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.

发育中的脊椎动物中的细胞分化过程是复杂的，尚未完全理解。作为关键器官系统的脉管系统是在早期的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（VSMC）产生的。使用遗传谱系追踪，我们确定VSMC是由Wilms的肿瘤蛋白1（WT1）在胚胎日（E）7.5-8.5小鼠胚胎中产生的。但是，WT1表达与VSMCS命运与分化之间的联系尚不清楚。为了分析这一点，我们正在利用一种称为胃lo子的新型体外实验系统：从小鼠胚胎干细胞（MESC）生长的类器官，该系统补充了E7.5和E9.5之间小鼠胚胎的体内分析。我们旨在对WT1+ VSMC祖细胞在分化过程中在细胞水平上经历的变化进行全面的了解。为了表征发育中的胚胎和胃类中的VSMC分化过程，我们建议使用高级分子表型技术和光（如果，HCR）和电子显微镜（串行块面扫描扫描电子显微镜（SBF-SEM））的多学科组合（if，hcr）和电子显微镜（SBF-SEM）。从每种方法的（互补）优势中综合信息将使我们能够提高对细胞特异性和杂细胞的生物学理解，从而确定组织和器官形态发生。该项目认识到探索分子和细胞特异性表型在组织/器官分化中的及时性，通过对X-，Y和Z轴（3D）中光和电子显微镜的数据进行综合询问。此外，相关光和电子显微镜（CLEM）的进步为回答重要的生物学问题提供了诱人的机会，尽管仍然需要解决所有实验场景的一般可转移性，包括检查3D胃lul虫，需要解决。我们将通过三个不同的工作包（WPS）：WP1：表达WT1的细胞的分子表型来解决这些挑战。学生将学习胃培养方法，制备小鼠胚胎和IF/HCR的处理，以获得表征VSMC分化过程的2D/3D图像。 WP2：使用SBF-SEM对VSMC特性的3D分析。该学生将接受有关NU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。将SBF-SEM固定方法的元素纳入IF/HCR的元素。到达新型工作流程，该工作流程允许使用SBF-SEM捕获荧光成像并捕获详细数据。