Undergraduate researchers: Donovan Lundy (lundydt@miamioh.edu); Biology/ Pre-med and Tyler Peace (peacetd@miamioh.edu)

Graduate Student Mentor: Jackson Riffe (riffejt@miamioh.edu) 

Faculty Mentor: Dr. Joyce Fernandes (joyce.fernandes@miamioh.edu) 

Abstract

Purpose: Ecdysteroid receptors (EcR) are proteins that receive hormonal signals to drive life-stage specific changes in neuronal development and reorganization of the nervous system. In the fruit-fly, these proteins drive the four-day process of metamorphosis and are important for the resulting neural plasticity - for example re-organization of layers of glial cells that surround abdominal nerves. Our project will test the hypothesis that EcR expressed by an external layer of glial cells (PG) initiates a signaling cascade that brings about degradation of an acellular layer, the Neural Lamella (NL). 

Expected Outcomes:  The presence of EcR in PG cells associated with abdominal nerves is unknown and will be a contribution to the field. Our hypothesis will be tested by genetically knocking down expression of EcRs in glial cells. Our project predicts that NL degradation will be prevented and will in turn affect formation of the adult pattern of abdominal nerve projections. 

Significance:  We will learn about the role of ecdysteroid receptors and the neural lamella in signaling events that bring about structural plasticity of abdominal nerves in a genetic model system.  This research is applicable to neural plasticity of humans, for example hormone-based changes during puberty that establish executive function in adolescents.

1. Drosophila as a Model Organism

75% of human disease genes are seen in fruit flies

Easy to observe and maintain

Short Life cycle and ease of genetic manipulation

Researched over 100 years

Nobel Prizes (Physiology/Medicine) have been awarded to Drosophila Researchers

2. Steroid Hormones in Organismal Development

Drosophila - Steroids known as ecdysteroids in the fruit fly, play a critical role in metamorphosis nervous system and motor development. Allowing the fly to go from a crawling larva to a flying adult.

Amphibians - exhibit extensive anatomical reorganization. Initially, a tadpole begins developing limbs and lungs allowing the animal to survive on land. At the adult stage, the tail is completely absorbed and the lungs are fully functional. [1]

Humans - Major effects during puberty. Secondary sex characteristics are developed and neural pathways are remodeled and further enhanced. [2]

4. Stage-specific nervous system structure

7. Hypothesis 

Ecdysteroid reception by perineurial glia cells initiate the degradation of the neural lamella.

1) Ecdysteroid receptors are present in the perineurial glia cells

2) Knockdown of EcRs at the onset of metamorphosis affects the development of adult specific motor functions

Methods

Tissue dissections/Immunofluorescent staining:

1) Late-stage larva, 0h APF and 6h APF animals will be dissected and stained for the presence of ecdysteroid receptors (EcR) and a PG nuclei reporter (GFP).

2) 6h APF, 24h APF and adult animals are dissected and stained for the presence of NL (laminin) and nerves (HRP).

Genetic Manipulation

At the onset of metamorphosis, animals undergo a controlled expression of the transgene (UAS-EcR RNAi) which knocks down all ecdysteroid receptors in the targeted cells. 3 driver lines were used. 2 PG specific driver lines: C527 Gal4, and C527 Gal4;Gal80ts  and a driver line specific to all glia, Repo Gal4.

Eclosion Monitoring

Larvae were collected and monitored throughout metamorphosis, and their ability to eclose was observed and tabulated.

9. Conclusion

Ecdysteroid receptors are present in the PG layer

Knockdown of ecdysteroid receptors showed no impact on development of eclosion behavior-tested in c527Gal4;Gal80ts

Next Steps: Determine status of NL during degradation

References 

1. Kikuyama S, Yamamoto K, Toyoda F, Kouki T, Okada R. Hormonal and pheromonal studies on amphibians with special reference to metamorphosis and reproductive behavior. Dev Growth Differ. 2023 Aug;65(6):321-336. doi: 10.1111/dgd.12868.

2. Weyandt LL, Clarkin CM, Holding EZ, et al. Neuroplasticity in children and adolescents in response to treatment intervention: A systematic review of the literature. Clinical and Translational Neuroscience. 2020;4(2). doi:10.1177/2514183X20974231

3. Subramanian A, Siefert M, Banerjee S, Vishal K, Bergmann KA, Curts CCM, Dorr M, Molina C, Fernandes J. Remodeling of peripheral nerve ensheathment during the larval-to-adult transition in Drosophila. Dev Neurobiol. 2017 Oct;77(10):1144-1160. doi: 10.1002/dneu.22502.

4. Stork T, Engelen D, Krudewig A, Silies M, Bainton RJ, Klämbt C. Organization and function of the blood-brain barrier in Drosophila. J Neurosci. 2008 Jan 16;28(3):587-97. doi: 10.1523/JNEUROSCI.4367-07.2008.

Career Readiness Prompt: 

Working in the lab this semester has had profound effects on our creative and critical-thinking skills. We generated experimental designs, analyzed data, and formed conclusions about our work, allowing us to practice these skills. Working alongside graduate students and professors has also taught us how to act/ work in a professional STEM setting. Our ultimate goal is to attend graduate school which means we will have to know how to work alongside professors and other researchers to be successful. Developing teamwork skills in a scientific context has proven invaluable and will continue to be useful throughout our careers. During our time in the lab, we gained hands-on experience using fluorescence microscopy, immunohistochemistry, and other advanced lab techniques that will prove useful in our future research experiences. Additionally, learning these techniques also requires critical thinking as they are often complex processes. We have also had the opportunity to present multiple posters of our research which has greatly improved our scientific communication skills. We have learned to effectively develop a theme for our research and then communicate our findings to other people.