Four-dimensional prediction and quantification of how physical forces impact organogenesis in zebrafish

项目概要/摘要 胚胎发育过程中程序化细胞形状改变的缺陷会破坏器官形态发生 导致结构性出生缺陷在我们对细胞如何改变其功能的理解上， 器官形成过程中的形状。虽然生化信号和形态梯度，帮助控制 器官发生是很好的研究，越来越多的证据表明，强大的控制器官的形式和功能往往也 依赖于多种机械机制，这些机制仍然知之甚少。因此，迫切需要 梳理出多种机制--包括组织规模的动力和细胞自主 收缩力-共同作用产生“机械梯度”， 器官形成过程中的形状。一个挑战是影响整个胚胎的机械扰动 通常导致相同的全局表型，使得很难确定每种机制的作用。我们长久以来- 我们的长期目标是开发一个结合细胞生物学和建模工具包，使我们能够预测细胞规模 表型和适当的扰动，可用于区分多种机械 机制等该项目使用Kupffer囊泡（KV），一种短暂的上皮器官， 斑马鱼胚胎的不对称性，作为一个模型系统。没有上游生化信号梯度 已经确定，调节KV细胞形状所需的左右图案，但多个机械 机制受到牵连。初步结果-来自（4D = 3D +时间）实验扰动和 单KV细胞形状的测量，以及模拟相互作用的3D组织的新型数学模型 结构，同时保持细胞尺度的分辨率-导致我们制定我们的中心假设，细胞形状 KV器官发生的关键变化是由机械梯度引起的，机械梯度是由细胞之间的相互作用产生的。 KV和周围组织结构以及KV内部的细胞自主收缩力。的目标 目的1是确定KV和脊索之间的相互作用如何影响细胞形状的变化。4D建模 结合活体成像和局部激光消融预测细胞形状和细胞运动 将决定如何不对称的力量所产生的棒状脊索的影响KV细胞形状的变化 在器官形成过程中。目的2的目标是了解肌动球蛋白收缩性在心肌细胞中的作用机制。 尾芽细胞周围及KV内部发生KV细胞形态变化。新的数学模型将 预测如何局部光学扰动尾芽力学，以及扰动体积和细胞- KV内细胞的自主收缩性，影响KV器官的形状。主要产出包括一个建模工具包 对于复杂的3D组织结构之间的动态相互作用的高通量模拟， 通过细胞生物学工具包，该工具包测试具有空间和时间调制的激活的模型预测， 生物力学和生物化学信号分子。这些结果将查明机械机制， 调节器官发生，并可能最终有助于预测或预防出生缺陷。

PROJECT SUMMARY/ABSTRACT Defects in programmed cell shape changes during embryonic development can disrupt organ morphogenesis and cause structural birth defects. There are fundamental gaps in our understanding of how cells change their shape during organ formation. While the biochemical signals and morphogen gradients that help govern organogenesis are well-studied, evidence is growing that robust control of organ form and function often also depends on multiple mechanical mechanisms that remain poorly understood. Thus, there is a critical need to tease apart how multiple mechanisms – including tissue-scale dynamic forces and cell-autonomous contractile forces – work together to generate “mechanical gradients” that program cell and organ shape during organ formation. A challenge is that mechanical perturbations that affect the entire embryo often result in the same global phenotype, making it difficult to pinpoint the role of each mechanism. Our long- term goal is to develop a combined cell biology and modeling toolkit that allows us to predict cell-scale phenotypes and appropriate perturbations that can be used to distinguish between multiple mechanical mechanisms. This project uses Kupffer’s vesicle (KV), a transient epithelial organ that establishes left-right asymmetry in the zebrafish embryo, as a model system. No upstream biochemical signaling gradients have been identified that regulate KV cell shapes as required for left-right patterning, but multiple mechanical mechanisms have been implicated. Preliminary results – from (4D = 3D + time) experimental perturbations and measurements of single KV cell shapes, and novel mathematical models that simulate interacting 3D tissue structures while retaining cell-scale resolution – lead us to formulate our central hypothesis that cell shape changes critical for KV organogenesis result from mechanical gradients generated by interactions between the KV and surrounding tissue structures as well as cell-autonomous contractile forces from inside KV. The goal of Aim 1 is to determine how interactions between KV and notochord impact cell shape changes. 4D modeling predictions for cell shapes and cell movement combined with live in vivo imaging and localized laser ablations will determine how asymmetric forces generated by the rod-like notochord impact KV cell shape changes during organogenesis. The goal of Aim 2 is to understand mechanisms by which actomyosin contractility in surrounding tailbud cells and inside KV generate KV cell shape changes. Novel mathematical models will predict how localized optical perturbations to tailbud mechanics, as well as perturbations to volume and cell- autonomous contractility in cells inside the KV, affect KV organ shape. Key outputs include a modeling toolkit for high-throughput simulations of dynamic interactions between complex 3D tissue structures complemented by a cell biology toolkit that tests model predictions with spatially and temporally modulated activation of biomechanical and biochemical signaling molecules. These results will pinpoint mechanical mechanisms that regulate organogenesis, and may ultimately aid in the prediction or prevention of birth defects.