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Temporal coordination of gene expression during development

Final Report Summary - CELLCOORDINATION (Temporal coordination of gene expression during development)

Publishable summary:
• Project Summary
The main goal of this proposal is to understand the temporal coordination of gene expression during embryonic development. Development of living multicellular organisms requires precise coordination of genetic programs both in time and in space. This coordination is regulated by the accurate interaction between intercellular signalling and gene regulatory networks, which modulate transcription and ultimately drive cell behaviour in the developing tissues. While much has been learnt over the last few years about the regulatory motifs driving gene expression at the cellular level and the spatial regulation of cells in a tissue through morphogens, little is known about the timing and coordination of gene expression in populations of cells. This is an open problem that requires of both quantitative experimental approaches that allow time-resolved measurement of individual cells and a theoretical framework that provides a mechanistic understanding. It has been the aim of this project to combination that I
to develop in the course of the fellowship.
• Description of work performed
In the 24 months since the start of the grant Dr Rue' (the Researcher) has made progresses towards the understanding of cell dynamics and temporal coordination during differentiation and development. (see Significant Results below). Although initially focused on the in vitro differentiation of Embryonic Stem (ES) cells into mesendoderm(ME)/Primitive Streak(PS)-like cells these contributions are not limited to them.
In collaboration with other experimentalists, the Researcher has studied successfully
1. the preliminary results described on the grant application on the PS-like differentiation of ES cells and the dynamics of Brachyury (T/Bra) have been expanded and published;
2. the differentiation dynamics and the role of signalling in the Embryonic vs. Extraembryonic cell fate decision in cultured ES cells;
3. the competition dynamics between the Neurectoderm (NE) and the ME fates as ES cells exit from pluripotency;
4. the dynamics of endocrine cell differentiation in the Developing pancreas.
The analysis of the role of signalling in the mechanisms of cell coordination during PS-like differentiation, which are the main aim of the application build on top of these published results.
Finally, during the study of in vitro ME differentiation, members of the host lab identified a population of cells with a transcriptional profile that is highly reminiscent of a NeuroMesodermal progenitor (NMp) that has been described in the embryo, an undifferentiated population of cells that at the end of gastrulation fuels the expansion and axial elongation of the embryo populating the spinal cord and the surrounding paraxial mesoderm. The Researcher has played a major role in the identification of the transcriptional signature of this population through data analysis of single cells.

• Significant results achieved since the start of the Fellowship
1. Gastrulation is triggered by the formation and elongation of the Primitive Streak, a population of cells from the epiblast that ingress and become mesendoderm, from which the mesoderm and endoderm will form. Live imaging of individual ES cells differentiated towards a PS-like fate in vitro has disclosed a remarkable resemblance between the behaviour of these cells and the Primitive Streak in the embryo during gastrulation. Combined quantitative analysis of cell motility and fluorescent reporters of gene expression through image processing showed transient T/Bra expression and increased motility through EMT-like movements, both controlled by Wnt/beta-Catenin signalling. The Researcher's contribution in the achievement of these results was pivotal.
2. During pancreas development, a pool of pancreatic progenitor cells expand and differentiate into all cell types present in the adult organ, including the whole endocrine lineage. In collaboration with the group of Anne Grapin-Botton, the Researcher has recently uncovered that these progenitor the dynamics of division and differentiation of these progenitors. The results support the idea the timing of initiation of that endocrine differentiation during the cell cycle determines the progenitor division mode. Data analysis and mathematical modelling provided by the Researcher was decisive in interpreting these results. Furthermore, the Researcher's theoretical model might also be of broader application to other differentiation processes.
3. In collaboration with members of the Martinez Arias lab, Dr Rue' showed that when ES cells exit pluripotency, they display an inherent bias towards an anterior NE fate with their ability to generate the alternative fate, ME, increasing with time. This can be represented as a biased ‘race for fates’ where cells can deviate from the neural fate if signals are given within a specific time frame. The Researcher also engaged into a collaboration with the group of Prof Gunawardena to understand how the network of transcription factors that fuels pluripotency is dynamically re-arranged as cells differentiate towards ME and NE fates respectively.
4. In another collaboration with the Martinez Arias lab, the Researcher has studied the differentiation dynamics and the role of signalling in the embryonic vs. extraembryonic cell fate decision in cultured ES cells. Quantitative analysis at the single-cell level of the transcriptional dynamics underlying the two fates together with mathematical model points to the existence of a mutual repression circuit that functions as a bistable switch with a switching threshold that is controlled by Fgf/MAPK signalling. This is a novel principle for the role of signalling in cell-fate decisions, and may be used to balance the proportions of cells with specific fates in several contexts.