Integration of molecular regulatory networks: The Drosophila insulin response and mouse embryonic stem cell differentiation dynamics

Systems biology relies on considering processes on multiple levels of the organism—e.g. tissues, transcripts and proteins, developmental stages, and age—to generate and test hypotheses that are not amenable to a single-gene approach. This is of particular importance in ageing research, which deals with convoluted relationships between multiple disparate aspects of biology and medicine, as summarized recently in “The Hallmarks of Aging” (López-Otín et al. 2013). In this thesis, I present results from two large collaborative efforts that generated, integrated, and analysed systems level data. These projects focus on two of the nine hallmarks of ageing—deregulated nutrient sensing and stem cell exhaustion—, but touch on several others. The discovery of downstream processes of delayed ageing in Drosophila insulin mutants. This project is further subdivided into four major parts: The tissue-specific proteome response to reduced insulin in the mNSC-ablation model of reduced insulin signalling (IIS). Furthermore, the dependency of these changes on the transcription factor dFOXO. As published in (Tain et al. 2017). The comparison of the tissue-specific proteome responses of mNSC-ablated flies and a second model of reduced IIS, dilp2-3,5 mutant flies. Furthermore, the dependency of the dilp2-3,5 response on the presence of the endosymbiont Wolbachia. The role of post-transcriptional regulation in the tissue-specific proteome to reduced IIS response of dilp2-3,5 mutants, through the analysis of matching RNAseq data. The age-dependent proteome response to reduced IIS in dilp2-3,5 mutants. Presented are preliminary results from two tissues (fat body and thorax). Understanding mouse embryonic stem cell pluripotency and early differentiation. The focus of this project is the analysis of RNAseq data from 74 differentiation defect mESC lines under conditions promoting naive pluripotency or early differentiation. We also present supporting analysis of the specific effect of LIF on naive mESCs and insights from a screen of a inhibitor/knockout screen. As the project is ongoing, results presented in this thesis are preliminary. Additionally, the appendix describes custom R tools that were developed to facilitate the analysis, visualization of results, and collaboration for these projects: CellPlot, SETHRO, and mESCexplorer

Cooperative genetic networks drive embryonic stem cell transition from naïve to formative pluripotency.

In the mammalian embryo, epiblast cells must exit the naïve state and acquire formative pluripotency. This cell state transition is recapitulated by mouse embryonic stem cells (ESCs), which undergo pluripotency progression in defined conditions in vitro. However, our understanding of the molecular cascades and gene networks involved in the exit from naïve pluripotency remains fragmentary. Here, we employed a combination of genetic screens in haploid ESCs, CRISPR/Cas9 gene disruption, large-scale transcriptomics and computational systems biology to delineate the regulatory circuits governing naïve state exit. Transcriptome profiles for 73 ESC lines deficient for regulators of the exit from naïve pluripotency predominantly manifest delays on the trajectory from naïve to formative epiblast. We find that gene networks operative in ESCs are also active during transition from pre- to post-implantation epiblast in utero. We identified 496 naïve state-associated genes tightly connected to the in vivo epiblast state transition and largely conserved in primate embryos. Integrated analysis of mutant transcriptomes revealed funnelling of multiple gene activities into discrete regulatory modules. Finally, we delineate how intersections with signalling pathways direct this pivotal mammalian cell state transition

Textural and rheological evolution of basalt flowing down a lava channel

International audienceThe Muliwai a Pele lava channel was emplaced during the final stage of Mauna Ulu’s 1969–1974 eruption (Kilauea, Hawaii). The event was fountain-fed and lasted for around 50 h, during which time a channelized flow system developed, in which a 6-km channel fed a zone of dispersed flow that extended a further 2.6 km. The channel was surrounded by initial rubble levees of ’a’a, capped by overflow units of limited extent. We sampled the uppermost overflow unit every 250 m down the entire channel length, collecting, and analyzing 27 air-quenched samples. Bulk chemistry, density and textural analyses were carried out on the sample interior, and glass chemistry and microlite crystallization analyses were completed on the quenched crust. Thermal and rheological parameters (cooling, crystallization rate, viscosity, and yield strength) were also calculated. Results show that all parameters experience a change around 4.5 km from the vent. At this point, there is a lava surface transition from pahoehoe to ’a’a. Lava density, microlite content, viscosity, and yield strength all increase down channel, but vesicle content and lava temperature decrease. Cooling rates were 6.7 °C/km, with crystallization rates increasing from 0.03 Фc/km proximally, to 0.14 Фc/km distally. Modeling of the channel was carried out using the FLOWGO thermo-rheological model and allowed fits for temperature, microlite content, and channel width when run using a three-phase viscosity model based on a temperature-dependent viscosity relation derived for this lava. The down flow velocity profile suggests an initial velocity of 27 m/s, declining to 1 m/s at the end of the channel. Down-channel, lava underwent cooling that induced crystallization, causing both the lava viscosity and yield strength to increase. Moreover, lava underwent degassing and a subsequent vesicularity decrease. This aided in increasing viscosity, with the subsequent increase in shearing promoting a transition to ’a’a

Pahoehoe to `a`a transition of Hawaiian lavas: an experimental study

International audienceBasaltic lavas collected at the Muliwai a Pele lava channel, built during 1974 as part of Mauna Ulu’s eruption on Kilauea’s east rift zone, have been studied to investigate the effect of cooling and crystallization on the rheological properties of the lava. We have quantified the viscosity-strain-rate dependence of lava during cooling and crystallization, using concentric cylinder viscometry. We measured the viscosity of the crystal-free liquid between 1600 and 1230 °C, where we observed a deviation from the expected viscosity trend, marking the liquidus. We then made rheology measurements at subliquidus temperatures of 1207, 1203, 1183, 1176, and 1169 °C, varying the applied strain rates at each temperature. While the crystal-free liquid behaved as a Newtonian fluid, crystallization changed the rheological response to pseudo-plastic behavior, even at the lowest crystal volume fraction of 0.025. Pseudo-plastic behavior was observed down to a temperature of 1183 °C, with a crystal fraction of 0.15. Between 1183 and 1176 °C, the two-phase suspension transitioned from a power-law fluid to a Herschel-Bulkley fluid. At temperatures of 1176 and 1169 °C, with crystal fractions of 0.33 and 0.42, respectively, we observed lobate surface textures on the experimental samples, which remained preserved until the end of the experiments. Measurements at these temperatures indicated yield strengths of 82 ± 16 and 238 ± 18 Pa, respectively. The yield strength resulted from the development of an interconnected crystal network of diopside and enstatite by 1176 °C. By 1169 °C, diopside and plagioclase microcrystals had also appeared, and the effective viscosity was between 2000 and 5000 Pa s, depending on the strain rate. Further cooling to 1164 °C resulted in a rapid viscosity increase, to an effective viscosity in excess of 105 Pa s that exceeded the measurement range of our apparatus. The yield strength varies with crystallinity in an exponential fashion, with yield strength in Pa given by σ y = 1.25e12.93Φ c, where Φ c is the crystal volume fraction. The physical effect of crystals on the relative viscosity of magma was assessed by removing the effects of changing residual liquid viscosity, due to changing composition and temperature. To do this, we analyzed, synthesized, and measured the most evolved residual liquid from the subliquidus experiments. The effect of crystals was best captured by the Einstein-Roscoe equation for polydisperse spherical inclusions. We also measured the viscosity of the same crystal-liquid mixtures at low temperatures and strain rates using parallel-plate viscometry. The effect of crystals on magma viscosity was slightly greater at low strain rates, in agreement with theoretical models, although no single model reproduced these results well. In our experiments, the transition from pahoehoe to `a`a occurred between 1200 and 1170 °C, at viscosities between 100 and 1000 Pa s, depending on strain rate

Tissue-specific modulation of gene expression in response to lowered insulin signalling in Drosophila

Reduced activity of the insulin/IGF signalling network increases health during ageing in multiple species. Diverse and tissue-specific mechanisms drive the health improvement. Here, we performed tissue-specific transcriptional and proteomic profiling of long-lived Drosophila dilp2-3,5 mutants, and identified tissue-specific regulation of >3600 transcripts and >3700 proteins. Most expression changes were regulated post-transcriptionally in the fat body, and only in mutants infected with the endosymbiotic bacteria, Wolbachia pipientis, which increases their lifespan. Bioinformatic analysis identified reduced co-translational ER targeting of secreted and membrane-associated proteins and increased DNA damage/repair response proteins. Accordingly, age-related DNA damage and genome instability were lower in fat body of the mutant, and overexpression of a minichromosome maintenance protein subunit extended lifespan. Proteins involved in carbohydrate metabolism showed altered expression in the mutant intestine, and gut-specific overexpression of a lysosomal mannosidase increased autophagy, gut homeostasis, and lifespan. These processes are candidates for combatting ageing-related decline in other organisms

NMD is required for timely cell fate transitions by fine-tuning gene expression and regulating translation

Cell fate transitions depend on balanced rewiring of transcription and translation programs to mediate ordered developmental progression. Components of the nonsense-mediated mRNA decay (NMD) pathway have been implicated in regulating embryonic stem cell (ESC) differentiation, but the exact mechanism is unclear. Here we show that NMD controls expression levels of the translation initiation factor Eif4a2 and its premature termination codon-encoding isoform (Eif4a2(PTC)). NMD deficiency leads to translation of the truncated eIF4A2(PTC) protein. eIF4A2(PTC) elicits increased mTORC1 activity and translation rates and causes differentiation delays. This establishes a previously unknown feedback loop between NMD and translation initiation. Furthermore, our results show a clear hierarchy in the severity of target deregulation and differentiation phenotypes between NMD effector KOs (Smg5 KO > Smg6 KO > Smg7 KO), which highlights heterodimer-independent functions for SMG5 and SMG7. Together, our findings expose an intricate link between mRNA homeostasis and mTORC1 activity that must be maintained for normal dynamics of cell state transitions