Pou4 Function in an Invertebrate Chordate

转录因子(Tf)是控制基因表达的蛋白质。这个项目使用了一种与我们这样的脊椎动物关系密切的简单动物--海鞘动物--来研究一种名为Pou4的转运蛋白，它对感觉神经元的发育非常重要，包括内耳的毛细胞。以前发现，在海鞘细胞类型中表达海鞘Pou4，通常不会发现这些细胞将这些细胞转化为感觉神经元。同样，强迫在错误的海鞘细胞类型中表达三个小鼠Pou4基因中的任何一个，也会将这些细胞转化为感觉神经元。然而，在小鼠中，在错误的细胞类型中表达Pou4并不能将这些细胞转化为感觉神经元。本项目研究海鞘和小鼠Pou4基因差异的机制基础，以更好地了解发育过程中导致新细胞类型(如脊椎动物感觉神经元)的进化变化。为了回答这些问题，研究人员正在调整一种新的基因编辑系统(CRISPR/Cas9)来破译Pou4功能背后的机制。更广泛的影响：圣地亚哥州立大学在经济和校园种族多样性方面跻身美国顶尖大学之列。拟议的研究将为本科生和研究生提供实验室研究机会。为了向南加州大学更多的本科生拓展转基因生物的功能和实验应用的整体理解，研究人员正在与他们的细胞和分子生物学入门课程的协调员合作，开发一种相对简单的“动手”练习，学生们可以在其中生成和分析转基因海鞘胚胎。在海鞘动物中，单一的Pou4(CiPou4)转录因子(Tf)是PNS的关键规则，该基因的异位表达产生了异位感觉神经元。在哺乳动物中，有三个Pou4；它们都主要在感觉神经元中表达。研究人员使用RNA-SEQ来确定CiPou4的转录靶点，其中许多已知在脊椎动物毛细胞中表达，这表明这些细胞类型之间存在深刻的进化联系。在哺乳动物中，毛细胞Pou4F3基因的异位表达不会产生新的毛细胞；但当在海鞘中表达时，会产生异位感觉神经元。这一观察表明，虽然蛋白质功能是保守的，但Pou4的转录靶点已经丢失和/或在脊椎动物中受到差异调控。在这里，我们将使用RNA-SEQ来确定与CiPou4相比，小鼠Pou4转录因子是否激活了类似的转录靶点。我们将测试CRISPR/Cas9介导的CiPou4敲除是否可以被小鼠Pou4基因拯救。这个项目具有变革性，因为海鞘幼虫提供了一种独特的系统来研究TF功能的进化，以及这种TF在一种新的脊椎动物细胞类型--感觉毛细胞的进化中所起的作用。

Transcription factors (TF) are proteins that control the expression of genes. This project uses a simple animal that is closely related to vertebrates like ourselves, the ascidian, to study a TF called Pou4 that is important for the development of sensory neurons, which include the hair cells of the inner ear. It was previously found that expressing ascidian Pou4 in ascidian cell types where it is not usually found converts those cells into sensory neurons. Likewise, forced expression of any of the three mouse Pou4 genes in the wrong ascidian cell types also converts those cells into sensory neurons. However, in mice, expressing Pou4 in the wrong cell type does not convert those cells into sensory neurons. The present project examines the mechanistic basis for the differences between the ascidian and mouse Pou4 genes in order to better understand evolutionary changes in development that give rise to new cell types like vertebrate sensory neurons. To answer these questions, the researchers are adapting a new gene editing system (CRISPR/Cas9) to the ascidian to decipher the mechanisms behind Pou4 function. Broader Impacts: San Diego State University ranks among the top US universities for economic and campus ethnic diversity. The proposed study will provide laboratory research opportunities for undergraduate and graduate students. To broaden the overall understanding of the function and experimental use of transgenic organisms to SDSU's larger undergraduate population, the researchers are working with the coordinator of their introductory cell and molecular biology courses to develop a relatively simple, "hands on" exercise where students can generate and assay transgenic ascidian embryos.In ascidians, the single Pou4 (CiPou4) transcription factor (TF) is a key regular of the PNS, and ectopic expression of this gene produces ectopic sensory neurons. In mammals, there are three Pou4s; all predominately expressed in sensory neurons. The investigators used RNA-SEQ to identify CiPou4 transcriptional targets and many are known to be expressed in vertebrate hair cells, suggesting a deep evolutionary linkage between these cells types. In mammals, ectopic expression of the hair cell Pou4F3 gene does not produce new hair cells; but when expressed in ascidians ectopic sensory neurons are generated. This observation suggests that while protein function is conserved, Pou4 transcriptional targets have been lost and/or are differentially regulated in vertebrates. Here we will use RNA-SEQ to determine if the mouse Pou4 TFs activate similar transcriptional targets compared to CiPou4. We will test if a CRISPR/Cas9-mediated CiPou4 knock-out can be rescued by the mouse Pou4 genes. This project is transformative because the ascidian larva provides a unique system to examine the evolution of TF function and the role this TF has played during the evolution of a novel vertebrate cell type, the sensory hair cell.