Preview

PDF, English Download (12MB) | Terms of use

Abstract

New phenotypes can arise in organisms as a response to changing environments. Changes in environmental conditions, such as temperature fluctuations, pressure changes, or nutrient availability, are translated into cellular processes, thus giving rise to new phenotypes without affecting the genotype. Metabolism can be a translator of such environmental changes. It is being recognized more and more how metabolism can not only have a bioenergetic role but also affect signaling, gene expression, epigenetics, post-translational modifications, etc. I have conducted this work in mouse embryos, where Hidenobu Miyazawa has observed a phenotype in developmental timing as a result of changes in glycolytic flux. Previous data shows that increasing glycolytic flux in the presomitic mesoderm (PSM) slows down the tempo of the segmentation clock (a molecular clock that controls the patterning of the PSM). Because of these previous findings, I was interested in investigating the link between glycolysis and signaling in the PSM. I addressed the questions in the first part of this work by applying a microfluidics-based entrainment approach. Entrainment is a fundamental property of oscillating systems, which occurs when an oscillator adjusts its phase and its period to an external periodic pertur- bation, thus getting entrained to it. It has been shown before that glucose can entrain the segmentation clock, so first, I expanded the approach of glycolytic entrainment and tested which glycolytic metabolites can entrain the segmentation clock. The segmentation clock consists of an intricate network of different signaling pathways, such as Notch, Wnt, and FGF. I attempted entrainment of Notch oscillations (which are the core oscillatory compo- nent of the segmentation clock) with fructose-6-phosphate (F6P), fructose-1,6-bisphosphate (FBP), and pyruvate. I quantified the efficiency of entrainment by F6P, FBP and pyruvate by using the entrainment phase and in-phase synchrony as readouts. I identified FBP as a Zeitgeber (an external cue that synchronizes an organism’s intrinsic clock), which entrained both Notch and Wnt oscillations based on the predefined criteria. I showed that FBP pulses change the phase of entrainment when pulses are applied at different periods (detunings). I saw a difference in timing to entrainment between Wnt and Notch during entrainment with FBP pulses, hinting at a differential relationship between FBP and these two signaling pathways. The segmentation clock did not get entrained by pulses of pyruvate. Therefore, I suggest that the part of glycolysis that is linked to signaling might be confined between FBP and pyruvate (excluding pyruvate). Furthermore, I investigated whether glycolysis has a signaling role in the segmentation clock and how glycolysis is functionally linked to signaling during mouse embryo segmen- tation. I tried to disentangle the functional link of FBP to signaling by entraining with combination pulses of FBP and signaling perturbations of Wnt and Notch. By using pre- dictions from theory of dynamical systems, I showed that FBP functions as a signal in entraining the segmentation clock, hinting at a role of FBP in the PSM that goes beyond its bioenergetic function. I made some predictions about the functional link between FBP and signaling based on the resulting phases of entrainment after applying combined pulses of FBP and signaling perturbations. In the second part of this work, I explored the presence of glycolytic oscillations in the PSM by using Ca2+ oscillations as indirect evidence. I discovered that there are Ca2+ oscillations present in the PSM, which are induced by glucose and F6P supplementation in the medium, whereas they are depleted by FBP and pyruvate supplementation. Thus, I gathered some first evidence, which suggests that it might be worth it to develop FBP sensors in mice in order to consolidate the presence of glycolytic rhythms in the PSM.