Cell-cell contacts specify cell fate Ascidians, or sea squirts, are marine invertebrate filter feeders with highly reproducible cellular events and invariant embryonic cell lineages. Guignard et al. studied the ascidian embryo to address the determinants of this cellular reproducibility. They introduce computational methods for the robust and automated segmentation, tracking, and analysis of whole-cell behaviors in high-throughput light-sheet microscopy datasets. This work shows that cell induction can be controlled by the contact area among cells. The range of cell signaling is proposed to set the scale at which animal embryonic reproducibility is observed. A high level of reproducibility of embryonic geometries may also counter-intuitively lift constraints on genome evolution, thereby contributing to the rapid molecular evolution observed in ascidians. Science, this issue p. eaar5663 A combination of advanced imaging and computational modeling reveals that a limited range of cell signaling constrains sea squirt development. INTRODUCTION Within each animal species, embryonic development is highly reproducible, ensuring the faithful production of a complex organism with precisely arranged and shaped organs. In most animal embryos, reproducibility is found at the tissue scale, the behaviors of individual cells being stochastic beyond the first cell divisions. Ascidians, a group of marine invertebrate chordates, show an extreme form of embryonic reproducibility: Homologous cells can be found across individual embryos, and early embryonic cell lineages are considered invariant. Embryonic geometries are even conserved between species, which diverged 400 million years ago and have very dissimilar genomes. Because of their evolutionary conservation of early embryonic development and ability to buffer genetic divergence, ascidians constitute attractive model systems to study the mechanisms driving cellular reproducibility. RATIONALE To quantify embryonic reproducibility in the ascidian Phallusia mammillata, we first built a high-resolution atlas of embryonic cell lineages, cell shapes, and cell interactions. We imaged 10 live embryos every 2 min up to the end of the neurula stages using multiview light-sheet microscopy. To systematically measure the developmental variability of a range of temporal and spatial cellular features, we developed a robust and scalable adaptive segmentation and tracking of embryonic cells procedure (ASTEC) compatible with high-throughput multiview light-sheet imaging datasets. We related these features to cell fate specification, which in ascidians is mainly controlled by differential sister cell inductions. Inspired by previous work indicating that the area of contact to signaling cells controls ascidian neural induction, we integrated our geometric description with a signaling gene expression atlas. This integration allowed us to test, through computational and experimental approaches, the hypothesis that contact area–dependent cell communication imposes constraints on embryonic geometries. RESULTS We found that, up to the neurula stages, Phallusia embryos develop without cell growth, programmed cell death, or cell neighbor exchanges. Beyond cell position, cell cycle duration, and cell lineages, we observed a high reproducibility of cell arrangements: 75% of cells shared at least 80% of their neighbors in all 10 embryos studied. Furthermore, the areas of contact between homologous cells varied by less than 20% across embryos. Mechanistically, we uncovered a tight link between the control of cell arrangements and asymmetric cell divisions, which give rise to sister cells of distinct fates. We then combined computational and experimental approaches to reveal that areas of cell contact between signaling and responding cells have sufficient encoding potential to explain all known early embryonic inductions, without the need to invoke gradients of ligand concentration. Finally, using geometrical perturbations of embryonic development we demonstrated that precise areas of cell-cell contact were important for mesendodermal and neural fate specification. CONCLUSION Our work establishes the highly reproducible ascidian embryo as a framework to bridge cell behaviors, morphogenesis, and the underlying regulatory program. The ASTEC pipeline allows systematic automated whole-cell segmentation and tracking across whole embryos in high-throughput light-sheet datasets. Second, we establish the geometric control of embryonic inductions as an alternative to classical morphogen gradients and suggest that the range of cell signaling events sets the scale at which embryonic reproducibility is observed. Finally, our study suggests that the high level of reproducibility of ascidian embryonic geometries may paradoxically lift constraints on the evolution of ascidian genomes, thereby contributing to rapid molecular evolution. Reconstruction and modeling of Phallusia embryogenesis. (Left) Quantitative analysis of Phallusia embryogenesis. We combined live light-sheet imaging of cell membranes (left images) with automated cell segmentation and tracking with color-coded cell fates (center images) to extract quantitative cell morphological properties (right images, color-coded by cell compactness). From top to bottom: embryo at the 64-cell, mid-gastrula, and late gastrula stages. (Right) Cell signaling model. We first made simplifying assumptions concerning the distribution and diffusion of signaling pathway components (top) and then integrated cell contact geometry with gene expression profiles to predict pathway activation levels in single cells (center) and binarized induction status (bottom). Marine invertebrate ascidians display embryonic reproducibility: Their early embryonic cell lineages are considered invariant and are conserved between distantly related species, despite rapid genomic divergence. Here, we address the drivers of this reproducibility. We used light-sheet imaging and automated cell segmentation and tracking procedures to systematically quantify the behavior of individual cells every 2 minutes during Phallusia mammillata embryogenesis. Interindividual reproducibility was observed down to the area of individual cell contacts. We found tight links between the reproducibility of embryonic geometries and asymmetric cell divisions, controlled by differential sister cell inductions. We combined modeling and experimental manipulations to show that the area of contact between signaling and responding cells is a key determinant of cell communication. Our work establishes the geometric control of embryonic inductions as an alternative to classical morphogen gradients and suggests that the range of cell signaling sets the scale at which embryonic reproducibility is observed.

中文翻译：

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接触面积依赖性细胞通讯和海鞘胚胎发生的形态不变性

细胞间接触决定细胞命运 海鞘或海鞘是海洋无脊椎动物滤食性动物，具有高度可重复的细胞事件和不变的胚胎细胞谱系。吉尼亚德等人。研究了海鞘胚胎以解决这种细胞再现性的决定因素。他们引入了计算方法，用于在高通量光片显微镜数据集中对全细胞行为进行稳健和自动化的分割、跟踪和分析。这项工作表明细胞诱导可以通过细胞之间的接触面积来控制。细胞信号的范围被提议用来设定观察动物胚胎再现性的尺度。胚胎几何形状的高度可重复性也可能违反直觉解除对基因组进化的限制，从而有助于在海鞘中观察到的快速分子进化。科学，这个问题 p。eaar5663 先进成像和计算模型的结合表明，有限范围的细胞信号限制了海鞘的发育。简介 在每个动物物种中，胚胎发育具有高度可重复性，确保忠实地生产出具有精确排列和形状器官的复杂有机体。在大多数动物胚胎中，在组织尺度上发现了可重复性，单个细胞的行为在第一次细胞分裂之后是随机的。海鞘是一组海洋无脊椎动物脊索动物，表现出一种极端形式的胚胎再现性：可以在单个胚胎中发现同源细胞，并且早期胚胎细胞谱系被认为是不变的。胚胎几何形状甚至在物种之间是保守的，这些物种在 4 亿年前分化并且具有非常不同的基因组。由于它们对早期胚胎发育的进化保守性和缓冲遗传差异的能力，海鞘构成了有吸引力的模型系统来研究驱动细胞可重复性的机制。基本原理为了量化海鞘 Phallusia mammillata 的胚胎再现性，我们首先构建了胚胎细胞谱系、细胞形状和细胞相互作用的高分辨率图谱。我们使用多视图光片显微镜每 2 分钟对 10 个活胚胎进行成像，直到神经胚阶段结束。为了系统地测量一系列时间和空间细胞特征的发育变异性，我们开发了一种强大且可扩展的自适应胚胎细胞分割和跟踪程序 (ASTEC)，与高通量多视图光片成像数据集兼容。我们将这些特征与细胞命运规范相关联，在海鞘中，其主要受差异姐妹细胞诱导控制。受先前工作的启发，表明与信号细胞的接触区域控制海鞘神经诱导，我们将几何描述与信号基因表达图谱相结合。这种整合使我们能够通过计算和实验方法来测试依赖于接触区域的细胞通讯对胚胎几何结构施加限制的假设。结果我们发现，直到神经胚阶段，假阴茎胚胎发育没有细胞生长、程序性细胞死亡或细胞邻居交换。除了细胞位置、细胞周期持续时间和细胞谱系外，我们还观察到细胞排列的高度可重复性：在研究的所有 10 个胚胎中，75% 的细胞共享至少 80% 的相邻细胞。此外，同源细胞之间的接触面积在胚胎中的差异不到 20%。从机制上讲，我们发现了细胞排列控制与不对称细胞分裂之间的紧密联系，这导致了具有不同命运的姐妹细胞。然后我们结合计算和实验方法来揭示信号和响应细胞之间的细胞接触区域具有足够的编码潜力来解释所有已知的早期胚胎诱导，而无需调用配体浓度梯度。最后，使用胚胎发育的几何扰动，我们证明了细胞 - 细胞接触的精确区域对于中内胚层和神经命运规范很重要。结论我们的工作建立了高度可重复的海鞘胚胎作为连接细胞行为、形态发生和潜在调节程序的框架。ASTEC 管道允许在高通量光片数据集中对整个胚胎进行系统的自动化全细胞分割和跟踪。其次，我们建立了胚胎诱导的几何控制，作为经典形态发生素梯度的替代方案，并建议细胞信号事件的范围设定了观察胚胎再现性的尺度。最后，我们的研究表明，海鞘胚胎几何形状的高度可重复性可能会矛盾地解除对海鞘基因组进化的限制，从而促进分子的快速进化。假阴茎胚胎发生的重建和建模。（左）假阴茎胚胎发生的定量分析。我们将细胞膜的实时光片成像（左图）与自动细胞分割和跟踪与颜色编码的细胞命运（中心图像）相结合，以提取定量的细胞形态学特性（右图，按细胞紧密度进行颜色编码）。从上到下：处于 64 细胞、原肠胚中期和原肠胚后期的胚胎。（右）细胞信号模型。我们首先对信号通路组分的分布和扩散（顶部）进行了简化假设，然后将细胞接触几何形状与基因表达谱相结合，以预测单细胞（中）和二值化诱导状态（底部）中的通路激活水平。海洋无脊椎动物海鞘显示胚胎可重复性：尽管基因组迅速分化，但它们的早期胚胎细胞谱系被认为是不变的，并且在远缘物种之间是保守的。在这里，我们解决了这种可重复性的驱动因素。我们使用光片成像和自动细胞分割和跟踪程序来系统地量化 Phallusia mammillata 胚胎发生过程中每 2 分钟单个细胞的行为。观察到个体间可重复性直至单个细胞接触的区域。我们发现胚胎几何形状的可重复性与不对称细胞分裂之间存在紧密联系，由差异姐妹细胞诱导控制。我们结合建模和实验操作来表明信号和响应细胞之间的接触区域是细胞通讯的关键决定因素。我们的工作建立了胚胎诱导的几何控制，作为经典形态发生素梯度的替代方案，并表明细胞信号的范围设定了观察胚胎再现性的尺度。我们结合建模和实验操作来表明信号和响应细胞之间的接触区域是细胞通讯的关键决定因素。我们的工作建立了胚胎诱导的几何控制，作为经典形态发生素梯度的替代方案，并表明细胞信号的范围设定了观察胚胎再现性的尺度。我们结合建模和实验操作来表明信号和响应细胞之间的接触区域是细胞通讯的关键决定因素。我们的工作建立了胚胎诱导的几何控制，作为经典形态发生素梯度的替代方案，并表明细胞信号的范围设定了观察胚胎再现性的尺度。