The Molecular Genetics of Early Neurogenesis

描述(由申请人提供)：在空间和时间上将基因表达定向到特定细胞类型的能力是再生医学的一个重要目标。例如，为了治疗背侧脊髓损伤，人们可能希望在局部诱导信号的控制下，如骨形态发生蛋白(BMPs)，引导分化的干细胞承担感觉或神经峰的命运。BMPS在神经诱导过程中发挥着高度保守的作用，建立背腹(DV)轴，区分表皮和中枢神经系统的细胞命运。随后，BMP决定了脊索背侧区域的细胞命运，在那里它们通过高度保守的效应基因发挥作用。在果蝇和脊椎动物中，中枢神经系统的背部细胞沿着产生BMP的表皮的边缘形成，并表达Msx1转录因子(在果蝇中为MSH)，而其他转录因子Pax6和GSH(在果蝇中为ind)和Nkx2.2(在果蝇中为vn)分别在中枢神经系统的外侧和腹侧区域表达。这些保守的“神经同一性”基因决定了它们在其中表达的细胞的命运，但在果蝇和脊椎动物中，BMPs可能会以不同的方式进行调控。因此，在果蝇中，遗传数据表明，BMP在神经诱导过程中以剂量依赖的方式抑制ind和MSH的表达。然而，在脊椎动物中，BMP被认为可以积极地调节Msx1等基因。分析BMP依赖的神经识别基因顺式调控具有广泛的进化意义，并有助于开发设计顺式调节模块(CRM)，以针对脊髓特定区域的神经元分化。在目前的资助中，我们建议开展一项CRM控制果蝇和脊椎动物神经识别基因BMP反应性表达的比较机制研究。在目标1中，我们将研究BMP介导的MSH表达抑制的顺式调控基础，与IND的顺式调控基础进行比较，IND被BMP更强烈地抑制。使用我们开发的尖端成像和定量方法，在单细胞分辨率下精确测量基因表达水平，我们还将研究IND和MSH表达结构域分解为相互排斥的相邻区域的机制。在目标2中，我们将鉴定和分析驱动斑马鱼胚胎神经板/神经管中神经识别基因差异表达的脊椎动物CRM。与Shannon Fisher的小组(Univ.Penn)，我们最近发现了斑马鱼MsxB和小鼠Msx1 CRM，它们准确地驱动报告基因在背部中枢的表达。我们将首先定义在背侧中枢神经系统中驱动MSX基因表达的最小CRM序列，然后在这些最小CRM中识别BMP响应序列并对其进行突变。在这样的CRM突变体中，我们将询问报告基因的表达是否丢失(即BMP正向调节CRM活性)或扩展到邻近的表皮结构域(即BMP抑制CRM活性)。我们将遵循类似的策略来识别和表征驱动侧向(Pax6)和腹向(Nkx2.2)表达的CRM。基因。

DESCRIPTION (provided by applicant): The ability to direct gene expression to specific cell types in space and time is an important goal for regenerative medicine. For example, to treat dorsal spinal chord injuries one may wish to direct differentiating stem cells to assume sensory or neural crest fates under the control of local inductive signals such as Bone morphogenetic proteins (BMPs). BMPs play a highly conserved role during neural induction to establish the dorsal-ventral (DV) axis and to distinguish epidermal from central nervous system cell fates. Subsequently, BMPs determine cell fates within dorsal regions of the spinal chord, where they act via highly conserved effector genes. In Drosophila and vertebrates alike, dorsal cells of the CNS form along the border of the BMP producing epidermis and express the Msx1 transcription factor (msh in flies), while other transcription factors Pax6 and Gsh (ind in flies) and Nkx2.2 (vn in flies) are expressed respectively in lateral and ventral domains of the CNS. These conserved "neural identity" genes determine the fates of cells in which they are expressed, but may be regulated differently by BMPs in flies and vertebrates. Thus, in flies, genetic data indicate that BMPs act as they do during neural induction to repress expression ind and msh in a dose-dependent fashion. In vertebrates, however, BMPs have been proposed to positively regulate genes such as Msx1. Analysis of BMP-dependent cis-regulation of neural identity genes has broad evolutionary implications and should aid in the development of designer cis-regulator modules (CRMs) to target neuronal differentiation to specific regions of the spinal chord. In the current grant, we propose to carry out a comparative mechanistic study of CRMs controlling BMP-responsive expression of neural identity genes in Drosophila and vertebrates. In Aim 1, we will examine the cis-regulatory basis for BMP-mediated repression of msh expression as compared to that of ind, which is more strongly repressed by BMPs. Using cutting edge imaging and quantitative methods we have developed for precisely measuring gene expression levels at single-cell resolution, we will also examine the mechanism by which the ind and msh expression domains resolve into mutually exclusive adjacent territories. In Aim 2, we will identify and analyze vertebrate CRMs driving differential expression of neural identity genes in the neural plate/tube of zebrafish embryos. In collaboration with Shannon Fisher's group (Univ. Penn), we have recently identified zebrafish msxB and mouse Msx1 CRMs that accurately drive reporter gene expression in the dorsal CNS. We will first define minimal CRM sequences driving expression of Msx genes in the dorsal CNS and then identify BMP-responsive sequences in these minimal CRMs and mutate them. In such CRM mutants we will then ask whether reporter gene expression is lost (i.e., BMPs positively regulate CRM activity) or is expanded into the adjacent epidermal domain (i.e., BMPs repress CRM activity). We will follow a similar strategy to identify and characterize CRMs driving expression of laterally (Pax6) and ventrally (Nkx2.2.) genes.