Developmental coordination of C. elegans embryos in temperature gradients

描述（由申请人提供）：受精后，人类胚胎经历一个戏剧性的自组装过程，最终形成具有数万亿细胞的可再生结构动物。这个过程需要做出大量的决定，每一个决定都有很大的错误率。因此，人类的受精卵如此频繁地孕育出一个合适的个体，这是值得注意的。虽然不断纠正这些错误的质量控制系统必须普遍存在，并且对人类胚胎发生至关重要，但实际上对这些系统的运作方式一无所知。我们建议通过研究秀丽隐杆线虫胚胎在温度梯度施加的不协调条件下的高可重复性发育，来研究这种高保真发育是如何实现的。推动上述研究的假设是，细胞分裂率通过纠正或补偿与标准的偏差而在不同的谱系中得到协调，从而确保细胞分裂事件的可重复顺序和关键阶段的定型细胞几何形状。在Aim 1中，我们将通过使用我们制造和验证的v1.0设备将胚胎置于陡峭的热梯度中来挑战胚胎的不协调条件，并将检查细胞分裂率，胚胎几何形状和生存能力的结果。我们将调查在胚胎发生的特定阶段或具有特定极性的检查点是否被用来监测和纠正不同谱系之间的不一致，正如我们的初步数据所表明的那样。在Aim 2中，我们将改进我们的v2.0微流控装置，该装置允许在热梯度和实时高分辨率成像中对胚胎进行高通量处理。我们将开发比例测温技术，使我们能够测量活胚胎的区域温度差异。在目标3中，我们将验证两细胞阶段胚胎极化是补偿不协调条件所必需的假设，并将研究间隙连接活性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病理的基础，包括出生缺陷和肿瘤发生过程中细胞增殖失调。通过揭示这些先前未被探索的确保细胞之间适当协调的机制，我们的研究可能会揭示癌症生物学的新见解，并更全面地了解高保真人类发育是如何实现的，这对健康组织和器官的形成和维持至关重要。

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.