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导向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（例如骨形态发生蛋白（BMP））的控制下引导分化干细胞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) 轴并区分表皮细胞和中枢神经系统细胞的命运。随后，BMP 决定脊髓背侧区域的细胞命运，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，中枢神经系统的背细胞沿着产生 BMP 的表皮边缘形成，并表达 Msx1 转录因子（果蝇中的 msh），而其他转录因子 Pax6 和 Gsh（果蝇中的 ind）和 Nkx2.2（果蝇中的 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”基因决定了它们表达的细胞的命运，但在果蝇和脊椎动物中可能受到 BMP 的不同调节。因此，在果蝇中，遗传数据表明 BMP 在神经诱导过程中发挥作用，以剂量依赖性方式抑制 ind 和 msh 的表达。然而，在脊椎动物中，BMP 被认为可以正向调节 Msx1 等基因。对神经同一基因的 BMP 依赖性顺式调节的分析具有广泛的进化意义，并且应该有助于开发设计顺式调节器模块 (CRM)，以将神经元分化定向到脊髓的特定区域。 在目前的资助中，我们建议对果蝇和脊椎动物中控制 BMP 反应性神经识别基因表达的 CRM 进行比较机制研究。在目标 1 中，我们将检查 BMP 介导的 msh 表达抑制的顺式调控基础，与 ind 的抑制相比，ind 受到 BMP 的抑制更强烈。使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 表达域解析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。我们与 Shannon Fisher 团队（宾夕法尼亚大学）合作，最近鉴定了斑马鱼 msxB 和小鼠 Msx1 CRM，它们可以准确驱动背侧 CNS 中的报告基因表达。我们将首先定义驱动背侧 CNS 中 Msx 基因表达的最小 CRM 序列，然后识别这些最小 CRM 中的 BMP 响应序列并对它们进行突变。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.