Transcriptional Control of Retinal Bipolar Neuron Diversification

描述（由申请人提供）：中枢神经系统（CNS）中的大部分神经元多样性存在于神经元亚型水平。脊椎动物视网膜由超过60个可分类的亚型组成，长期以来一直被用作研究多能祖细胞产生不同细胞类型的模型。视网膜双极神经元正在成为研究亚型多样化的一个特别强大的系统。它们出生在小鼠的产后时期，因此是一种独特的易受电穿孔和逆转录病毒感染干扰的人群。视网膜双极神经元由大约12个亚型代表，这些亚型在不同程度上具有免疫组织化学性质、形态和电生理学特征。通过详细研究单个亚型的规范，可以了解这些亚型彼此区分的机制。2型锥体双极神经元（CBP2s）是一个很好的候选进一步研究。它由几个独特的标记物和转录因子Bhlhb5的表达来定义，Bhlhb5在其他双极神经元中不表达，并在分化后维持。Bhlhb5敲除小鼠缺乏CBP2s，但该基因在确定双相亚型中的功能尚未被探索。Bhlhb5是决定脊髓中间神经元亚型的中心因子。本提案的目的是通过关注Bhlhb5的作用，探索定义单一双相亚型CBP2的基因调控网络。我们提出以下目标：1)确定Bhlhb5靶向CBP2中的差异表达基因，2)确定Bhlhb5在维持亚型特异性基因表达中的作用，3)确定Bhlhb5改变双相前体命运的能力。从研究双极神经元多样化中产生的原理可能与整个中枢神经系统中神经元亚型的形成有关。此外，这项工作与人类健康有关，因为在小鼠视网膜中观察到的双极神经元的多样性与在人类视网膜中观察到的情况基本一致。特别是，这里提出的顺式调控分析可以用来产生新的驱动程序，用于基于双极神经元中光激活通道表达的基因治疗。此外，发现的细胞命运调节机制可能会改善干细胞向视网膜组织的定向分化，最终目标是再生功能性视觉回路。

DESCRIPTION (provided by applicant): Much of the neuronal diversity in the central nervous system (CNS) exists at the level of neuronal subtypes. The vertebrate retina is composed of greater than 60 classifiable subtypes, and has long served as a model to study the generation of diverse cell types from multipotent progenitors. Retinal bipolar neurons are emerging as a particularly strong system for studying subtype diversification. They are born in the postnatal period in mouse, and are therefore a uniquely accessible population for perturbation by electroporation and retroviral infection. Retinal bipolar neurons are represented by approximately 12 subtypes characterized to different extents by immunohistochemical properties, morphology, and electrophysiology. An understanding of the mechanisms by which these subtypes are distinguished from one another can emerge from studying the specification of a single subtype in detail. The type 2 cone bipolar neuron (CBP2s) is an excellent candidate for further investigation. It is defined by several unique markers and expression of a transcription factor, Bhlhb5, which is expressed in no other bipolar neurons and is maintained after differentiation. Bhlhb5 knockout mice lack CBP2s, but the function of this gene in bipolar subtype determination is otherwise unexplored. Bhlhb5 is a central factor in determination of interneuron subtypes in the spinal cord. The objective of this proposal is to explore the gene regulatory network that defines a single bipolar subtype, CBP2, by focusing on the role of Bhlhb5. The following aims are proposed: 1) Identify differentially expressed genes in CBP2 targeted by Bhlhb5, 2) Determine the role of Bhlhb5 in maintaining subtype-specific gene expression and 3) Determine the ability of Bhlhb5 to alter the fates of bipolar precursors. The principles that emerge from studying bipolar neuron diversification are likely to have relevance to the formation of neuronal subtypes throughout the CNS. Furthermore, this work has relevance to human health, as the diversity of bipolar neurons observed in the mouse retina is largely consistent with what has been observed in human retina. In particular, cis-regulatory analyses proposed here can be used to generate new drivers for use in gene therapies based on expression of light-activated channels in bipolar neurons. Furthermore, the mechanisms of cell fate regulation discovered may improve directed differentiation of stem cells into retinal tissue, with the ultimate goal of regenerating functional visual circuits.