Changes in Metabolism during Zebrafish Development, Wound Healing and Regeneration

Abstract

An interest in metabolism dates back centuries, with Charles Manning Child being the first to propose a role for metabolism in specifying developing structures during embryogenesis in the 1900s. Modern tools and techniques are now available to allow the detailed analysis of patterns and changes in metabolites, such as with genetically encoded biosensors, which permit the study of metabolism with spatial and temporal resolution in vivo. In this thesis, I ask the central question: Can genetically encoded biosensors be used in zebrafish to observe changes in metabolism during development or regeneration and wound healing? If changes occur, is this metabolic reprogramming important and consequential for these systems? Evidence of metabolites such as hydrogen peroxide (H₂O₂) and lactate acting as second messengers or influencing signalling pathways are becoming increasingly apparent. An initial burst and subsequent oscillations of mitochondrial H₂O₂ _have been shown to be important for the cleavage stage Xenopus embryo, while H₂O₂ _and a switch to glycolytic metabolism are essential for successful regeneration, with blastemal cells displaying up-regulated expression of glycolytic genes. Inhibition of either H₂O₂ _production or glycolysis reduces the number of proliferating cells in the regenerating zebrafish fin and heart, and this link leads us to postulate that the Warburg effect—associated with providing molecules for the generation of biomass in highly proliferative systems—may be activated in regeneration. Development involves significant growth, and is therefore also a promising candidate for metabolic reprogramming and the Warburg effect. This project aims to determine whether a more diverse role for metabolic alteration in development and wound healing can be distinguished using genetically encoded biosensors in the zebrafish model. The sensors were sub-cloned into constructs for testing in vivo via mRNA injections, followed by the generation of transgenic lines to yield brightly expressing embryos at both early developmental stages and two days post fertilisation for larval fin amputations. I establish that the sensors Laconic, for lactate detection, and SoNar, for monitoring NADH/ NAD+ ratio, are viable in zebrafish embryos; using transgenic lines of these two sensors and HyPer, for H₂O₂, combined with pharmacological treatment, I demonstrate that lactate increases following the midblastula transition, suggesting possible employment of Warburg metabolism upon increased growth of the embryo, while an increase in lactate occurs immediately following amputation, putatively to generate rapid ATP for the contraction of the actomyosin cable responsible for wound closure. Inhibition of glycolysis over the entire period of regeneration results in attenuated regeneration, concurring with existing literature citing the importance of glycolysis in zebrafish regeneration. I also investigate H₂O₂, calcium, lactate, and NADH/NAD+ ratio in a zebrafish mutant for hai1a and show the phenotype shares similarities with a constitutively healing wound, including high H₂O₂ _levels, sterile inflammation, and elevated lactate levels in abnormally proliferative keratinocyte aggregates. The work in this thesis contributes further evidence of the importance of glycolytic metabolism in a systems biology context with a function besides ATP generation, forming a foundation for future work, potentially presenting a target for regenerative therapies.

Date of Award	1 Aug 2021
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Christoph Ballestrem (Supervisor) & Enrique Amaya (Supervisor)