2009 - DISRUPTING THE MASTER REGULATORS OF BLOOD EMERGENCE IN THE MOUSE EMBRYO

Abstract

Brachyury (T) and Tal1 are well-known regulators of Mesoderm and Blood, respectively. However, their molecular role in shaping embryonic transcriptional landscapes and in driving the emergence of these lineages during gastrulation has been challenging to elucidate, due to knockout embryo lethality and malformations. We have recently mapped mammalian gastrulation, by transcriptionally profiling 116,312 single cells from mouse embryos collected at nine sequential time-points between 6.5 and 8.5 days post-fertilisation. This atlas reveals the putative transcriptional trajectories from the pluripotent epiblast to all embryonic lineages, including the first haematopoietic cells, and thus provides a baseline to analyse transcriptional datasets of developmental perturbations. We have now performed single-cell RNA sequencing in whole chimeric embryos generated by injecting T-/- and Tal1-/- tdTomato+ mouse embryonic stem cells into wildtype blastocysts. In the respective resulting chimeras, wildtype cells rescue the severe knockout phenotype, providing an unprecedented opportunity to dissect the cell-autonomous molecular functions of these key transcription factors at the single-cell level. Comprehensive computational analysis of the haematoendothelial lineage trajectories unravelled the precise blockage point in Tal1-/- cells, devoid of haematopoietic potential. In the case of T-/- cells, although no significant quantitative defect was observed in haematopoiesis, profound qualitative and quantitative defects were seen in other mesodermal lineages. Ongoing work is aiming to characterize the molecular networks deregulated in Tal1-/- and T-/- cells during gastrulation, to provide a functional underpinning of computationally inferred developmental trajectories, and thus obtain new mechanistic insights into the emergence of the first embryonic blood.