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

开发新技术来分析体内和离体胚胎发育过程中的细胞分化过程

• 批准号: 2888341
• 金额: --
• 依托单位: University of Liverpool
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2023
• 资助国家: 英国
• 起止时间: 2023 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2888341
• 关键词: Developing; novel; technologies; analyse; cellular

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.

发育中的脊椎动物的细胞分化过程很复杂且尚未完全了解。脉管系统作为关键器官系统，在早期胚胎发育过程中由内皮祖细胞和血管平滑肌细胞（vSMC）祖细胞产生。通过遗传谱系追踪，我们确定 vSMC 源自胚胎日 (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。为了对此进行分析，我们正在利用一种称为原肠胚样的新型体外实验系统：从小鼠胚胎干细胞 (mESC) 中生长的类器官，可补充 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经历的变化。为了表征发育中胚胎和类原肠胚中的 vSMC 分化过程，我们建议使用先进分子表型技术和光（IF、HCR）和电子显微镜（串行块面扫描电子显微镜（SBF-SEM））的多学科组合。整合每种方法（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。该项目认识到通过对光学和电子显微镜在 x、y 和 z 轴 (3D) 上积累的数据进行综合询问来探索参与组织/器官分化的分子和细胞特异性表型的及时性。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，尽管对于所有实验场景（包括 3D 原肠胚的检查）的一般可转移性仍然存在需要解决的警告和限制。我们将通过三个不同的工作包 (WP) 应对这些挑战： WP1：针对 vSMC 特征对 Wt1 表达细胞进行分子表型分析。学生将学习原肠胚培养方法、小鼠胚胎的制备和 IF/HCR 处理，以获得表征 vSMC 分化过程的 2D/3D 图像。 WP2：使用 SBF-SEM 对 vSMC 特性进行 3D 分析。学生将在 NU 接受有关 SBF-SEM 的所有相关技术培训，以便对组织结构（毛细血管）内的 vSMC 及其与其他细胞的空间关系进行 3D 表征。WP3：修改 WP1 和 WP2 的方法，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 的固定方法中。实现一种新颖的工作流程，允许使用 SBF-SEM 进行荧光成像和捕获详细数据。