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，人们可能希望引导分化中的干细胞在局部诱导信号如骨形态发生蛋白（BMP）的控制下呈现感觉或神经嵴命运。BMP在神经诱导过程中发挥高度保守的作用，以建立背腹（DV）轴和区分表皮和中枢神经系统细胞的命运。随后，BMP决定脊髓背侧区域内的细胞命运，在那里它们通过高度保守的效应基因起作用。在果蝇和脊椎动物中，CNS的背侧细胞沿着产生BMP的表皮的边界形成，并表达Msx 1转录因子（苍蝇中的msh），而其他转录因子Pax 6和Gsh（苍蝇中的ind）和Nkx 2.2（苍蝇中的vn）分别在CNS的侧部和腹部区域中表达。这些保守的“神经身份”基因决定了它们表达的细胞的命运，但在苍蝇和脊椎动物中可能受到BMP的不同调节。因此，在苍蝇中，遗传数据表明，骨形态发生蛋白的作用，因为他们在神经诱导抑制表达ind和msh在剂量依赖性的方式。然而，在脊椎动物中，BMPs已被提出积极调节基因，如Msx 1。BMP依赖的顺式调节神经身份基因的分析具有广泛的进化意义，并应有助于设计师顺式调节模块（CRM）的发展，以靶向神经元分化的特定区域的脊髓。 在目前的补助金，我们建议进行比较机制研究的标准物质控制骨形态发生蛋白反应表达的神经身份基因在果蝇和脊椎动物。在目的1中，我们将研究BMP介导的抑制msh表达的顺式调节基础相比，ind，这是更强烈的抑制BMP。利用我们已经开发的用于精确测量单细胞分辨率的基因表达水平的尖端成像和定量方法，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/管中神经身份基因差异表达的脊椎动物CRMs。在与香农费舍尔的小组（宾夕法尼亚大学）合作，我们最近确定了斑马鱼msxB和小鼠Msx 1 CRM，准确地驱动报告基因在背侧中枢神经系统的表达。我们将首先定义最小CRM序列驱动Msx基因在背侧CNS中的表达，然后在这些最小CRM中鉴定BMP响应序列并使其突变。在这样的CRM突变体中，我们将询问报告基因表达是否丢失（即，BMP正调节CRM活性）或扩展到邻近的表皮结构域（即，BMP抑制CRM活动）。我们将遵循类似的策略来识别和表征驱动横向（Pax 6）和腹侧（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.