Interpreting Bone Morphogenetic Protein Gradients in Vertebrate Development

项目摘要/摘要 动物的发育包括通过细胞间通讯进行复杂的细胞协调和重排。 祖细胞如何解释影响细胞命运决定的相邻细胞信号，目前还不是很清楚。一 动物发育中的关键信号元件是形态原信号。骨形态发生蛋白(BMP) 在脊椎动物发育过程中，BMP在背腹(DV)轴上起到形态形成的作用，其中BMP 浓度梯度与不同的细胞特性相关。为了研究祖细胞在其中的机制 解读BMP渐变，我期待斑马鱼的发育。斑马鱼在胚胎和胚胎期间是透明的 幼虫阶段，便于用高分辨率显微镜观察细胞命运的变化和迁移模式。斑马鱼份额 它们70%以上的基因与人类有关，使它们成为人类发育和 疾病研究。使用斑马鱼胚胎，我将操纵BMP浓度、祖细胞位置和 BMP信号在中胚层祖细胞内的持续时间，并决定形态发生的结果。在目标1中 本项目，我将研究BMP信号协调其迁移和细胞命运基因的机制 导致其特有的形态发生输出的表达。利用DV BMP形态发生信号，细胞 可能位于微环境影响二元命运开关的胚胎区域。回答 我的问题是，我将使用转基因胚胎有条件地过度表达异位BMP信号。移栽 在原肠发育阶段之前将转基因细胞转化为野生型(WT)胚胎将允许进行转基因分析 WT环境中的细胞命运决定。我将分析发育胚胎的时间流逝，其中包含 这些转基因细胞使用旋转圆盘共聚焦显微镜。细胞命运将使用In进行遗传评估 原肠前、中、后的原位杂交和命运特异性荧光探针。在《目标2》中，我将成为 确定BMP信号持续时间对细胞命运决定的作用。我将利用生长素诱导 Degron(AID)系统创建特定持续时间的BMP信号和BMP靶基因表达，以便 确定信号持续时间如何影响命运。因为直接的BMP靶蛋白抑制转录 细胞命运特异的转录因子，我假设BMP信号的持续时间会改变 该靶基因存在的时间长度，允许细胞命运基因在正常情况下不结合 因此可以改变细胞的命运。结合使用细胞自主报告鱼线，移植， 基因表达分析、利用AID系统的转基因FISH和旋转圆盘共聚焦显微镜将 请允许我测试一下我的假设。

Project Summary/Abstract Animal development consists of complex cell coordination and rearrangement via intercellular communications. It is not well understood how progenitor cells interpret neighbor cell signals that impact cell fate decisions. One critical signaling element in animal development is morphogen signaling. Bone morphogenetic protein (BMP) acts as a morphogen to pattern the dorsoventral (DV) axis during vertebrate development, where BMP concentration gradients correlate with different cell identities. To study the mechanism in which progenitors interpret BMP gradients, I look to zebrafish development. Zebrafish are transparent during embryonic and larval stages, facilitating high resolution microscopy of cell fate change and migration patterns. Zebrafish share more than 70% of their genes with humans, making them excellent models for human development and disease research. Using zebrafish embryos, I will manipulate BMP concentration, progenitor cell location, and BMP signal duration within mesodermal progenitors and determine the morphogenetic outcome. In Aim 1 of this project, I will investigate the mechanism in which BMP signal coordinates its migration and cell fate gene expression that results in its characteristic morphogenetic output. With DV BMP morphogenetic signaling, cells may be positioned in embryonic domains where the microenvironment affects a binary fate switch. To answer my question, I will be using transgenic embryos to conditionally overexpress ectopic BMP signal. Transplanting transgenic cells into wildtype (WT) embryos prior to gastrulation stages will allow for the analysis of transgenic cell fate decisions within the WT environment. I will analyze time lapses of developing embryos containing these transgenic cells using spinning disk confocal microscopy. Cell fates will be genetically assessed using in situ hybridization and fate-specific fluorescent probes before, during and after gastrulation. In Aim 2, I will be determining the role that BMP signal duration has on cell fate decisions. I will be utilizing the Auxin Inducible Degron (AID) system to create specific durations of BMP signaling and BMP target gene expression in order to determine how signal duration impacts fate. Because a direct BMP target protein inhibits the transcription of cell fate specific transcription factors, I hypothesize that the duration of BMP signaling would change the amount of time that this target gene is present, allowing cell fate genes to bind when they normally would not be able to, thus altering cell fates. The combined use of cell autonomous reporter fish lines, transplantation, gene expression analysis, transgenic fish utilizing the AID system, and spinning disk confocal microscopy will allow me to test my hypotheses.