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

哺乳动物是“双侧动物”，这意味着它们的身体分为左右两侧，它们是“从头到脚”（正前方；AP）轴的镜像。这种分裂出现在发育早期，随着中线的形成而出现，中线是沿胚胎AP轴的一条虚拟条纹。沿着中线形成的结构是指示胚胎前后轴（背腹轴；DV）和左右轴（中外侧轴；ML）组织的重要信号来源。中线是组织和器官放置的关键参考点。功能不正常的中线会导致先天性畸形，慢性出生缺陷和心脏病。因此，了解胚胎使用的生物机制（遗传的、化学的、机械的）来精确定义中线的位置和方式，控制中线宽度的因素，以及中线结构是如何产生的，是很重要的。尽管使用遗传方法（去除/增强基因功能）的传统动物实验已经揭示了指定AP轴的信号和模式，但关于指导胚胎细胞形成中线并控制其边界的机制（化学，机械，机械化学）知之甚少。这些都是根本性的、尚未解决的问题，本项目将采用一种创新的方法来解决，使用我们先进的、最先进的实验系统，称为“肠胃液”。原肠样体是胚胎干细胞的三维聚集体，发育3个胚胎轴，模仿许多早期胚胎模式事件。重要的是，类原胃发育中线，为研究中线规范、形成和形态发生的动态过程提供了一种简便的方法。我们的方法，将类胃原体与定量生物学技术和数学建模相结合，使我们能够进行昂贵、困难或不可能单独在胚胎中进行的实验。首先，我们将利用测量转录物和蛋白质的技术，在多个中线标记表达之前和期间表征胃原体中线。我们将通过结合化学和基因功能损失/增益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定位关键信号通路，并确定哪些信号对控制细胞分配和中线边界是重要的。实时延时成像实验将使我们能够定量测量中线规范对施加或抑制化学信号的响应动态，使我们能够确定初始细胞位置与其在中线中的最终位置之间是否存在相关性。其次，我们将通过将类胃原体嵌入一种专门的化学定义的“凝胶”中，精确地扰乱其所处的机械化学信号环境。这些凝胶的配方将具有明确的性能和成分，确保每种配方的抗拉强度和弹性保持不变，以免增加额外的复杂因素。这将使我们能够理清影响中线形成和位置的机械或生化信号的作用。我们将通过活体显微镜和上述定量生化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联系起来。最后将采用数学方法，建立模型来探讨中线地层的理论基础。我们将推断可能驱动中线模式出现的遗传机制。我们将探索中线模式的替代机制，产生明确的假设和预测，关键的是，这些假设和预测将在类胃原体中进行实验测试。我们的多学科方法将提供关于中线组织如何在类胃组织中被指定以及机械化学信号如何微调中线命运的详细理解。

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.