Mesoderm diversification during mouse embryonic development

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During early mouse embryonic development, a single cell, the fertilised egg, will give rise to a wide range of cell types that become specialised at a functional and molecular level. Gastrulation and early organogenesis are two of the most critical events during these early stages, when pluripotent cells, able to generate any cell in the embryo, proliferate and become lineage-restricted into the progenitors of the major organs. The vast amount of cell fate decisions taking place in this 48-hour window makes these stages a suitable paradigm to study cell type diversification. Nevertheless, the low cell numbers in early mouse embryos and the limited strategies to isolate homogenous cell populations have restricted the study of the transcriptional programs and regulatory processes that underlie these processes in vivo.

With the advent of high-throughput single-cell genome-wide technologies, it is now possible to obtain the molecular profiles of hundreds of individual cells at once, thus opening a new window for the study of early embryogenesis. To delineate the molecular events underlying gastrulation and early organogenesis, we have therefore generated a comprehensive single-cell transcriptomic atlas of these stages. In Chapter 3, I introduce this atlas and give a general overview of the lineages that have been captured.

Due to the importance of the haemato-endothelial lineages to establish the circulatory system in the embryo for appropriate oxygenation, in Chapter 4, I characterise their emergence using the atlas. My analyses uncover a rapid formation of primitive erythrocytes that do not transition through mature endothelium. Furthermore, I report the transcriptomes of megakaryocytic and myeloid progenitors as well as show that endothelial cells from different embryonic locations present distinctive transcriptional signatures.

Getting a better characterisation of embryogenesis gives us a solid baseline to understand the consequences of genetic mutations. In Chapter 5, I explore the effects of disrupting the blood regulator Tal1 using mouse embryonic chimaeras and reveal that endothelial cells are transcriptionally aberrant at early organogenesis and express genes characteristic of other mesodermal lineages.

Although single-cell transcriptomics unveils the molecular programs defining each cell type, studying gene expression is not enough if we want to highlight the regulatory events behind cell type diversification. Therefore, in Chapter 5, I examine the use of single-cell transcriptomics to detect RNAs at enhancers, which may represent a surrogate for enhancer activity. Due to the limitations encountered, in Chapter 7, I perform single-nucleus ATAC-seq in cells at early organogenesis, a time-point by which all major progenitors are established. Analysing the resulting chromatin accessibility maps together with subsequent in vivo validation experiments have allowed the discovery of two novel endothelial enhancers as well as a previously unrecognised role for the ETS transcription factor FEV in the establishment of haemato-endothelial lineages.

In conclusion, single-cell genome-wide technologies have permitted the comprehensive characterisation of the molecular programs and regulatory events underlying gastrulation and the start of organogenesis in the early mouse embryo. Having acquired this information has not only contributed to our understanding of embryonic development, but it will also help the optimisation of in vitro differentiation protocols in the future.

Göttgens, Berthold
Developmental Biology, Embryonic Development, Gastrulation, Organogenesis, Single-cell, Transcriptomics, RNA-seq, Chromatin, ATAC-seq, eRNA, Blood, Endothelium, Haemato-endothelium, Erythrocytes, Chimaera, Scl, Tal1, Enhancer, Fev, Etv2, Etsrp, Mouse, Zebrafish
Doctor of Philosophy (PhD)
Awarding Institution
University of Cambridge
Wellcome Trust 4-Year PhD Programme in Stem Cell Biology and Medicine and the University of Cambridge, United Kingdom.