Transcriptional Control of Retinal Bipolar Neuron Diversification

描述(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的水平上。脊椎动物视网膜由60多个可分类的亚型组成，长期以来一直被用作研究从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。它们出生在小鼠出生后的时期，因此是唯一可以通过电穿孔和逆转录病毒感染进行扰动的种群。视网膜双极神经元由大约12个亚型代表，在免疫组织化学特性、形态和电生理学方面具有不同程度的特征。通过详细研究单个子类型的规范，可以理解这些子类型相互区别的机制。2型锥体双极神经元(CBP2s)是一种很好的候选细胞，值得进一步研究。它由几个独特的标记和转录因子Bhlhb5的表达所定义，该转录因子在其他双极神经元中不表达，并在分化后保持不变。Bhlhb5基因敲除小鼠缺乏CBP2s，但该基因在双极亚型确定中的作用尚不清楚。Bhlhb5是决定脊髓中间神经元亚型的中心因子。这项建议的目的是通过关注Bhlhb5的作用来探索定义单一双极亚型CBP2的基因调控网络。目的如下：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改变双极前体命运的能力。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因素，用于基于双相神经元中光激活通道的表达进行基因治疗。此外，已发现的细胞命运调控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.