Decoding neural cell fate diversity

项目摘要/摘要 细胞命运多样化是产生许多神经元亚型所需的关键一步 脊椎动物大脑中的功能回路。我们知道相当多关于神经性 祖细胞使命运多样化，形成不同的子代神经元。然而，我们知道的要少得多。 关于后来改变有丝分裂后神经元命运的过程。其中一个原因是 知识差距是在大多数脊椎动物模型中，不可能识别和研究 在不同的个体中，完全相同的神经元，限制了预测和统计能力。我们 通过研究两个相邻的、可单独识别的、 脊髓运动神经元。我们发现，这些神经元最初是等价的，而且 它们之间的相互作用，除了与一组已识别的肌肉纤维相互作用外， 打破了两个神经元之间的等价性，导致它们采用不同的命运。 此外，这些神经元中的一个通常会死亡，这是塑造大脑的重要命运 架构和电路。在脊椎动物中，这是一种独特的情况，我们可以在其中观察命运 在活胚胎中发生的多样性，并预测神经元将在很久以前死亡 这个过程发生了，但我们仍然不知道潜在的分子事件。我们会克服的 这一屏障使用各种工具，使我们能够操纵这两个神经元的命运 以及发育过程中特定阶段的单细胞RNA测序。这 两者的结合使我们能够发现参与神经细胞命运多样化的基因 以及预测神经细胞死亡和存活的基因。我们计划验证这些基因 采用定量RNA原位杂交技术。然后我们将测试经过验证的候选人 使用创新的F0 CRISPR屏幕。我们提出的研究将揭示以前的基因 在神经细胞命运多样化、存活和死亡过程中功能未知。如果成功， 他们将史无前例地揭示神经细胞命运多样化的遗传机制 精确度，从而将开辟新的调查途径，并加深我们对 神经发育机制。

PROJECT SUMMARY/ABSTRACT Cell fate diversification is a critical step in producing the many neuronal subtypes required for functional circuitry in the vertebrate brain. We know a considerable amount about how neural progenitors diversify fates to form distinct daughter neurons. However, much less is known about later processes that diversity the fates of postmitotic neurons. One reason for this knowledge gap is that in most vertebrate models it is impossible to recognize and study precisely the same neurons in separate individuals, limiting predictive and statistical power. We can overcome these limitations in zebrafish by studying two adjacent, individually identifiable, spinal motoneurons. We discovered that these neurons are initially equivalent, and that interactions between them, in addition to interactions with an identified set of muscle fibers, breaks the equivalence between the two neurons and causes them to adopt distinct fates. Moreover, one of these neurons then typically dies, an important fate for sculpting brain architecture and circuitry. This is a unique situation in vertebrates, in which we can observe fate diversification as it is occurring in living embryos and predict that a neuron will die well before the process occurs, yet we still do not know the underlying molecular events. We will overcome this barrier using a variety of tools that enable us to manipulate the fates of these two neurons and single cell RNA sequencing at defined stages during the developmental process. This combination with enable us to discover genes involved in neuronal cell fate diversification as well as genes that predict neuronal cell death and survival. We plan to validate these genes using quantitative RNA in situ hybridization techniques. We will then test validated candidates using an innovative F0 CRISPR screen. Our proposed studies will reveal genes previously unknown to function during neuronal cell fate diversification, survival, and death. If successful, they will uncover genetic mechanisms of neuronal cell fate diversification with unprecedented precision, and thus will open new avenues of inquiry and deepen our understanding of neurodevelopmental mechanisms.