NEURON DIFFERENTIATION IN C ELEGANS

描述(摘自申请人的摘要)：这项申请研究了神经元是如何被指定来表达一组明确的差异化特征的。埃蒙斯博士一直在研究线虫雄性尾巴的形态发生。雄性尾巴由9对形态截然不同的射线组成，呈无细胞扇形。每条射线的3个细胞、2个神经元和一个结构细胞来自射线前体细胞Rn。通过对具有异常尾部形态的突变体进行特征分析，已经确定了几个与尾部形态发生有关的基因。射线亚系的重复表达依赖于LIN-32的表达，LIN-32是Ahaete/Scute家族的bHLH转录因子。射线之间的差异取决于线虫Hox簇的几个转录因子，MAB-5，EGL-5和Pax-6。这些转录因子的表达可能受细胞外因子的调节。参与射线5、7和9形成的一个细胞外因子是DBL-1，它编码BMP2同源物。DBL-1可能分别通过SMA-6和DAF-4、I型和II型BMP受体以及SMAD蛋白SMA-2、SMA-3和SMA-4的产物发挥作用。神经元的分化特征之一是它们的递质互补的表达。埃蒙斯博士建议将重点放在神经递质表型是如何指定的，并将研究雄性尾巴射线5、7和9的三对双侧感觉神经元中多巴胺(DA)的规格。携带TH：：GFP报告结构的转基因动物在DBL-1、SMA-3、EGL-5和MAb-21的功能丧失背景中进行了检测。这些动物在其他射线中异位表达GFP，这表明DA的表达不依赖于BMP，但DA的模式依赖于BMP。通过hs：：DBL-1转基因过表达的DBL-1蛋白导致了GFP在射线3-9中的表达，但在射线1和射线2中没有表达。这些数据表明DBL-1可能提供了有益的线索。因为在射线1或射线2中看不到表达，所以这些射线可能不能响应DBL-1信号。研究人员提出了DA规范基于三个图案化过程的模型：配基的图案化表达(DBL-1)，EGL-5和其他未知基因对细胞能力的预图案化，以及侧向抑制。本研究的目的是：(1)研究DBL-1、HOX等基因在诱导DA表型中的作用。将在BMP和HOX基因突变的动物身上检测TH：：GFP的表达。这将决定是否所有细胞都具有同等的能力来呈现DA表型，区分射线细胞是否预先构型以响应BMP，或者是否空间构型为BMP，并确定Hox基因是否在建立细胞响应BMP信号的能力方面发挥作用。还将确定是否存在限制DA表达的抑制途径。编码一种新蛋白质的mab-21突变的动物将接受TH：：GFP表达检查；野生型动物也将被切除不同的射线前体细胞。(2)确定参与DA合成的基因中是否存在共同的顺式调控元件。射线中DA表达所需的启动子区域将被划定并检查保守基序。(3)筛选TH：：GFP表达缺失的突变体(包括ts等位基因)，并对相应的基因进行分子特征分析。(4)寻找与DA神经元轴突连接有关的基因。

DESCRIPTION (from applicant's abstract): This application examines how a neuron is specified to express a defined set of differentiated characteristics. Dr. Emmons has been examining the morphogenesis of the male tail in the nematode C. elegans. The male tail is composed of nine morphologically distinct pairs of rays in an acellular fan. The 3 cells of each ray, 2 neurons and a structural cell are derived from a ray precursor cell, Rn. By characterizing mutants with abnormal tail morphologies, several genes responsible for tail morphogenesis have been identified. Repeated expression of the ray sublineage is dependent on expression of lin-32, a bHLH transcription factor of the achaete/scute family. Differences among the rays are dependent on several transcription factors of the C. elegans Hox cluster, mab-5, egl-5, and Pax-6. The expression of these transcription factors may be regulated by extracellular factors. One extracellular factor that is involved in ray 5, 7, and 9 formation is dbl-1, which encodes a BMP2 homolog. Dbl-1 may act through SMA-6 and DAF-4, type I and type II BMP receptors, respectively, and the products of the sma-2, sma-3, and sma-4, which are SMAD proteins. Among the differentiated characteristics of neurons is the expression of their complement of transmitters. Dr. Emmons proposes to focus on how neurotransmitter phenotype is specified, and will examine the specification of dopamine (DA) in three bilateral pairs of sensory neurons in rays 5, 7, and 9 of the male tail. Transgenic animals carrying a TH::GFP reporter construct were examined in dbl-1, sma-3, egl-5, and mab-21 loss of function backgrounds. These animals expressed GFP ectopically in other rays, suggesting that the expression of DA is independent of BMP but DA patterning is dependent on BMP. Overexpression of the DBL-1 protein by a hs::dbl-1 transgene resulted in GFP expression in rays 3-9, but never in rays 1 or 2. These data suggest that DBL-1 may provide instructive cues. Since no expression is seen in rays 1 or 2, these rays may be incompetent to respond to the DBL-1 signal. The investigator proposes the model that DA specification is based on three patterning processes: patterned expression of the ligand (DBL-1), pre-patterning of cell competence by egl-5 and some other unidentified gene, and lateral inhibition. The aims of this application are to: (1) examine the role of DBL-1, Hox genes, and other genes in inducing DA phenotype. TH::GFP expression will be examined in animals with mutations in BMP and Hox genes. This will determine whether all cells have equal competence to take on a DA phenotype, to distinguish between whether ray cells are prepatterned to respond to BMP or whether BMP is spatially patterned, and to determine whether Hox genes play a role in establishing competence to cells to respond to the BMP signal. It will also be determined whether there is an inhibitory pathway restricting DA expression. Animals with mutations in mab-21, which encodes a novel protein, will be examined for TH::GFP expression; wild-type animals will also be ablated for different ray precursor cells. (2) determine whether common cis-regulatory elements are present in genes involved in DA synthesis. Promoter regions necessary for DA expression in rays will be delimited and examined for conserved motifs. (3) screen for mutants (including ts alleles) in which TH::GFP expression is lost and characterize the corresponding genes molecularly. (4) identify genes involved in the axon connectivity of DA neurons.