Mapping cell fate flow and feedback control on vertebrate embryonic landscapes

项目摘要 从受孕的那一刻起直到死亡，后生动物面临着对它们的组织，细胞和它们的细胞的无情攻击。 基因组错综复杂的生物组织能够动态地承受这些挑战是至关重要的 但首先--也许是最关键的--在胚胎发育期间进行测试。早期失败 胚胎模式化可导致快速和过早的死亡，长期的出生缺陷，和/或毁灭性的 发育障碍多年来，发育生物学家在理解 许多连接环境和遗传干扰与其在胚胎中的后果的联系， 通过应用简化方法（例如单基因突变表型，单克隆命运作图）。但 很大一部分人类怀孕仍然导致发育缺陷或不明原因流产。 目前，我们也经常无法理解为什么某些扰动会导致一些胚胎发生失败， 个人，而不是其他人。一个持续存在的挑战是，任何发展模式的模型必须 同时考虑到一个复杂系统的许多元素：多种细胞类型，每种细胞类型都发挥两种作用， 内在和非自主行为，细胞周转动力学，谱系关系，以及数百种 遗传因素 为了应对这些挑战，我们正在建立一个新的实验范式， 发育生物学，利用微流体，单细胞基因组学，计算生物学，和一个强大的， 脊椎动物胚胎发生的体内分子遗传模型，实验室斑马鱼（Danio rerio）。根据本 范式，我们寻求一种新形式的生物还原论：全面的，分子分解的 脊椎动物胚胎组织转化为它们的组成细胞状态，在实验规模上， 发育生物学家可获得的（例如，每个实验104-106个单细胞测量，结合 全基因组或全转录组分辨率）。在本申请中，我们提出了我们的动机和愿景， 全胚胎单细胞状态景观作为一个多功能的实验和概念平台的系统- 脊椎动物胚胎发生的水平询问。使用这种景观方法，我们将系统地量化 所有胚胎组织的发育稳健性（即“渠道化”），以识别重复出现的瓶颈， 导致人类出生缺陷的脆弱性。与此同时，我们将剖析细胞命运控制的机制， 引导细胞的“流动”沿着胚胎的景观，特别是在个体细胞在其功能上不同的情况下， 遗传和/或发育适应性。因此，我们的实验愿景将重新审视 发育生物学，近80年前制定的，但一直坚持到现在作为抽象的原则， 而不是作为可检验的假设。我们预计这项研究计划将加速我们的集体努力， 更好地理解健康和疾病中脊椎动物个体发育的组织原则。

PROJECT SUMMARY From the moment of conception until death, metazoans face relentless assault on their tissues, their cells, and their genomes. The ability of intricately patterned biological tissues to dynamically withstand these challenges is essential for adult life but is tested first – and perhaps most critically – during embryonic development. Early failures of embryonic patterning can lead to rapid and premature lethality, long-lasting birth defects, and/or devastating developmental disorders. Over the years, developmental biologists have made great progress in understanding many of the links connecting environmental and genetic perturbations to their consequences in embryos, generally by applying reductionist approaches (e.g. single-gene mutant phenotypes, single-clone fate mapping). However, a significant proportion of human pregnancies still result in developmental defects or miscarriages of unknown cause. At present, we also often fail to understand why certain perturbations result in failed embryogenesis in some individuals, but not in others. One persisting challenge is that any model of developmental patterning must simultaneously take into account many elements of a complex system: multiple cell types, each exerting both intrinsic and non-autonomous behaviors, cell turnover dynamics, lineage relationships, and many hundreds of genetic factors. To address these challenges, we are establishing a new experimental paradigm for studies of developmental biology that leverages microfluidics, single-cell genomics, computational biology, and a powerful in vivo molecular genetic model of vertebrate embryogenesis, the laboratory zebrafish (Danio rerio). Under this paradigm, we seek a new form of biological reductionism: comprehensive, molecular decomposition of vertebrate embryonic tissues into their constituent cell states, at an experimental scale not previously accessible to developmental biologists (e.g. 104–106 single-cell measurements per experiment, combined with whole-genome or whole-transcriptome resolution). In this application, we present our motivation and our vision for the whole-embryo single-cell state landscape as a versatile experimental and conceptual platform for systems- level interrogation of vertebrate embryogenesis. Using this landscape approach, we will systematically quantify developmental robustness (i.e. “canalization”) of all embryonic tissues to identify reoccurring bottlenecks and vulnerabilities that drive human birth defects. In parallel, we will dissect mechanisms for cell fate control that direct the “flow” of cells down the embryonic landscape, particularly in contexts where individual cells differ in their genetic and/or developmental fitness. Our experimental vision will therefore revisit fundamental questions in developmental biology, formulated nearly 8 decades ago, but which have persisted until now as abstract principles rather than as testable hypotheses. We anticipate this research program will accelerate our collective efforts to better understand organizing principles of developmental ontogeny in vertebrates in health and disease.