Developmental coordination of C. elegans embryos in temperature gradients

描述(申请人提供)：受精后，人类胚胎经历了一个戏剧性的自我组装过程，最终形成一个具有数万亿细胞的可复制结构的动物。这一过程需要大量的决策，每个决策都背负着相当大的错误率。因此，人类受精卵如此频繁地产生一个合适的个体，这是值得注意的。尽管持续纠正这些错误的质量控制系统必须无处不在，而且对人类胚胎发育至关重要，但实际上人们对这些系统是如何运作的一无所知。我们建议通过研究线虫胚胎对温度(T)梯度施加的不协调条件的高度可重复性发育来研究这种高保真发育是如何实现的。推动这项研究的假设是，通过纠正或补偿对正常水平的偏离，细胞分裂速率在不同的谱系之间得到协调，从而确保关键阶段细胞分裂事件的可重复顺序和刻板印象的细胞几何形状。在目标1中，我们将用我们制造和验证的v1.0设备将胚胎置于陡峭的温度梯度，以挑战不协调的条件，并将检查细胞分裂率、胚胎几何形状和存活率的结果。我们将调查在胚胎发育的特定阶段或具有特定极性的检查点是否被用于监测和纠正不同血统之间的不一致性，正如我们的初步数据所表明的那样。在目标2中，我们将改进我们的v2.0微流控设备，允许高通量处理温度梯度中的胚胎和实时高分辨率成像。我们将开发比率测温技术，使我们能够测量活胚胎中的区域T差异。在目标3中，我们将检验两细胞阶段胚胎极化是补偿不协调条件所必需的假设，并将探讨缝隙连接活动在这一过程中的作用。确保胚胎保真度的细胞过程缺陷是广泛的病理基础，包括出生缺陷和肿瘤发生过程中细胞增殖的失调。通过揭示这些以前未探索过的确保细胞之间适当协调的机制，我们的研究可能会揭示对癌症生物学的新见解，并更全面地了解人类高保真发育是如何实现的，这对健康组织和器官的形成和维护至关重要。

DESCRIPTION (provided by applicant): Following fertilization, human embryos undergo a dramatic self-assembly process, culminating in a reproducibly structured animal with trillions of cells. An enormous number of decisions are required for this process, each burdened with a significant rate of error. It is thus remarkable that human zygotes so frequently give rise to a proper individual. While quality control systems that continually correct these errors must be pervasive and are critically important for human embryogenesis, virtually nothing is known about how such systems operate. We propose to investigate how such high-fidelity development is achieved by investigating the highly reproducible development of C. elegans embryos in response to discordant conditions imposed by temperature (T) gradients. The hypothesis driving the proposed studies is that cell division rates are coordinated across disparate lineages by correcting or compensating for deviations from the norm, thereby ensuring a reproducible order of cell division events and a stereotyped cellular geometry at key stages. In Aim 1, we will challenge embryos to discordant conditions by subjecting them to a steep thermal gradient with a v1.0 device that we have manufactured and validated, and will examine the outcome on cell division rates, embryo geometry, and viability. We will investigate whether checkpoints operating at particular stages in embryogenesis or with a particular polarity are used to monitor and correct for discordance between disparate lineages, as has been suggested by our preliminary data. In Aim 2, we will refine our v2.0 microfluidic device, which allows high-throughput processing of embryos in thermal gradients and high-resolution imaging in real time. We will develop ratiometric thermometry techniques that allow us to measure regional T differences in living embryos. In Aim 3, we will test the hypothesis that polarization of the embryo at the two-cell stage is required for compensation to discordant conditions and will investigate the role of gap junction activity in this process. Defects in cellular processes that ensure fidelity in embryos underlie wide-ranging pathologies including birth defects and dysregulated cell proliferation in the genesis of tumors. By revealing these previously unexplored mechanisms that ensure proper coordination between cells, our studies may reveal new insights into cancer biology and a more complete understanding of how high-fidelity human development is achieved, which is crucially important for the formation and maintenance of healthy tissues and organs.