Analysis of the function and regulation of the conserved Mef2 transcription factor in muscle differentiation and maintenance in Drosophila melanogaster

Mitchell-Gee, Robert 2024. Analysis of the function and regulation of the conserved Mef2 transcription factor in muscle differentiation and maintenance in Drosophila melanogaster. PhD Thesis, Cardiff University. Item availability restricted.

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Abstract

Understanding the mechanisms that govern the transition from undifferentiated precursor cell types, to fully differentiated, functioning tissues is critical to many aspects of biology. These mechanisms include cell signalling, cell migration, morphogenesis, tissue patterning and growth regulation; and form the basis for research into stem cells, regeneration, wound healing, cancer and other diseases. At the molecular level, determining how transcription factors regulate gene expression is fundamental to understanding their physiological role in ensuring correct developmental patterning and specifying cell types. Clear links exist between many human diseases and transcription factor dysregulation, thus continued efforts into understanding how they function, and how their activity is modulated, is paramount. Here, I use the model organism Drosophila melanogaster to investigate several aspects of the function and regulation of the conserved transcription factor Mef2, during the process of muscle development. I describe Mef2’s expression pattern in the wing imaginal disc-associated adult muscle progenitor cell (AMP) population, and then go on to show that overexpression of Mef2 in these cells results in a striking phenotype: the precocious formation of ‘mini-muscles’ as a result of premature AMP differentiation. These prematurely developed myofibres express a panel of sarcomeric proteins, and are resemblant of differentiated muscle. Using this system, I have developed an assay to quantify Mef2’s transcription factor activity, which can be used to determine how addition of co-factors/repressors, or mutation to conserved residues of interest, can impact upon its behaviour. One route by which Mef2 activity is thought to be modulated is through Him, an inhibitor of myogenesis. In wing disc associated AMPs, I demonstrate that the Him protein is expressed in a highly localized pattern, which potentially represent a particularly naïve subpopulation of progenitor cells. Overexpression of Him can inhibit Mef2-overexpression induced premature differentiation, demonstrating that Him can repress Mef2 activity in this cell-type. I show that this is likely through a direct protein-protein interaction, as in a Yeast2Hybrid assay, these two proteins can physically interact with one another. I then explored the role of Him through loss-offunction experiments, describing novel jump muscle and pericardial cell patterning phenotypes. vi Sumoylation is an additional mechanism by which Mef2’s transcription factor activity can be altered. For the first time in an animal model, I demonstrate that myogenesis cannot proceed without the sumoylation pathway through RNAi-knockdown experiments. To explore Mef2-specific effects, I generated a series of overexpression constructs in which a well conserved Mef2 C-terminal sumoylation motif was mutated. Using this novel toolkit, I demonstrate that sumoylation can significantly repress Mef2 activity, in both the context of the wing imaginal disc premature differentiation assay, and during the process of flight muscle development. The recent discovery of muscle satellite cells (MuSCs) in Drosophila, inspired experimentation into Mef2 function in muscle homeostasis and repair; a separate but related process to muscle development. I have identified Him as just the second marker of MuSCs in Drosophila, and describe a putative leg muscle-associated MuSC population; two core results which will help drive the field forwards. Mef2 function in these cells was explored by MuSC-specific knockdown, which resulted in an exacerbation of age-associated decline in flight ability, suggestive of a novel function for Mef2 in Drosophila muscle maintenance. The findings presented here are directly relevant to mammalian biology, because of the general conservation of the underlying mechanisms that underpin muscle tissue differentiation. In particular, I build on the current understanding of Mef2 regulation, whose function is central to both Drosophila and vertebrate muscle development. Moreover, I further establish Drosophila as a model for studying how muscle is maintained post-developmentally. This newly emerging model could prove an effective direction for building on the field of muscle satellite cell biology.

Item Type:	Thesis (PhD)
Date Type:	Completion
Status:	Unpublished
Schools:	Schools > Biosciences
Subjects:	Q Science > Q Science (General)
Date of First Compliant Deposit:	11 March 2025
Last Modified:	13 Mar 2025 15:54
URI:	https://orca.cardiff.ac.uk/id/eprint/176798

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