Developing novel technologies to analyse cellular differentiation processes during embryonic development in vivo and ex vivo

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Cellular differentiation processes in developing vertebrates are complex and not fully understood. The vasculature as a key organ system arises during early embryonic development from endothelial progenitor cells and progenitors of vascular smooth muscle cells (vSMCs). Using genetic lineage tracing, we have determined that vSMCs arise from Wilms' tumour protein 1 (Wt1)-expressing cells in embryonic day (E) 7.5-8.5 mouse embryos. However, the link between Wt1 expression and vSMCs fate and differentiation is unclear. To analyse this, we are utilising a novel in vitro experimental system called Gastruloids: organoids grown from mouse embryonic stem cells (mESCs) that complement in vivo analysis of mouse embryos between E7.5 and E9.5. We aim to develop a comprehensive understanding of the changes the Wt1+ vSMC progenitor cells undergo at the cellular level during their differentiation processes. To characterise the vSMC differentiation processes in the developing embryos and gastruloids, we propose to use a multi-disciplinary combination of advanced molecular phenotypic techniques and light (IF, HCR) and electron microscopy (Serial Block Face Scanning Electron Microscopy (SBF-SEM)). Integrating information from the (complementary) advantages of each approach will enable us to advance biological understanding of the cell-specific, and heterocellular, arrangements determining tissue and organ morphogenesis. This project recognises the timeliness to explore molecular- and cell-specific phenotype involvement in tissue/organ differentiation by an integrated interrogation of data accrued with both light and electron microscopy in the x-, y- and z-axes (3D). In addition, advances in correlative light and electron microscopy (CLEM) offer tantalising opportunities for answering important biological questions although there remain caveats and limitations for general transferability to all experimental scenarios including the examination of 3D gastruloids, that require to be resolved. We will address these challenges through three distinct work-packages (WPs): WP1: Molecular phenotyping of Wt1-expressing cells for vSMC characteristics. The student will learn gastruloid culture methods, preparation of mouse embryos and processing for IF/HCR in order to obtain 2D/3D images that characterise the vSMC differentiation processes. WP2: 3D analysis of vSMC characteristics using SBF-SEM. The student will be trained in all relevant techniques regarding SBF-SEM at NU to enable 3D characterisation of vSMCs within tissue structures (capillaries) and their spatial relationship to other cells.WP3: Modifications of methods from WP1 and WP2 to achieve molecular and cellular vSMC characterisation. Incorporate elements of SBF-SEM fixation methods into those for IF/HCR. Arrive at a novel workflow that allows fluorescence imaging and the capture of detailed data using SBF-SEM.