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

使用原肠胚来定义哺乳动物中线规格动态的多学科方法

• 批准号: BB/X000907/1
• 负责人: David Turner
• 金额: $ 92.07万
• 依托单位: University of Liverpool
• 依托单位国家: 英国
• 项目类别: Research Grant
• 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=BB%2FX000907%2F1
• 关键词: multidisciplinary; approach; define; dynamics; mammalian

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.

哺乳动物是“两侧对称动物”，这意味着它们的身体分为左右两侧，沿着“从头到脚”（前后轴；AP）轴呈镜像。这种分裂出现在中线形成后的早期发育中，中线是沿着胚胎 AP 轴的虚拟条纹。沿中线形成的结构是指导胚胎前后轴（背腹轴；DV）和左右轴（中外侧轴；ML）组织的重要信号源。中线是放置组织和器官的重要参考点。中线功能不正常可能导致先天畸形、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、化学、机械）来精确定义中线的位置和方式、控制其宽度的因素以及中线结构如何从中产生非常重要。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和模式化 AP 轴的信号，但对于引导胚胎细胞形成中线并控制其的机制（化学、机械、机械化学）知之甚少。这些都是尚未解决的基本问题，该项目将通过采用创新方法，使用我们先进的、最先进的称为“原肠胚”的实验系统来解决这些问题。原肠胚是胚胎干细胞的 3D 聚集体，发育出 3 个胚胎轴，模仿许多早期胚胎模式事件。重要的是，类原肠胚发育出一条中线，为研究中线规范、形成和形态发生所涉及的动态过程提供了一种易于处理的方法。我们的方法将原肠胚与定量生物技术和数学建模相结合，使我们能够进行原本在胚胎中成本高昂、困难或不可能进行的实验。首先，我们将在多个中线表达之前和期间表征原肠胚中线使用测量转录物和蛋白质的技术进行标记。我们将通过化学和遗传功能丧失/获得实验的结合来确定化学信号在中线规范中发挥的作用。我们将在精确的时间点瞄准关键信号通路，并确定哪些信号对于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范响应施加或抑制的化学信号的动态，从而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。 ，我们将通过将类原肠胚嵌入专门的化学定义的“凝胶”中来精确扰动类原肠胚所在的机械化学信号环境。这些凝胶将按照规定的特性和成分配制，确保每种配方的拉伸强度和弹性保持相同，以免增加额外的复杂因素。这将使我们能够理清机械或生化信号在影响中线形成和位置方面的作用。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、成分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。我们将探索中线模式的替代机制，产生明确的假设和预测，重要的是，这些假设和预测将在类原肠胚中进行实验测试。我们的多学科方法将详细了解原肠胚中的中线组织是如何指定的以及机械化学信号如何微调中线命运。

项目成果

Negative feedback on Retinoic Acid by Brachyury guides gastruloid symmetry-breaking

Brachyury 对视黄酸的负反馈引导原肠胚对称性破坏

• DOI: http://dx.10.1101/2023.06.02.543388
• 发表时间: 2023
• 作者: Hennessy M

Modifying gastruloids to dissect mechanisms of tissue-specific induction.

修改原肠胚来剖析组织特异性诱导的机制。

• DOI: http://dx.10.1016/j.gde.2023.102130
• 发表时间: 2023
• 期刊: Current opinion in genetics & development
• 影响因子: 4
• 作者: Turner DA