NEURON DIFFERENTIATION IN C ELEGANS

描述（来自申请人的摘要）：本申请检查如何指定神经元以表达定义的一组差异化特征。 埃蒙斯博士一直在研究C线虫雄性尾巴的形态发生。优雅 雄尾由9对形态上不同的鳍条组成，呈无细胞扇形。 每条射线的3个细胞、2个神经元和1个结构细胞均来源于射线前体细胞Rn。通过表征具有异常尾部形态的突变体，已经鉴定了负责尾部形态发生的几个基因。 射线亚系的重复表达依赖于lin-32的表达，lin-32是一种无毛/盾叶家族的bHLH转录因子。 射线之间的差异取决于C. elegans Hox簇，mAb-5，egl-5和Pax-6。 这些转录因子的表达可能受到细胞外因子的调控。 参与射线5、7和9形成的一种细胞外因子是dbl-1，其编码BMP 2同源物。 Dbl-1可分别通过SMA-6和SMA-4（I型和II型BMP受体）以及SMA-2、SMA-3和SMA-4的产物（SMAD蛋白）起作用。神经元的分化特征之一是其递质补体的表达。 Emmons博士建议专注于神经递质表型是如何指定的，并将检查多巴胺（DA）在男性尾巴的5，7和9条射线中的三对双边感觉神经元中的指定。 在dbl-1、sma-3、egl-5和mab-21功能丧失背景下检查携带TH：：GFP报告基因构建体的转基因动物。这些动物在其他射线中异位表达GFP，表明DA的表达不依赖于BMP，但DA模式依赖于BMP。通过hs：：dbl-1转基因过表达DBL-1蛋白质导致在射线3-9中GFP表达，但在射线1或2中从不表达。 这些数据表明，DBL-1可能提供有益的线索。 由于在射线1或2中没有看到表达，因此这些射线可能不能对DBL-1信号做出响应。 研究者提出了模型，DA规格是基于三个图案化过程：图案化的表达的配体（DBL-1），预图案化的细胞能力的egl-5和其他一些未知的基因，和侧抑制。 本申请的目的是：（1）检测DBL-1、Hox基因和其他基因在诱导DA表型中的作用。 TH：：将在BMP和Hox基因突变的动物中检查GFP表达。这将确定是否所有细胞都具有同等的能力来呈现DA表型，以区分射线细胞是否被预图案化以响应BMP或BMP是否被空间图案化，并确定Hox基因是否在建立细胞响应BMP信号的能力中起作用。 还将确定是否存在限制DA表达的抑制途径。 将检查编码新蛋白质的mAb-21中具有突变的动物的TH：：GFP表达;还将消融野生型动物的不同射线前体细胞。 (2)确定共同的顺式调控元件是否存在于参与DA合成的基因中。启动子区所需的DA表达射线将划定和检查保守的图案。 (3)筛选TH：：GFP表达缺失的突变体（包括ts等位基因），并从分子上表征相应的基因。 (4)确定参与DA神经元轴突连接的基因。

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.