DOI: 10.18742/20109767.v1
title: Data supporting the thesis, Dissection of the key molecular pathways for muscle regeneration in the zebrafish in vivo model

description: Descriptions of the .avi and .xlsx files are included in the thesis, Dissection of the key molecular pathways for muscle regeneration in the zebrafish in vivo model, as part of the Appendix, pp.327-329, and are also included here. ----- Thesis Abstract In mouse, Notch signalling is important for the maintenance of the muscle stem cell (muSC) niche during muscle development and regeneration. Inhibition of Notch signalling in Pax7+ cells leads to the loss of the muSC pool due to premature differentiation, perturbing muscle repair. Following muscle damage, an influx of immune cells generates a pro-inflammatory microenvironment. Murine macrophage ablation studies have demonstrated the importance of macrophages in successful muscle repair. Following macrophage ablation, neutrophilic mediated inflammatory microenvironment is prolonged, leading to increased muscular necrosis and fibrosis, not seen in normal regeneration. Furthermore, it has been shown that proinflammatory macrophages inhibit myogenic precursor cell (MPC) fusion and promote proliferation, whereas anti-inflammatory macrophages promote differentiation. Moreover, it has been shown in mouse that both anti- and proinflammatory macrophages inhibit Notch activity in muSCs. The activity of Notch signalling in muSC during muscle repair is still poorly defined in vivo, and it is not understood if Notch regulates muscle regeneration in zebrafish. Moreover, it is not known if infiltrating immune cells, macrophages and neutrophils, and the inflammatory microenvironment are important for regulating Notch activity during regeneration. Therefore, I aim to determine how Notch signalling regulates muSC biology in zebrafish. Additionally, I aim to understand if the inflammatory microenvironment influences the Notch-dependent regulation of muSCs. To understand the importance of Notch signalling for the muSC response to injury in zebrafish, I used transgenic zebrafish to visualise muSCs in conjunction with pharmacological (DAPT) and genetic (dnsu[h]) tools to globally inhibit Notch. I found that fewer proliferating pax7a-expressing muSCs were present following injury in the absence of Notch, which was associated with an increase in the proportion of differentiating muSCs. Moreover, I found that in the myosepta, described as the muSC niche, there was an increase in the number of proliferating pax7a-expressing muSCs in the absence of injury. This suggests that Notch signalling is important to promote proliferation following injury, and limit proliferation in the absence of injury. A dynamic reporter of Notch activity revealed that proliferating pax7a+ cells express Notch (Tp1:VenusPEST). Additionally, by immunolabelling I also found that very few Pax7+ cells express a stable reporter for Notch activity (Tp1:H2BmCherry) at 24 hpi. Moreover, the proportion of Tp1:H2BmCherry+ cells expressing pax7a increased over time following injury. Taken together, I concluded that the majority of Tp1+ nuclei reside in myofibers and do not contribute to muscle repair. Contrastingly, Tp1+ cells which upregulate pax7a expression, respond to muscle injury. Moreover, I found that these Tp1:H2BmCherry+ cells are controlled by Notch signalling. DAPT treatment resulted in more Tp1:H2BmCherry+ cells after injury. Using a pharmacogenetic method to ablate macrophages, DEX (steroid) and a TNF-α receptor mutant zebrafish line, I showed that reducing inflammatory signalling following injury resulted in more Pax7+ muSCs. Additionally, I found that macrophages express DeltaD suggesting that macrophages can signal to adjacent cells via Notch. Together my data shows that muSCs are regulated by Notch signalling and the inflammatory microenvironment. I propose that pro-inflammatory macrophages signal via DaltaD to limit Pax7+ and Tp1+ cell proliferation. Moreover, as macrophage ablation and Notch inhibition have opposing effects on the Pax7+ cell population, Notch activity is also important to limit Pax7-expressing muSC differentiation and promote proliferation.