Input-output decoding of oscillatory gene expression during neurogenesis

在2-5小时的短时间内，基因表达振荡对于细胞-状态转换具有重要的功能，但下游靶标和解码机制尚不清楚。在这里，我们将使用内源性蛋白的单细胞实时成像的创新组合-融合-记者与操纵动力学的力量来解决这些问题。我们将关注转录因子Neurogenin2，Ngn2及其下游靶标NeuroD的动态。我们的模型系统将是体外神经分化，采用成熟的已发表的人类胚胎干细胞(HESCs)系的方案，沿着脊髓腹侧神经通路，用于活体成像和分子研究。在第一个目标中，我们将询问Ngn2振荡是如何在神经D的动力学中产生和解释的。由于Ngn2表现出动态表达，在第二个目的中，我们将操纵Ngn2的动态，并询问哪些基因被振荡和持续表达的Ngn2差异激活。我们的研究将剖析导致细胞状态转变的分子事件序列，并在这样做的过程中提供对基本生物过程的机械性洞察，这是分化细胞转变的先驱，是发育、再生和癌症的基础。

Gene expression oscillations in a short time scale of 2-5hrs are functionally important for cell-state transitions but the downstream targets and the decoding mechanisms are not understood. Here, we will use innovative combination of single cell live imaging of endogenous protein-fusion-reporters with the power of manipulating dynamics to address these questions. We will focus on the dynamics of a transcription factor Neurogenin2, Ngn2 and its downstream target, NeuroD. Our model system will be in vitro neural differentiation with well-established published protocols of established lines of human Embryonic Stem Cells (hESCs) , along a ventral spinal cord neuronal pathway, for live imaging and molecular studies. In the first aim we will ask how Ngn2 oscillations generated and interpreted in the dynamics of NeuroD. Since Ngn2 shows dynamical expression, in the second aim, we will manipulate the Ngn2 dynamics and ask what genes are differentially activated by oscillatory versus sustained Ngn2 expression. Our research will dissect the sequence of molecular events that lead to cell-state transitions and in doing so will provide mechanistic insight into a fundamental biological process, the progenitor to differentiated cell transition, that underlies development, regeneration and cancer.