Fly Anomalies: Examining Differences

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1 Drosophila Anomalies: Examining Differences Among Wild and Nf1 Mutant Drosophila By: Sarah Riley Title Fly Anomalies: Examining Differences 1

2 Title Among Wild and Nf1 Mutant Drosophila Researchable Question Do Drosophila afflicted with Neurofibromatosis Type 1 through an Nf1 gene mutation behave differently than wild type Drosophila as measured by various behavioral assays? 2

3 Hypothesis It is hypothesized that Drosophila carrying an Nf1 gene mutation will present measurable behavioral differences when compared with wild type Drosophila in the Larval Crawling Assay, Mating and Courtship Assay, and RING Assay. Purpose NF1 is one of the world s most common genetic diseases in humans, and is caused by lack of neurofibromin due to a mutation on the NF1 gene on chromosome 17. People inflicted with these diseases have limited options to treatments, many of which are specific to only a part of the population of people with the disease (for example, treatments for a disease such as cystic fibrosis that can be caused by many mutations is available to a somewhat limited portion of the population of people with the disease). My project will focus on the Nf1 (neurofibromin 1) in Drosophila in order to observe differences in the results of behavioral assays (Larval Crawling Assay, Mating and Courting Assay, and the Rapid Iterative Negative Geotaxis [RING] Array). 3

4 Background Information Neurofibromatosis Type 1 (NF1) Frequency of disease: 1 in 3,500 people around the world have NF1 Symptoms of disease: Scoliosis, café en late spots, neurofibromas (tumors, usually benign but rarely malignant) Symptoms due to decreased production of neurofibromin 1 from NF1 gene mutation Drosophila Melanogaster (Drosophila) Have been used as models for a multitude of genetic diseases, such as Alzheimer's disease, Parkinson s disease, and von Recklinghausen s disease (otherwise known as Neurofibromatosis type 1) due to their relatively similar genetic makeup to that of humans Materials Material: Quantity: Wild-Type Drosophila Preliminary Mutant Drosophila (Ipo/Aldox) First Nf1 Mutant (Pbac{PB}Nf1[c00617]) Second Nf1 Mutant (Mi{MIC}Nf1[MI01388]) Larval Crawling Assay Apparatus 1 Mating and Crawling Assay Mating Wheel 1 RING Assay Apparatus 1 Arduino Uno 1 Arduino BH1750 Light Sensor 1 Arduino MicroSD Card Adapter 1 Arduino DHT22 Temperature/Humidity Sensor 1 Arduino DS3231 RTC 1 Cooler/Container for Incubator 1 4

5 Larval Crawling Assay Setup 1. Put the male and female Drosophila into an 8 ounce bottle 2. Allow the Drosophila to lay their eggs for a full 24 hours 3. Empty bottle of adult Drosophila 4. Incubate the bottle full of eggs until the larvae are in third instar phase 5. Add 20% sucrose to the bottle that contains the Drosophila that were observed in step 4 6. Let the 20% sucrose sit in the specified bottle for about 20 minutes 7. Collect the larvae with a serological pipette 8. Put the Drosophila into the mesh basket, wash larvae twice with deionized H2O 9. Use a brush to bring Drosophila to beaker with 5% sucrose 10. Wait about 15 minutes for the larvae to eat 11. Place the larvae back into the mesh basket 12. Rinse the larvae with water Larval Crawling Assay Procedure 1. Place petri dish with 2% agarose over graph paper 2. Set up camera for taking pictures of the Drosophila as they complete the assay 3. Move larvae to a petri dish with 2% agarose in it by using a brush 4. Prepare timer (so that when ready, the timer can be pressed and the Drosophila can be observed as soon as possible) 5. Begin timer 6. Observe the behavior of the Drosophila (take note of how many contractions occur within the time frame of observing the Drosophila) 7. Repeat steps 1-16 as needed in relation to how many trials are needed for experimentation 5

6 Mating and Courtship Assay Procedure 1. Put one female inside of the mating wheel 2. Put one male inside of the same mating wheel 3. Set camera up 4. Look into the dissection microscope 5. Take note of what is observed regarding the six stages of Drosophila mating and courting (orientation, tapping, wing song, licking, curling, copulation) for about 10 minutes (or until successful copulation occurs) Rapid Iterative Negative Geotaxis (RING) Assay Procedure 1. Put 25 of the young adult male Drosophila into polystyrene vials 2. Place vials with Drosophila into the RING apparatus 3. Wait about 20 minutes for the Drosophila to adjust to their new environment and place the digital camera about a meter in front of the RING apparatus 4. Zoom the camera so that the Drosophila can be seen through its lenses 5. Set camera timer and self-timer for 3 seconds 6. Tap the apparatus down onto the surface of the bench at which the assay is being performed on 3 times 7. Wait 1 minute, preparing for another shaking round (complete step 7 during this minute of waiting time) 8. Repeat 8-9 at least 5 times 9. Upload images taken by camera onto a computer 6

7 Variables and Constants Variables: Measurements of a given assay (for example, different genotypes of Drosophila could tend do have longer/shorter courtship time, etc.) Constants: Drosophila genotype (based on which genotype is being observed at a given point in time) Living conditions (incubator) Results Larval Crawling Assay Measured number of contractions Mating and Courtship Assay Courtship latency Courtship duration Time courting Courtship index Copulation duration Fraction courting Fraction copulating RING Assay Mean height climbed 7

8 Possible Sources of Error Alteration in living conditions How the error due to such alteration is being minimized: The incubator will be logging data that is collected from each of the sensors every three seconds, and can be uploaded onto a computer and into Microsoft Office Excel, so outliers in the data from the sensors can be observed and can be fixed Inaccuracies in assay readings How the error due to such inaccuracies is being minimized: Video tape/take pictures of every trial of each assay that is conducted so that they are available for reexamination if the data readings are very off Data Analysis Larval Crawling Assay The larval crawling assay is performed in order to help determine the typical activity levels of Drosophila with a certain genotype Mating and Courtship Assay The mating and courtship assay helps to determine the fine motor coordination of Drosophila with a certain genotype. This assay has also be used to gain insight into the sensory processes that occur within Drosophila with different genotypes Both of these analyses combine to help examine the behavior of Drosophila with different genotypes and potentially gain more insight into human diseases through such Drosophila with them RING Assay The RING assay assesses the reaction of Drosophila of different genotypes to shaking (seeing whether or not the Drosophila climb to the top of the vial that they are in for the duration of the assay, etc.) 8

9 Future Extensions Due to Drosophila being frequently used as human disease models, future research could surround trying to make new connections between Nf1 mutations in Drosophila and NF1 mutations in humans My original project idea, in which I try to use CRISPR to alter the genetic makeup of Drosophila and rid previously Nf1 mutant Drosophila of symptoms of Neurofibromin 1 deficiency (through RNA alteration) Timeline December Fair Winter Break January February Have a complete description of the process for each behavioral assay Have completed Drosophila incubator Obtain wild-type Drosophila Have materials for all behavioral assays Finish all behavioral assays with wild-type Drosophila Finish all behavioral assays with mutated Nf1 Drosophila Complete data analysis Potentially begin to work on CRISPR sequencing Potentially finish CRISPR sequencing Complete presentation for STEM fair 9