Morphomic Analysis of a Simple Chordate

最初的申请提出了对 在野生型和实验操作的胚胎中的形态发生。重点 被放置在表征形态发生在脊索和中央 神经系统我们还建议对实验操作的胚胎进行成像， 哪些关键的胚胎发生基因被敲除。在这项为期两年的研究中，我们 将把我们的努力限制在野生型胚胎中发育的脊索上。我们的目标 以脊索为重点是建立图像捕获的方法， 细分和分析，这将是最广泛的潜在用途，我们和其他人。在 收集胚胎图像进行分析，我们将捕捉胚胎的全部。 和高分辨率。因此，当我们将重点放在脊索，图像集 在未来的研究中，将适用于其他组织的分析。脊索会 作为发展这些方法的理想模式。除了普遍的重要性 在脊索动物发育的脊索中，海鞘脊索呈现出一系列的 计算机分割和分析的挑战，但它可能是最容易处理的 由于细胞数量少且固定， 一旦完成电池嵌入，电池的整体布置不 变化在初步结果部分，我们展示了3D延时成像， 通过DIC和荧光显微镜观察活胚胎。在本修订提案中，我们将 继续使用这两种成像方法。与最初的建议一样， 脊索细胞分割的方法将被追求和优化。我们有 提出了两种非常不同的细胞边界的方法的初步结果 检测：DIC图像的网络蛇;以及基于分水岭的共聚焦方法 用荧光膜染料染色的活胚胎的图像。我们预计 专注于脊索将使我们能够在两个方面做出实质性的贡献， 这些工具可以在以后扩展到更具挑战性的组织。 最后，我们将开发工具的形态分析和可视化的 脊索数据

The original application proposed a comprehensive characterization of morphogenesis in both wild type and experimentally manipulated embryos. Emphasis was to be placed on characterizing morphogenesis in both the notochord and the central nervous system. We also proposed to image experimentally manipulated embryos in which key embryogenesis genes were knocked down. In this revised two-year study we will restrict our efforts to the developing notochord in wild-type embryos. Our goal in focusing on the notochord is to establish the methodologies for image capture, segmentation and analysis that will be of broadest potential use to us, and others. In collecting the embryo images for this analysis we will be capturing the embryos in toto and in high resolution. Thus, while we will be focusing on the notochord, the image sets will be amenable to the analysis of other tissues in future studies. The notochord will serve as an ideal model for developing these methods. Besides the universal importance of the notochord for chordate development, the ascidian notochord presents a range of challenges for computer segmentation and analysis, yet it is probably the most tractable tissues in the embryo because of the low and fixed numbers of cells, and the fact that once cell intercalation is completed, the overall arrangement of the cells does not change. In the Preliminary Results section we demonstrated 3D time-lapse imaging of live embryos by both DIC and fluorescence microscopy. In this revised proposal we will continue with both of these imaging methods. As in the original proposal, several approaches for notochord cell segmentation will be pursued and optimized. We have presented preliminary results with two very different approaches for cell boundary detection: network snakes for DIC images; and a watershed-based method for confocal images of live embryos stained with a fluorescent membrane dye. We expect that concentrating on the notochord will allow us to make substantial contributions in only two years while building tools that can later be extended to more challenging tissues. Finally, we will develop tools for the morphometric analysis and visualization of the notochord data.