Cell sorting and lineage specification at the midbrain-hindbrain boundary

在发育过程中，胚胎细胞逐渐组织成组织和器官，具有高度特征性的细胞补充和排列。在脊椎动物的发育中，这是以一种循序渐进的方式发生的，在被称为组织中心的细胞组的控制下，组织中心使用细胞外信号来指示邻近细胞关于它们的细胞身份。中脑-后脑边界(MHB)包含一个位于正在形成的中脑和后脑区域之间的交界处的聚合组织者细胞群。胚胎学研究表明，以适当的空间顺序安排和维持组织者细胞群体本身和周围细胞是至关重要的，但这是如何从机械上实现的还不是很清楚。具体地说，我们和其他人已经证明，MHB是一个谱系限制边界，可以防止预期的中脑和后脑细胞在胚胎发育期间混合，但其背后的分子机制尚不清楚。在这个项目中，我们结合使用了多个内部生成的基于CRISPR的敲入报告线，用于MHB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，以显示预期的中脑和后脑前体细胞在早期发育阶段表现出不同的黏附和拉伸特性。此外，Eph-ephin信号的中断导致了整个MHB细胞的错误分类。这些观察结果表明，Eph-ephin信号可能对导致不同黏附和张力的分子机制起作用。在这些结果的基础上，拟议的项目旨在确定观察到的差异黏附和张力是否对MHB形成和维持过程中的细胞分选和限制至关重要。具体地说，我们建议(I)使用遗传和光遗传学工具，结合转基因报告和Cre重组酶驱动系，在预期的中脑和后脑细胞中特异性地靶向肌球蛋白和细胞骨架成分，并可视化它们对MHB形成的影响。(Ii)第二，我们将探讨边界细胞的性质和细胞命运的可能性，使用细胞类型特异性消融和活体成像来追踪早期OTX-GBX重叠的边界细胞，分别标记中脑和后脑的发育，并对这些细胞和组织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以及在药物阻断Eph-ePhin信号后，结合分子力传感器，确定Eph-ePhin信号在建立跨MHb的差异黏附和张力方面的功能。这些结果将使人们更好地理解在胚胎发育过程中以如此惊人的精度发生的有序组织形成、边界形成和图案形成。

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.