Analysing cardiac specification and morphogenesis using synthetic mouse embryos

Background:

This project will address how embryonic cells develop into distinct cardiac lineages and form a heart during morphogenesis. Addressing this question has important implications for our understanding of congenital heart diseases -affecting ~1% of newborns- and the design of regenerative methods targeting specific populations of heart cells.

Our heart is composed of four chambers, two ventricles and two atria arranged in parallel and driving blood from the body to the lungs and through the rest of the body. We recently established a fate map demonstrating ventricular and atrial cardiac progenitors have distinct spatial and temporal origins in the primitive streak (the early embryonic structure giving rise to mesodermal and endodermal tissues) (Ivanovitch et al., 2021). We further demonstrated that the primitive streak cells contributing to the ventricles have a distinct molecular signature from those forming the atria. These observations suggest cardiac progenitors are already prespecified in the streak; and this may prefigure their allocation to distinct anatomical structures of the heart.

The student will exploit a 3D mouse stem cell-based model resembling natural embryos (synthetic embryos) currently developed by Prof. Zernicka-Goetz's team to address this question (Amadei et al.,2021, Harrison et al., 2017). Remarkably, these synthetic embryos can now develop to a stage they form a beating heart and may present a unique opportunity to study the molecular mechanisms regulating cell fate decision and morphogenesis in vitro as an alternative/complement to in vivo experiments.

Objectives & Methods:

The project's objective is to uncover the mechanisms underlying the specification and morphogenesis of chamber specific cardiac cell types. We recently found that cardiac progenitors are assigned to specific compartments of the heart already in the primitive streak. Anterior regions of the primitive streak -expressing low levels of the T-box transcription factor T- contribute to the ventricles. In contrast, the posterior primitive streak -expressing high levels of T- contributes to the atria.

1) The student will test the hypothesis that ventricular or atrial cardiomyocytes (transcriptionally and functionally distinct) specification depends on initial molecular conditions set in the primitive streak.

They will first generate distinct populations of primitive streak-like cells expressing low or high levels of T using mES cells in 2D culture. They will test their ventricular and atrial differentiation potential. In the low T condition, they will then overexpress T using an inducible overexpressing cell line (TTET-ON line) and analyse the putative changes in differentiation potential induced by T overexpression. They may extend the analysis to other genes differentially expressed in the Tlow versus Thigh primitive streak.

2) Using the synthetic embryos -currently developed by Prof. Zernicka-Goetz's team, they will continue analysing the emergence of the cardiac lineages from the primitive streak. The synthetic embryos undergo morphogenesis forming a beating looped heart as it occurs in natural embryos. They will assess reproducibility and similarities with natural embryos in detail.

3) Using the synthetic embryos, they will then test if overexpression of key transcription factors in the primitive streak (e.g. T or others) can modulate the proportions of ventricular vs atrial cardiomyocytes and consequently disrupt heart morphogenesis. Therefore, the student will implement synthetic embryos as a novel tool for studying the molecular and genetic basis of cardiac lineage specification, morphogenesis and congenital heart diseases.

For this project, the student will work under the supervision of Dr Ivanovitch (project lead) and collaborate with Prof Magdalena Zernicka-Goetz's team at the University of Cambridge. More information on their work can be found here: ivanovitch-lab.com and zernickagoetzlab.com.   

References:

1. Ivanovitch K et al. Ventricular, atrial and outflow tract heart progenitors arise from spatially and molecularly distinct regions of the primitive streak. PLOS Biology doi.org/10.1371/journal.pbio.3001200

2. Amadei G et al. Inducible Stem-Cell-Derived Embryos Capture Mouse Morphogenetic Events In Vitro. Dev Cell. 56(3):366-382.e9. (2021).

3. Harrison SE et al. (2017) Assembly of embryonic and extraembryonic stem cells to mimic embryogenesis in vitro. Science. Apr 14;356(6334):eaal1810.