| Abstract |
Like all living systems, developing embryos use metabolic pathways to convert energy and to build cellular structure. Upon fertilization, embryos undergo a series of cleavage divisions followed by the maternal zygotic transition prior to gastrulation. During cleavage divisions, cells divide rapidly without growth in volume in most animal species. Global metabolic measurements including oxygen consumption and heat dissipation rate measurements during cleavage development in different species have shown that the overall energy expenditure increases despite the absence of growth. This raises the question of how embryonic metabolism satisfies the increasing energy demands. Mitochondrial metabolism is essential for providing ATP, the most fundamental energy currency of cells. A large pool of fragmented and immature mitochondria is maternally deposited into the oocyte, where they remain in a quiescent state until fertilization. During cleavage, mitochondrial content in terms of numbers remains constant and mitochondria are equally distributed between blastomeres to ensure proper development in most species. Thus, while the embryo’s energy demand increases, its mitochondrial content remains constant, leading to the central question of this thesis: How is mitochondrial activity regulated in time and space to meet the increase in energy demands of early cleavage development? In this thesis, we address this question by analyzing mitochondrial activity, function and morphology in zebrafish cleavage stage embryos using live imaging, electron microscopy and a theoretical modeling approach, combined with heat dissipation and oxygen consumption measurements. We have established a live imaging approach and image analysis pipeline to visualize and quantify mitochondrial activity and NADH fluorescence lifetime in early zebrafish embryos We found a striking spatial pattern of mitochondrial activity with high activity at the cell periphery gradually decreasing towards the cell center, while mitochondrial distribution is rather homogenous. This suggests that mitochondria are activated from the cell surface. As the increase in energy expenditure increases proportional to the increase in surface area, mitochondrial activation from the plasma membrane might be the underlying mechanism to gradually increase ATP supply to meet the embryo’s energy demand. By further characterizing the mitochondrial activity gradient we found that the fraction of active mitochondria lies in a range of ~ 20 ± 5 µm from the plasma membrane during early cleavage stages, independent of the stage and cell size. Using a theoretical model, we showed that the gradient fits a diffusion-degradation model, suggesting that a molecular reaction-diffusion mechanism may regulate mitochondrial activation. Analyzing mitochondrial activity in embryos of other species revealed that mitochondrial activation from the cell periphery occurs beyond zebrafish, except in mouse embryos, indicating species-specific and cell size-dependent differences. To further explore the relationship between mitochondrial activity and structure, we analyzed mitochondrial morphology using electron microscopy in zebrafish embryos. These data revealed that mitochondria remain round and fragmented throughout cleavage divisions and contain large cristae structures. We found that mitochondrial area is larger close to the plasma membrane where mitochondria display increased activity and the overall size increases over time suggesting that mitochondria undergo size maturation. Analyzing potential mechanisms upstream of mitochondrial activation, we found that Ca²⁺ waves are required to induce activity. Live imaging using genetically encoded Ca²⁺ sensors revealed that dynamic Ca²⁺ waves originating from the cleavage furrow precede mitochondrial activation. Pharmacological inhibition of Ca²⁺ release and uptake from the ER disrupted mitochondrial activation, indicating that Ca²⁺ signaling is necessary for initiating mitochondrial activity. In the last part, we explored potential non-energetic functions of mitochondria during cleavage-stage development, particularly their involvement in regulating zygotic genome activation (ZGA). We observed that active mitochondria begin to form transient contacts with the nucleus at later cleavage stages, just prior to the onset of ZGA. These findings suggest that mitochondrial-nuclear interactions may facilitate the transfer of metabolites, signaling molecules, or metabolic enzymes needed for transcriptional activation in zebrafish embryos. Overall, this thesis reveals that mitochondrial metabolism in early embryos is spatially and temporally organized within the large embryonic cells. The gradual activation of mitochondria from the plasma membrane might serve as a mechanism to meet the increasing energetic demands of early development. More broadly, our results might help to further understand how mitochondrial metabolism transitions from a quiescent state to an activated state. This work contributes to a deeper understanding of how mitochondria organize and distribute bioenergetic and regulatory tasks within the subcellular environment and demonstrates their crucial role in early embryonic development. |