reproductive tract.
Hence, collecting virgin females ensures consistency in experimental setups, so genetic studies have accuracy and
reproducibility.

How to Distinguish between Male and Female Pupae?

The presence of sex combs on the forelegs is the key characteristic that distinguishes male and female Drosophila
pupae. If sex combs are absent, the pupa is female; if they are present, it is male. Sex combs appear as two black dots
on the forelegs of the pupae. Other differences include enlarged male gonads in third-instar pupae, whereas female
gonads are small.
 Precautions to take while separating Male and Female Pupae

1. Keep the pupae moist.

2. Only pick up the matured pupae from the vial for sorting, and leave the immature ones.
3. Use the brush lightly to avoid accidentally damaging the pupae.
4. Wet the brush slightly before picking up the pupae.
5. Wipe all the instruments used and the table with alcohol after completing the experiment to ensure no leftover
pupae or flies are left, which can introduce contamination.
6. Complete sex identification and separation quickly, as the pupae will start eclosing and turning into flies,
contaminating the setup, or the flies will escape.

Drosophila Head Decapitation

▪ The flies are first etherized.

▪ To obtain a clear view of the flies, a dissecting microscope is used with the appropriate magnification.
▪ PBS is put on the flies during dissection.
▪ The heads are dissected from the body using fine needles, ensuring an appropriate amount of PBS is present.
▪ The proboscis is then removed from the head.
▪ The decapitated heads are immersed in PBS solution.

PBS (Phosphate-buffered Saline solution) is a buffer solution (pH 7.4). The buffer helps to maintain a constant pH. PBS
solution maintains the viability of cells during transportation and storage, and helps to avoid desiccation. The proboscis
and other tissues associated with the head are removed to prevent interference while imaging the brain.

DROSOPHILA HEAD

Modifiers and Balancers in Drosophila

Modifiers: Modifiers are genes that influence the phenotypic expressions of other genes. They can enhance, suppress
or change the effects of other genes.
Suppressors are modifiers that reduce the expression of a mutant phenotype. For example, if a mutation in gene A
causes a defect, a suppressor mutation in gene B might restore the normal phenotype.
Enhancers work opposite to suppressors, they increase the severity or expression of a mutant phenotype. For instance,
if a mutation in gene C causes a mild defect, an enhancer mutation in gene D might worsen this defect.

Balancers: Balancers are special types of chromosomes used to maintain deleterious mutations in populations, without
the mutations being lost.Balancer chromosomes contain multiple inversions that reduce recombination.This ensures
deleterious mutations are not lost or altered through recombination. Flies with two copies of the balancer are inviable
due to recessive lethal mutations. Flies with two copies of the mutation are non-viable or sterile, ensuring that only
heterozygous flies survive.(Daniel St Johnston - Nature reviews, Genetics)
Balancer chromosomes carry dominant visible markers (such as curly wings, stubble bristles, or specific eye colours)
and recessive lethal mutations. These markers make it easy to identify flies carrying the balancer. Common balancers
with markers: Cyo (curly wing marker), Tb (tubby body shape), dCO2 (dark body colour), Sp (sternopleural bristles).

UAS-GAL4 Lines
The GAL4-UAS System is used to study gene expression in Drosophila. This system has two parts: GAL4 which is a yeast
transcription factor and UAS (upstream activating sequence). GAL4 is used as a driver. It can be expressed under the
control of tissue-specific or inducible promoters.
UAS is a DNA sequence that contains multiple GAL4 binding sites. It is typically placed upstream of a gene of interest.
When GAL4 binds to the UAS sequence, it activates the transcription of the gene of interest resulting in the expression
of the gene.
GAL4-Tissue-specific site (like GMR - eye-specific or Elav - brain specific) x UAS-GOI (like GFP)
↓
𝑼𝑨𝑺−𝑮𝑶𝑰
expression of GOI at the tissue-specific site
𝑮𝑴𝑹−𝑮𝑨𝑳𝟒

ELAV
ELAV (Embryonic Lethal Abnormal Vision) is a gene that encodes a protein involved in the development of the nervous
system in Drosophila. Researchers can use the ELAV promoter to drive the expression of diseases. Since it is expressed
in the eye it is easy to visualize as an abnormality in the eye.

Genetic Crosses
Types of lines:
Introgressed Lines: Developed by introducing genes from one species into another through repeated backcrossing.
Retrogressed Lines: Lines in which desirable traits that were lost are reintroduced, through backcrossing.
Recombinant Lines: Recombination of genetic material from different lines, resulting in new gene combinations and
traits.

Spectra Processing using Metaboanalyst 6.0

Metabolomics is the scientific study of chemical processes involving metabolites, molecular substrates, intermediates,
and products of cell metabolism. Therefore, metabolomics is the study of the unique chemical fingerprints that specific
cellular processes leave behind. We are analyzing LC-MS Spectra (mzML, mzXML or mzData files). In the resulting

analysis we get a mass chromatogram which is a representation of mass spectrometry data, where the X-axis
represents time and the Y-axis represents signal intensity.
While liquid chromatography separates mixtures with multiple components, mass spectrometry provides spectral
information to identify each separated component. These two processes combined contribute to giving us a LC-MS
(Liquid chromatography-Mass spectrometry) spectra.
Mass spectrometry measures mass to charge ratio (m/z) of charged particles (ions).Components of a mass
spectrometer include an ion source in which components of the sample are ionized using electron beams, UV lights or
lazer,a mass analyzer, which applies electric and magnetic fields to sort the ions by their masses, and a detector which
calculates abundances of each mass-resolved ion.

Prominent frequencies present in a signal are called spectral peaks. These peaks represent the dominant components of
a signal.
Retention time in LC-MS is the duration that a specific analyte takes to pass through the chromatography column from
the time of injection to the time it is detected by the mass spectrometer. Different compounds typically have unique
retention times.

Total Ion Chromatogram (TIC)

It is a plot that shows the total ion current detected in a mass spectrometer over a period of time. The X-axis shows
retention time (elution time). The y-axis represents the sum of all ion intensities (the abundance of ions detected by a
mass spectrometer at a given time) detected at each point in time.
Interpretation: Each peak in TIC corresponds to a compound that has been separated and detected. The height of the
peak reflects ion intensity, which is related to the concentration of the compound.

Base Peak Ion Chromatogram (BPI)

It is a plot that shows the intensity of the most intense ion (the base peak) detected in each mass spectrum as a
function of time. The X-axis shows the time or scan number, and the Y-axis shows the intensity of the base peak ion.
Interpretation: Peaks in the BPI chromatogram correspond to times when a significant compound elutes from the
chromatographic column.

REFERENCES
L. McGurk et al (2015), S. Tandon et al. (2024), Daniel St Johnston- Nature reviews, Genetics, Atlas of Drosophila
Morphology- Sylwester Chyb, Classical genetics simulator- CGSlab.com, Metaboanalyst 6.0