Cell sorting and lineage specification at the midbrain-hindbrain boundary

中脑-后脑边界的细胞分选和谱系规范

• 批准号: 251970880
• 负责人: Professor Dr. Michael Brand
• 金额: --
• 依托单位: Zentrum für Regenerative Therapien Dresden - CRTD
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/251970880?language=en
• 关键词: Cell; sorting; lineage; specification; midbrain

During development, embryonic cells progressively organize into tissues and organs, with a highly characteristic complement and arrangement of cells. In developing vertebrates, this occurs in a stepwise fashion, under the control of cell groups known as organizing centers that use extracellular signals to instruct neighbouring cells about their cell identity. The midbrain-hindbrain boundary (MHB) contains a paradigmatic organizer cell population located at the interface between the forming midbrain and hindbrain territories. Embryological studies showed previously that it is of crucial importance to arrange and maintain both the organizer cell population itself, and the surrounding cells, in proper spatial order, but how this is achieved mechanistically is not well understood. Specifically, we and others have shown that the MHB is a lineage restriction boundary that prevents the intermingling of prospective midbrain and hindbrain cells during embryonic development, but the molecular mechanisms underlying this are unclear. In this project, we have used a combination of multiple, in-house-generated CRISPR-based knock-in reporter lines for genes expressed at the MHB, atomic force microscopy of single cells, and molecular force sensors in vivo, to show that prospective midbrain and hindbrain progenitors display differential adhesive and tensile properties during the early developmental stages. Further, disruption of Eph-ephrin signalling resulted in mis-sorting of cells across the MHB. These observations imply that, Eph-ephrin signalling may contribute to the molecular mechanisms leading to differential adhesion and tension. Building on these results, the proposed project aims to determine if the observed differential adhesion and tension are essential for cell sorting and restriction during MHB formation and maintenance. Specifically, we propose to (i) use genetic and optogenetic tools, in combination with transgenic reporter and Cre-recombinase driver lines, to target actomyosin and cytoskeletal components specifically in prospective midbrain and hindbrain cells, and to visualize the effects of their disruption on MHB formation. (ii) Second, we will address the nature and cell-fate potential of the boundary cells, using cell type-specific ablation and live imaging to track early Otx-Gbx overlapping boundary cells marking midbrain and hindbrain development, respectively, and carry out lineage tracing experiments for these cells, as well as organizer cells. Finally, we will (iii) determine the function of Eph-ephrin signalling for establishing differential adhesion and tension across the MHB, by evaluating the phenotype of mutants and after pharmacological blockade of Eph-ephrin signalling, in combination with molecular force sensors. The results will allow better understanding of ordered tissue formation, boundary formation and patterning which occur with such marvelous precision during embryonic development.

在发育过程中，胚胎细胞逐渐形成组织和器官，具有高度特征性的细胞补充和排列。在发育中的脊椎动物中，这种过程是在被称为组织中心的细胞群的控制下逐步发生的，组织中心使用细胞外信号来指示邻近细胞的细胞身份。中脑-后脑边界（MHB）包含一个典型的组织细胞群，位于形成的中脑和后脑区域之间的界面。胚胎学研究表明，在适当的空间顺序中安排和维持组织者细胞群本身和周围细胞是至关重要的，但这是如何实现的机制尚不清楚。具体来说，我们和其他人已经证明MHB是一个谱系限制边界，可以防止胚胎发育过程中未来的中脑和后脑细胞混合，但其分子机制尚不清楚。在这个项目中，我们结合了多个内部生成的基于crispr的敲入报告系，用于MHB上表达的基因，单细胞的原子力显微镜，以及体内的分子力传感器，来显示潜在的中脑和后脑祖细胞在早期发育阶段表现出不同的粘附和拉伸特性。此外，ephrin信号的破坏导致细胞在MHB上的错误分类。这些观察结果表明，ephrin信号传导可能参与了导致粘附和张力差异的分子机制。基于这些结果，拟议的项目旨在确定在MHB形成和维持过程中观察到的差异粘附和张力是否对细胞分选和限制至关重要。具体而言，我们建议(i)使用遗传和光遗传学工具，结合转基因报告基因和cree重组酶驱动系，在中脑和后脑细胞中特异性靶向肌动球蛋白和细胞骨架成分，并可视化其破坏对MHB形成的影响。（ii）其次，我们将解决边界细胞的性质和细胞命运潜力，使用细胞类型特异性消融和实时成像来跟踪早期Otx-Gbx重叠边界细胞，分别标记中脑和后脑发育，并对这些细胞以及组织者细胞进行谱系追踪实验。最后，我们将（iii）通过评估突变体的表型，并结合分子力传感器对ephrin信号进行药物阻断后，确定epf -ephrin信号在MHB上建立差异粘附和张力的功能。结果将有助于更好地理解有序的组织形成，边界形成和模式发生在胚胎发育过程中如此惊人的精度。