Investigating a Role for Ecdysteroid Reception in Glia of Peripheral Nerves During Drosophila Metamorphosis
Christian Sohner, Mason Welch, Donovan Lundy, Jackson Riffe, Yoshitha Bhargav and Joyce J Fernandes
Department of Biology, Miami University, Oxford, Ohio
Impact on Student Learning: NACE competencies
Completing research in the lab has taught us a lot of transferable skills that we can carry on throughout the rest of our time in undergraduate school and into graduate school and the professional workspace. Through this research, we have expanded our scientific communication skills through weekly lab meetings, presentations, and coordinating experiments. We have also developed crucial STEM skills, such as critical thinking and problem-solving.
1) Drosophila exhibits stage specific motor behaviors and CNS structure during its 10-day life cycle
EMBRYO: Day 1
Squirming, hatching
Nerve cord along the length
Short segmental nerves
LARVA: Day 2-5
Crawling, feeding
Nerve cord at anterior
Long peripheral nerves
ADULT: Day 6-10
Walking, flight, reproduction; CNS in the thorax
Fused posterior nerves
2) The changing structure of the nervous system during metamorphosis
Extensive neural restructuring initiated by steroid hormones occurs during metamorphosis to support adult-specific behaviors. The thoracic ganglion expands to accommodate flight and walking, whereas the abdominal ganglion shrinks. Although the abdomen does not contribute to locomotion, this is the seat forbehaviors such as eclosion and reproduction.
Image Credit: Camillo Molina
Formation of the Terminal Nerve Trunk, TNT
Eight pairs of abdominal nerves (A) begin fusing at the posterior (B) at 28h APF, leading to formation of the TNT by 48h APF (C) which is evident in the adult (D). Subramanian et al., 2017.
Glia ensheath larval nerves
Neural lamella (NL), is acellular followed by perineurial glia (PG), the blood-brain barrier created by sub-perineurial glia (SPG), and wrapping glial (WG). PG cells are the most abundant glia. Stork et al., 2008.
Stork et al., 2008
HYPOTHESIS
Reception of ecdysteroids in the PG layer impacts the behavioral and structural plasticity that occurs during the transition from the larva to the adult.
The driver line serves two purposes:
(1) Spatial control: C527Gal4 targets transgene expression to PG cells (McGuire et al., 2004).
(2) Temporal control: Gal80ts represses expression of transgenes at 18℃ and allows expression at 29℃.
Gal4; Gal80ts System
Heat Shock Timings with EcR Isoform Expression
5) EcR Expression in PG cells
Line: 4776 (GFPnls) x 90391 (C527Gal4)
Drives the expression of GFP labelled PG cells, stained with anti-EcR antibodies.
Each abdominal larval nerve is ensheathed by approximately 10 perineurial glia.
88.52% of PG nuclei (green) in A1-A8 express Ecdysteroid receptor B1 (red).
Truman and Riddiford, 2023
Image Credit: Jackson Riffe
Image Credit: Yoshitha Bhargav
EcR-A is not expressed in the PG cells of larval abdominal nerves.
6) Monitoring eclosion in manipulated animals
The B isoform of Ecdysteroid receptors, but not the A isoform, is expressed by PG cells at the larval stage.
Knockdown of ecdysteroid receptors showed no impact on development of eclosion behavior-tested in EcR-RNAi. B1 mutants had an impact on eclosion.
Next Steps:
Examine status of NL degradation, nerve fusion, and TNT structure in manipulated animals.
Different heat shock timings: 24-48h APF, 48-72h APF.
Acknowledgements:
Fly Stocks: The Bloomington Stock center & Fly community
Funding: Dean’s Scholar Award, Honors College & the Biology department supported the work of Donovan Lundy. Biology Department supported the work of Christian Sohner and Mason Welch
Imaging was conducted at the Center for Advanced Microscopy and Imaging (CAMI). We thank Matt Duley for assistance.
References
S. Paul Bainbridge, Mary Bownes; Staging the metamorphosis of Drosophila melanogaster. Development 1 December 1981; 66 (1): 57–80. doi: https://doi.org/10.1242/dev.66.1.57
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.
Subramanian A, Siefet 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.
Truman JW, Riddiford LM. Drosophila postembryonic nervous system development: a model for the endocrine control of development. Genetics. 2023 Mar 2;223(3):iyac184. doi: 10.1093/genetics/iyac184. PMID: 36645270; PMCID: PMC9991519.
