A multidisciplinary approach to define the dynamics of mammalian midline specification using Gastruloids

哺乳动物是“两侧对称”的，这意味着它们的身体被分为左右两侧，这是沿着“头到脚”（前后; AP）轴的镜像。这种分裂发生在胚胎发育的早期，伴随着中线的形成，中线是一条沿着胚胎的AP轴的虚拟条纹。沿着中线形成的结构是指示胚胎的后-前（背腹; DV）和左右（中间-外侧; ML）轴组织的重要信号来源。中线是组织和器官放置的重要参考点。中线功能不正常可能导致先天性畸形，慢性出生缺陷和心脏病。因此，了解胚胎使用的生物学机制非常重要。（遗传，化学，机械）来精确地定义中线在哪里和如何放置，是什么控制了它的宽度，以及中线结构是如何从它产生的。（去除/增强基因功能）已经揭示了指定和模式AP轴的信号，关于机制知之甚少。这些都是基本的，未解决的问题，该项目将通过采用创新的方法，使用我们先进的，最先进的实验系统称为'gastruloids'来解决。胚胎干细胞的3D聚集体Gastruloids发育3个胚胎轴，模仿许多早期胚胎图案化事件。重要的是，胃状体形成中线，提供了一种易于处理的方法来研究中线特化，形成和形态发生中涉及的动态过程。我们的方法，耦合gastruloids与定量生物技术和数学建模，使我们能够执行实验，否则将是昂贵的，困难的或不可能单独在胚胎中做。首先，我们将使用测量转录本和蛋白质的技术，在多个中线标记物的表达之前和期间，对gastruloids中线进行标记。我们将通过结合化学和遗传功能丧失/获得实验来确定化学信号在中线规范中的作用。我们将在精确的时间点瞄准关键的信号通路，并确定哪些信号对控制细胞分配和中线边界很重要。实时延时成像实验将使我们能够定量测量中线规格响应于施加或抑制的化学信号的动态变化，使我们能够确定初始细胞位置与其在中线的最终位置之间是否存在相关性。其次，我们将通过将胃状体嵌入到专门的化学定义的“凝胶”中来精确扰乱胃状体所在的机械化学信号环境。这些凝胶将以定义的特性和组成配制，确保每种配方的拉伸强度和弹性保持不变，以免增加额外的复杂因素。这将使我们能够解开机械或生化信号在影响中线形成和放置中的作用。我们将通过活体显微镜和上述定量生物化学技术将这些凝胶的时间、组成和性质与中线命运和形态的规格和位置相关联。最后将采用数学方法，制定模型来探索中线形成的理论基础。我们将推断可能驱动中线图案出现的遗传机制。我们将探讨中线图案产生明确的假设和预测，关键是，将在gastruloids实验测试的替代机制。我们的多学科方法将详细了解中线组织如何在类胃体中指定以及机械化学信号如何微调中线命运。

Mammals are 'bilaterians', meaning their body is divided into a left & right side that are mirror images along the 'head-to-toe' (anteroposterior; AP) axis. This division arises in early development following the formation of the midline, a virtual stripe along the AP axis of the embryo. Structures that form along the midline are important sources of signals that instruct the organisation of the embryo's back-front (dorsoventral; DV) & left-right (medio-lateral; ML) axes. The midline is a crucial reference point for the placement of tissues & organs. An improperly functioning midline can result in congenital malformations, chronic birth defects & heart conditions. Therefore, it is important to understand the biological mechanisms the embryo uses (genetic, chemical, mechanical) to precisely define where & how the midline is laid down, what controls its width, and how midline structures arise from it. Although traditional animal experiments using genetic approaches (remove/enhance gene function) have revealed the signals that specify & pattern the AP axis, less is known regarding the mechanisms (chemical, mechanical;mechanochemical) that direct embryonic cells to form the midline and control its boundaries.These are fundamental, unresolved issues which this project will resolve by taking an innovative approach, using our advanced, state-of-the-art experimental system called 'gastruloids'. Gastruloids, 3D aggregates of embryonic stem cells, develop the 3 embryonic axes, mimicking many early embryonic patterning events. Importantly, gastruloids develop a midline, offering a tractable means to study the dynamic processes involved in midline specification, formation & morphogenesis. Our approach, coupling gastruloids with quantitative biological techniques and mathematical modelling, allows us to perform experiments that would otherwise be costly, difficult or impossible to do in embryos alone.Firstly, we will characterise the gastruloid midline prior to & during the expression of multiple midline markers using techniques measuring transcript and protein. We will determine the role chemical signalling plays in midline specification through combination of chemical & genetic loss/gain of function experiments. We will target key signalling pathways at precise time-points, and establish which signals are important for controlling cell allocation and midline boundaries. Live, time-lapse imaging experiments will allow us to quantitatively measure the dynamics of midline specification in response to applied or inhibited chemical signals, allowing us to determine whether there is a correlation between the initial cell location and its final position in the midline.Secondly, we will precisely perturb the mechanochemical signalling environment in which gastruloids reside by embedding them in a specialised chemically-defined 'gel'. These gels will be formulated with defined properties and composition, making sure the tensile strength & elasticity is kept the same for each formulation so as not to add additional complicating factors. This will allow us to disentangle the role of mechanical or biochemical signals in influencing the formation and placement of the midline. We will correlate the timing, composition & properties of these gels with the specification and placement of midline fates & morphology by live microscopy and the aforementioned quantitative biochemical techniques.Finally will take a mathematical approach, formulating models to explore the theoretical basis for midline formation. We will infer the genetic mechanisms that could drive the emergence of midline patterning. We will explore alternative mechanisms for midline patterning generating explicit hypotheses & predictions that, critically, will be tested experimentally in gastruloids. Our multidisciplinary approach will deliver a detailed understanding on how midline tissues are specified in gastruloids & how mechanochemical signalling fine-tunes midline fates.