In vivo cell signaling dynamics in spinal cord patterning

多细胞生物的发展依赖于广泛的细胞间通讯，这种通讯是由少量保守的细胞信号通路介导的。过去30年的研究揭示了不同的信号成分和信号策略。然而，关于细胞信号的经典工作要么是在活体外的组织培养中进行的，要么是不得不依赖于解释静态发育时间点的事件(快照)。这些方法未能捕捉到发育的高度动态本质，在这种情况下，细胞与许多其他细胞相互作用，并随着时间的推移遇到各种信号分子，特别是因为组织形态发生往往会导致细胞的广泛运动。因此，开发体内系统以了解细胞如何正确整合多种信号，然后在发育中的组织中产生特定和适当的细胞反应是至关重要的。 在我之前发表的工作中，我已经使用斑马鱼脊髓作为模型系统来解决细胞信号动力学在发育中的重要性。斑马鱼胚胎的可及性和光学半透明性使得活体动物的细胞动力学和细胞-细胞相互作用的高分辨率实时成像成为可能。我已经证明，Notch和Hedgehog(HH)信号的动态部署是动物发育所需的两个关键信号通路，控制着神经前体细胞的更新和分化。我还开发了强大的活体技术，使我们能够可视化和操纵活动物的细胞信号动力学。在我们提出的研究计划中，我们将利用体内工具来剖析细胞信号动力学是如何在分子和细胞水平上调节的。在目标一中，我们将确定HH信号是如何在神经元分化的分子水平上终止的。在第二个目标中，我们将定位活胚胎中Notch信号的动态变化。我们研究计划的长期目标是阐明细胞信号动力学是如何以精确和刻板的方式调节的，以实现在活体动物中可复制的模式形成。

Development of multicellular organisms relies on extensive cell-to-cell communication, which is mediated by a small number of conserved cell signaling pathways. Studies in the past 30 years have revealed distinct signaling components and signaling strategies. However, classical work on cell signaling has been either carried out in tissue culture outside of living organisms or has had to rely on interpreting events at static developmental time-points (snapshots). These approaches fail to capture the highly dynamic nature of development, where cells interact with many other cells and encounter a variety of signaling molecules over time, especially since tissue morphogenesis often results in extensive cell movements. It is therefore essential to develop in vivo systems to understand how cells can correctly integrate multiple signals and then produce a specific and appropriate cellular response in developing tissues. In my previous published work, I have used the zebrafish spinal cord as a model system to address the importance of cell signaling dynamics in development. Accessibility and optical translucency of zebrafish embryos allows high resolution in vivo real-time imaging of cellular dynamics and cell-cell interactions in live animals. I have demonstrated that the dynamic deployment of both Notch and Hedgehog (Hh) signaling, two key signaling pathways required for animal development, controls the renewal and differentiation of neural progenitors. I have also developed powerful in vivo techniques that allow us to visualize and manipulate cell signaling dynamics in live animals. In our proposed research program, we will utilize in vivo tools to dissect how cell signaling dynamics is regulated at both the molecular and cellular level. In Objective One, we will determine how Hh signaling is terminated at the molecular level upon neuronal differentiation. In Objective Two, we will map the dynamics of Notch signaling in live embryos. The long-term goal of our research program is to elucidate how cell signaling dynamics is regulated in a precise and stereotypic manner to achieve reproducible pattern formation within a living animal.