CAREER: Investigating the Role of Clonal Cell Competition in Zebrafish Neural Development using in Vivo Multicolor Imaging

大脑的显著功能需要在胚胎中适当的生长和形成。首先，一小群细胞数量急剧增加，然后转变成一个组织精巧、连接模式复杂的器官。大多数情况下，这一过程会导致大脑完全正常，神经细胞数量正常。然而，令人惊讶的是，人们对发育中的大脑如何决定产生多少细胞知之甚少。这个研究项目将研究透明斑马鱼在生命的最初几天里生长的大脑中细胞数量的控制。PI已经开发了一种技术，可以在一种特殊的斑马鱼胚胎中观察分裂的细胞簇，这种胚胎的脑细胞会发出独特的颜色组合（称为“彩虹”）。当新细胞从母细胞中分裂出来时，这种颜色允许新细胞跟随。到目前为止，似乎细胞家族（母细胞和她的一组子细胞）在发育中的大脑中与其他家族竞争生存。这种类型的竞争以前从未在大脑中发现过，而且可能负责控制生长——不仅在大脑中，而且在其他器官中也是如此。PI将测试哪些基因对细胞家族在竞争中生存是重要的。这项工作将产生一些新的研究工具，将与科学界共享。本科生将亲自执行和分析实验，为发展科学家的职业生涯提供丰富的早期研究训练机会。通过将他们个人收集的数据转换成一个互动的教育网站，学生们将学习各种数字媒体方法，使传统和非传统受众都能理解科学材料。这项研究产生的彩色图像对科学家和非科学家都有很大的吸引力，使学生更容易学会如何让公众参与到他们的工作中来。为了研究活体大脑中的动态细胞行为，PI开发了一种使用斑马鱼体内延时共聚焦成像和多色荧光蛋白（Brainbow）表达的方法。利用这种技术，PI已经表明，程序性细胞死亡在活的大脑中非随机发生，特定的神经祖细胞及其神经元后代（整个克隆）以协调的方式发生细胞死亡，而邻近的克隆则表现正常。如果分裂细胞的整个克隆实际上是相互竞争的，那么细胞适应性的增加或减少应该会影响克隆的竞争优势。计划中的研究将使用马赛克方法来改变单个细胞中的基因表达，再加上全局脑虹表达，同时跟踪多个分裂细胞克隆的动态。细胞适应性将通过细胞内在机制（细胞周期，c-myc活性）和细胞外在机制（获取细胞外BMP信号）来靶向。这些实验将直接测试细胞适应性是否会影响克隆？在发育中的大脑中生存的能力。总的来说，这可能支持一个假设，即分裂细胞克隆之间的竞争有助于调节神经系统发育中神经元的产生和生长。

The remarkable function of the brain requires proper growth and formation in the embryo. First, a small cluster of cells increases dramatically in number, then transforms into an exquisitely organized organ with a complicated pattern of connections. Most of the time, this process results in brains that are perfectly normal, with the right number of nerve cells. Surprisingly little is known, though, about how the growing brain decides how many cells to produce. This research project will look at the control of cell number in the growing brains of transparent living zebrafish during the first few days of life. The PI has developed techniques for watching clusters of dividing cells over time in special zebrafish embryos whose brain cells glow with unique combinations of colors (called "Brainbow"). This coloring allows new cells to be followed as they divide off from their mother cell. So far it appears that families of cells (mother cell with her set of daughter cells) compete with other families to survive in the growing brain. This type of competition has not been seen before in the brain, and may be responsible for controlling growth - not only in the brain, but in other organs as well. The PI will test which genes are important for cell families to survive this competition. The work will generate a number of new research tools that will be shared with the scientific community. Undergraduate students will perform and analyze the experiments themselves, providing rich opportunities for research training early in developing scientists' careers. By also transforming the data they have personally collected into an interactive, educational website, students will learn various digital media approaches to making scientific material understandable to traditional and non-traditional audiences. The colorful images produced by this research have great appeal to both scientists and non-scientists, making it easier for students to learn how to engage the public with their work. To study dynamic cellular behavior in the living brain, the PI has developed an approach using in vivo time-lapse confocal imaging and multicolor fluorescent protein (Brainbow) expression in zebrafish. Using this technique, the PI has shown that programmed cell death occurs non-randomly in the living brain, with specific neural progenitor cells and their neuronal progeny (entire clones) undergoing cell death in a coordinated manner, while neighboring clones appear normal. If whole clones of dividing cells are in fact competing with one another, increases or decreases in cellular fitness should influence a clone's competitive edge. The planned research will use a mosaic approach to alter gene expression in individual cells, coupled with global Brainbow expression to simultaneously follow dynamics within multiple clones of dividing cells. Cellular fitness will be targeted via both cell-intrinsic mechanisms (cell cycle, c-myc activity) and cell-extrinsic mechanisms (access to extracellular BMP signaling). These experiments will test directly whether cellular fitness influences a clone?s ability to survive in the developing brain. Overall this may support the hypothesis that competition among clones of dividing cells helps regulate neuronal production and growth in the developing nervous system.