Dorsal midline patterning in the vertebrate CNS

描述（由申请人提供）：本提案的目标是定义背内侧生发区组织和调控的一般原则，使用Lmx1a作为分子切入点。脑背生发区，如小脑菱形唇区和端脑皮质边缘区，对中枢神经系统的神经元多样性有重要贡献，但驱动其神经发生的机制尚不明确。通过遗传命运定位和突变分析，我们已经证明，在小脑发育过程中，lim同源结构域转录因子Lmx1a 1)将顶板谱系从神经元菱形唇衍生物中分离出来，2)是维持整个胚胎晚期菱形唇所必需的，3)赋予一小部分菱形唇细胞后体身份。这些实验表明，小脑菱形唇是一个异质性的祖群体，在很早的阶段就有特定的命运，但我们对菱形唇祖的细胞和分子特征几乎一无所知。在本提案的Aim 1中，我们将对野生型和Lmx1a-/-动物的菱形唇祖基因表达和细胞周期参数进行广泛的分析，以表征菱形唇内组织，发育机制和赋予细胞命运的分子途径。通过对野生型和Lmx1a-/-动物的e13.5小脑菱形唇的微解剖分析，我们鉴定了几种Lmx1a候选效应物，我们将通过分析现存的小鼠突变体和在子宫内小鼠电穿孔操作基因表达来表征它们在菱形唇发育中的作用。我们的初步数据表明，Lmx1a也调节端脑皮质边缘的神经发生，其中Lmx1a的缺失导致皮质选择基因Lhx2的异常表达，从而导致海马细胞而不是Cajal-Retzius细胞的过量产生。在Aim 2中，我们提出了一系列遗传命运定位和体外外植体分析，以测试Lmx1a是否内在地作用于皮质下丘脑调节皮质下丘脑神经发生，以及Lmx1a表达的邻近脑端脉络丛是否也影响下丘脑发育。我们还将进行表达分析，以确定lmx1a下游效应物是否在背内侧生发区保守。最后，在Aim 3中，我们将探讨Lmx1a和Lmx1b功能去除后背中线异常的基础。这些基本的神经发育分析与人类中枢神经系统的结构畸形有直接的翻译相关性，并有可能揭示哺乳动物皮层大小的戏剧性进化的新见解。

DESCRIPTION (provided by applicant): The goal of this proposal is to define the general principles of dorsal medial germinal zone organization and regulation, using Lmx1a as a molecular entry point. Dorsal brain germinal zones, such as the cerebellar rhombic lip and telencephalic cortical hem, contribute substantially to neuronal diversity in the CNS, but the mechanisms that drive their neurogenesis are ill-defined. Using genetic fate mapping and mutant analysis we have demonstrated that during cerebellar development, the LIM-homeodomain transcription factor Lmx1a 1) segregates the roof plate lineage from neuronal rhombic lip derivatives, 2) is required for maintenance of the entire late embryonic rhombic lip and 3) confers posterior vermis identity to a subset of rhombic lip cells. These experiments demonstrate that the cerebellar rhombic lip is a heterogeneous progenitor population with fates specified at very early stages, yet we know nearly nothing about the cellular and molecular characteristics of rhombic lip progenitors. In Aim 1 of this proposal we will conduct an extensive analysis of rhombic lip progenitor gene expression and cell cycle parameters in wild-type and Lmx1a-/- animals to characterize the organization, developmental mechanisms and molecular pathways conferring cell fate within the rhombic lip. Using expression microarray analysis of microdissected e13.5 cerebellar rhombic lip from wild-type and Lmx1a-/- animals, we have identified several Lmx1a-candidate effectors and we will characterize their roles in rhombic lip development through analysis of extant mouse mutants and manipulation of gene expression using in utero mouse electroporation. Our preliminary data demonstrate that Lmx1a also regulates neurogenesis in the telencephalic cortical hem, where loss of Lmx1a results in aberrant expression of the cortical selector gene Lhx2, which leads to excessive production of hippocampal cells instead of Cajal-Retzius cells. In Aim 2 we propose a series of genetic fate mapping and in vitro explant analyses to test if Lmx1a acts intrinsically in the cortical hem to regulate cortical hem neurogenesis and if the adjacent telencephalic choroid plexus, where Lmx1a is also expressed, also influences hem development. We will also conduct expression analyses to determine if Lmx1a-downstream effectors are conserved across dorso-medial germinal zones. Finally in Aim 3, we will explore the basis of dorsal midline abnormalities when both Lmx1a and Lmx1b function removed. These basic neurodevelopmental analyses have direct translational relevance to human structural malformations of the CNS and have potential to reveal new insights into the dramatic evolution of cortical size in mammals. PUBLIC HEALTH RELEVANCE: With recent advances in imaging and genetics, malformations of the human brain are more frequently recognized. These malformations cause neurodevelopmental and motor delays in affected children, yet we know very little regarding which processes that go awry while the brain is developing or the underlying genetic causes. This grant uses genetic manipulations in the mouse to define the organizational principles of the embryonic stem cell populations that reside on the dorsal midline of the brain and generate vast numbers of neurons in the adult brain responsible for memory and motor coordination. When these stem cell populations develop abnormally, birth defects of the brain including cerebellar and cerebral malformations and hydrocephalus result. An improved understanding of their basic developmental biology will ultimately lead to improved diagnosis and eventually treatment for these devastating brain disorders.