Morphomic Analysis of a Simple Chordate

最初的申请提出了一个全面的特征 野生型和实验型胚胎的形态发生。重点 将重点放在表征脊索和中央的形态发生 神经系统。我们还建议将实验操作的胚胎成像在 哪些关键的胚胎发生基因被打倒了。在这项为期两年的修订研究中，我们 将把我们的努力限制在野生型胚胎的发育中的脊索。我们的目标是 专注于脊索是为了建立图像捕获的方法， 细分和分析将对我们和其他人具有最广泛的潜在用途。在……里面 收集用于分析的胚胎图像，我们将捕获全部胚胎 而且分辨率很高。因此，虽然我们将重点放在脊索上，但图像集 在未来的研究中将服从于对其他组织的分析。主弦乐团将 是开发这些方法的理想模型。除了普遍的重要性之外 对于脊索发育的脊索，海鞘脊索呈现的范围是 计算机分割和分析的挑战，但它可能是最容易处理的 胚胎中的组织，因为细胞数量少且固定，而且事实是 一旦细胞嵌入完成，细胞的总体排列不会 变化。在初步结果部分，我们演示了3D时间推移成像 用DIC和荧光显微镜观察活体胚胎。在这份修订后的提案中，我们将 继续使用这两种成像方法。与最初的提案一样，有几个 脊索细胞分割的方法将被追求和优化。我们有 提供了两种截然不同的细胞边界方法的初步结果 DIC图像的网络蛇检测和基于分水岭的共聚焦方法 用荧光膜染料染色的活体胚胎的图像。我们期待着 专注于脊索将使我们能够在两个月内做出实质性的贡献 数年的时间，同时制造工具，以后可以扩展到更具挑战性的组织。 最后，我们将开发用于形态测量分析和可视化的工具 Notochord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.