Defining the mechanisms of Hox gene repression during motor neuron development

描述(申请人提供)：运动是脊椎动物的一种基本行为，运动神经元(MN)通过在中枢神经系统和外周之间建立精确的联系而发挥核心作用。HOX转录因子在确定MNS的细胞类型特异性分子同一性方面是必不可少的，这是通过对HOX基因表达的时空调节来实现的。Polycomb group(PcG)蛋白最初是在果蝇胚胎中发现的，是Hox基因的调节者，其表达决定了身体的结构和附肢的位置，在脊椎动物系统中是保守的。PCG蛋白组装成两个多聚体复合体，Prc1和2，并通过染色质修饰维持基因抑制。然而，PRC1在神经元命运决定中的具体作用还知之甚少。我们最近发现，PRC1组分Bmi1/PCGF4在运动神经元(MN)亚型分化中起重要作用，它通过在有丝分裂后水平上沿脊髓吻尾轴产生PRC1活性梯度和确定MN柱中Hox基因表达的嘴边界而发挥重要作用。然而，PRC1的具体作用机制仍未得到解答。这一建议试图回答以下问题：(1)PRC1-Bmi1/PCGF4复合体的哪些亚基参与了PRC1的活性梯度？(2)额外的含有PCGF1、2、3、5或6的PRC1复合体在MN发育过程中是否在控制Hox表达方面发挥了作用？为了解决这些问题，我将首先通过原位杂交和免疫荧光来确定Prc1亚基的表达谱，并通过染色质免疫沉淀来确定它们在柱状定义-Hox基因座的定位。此外，我还将通过体内电穿孔的方法在鸡胚胎发育过程中耗尽和过度表达Prc1复合体，从而在建立Hox表达模式中发挥作用。本项目的总体目标是(1)剖析在发育中的脊髓中保持柱状定义的HOX基因表达的机制，(2)以MN规范作为模型系统，扩大我们对多梳状抑制在发育中的胚胎的功能和机制的总体理解。

DESCRIPTION (provided by applicant): Locomotion is a fundamental behavior in vertebrates, in which motor neurons (MNs) play a central role by establishing precise connections between the central nervous system and the periphery. Hox transcription factors are essential in defining the cell type specific molecular identity of MNs, which is achieved through temporal and spatial regulation of Hox gene expression. Polycomb group (PcG) proteins were initially discovered in Drosophila embryo as regulators of Hox genes, whose expression defines body structures and appendage position, and are conserved in the vertebrate system. PcG proteins assemble into two multimeric complexes, PRC1 and 2, and maintain gene repression through chromatin modifications. However, specific roles of PRC1 in neuronal fate determination are poorly understood. We have recently found that a PRC1 component, Bmi1/PCGF4, plays an essential role in motor neuron (MN) subtype differentiation, by creating gradient of PRC1 activity along rostrocaudal axis of spinal cord and defining the rostral boundary of Hox gene expression in MN columns at the post-mitotic level. However, the specific mechanisms of PRC1 action remain unanswered. This proposal attempts to answer the following questions: (1) Which subunits of PRC1- Bmi1/PCGF4 complex contribute to the PRC1 activity gradient? (2) Do additional PRC1 complexes containing PCGF1,2,3,5 or 6 play any role in controlling Hox expression during MN development? To address these questions I will first define the expression profiles of PRC1 subunits by in situ hybridization and immunofluorescence and determine their localization to column defining-Hox loci by chromatin immunoprecipitation. In addition I will address the function of PRC1 complexes in establishing Hox expression pattern by depleting and overexpressing its components in developing chick embryo by in ovo electroporation. The overall goal of this project is (1) to dissect the mechanism in which columnar-defining Hox gene expression is maintained in the developing spinal cord, and (2) to extend our general understanding of the functions and mechanisms of Polycomb repression in the developing embryo, using MN specification as a model system.