REGULATION OF CELL PHENOTYPE IN THE DEVELOPING NEOCORTEX

发育中新皮质细胞表型的调节

基本信息

批准号：
6243577
负责人：
Susan Hockfield
金额：
$ 16.31万
依托单位：
YALE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
1997
资助国家：
美国
起止时间：
1997-03-01 至 1998-02-28
项目状态：
已结题

项目摘要

During neural development, a population of morphologically homogenous, mitotically active precursor cells gives rise to all the neuronal and glial cells of the adult cerebral cortex. Postmitotic neurons extend processes by which they migrate to their adult positions and begin the elaboration of axonal and dendritic arbors. To understand the molecular events that permit a cell to assume neuronal properties, we previously used 2- dimensional gel electrophoresis to identify 15 proteins that are differentially regulated over the course of corticogenesis. One of these, TOAD-64, is a 64,000 dalton protein that is up regulated nearly 7-fold over the course of late embryonic cortical development and is subsequently down regulated during postnatal life. The sequence of a full length cDNA of TOAD-64 is homologous to the unc-33 gene from C. elegans, mutations in which lead to defective patterns of axon outgrowth. Several lines of evidence suggest that TOAD-64 may, similarly, play a role in axonogenesis in the rodent. First, TOAD-64 is re-expressed in adult motor neurons as they extend axons following a peripheral nerve lesion. Second, TOAD-64 expression increases coincident with neuritogenesis in two cell lines, pC12 and p19. Finally, TOAD-64 protein is present at the most peripheral portions of the growth cone, in lamellipodia and filopodia. Together, these data support the possibility that TOAD-64 is part of the intracellular machinery by which growing neurons elaborate axons. The experiments proposed here are designed to further characterize TOAD-64 and will also address the possible functions of this new protein in neurite outgrowth. The first aim is to further characterize TOAD-64. A possible role for TOAD-64 in neuronal remodeling in the adult will be investigated. A new panel of antibodies to primate TOAD-64 will be generated to permit studies of early corticogenesis in the monkey. For studies of the mechanisms by which the TOAD-64 gene is regulated, a genomic clone will be isolated for future experiments to identify its regulatory sequences. The second aim is to determine the function of TOAD-64. We will explore the role TOAD-64 may play in axonogenesis by changing levels of protein expression in primary neuronal cultures and in two cell lines. To test whether TOAD-64 is required for a cell to elaborate neurites, antisense oligonucleotides will be used to reduce TOAD-64 expression in neurons in primary culture. We will also determine whether a reduction in TOAD-64 can alter the response of growth cones and neurites to repulsive substrates. To determine whether TOAD-64 is critical for the elaboration of neurites in PC12 and P19 cells, expression of TOAD-64 will be reduced or increased using stable transfection.

在神经发育期间，形态学同质的人群，有丝分裂活性前体细胞产生所有神经元和神经胶质成年大脑皮层的细胞。蒙辛后神经元扩展过程他们迁移到成人职位并开始阐述轴突和树突状乔木。了解分子事件允许细胞假设神经元特性，我们先前使用了2- 尺寸凝胶电泳可鉴定15种蛋白在整个皮质生成过程中受到差异调节。其中之一 Toad-64是64,000个道尔顿蛋白，受到近7倍的调节后期胚胎皮质发育的过程，随后下降在产后生活期间受到监管。全长cDNA的序列 Toad-64与秀丽隐杆线虫的UNC-33基因同源，突变这导致轴突生长的缺陷模式。几行有证据表明，TOAD-64可能同样可以在轴突发生中发挥作用在啮齿动物中。首先，TOAD-64在成人运动神经元中重新表达它们在周围神经病变后延伸轴突。第二，Toad-64 表达与两种细胞系中的神经发生相吻合，PC12 和P19。最后，TOAD-64蛋白在最外围存在生长锥的一部分，薄片和丝状。一起，这些数据支持TOAD-64是细胞内机械通过，生长神经元精心轴突。这此处提出的实验旨在进一步表征Toad-64和还将解决该新蛋白在神经突的可能功能出生。第一个目的是进一步描述Toad-64。可能的角色将研究成人的神经元重塑中的Toad-64。一个新将生成针对灵长类动物TOAD-64的抗体面板以允许研究猴子早期的皮质生成。用于研究机制蟾蜍64基因的调节，将分离出基因组克隆未来的实验以识别其调节序列。第二个目的是确定蟾蜍64的功能。我们将探索 Toad-64的角色可能通过改变蛋白质水平来在轴突发生中起作用原代神经元培养物和两个细胞系中的表达。测试细胞是否需要toad-64才能精致的神经突，反义寡核苷酸将用于降低神经元中的TOAD-64表达初级文化。我们还将确定减少Toad-64是否可以改变生长锥和神经突对排斥底物的反应。确定Toad-64是否对神经突的阐述至关重要 PC12和P19细胞，TOAD-64的表达将降低或增加使用稳定的转染。