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引导到特定细胞类型的能力是再生医学的一个重要目标。例如，为了治疗脊髓背部损伤，人们可能希望引导分化干细胞在局部诱导信号（如骨形态发生蛋白（BMPs））的控制下承担感觉或神经嵴的命运。bmp在神经诱导建立背腹（DV）轴和区分表皮和中枢神经系统细胞命运的过程中发挥高度保守的作用。随后，bmp通过高度保守的效应基因决定脊髓背侧区域的细胞命运。在果蝇和脊椎动物中，中枢神经系统的背侧细胞沿产生BMP的表皮边缘形成，并表达Msx1转录因子（果蝇中为msh），而其他转录因子Pax6和Gsh（果蝇中为ind）以及Nkx2.2（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“神经身份”基因决定了它们在细胞中表达的命运，但在果蝇和脊椎动物中，它们可能受到bmp的不同调节。因此，在果蝇中，遗传数据表明bmp的作用就像它们在神经诱导过程中一样，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被认为可以积极调节Msx1等基因。分析bmp依赖的神经识别基因的顺式调控具有广泛的进化意义，并有助于设计顺式调控模块（crm），以将神经元分化靶定在脊髓的特定区域。在目前的拨款中，我们建议在果蝇和脊椎动物中开展一项CRMs控制bmp应答性神经身份基因表达的比较机制研究。在目的1中，我们将研究bmp介导的msh表达抑制的顺式调控基础，而ind则被bmp更强烈地抑制。利用我们开发的用于在单细胞分辨率下精确测量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表达域分解成相互排斥的相邻区域的机制。在Aim 2中，我们将鉴定和分析驱动斑马鱼胚胎神经板/管中神经识别基因差异表达的脊椎动物crm。通过与宾夕法尼亚大学的Shannon Fisher小组的合作，我们最近发现了斑马鱼的msxB和小鼠的Msx1基因，它们可以准确地驱动背中枢神经系统中报告基因的表达。我们将首先定义驱动背中枢神经系统中Msx基因表达的最小CRM序列，然后在这些最小CRM中鉴定bmp应答序列并使其突变。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，BMPs积极调节CRM活性）或扩展到邻近的表皮域（即，BMPs抑制CRM活性）。我们将采用类似的策略来识别和表征驱动侧侧（Pax6）和腹侧（Nkx2.2）基因表达的crm。

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.