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

发育中的脊椎动物的细胞分化过程是复杂的，尚未完全理解。血管系统作为一个关键的器官系统，在早期胚胎发育过程中从内皮祖细胞和血管平滑肌细胞（vSMC）的祖细胞产生。使用遗传谱系追踪，我们已经确定vSMC来自胚胎日（E）7.5 - 8.5小鼠胚胎中表达Wilms肿瘤蛋白1（Wt1）的细胞。然而，Wt1表达与vSMC命运和分化之间的联系尚不清楚。为了分析这一点，我们正在利用一种名为Gastruloids的新型体外实验系统：从小鼠胚胎干细胞（mESC）生长的类器官，补充E7.5和E9.5之间小鼠胚胎的体内分析。我们的目标是发展一个全面的了解的变化，Wt1 + vSMC祖细胞在细胞水平上经历在其分化过程中。为了验证发育中的胚胎和类胃窦中的vSMC分化过程，我们建议使用先进的分子表型技术和光（IF，HCR）和电子显微镜（连续块面扫描电子显微镜（SBF-SEM））的多学科组合。从每种方法的（互补）优势中整合信息将使我们能够推进对细胞特异性和异细胞排列的生物学理解，这些排列决定组织和器官的形态发生。该项目认识到及时探索分子和细胞特异性表型参与组织/器官分化的综合询问的数据与光和电子显微镜在x，y和z轴（3D）。此外，在相关的光和电子显微镜（CLEM）的进步提供了诱人的机会，回答重要的生物学问题，虽然仍然存在警告和限制，一般可转移到所有的实验方案，包括检查的3D gastruloids，需要解决。我们将通过三个不同的工作包（WP）来解决这些挑战：WP 1：表达Wt1细胞的vSMC特征的分子表型。学生将学习gastruloid培养方法，小鼠胚胎的制备和IF/HCR的处理，以获得2D/3D图像，从而验证vSMC分化过程。WP 2：使用SBF-SEM对vSMC特征进行3D分析。学生将在NU接受关于SBF-SEM的所有相关技术的培训，以实现组织结构（毛细血管）内vSMC的3D表征及其与其他细胞的空间关系。WP 3：WP 1和WP 2方法的修改，以实现分子和细胞vSMC表征。将SBF-SEM固定方法的要素纳入IF/HCR固定方法中。使用SBF-SEM实现一种新的工作流程，允许荧光成像和捕获详细数据。

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.