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，尚未完全了解。血管系统是胚胎发育早期内皮祖细胞和血管平滑肌细胞（vSMCs）祖细胞形成的重要器官系统。通过遗传谱系追踪，我们已经确定vSMCs起源于胚胎日(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vSMCs命运和分化之间的联系尚不清楚。为了分析这一点，我们利用了一种新的体外实验系统，称为gastruloid：从小鼠胚胎干细胞（mESCs）中生长的类器官，补充了E7.5和E9.5之间小鼠胚胎的体内分析。我们的目标是全面了解Wt1+ vSMC祖细胞在分化过程中在细胞水平上发生的变化。为了描述发育中的胚胎和原胃样细胞的vSMC分化过程，我们建议使用先进的分子表型技术、光（IF， HCR）和电子显微镜（连续块面扫描电子显微镜（SBF-SEM））的多学科结合。整合来自每种方法（互补）优势的信息将使我们能够推进对决定组织和器官形态发生的细胞特异性和异细胞排列的生物学理解。该项目通过对x， y和z轴（3D）的光和电子显微镜积累的数据进行综合分析，认识到探索分子和细胞特异性表型参与组织/器官分化的及时性。此外，相关光学和电子显微镜（CLEM）的进步为回答重要的生物学问题提供了诱人的机会，尽管仍然存在需要解决的所有实验场景（包括3D胃样物质的检查）的一般可转移性的警告和限制。我们将通过三个不同的工作包（WPs）来解决这些挑战：WP1：表达wt1的细胞的vSMC特征的分子表型。学生将学习胃原质培养方法，小鼠胚胎的制备和IF/HCR的处理，以获得表征vSMC分化过程的2D/3D图像。WP2：利用SBF-SEM对vSMC特征进行三维分析。该学生将在北大接受SBF-SEM相关技术的培训，以实现组织结构（毛细血管）内vSMCs的3D表征及其与其他细胞的空间关系。WP3：对WP1和WP2的方法进行修改，以实现vSMC的分子和细胞表征。将SBF-SEM固定方法的元素纳入IF/HCR固定方法。到达一个新的工作流程，允许荧光成像和使用SBF-SEM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.