Investigation on the Development of Motor Neurons in the Early Larval Stages of Drosophila Melanogaster. Marguerite Généreux and Maddie Thies. Deerfield Academy. 7 Boyden Lane, Deerfield, MA, 01342. Discussion Introduction We placed our two strains of flies, 42732 (UAS-GFP) and 64052 (dopamine receptor 1-GAL4), into two new, separate vials filled with the Nutri-Fly Formula. We then performed a cross between the two strains, placing two male 42732 (UAS-GFP-strain) drosophila and two virgin female 64052 (dopamine receptor 1-GAL4) drosophila into a new vial. We then performed a cross between the two strains, but instead we crossed two virgin female 42732 (UAS-GFP-strain) drosophila and two male 64052 (dopamine receptor 1-GAL4) drosophila. We performed a cross between 42732 (UAS-GFP) and 42734 (GAL4-tubulin), so that we have a positive control where GFP should be present in the whole body of the larvae since tubulin is present practically throughout the whole body. We also performed a cross between 64052 (dopamine receptor 1-GAL4) males and females so that we have a negative control where GFP should not be visible. We will then place larvae from our different crosses of different ages and from different stages of larval development in petri dishes under the dissecting microscope and exposed to blue light from our LED device to observe whether or not the larvae’s motor neurons are made fluorescent thanks to the GFP. We are doing this in the hopes of knowing at which larval development stage the motor neurons develop. We will then test larvae from the 64052 strain as a negative control. For our positive control, we will cross a male of the 42734 strain that is Tubulin-Gal4 with a female of the 42732, UAS-GFP. In this crossing the whole larvae should be fluorescent when observed under blue light because tubulin is an important component in the cytoskeleton of Drosophila melanogaster. Once we see the appearance of larvae in the vials, we will isolate larvae of the same age/developmental stage and of every strain we are experimenting with (male 64052 crossed with female 42734, male 64052 crossed with female 42732, male 42732 crossed with female and 64052 Males) in a vial and expose them under blue light. We will enter our data in the tables outlined in Results, and by keeping track of the days and the age of the drosophila, we can deduce at which larval stage the drosophila develop their motor neurons. Figures 3[8]  to the right will be helpful in identifying the age and developmental stages of all our larvae so we can correctly identify when the GFP is visible in their motor neurons or throughout their whole bodies. Larva hatches within a day of the egg being fertilized and from then on, the flies go through the larval stages. In this experiment, we worked to be able to successfully see GFP present in motor neurons in our different crosses. By doing so we were able to observe the stages of motor neuron development in transgenic Drosophila melanogaster larvae. We had our positive control (42732 x 42734) to see if we could successfully produce a cross that expresses GFP in tubulin since it is found throughout the whole body of the larvae. We had our negative control (64052) that did not express GFP to compare with the GFP expressing larvae. We hypothesized that motor neurons would be visible during the 2nd larval stage after doing our research and finding that other experiments had found that motor neurons first appeared approximately 50 hours after the fertilization of the egg and continued to appear and develop for 40 more hours. [1] In our observations, we hopefully will see that our larvae started to exhibit GFP in motor neurons from as 2 days to 3 days, which matches our prediction that motor neurons first start to appear at about 50 hours. If we were to continue this experiment, we would have to be able to observe the flies more closely and consistently to find the exact hour the larvae developed The movement of many living organisms is dependent on motor neurons that target specific muscles and organs. The excitation and contraction of muscles by motor neurons is necessary for the locomotion of animals.[1] In many vertebrates, motor systems include the motor neurons, whose cell bodies are located in the spinal cord, and the organs, muscles and glands they carry signal to which causes various effects throughout the body.[2] In adult Drosophila melanogaster, the common fruit fly, the motor system is grouped by muscle, which creates a certain map of the fly’s central nervous system. In larval stage Drosophila, the motor neurons are present, but their positioning within the larvae’s bodies isn’t grouped in the same way as the adults’ and their development in the larval stages are important in understanding the adult’s motor system.[1] Green fluorescent protein (GFP) is a protein made up of 238 amino acids that absorbs blue light and emits green light.[3] GFP is often used in biology to report expression of specific genes or presence of neurons since it can conveniently emit color without any other cofactors or enzymes.[4] GFP is extremely useful in biology research because it can be attached to many parts of an organism or a cell and can be easily tracked and seen within organisms when a blue light is shone on it.[5] For these reasons, we are using a transgenic fly cross in which GFP will be expressed and attached to the motor neurons of Drosophila melanogaster to evaluate at which point in the larval development the motor neurons are activated. Under blue light exposure, the motor neurons of the drosophila should emit green similar to the larvae pictured below if the motor neurons are present and activated at the time. In this experiment, we will be crossing male flies of the 64052 strain with female flies of a UAS-GFP (42732). These males’ genes contain Dopamine 1-like receptor 1 (Dop1R1), expressed in the motor neurons, glutamate response elements (GRE) and GAL4, while the female’s genes contain upstream activation sequence (UAS) and green fluorescent protein. We will observe the results of these crosses at the larval stage, and we expect to these transgenic flies’ genes to express the green fluorescent protein since transcription factors bind to GRE which activates GAL4 that activates UAS which in turn activates the expression of the GFP[6]. Below, Figure 1 illustrates the Gal4 UAS system in drosophila. Throughout this experiment, we want to observe the development of motor neurons in the transgenic drosophila melanogaster. Our hypothesis is that motor neurons in Drosophila melanogaster will be visible under blue light-exposure thanks to the GFP and these neurons will develop and be visible at the second instar larval stage of the drosophila, which occurs approximately two or three days after the fertilization of an egg. Figure 3: Life Cycle of Drosophila. Drosophila are a model organism to work with since their life cycle is short[10] and by looking at this figure, we will be able to identify or larvae and their level of development. motor neurons because the larval stages occur rapidly within hours. We could attempt to observe the age/ developmental stage the larvae are at when their sensory neurons develop and when their interneurons develop. We could cross the same 42732-strain flies (UAS-GFP) with fly strains containing Gal4, GRE and other genes associated to sensory neurons or interneurons as Dop1R1 is associated to motor neurons in this experiment. As will be shown in the Figures 2a and 2b, the observed data supports our conclusion, that the cross between 64052 x 42732 would have larvae that express GFP in motor neurons and would start to appear after 50 hours since the majority of our flies should exhibit GFP on the 2nd and 3rd days. As will be shown in Figure 2d, in the positive control cross of 42732 x 42734, GFP should appear throughout the larva's body on the 1st and 2nd day, meaning our cross was successful and the GFP worked by illuminating within the tubulin. We suspect that the GFP appeared earlier within these crosses because tubulin is one of the major components of the cytoskeleton and a globular protein, which may be functioning before there are motor neurons. [13] As will be shown in Figure 2c, in the negative control there should be no signs of GFP since there was no inserted GFP protein expressed in one of the genes. The main transgenic larvae under our observation, the 64052 x 42732, should show GFP in motor neurons by having the UAS-GFP bind to the dopamine receptor in motor neurons so that UAS will be activated to produce GFP within the motor neurons the dopamine receptor has bound to. We believe these flies’ motor neurons will develop some time around the second instar larval stage. Although we can observe the time and developmental stage at which the motor neurons in these flies develop thanks to GFP, not much is known about the neuronal circuits and their development in Drosophila.[11] Since the larval nervous system in Drosophila is a useful model in optogenetics, it would be useful to fully understand this system’s development fully. This experiment leaves us to speculate about the mechanism of motor neuron development and neuronal circuit development in Drosophila. There are 30 muscles and around 40 motor neurons in each abdominal hemi-segment of the larva’s body[11] and it is possible that that these muscles and neurons develop at the same larval stage or developmental event. Knowing the stages at which neuronal circuits develop and at which motor axon terminals differentiate[12] could be helpful in understanding the organization, the function and the patterning of the motor neurons and the nervous system within drosophila larvae which could lead to developments in future optogenetic research.   Results a) b) References and Notes [1 ]Baek, Myungin, and Richard S. Mann. "Lineage and Birth Date Specify Motor Neuron Targeting and  Dendritic Architecture in Adult Drosophila." J Neurosci, 27 May 2009, www.jneurosci.org/content/29/21/6904. [2] “Motor Neurons” Wikipedia. Last modified January 31, 2017. https://en.wikipedia.org/wiki/Motor_neuron. [3] Chalfie, M. Tu, Y, Euskichen, G. Ward, WW. Prasher, DC. “Green fluorescent protein as a marker for gen expression.” Science. 11 Feb 1994. http://science.sciencemag.org/content/sci/263/5148/802.full.pdf [4] “Green Fluorescent Protein.” Wikipedia. Last modified January 11, 2017, https://en.wikipedia.org/wiki/Green_fluorescent_protein. [5] Goodsel, David. “Green Fluorescent Protein.” RCSB PDB Molecule of the Month. June 2003  https://pdb101.rcsb.org/motm/42. [6] “GAL-4 UAS System.” Wikipedia. Last modified July 13, 2016. https://en.wikipedia.org/wiki/GAL4/UAS_system. [8] “Development of the Fruit Fly.” Accessed February 28, 2017. http://web.as.uky.edu/Biology/faculty/cooper/Population%20dynamics%20examples%20with%20fruit%20flies/08Drosophila.pdf. [10] “Model Organisms.” BiosJayChemist. Last modified September 26, 2013. Accessed March 7, 2017. https://biosjaychemist.wordpress.com/tag/drosophila-melanogaster/.   [11] Kohsaka, Hiroshi. Okusawa, Satoko. Irakura, Yuki. Fushiki, Akira. Nose, Akinao. “Development of larval motor circuits in Drosophila.” Wiley Online Library. Last Modified April 24, 2012. Accessed March 8, 2017. http://onlinelibrary.wiley.com/doi/10.1111/j.1440-169X.2012.01347.x/full. [12] Kim, Michael. Wen, Yuhui. Jan, Yuh-Nung. “Patterning and Organization of Motor Neuron Dendrites in the Drosophila Larva.” NCBI. Last Modified December 15, 2009. Accessed March 8, 2017. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2785125/. [13]  "Tubulin." Wikipedia. Last modified March 8, 2017. https://en.wikipedia.org/wiki/Tubulin . Figure 1. The Gal4 UAS System in Drosophila. Fly strains expressing Gal4 proteins under the control of enhancers are genetically crossed with fly strains bearing a gene expressing Chanelrhodopsin-2 downstream of UAS. Motor neurons, activated by Dop1R1, become fluorescent under blue light because of the green fluorescent protein (GFP). c) d) Methods and Materials Figure 2: Bar Graphs of Time (days) for GFP to be observed in the Drosophila larvae. Using the results of our observed data, these bar graphs will show the number of flies of different strains and transgenic crosses that develop motor neurons at specific ages/ times in their development. a) Number of flies with visible GFP in motor neurons vs. time (days) in flies from a transgenic cross of male 64052-strain flies and female 42732-strain flies. b) Number of flies with visible GFP in motor neurons vs. time (days) in flies from a transgenic cross of male 42732-strain flies and female 64052-strain flies. c) Number of flies with visible GFP in motor neurons vs. time (days) in flies of the 64052 strain. Since this is our negative control, we shouldn’t be observing any GFP. d) Number of flies with visible GFP in their body vs. time (days) in flies of the transgenic cross of male 42734-strain flies and female 42732-strain flies. Since this is our positive control, we should be observing GFP through their whole bodies. To begin this experiment, we made the Nutri-Fly Formula. In order to do so, we emptied the packet that contained the powder into a beaker and added water. We heated this mixture to a boil and then added propionic acid. The final step of this process was pipetting the mixture into vials and placing them in the fridge to solidify. We also built an LED device that can emit light on our drosophila larvae using an LED, a heat sink, an objective lens and a 9volt battery. To do so, we soldered a 9V battery snap connector, a resistor, and a side-emitting LED to a black box. We then attached these parts to a foam base and also attached a battery and an ocular objective to this base. Deerfield Academy Science, Math & Technology Symposium – Spring 2017