Dorsal midline patterning in the vertebrate CNS

描述(申请人提供)：本提案的目标是以Lmx1a为分子切入点，确定背侧内侧生发区组织和调控的一般原则。大脑背侧生发区，如小脑菱形唇区和端脑皮质区，对中枢神经系统神经元多样性有很大贡献，但驱动其神经发生的机制尚不清楚。利用遗传命运图谱和突变分析，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)将顶板谱系与神经元的菱形唇衍生物分开，2)整个胚胎后期的菱形唇细胞的维持所必需的，以及3)赋予菱形唇细胞亚群的后部蠕虫身份。这些实验表明，小脑菱形嘴唇是一个异质性的祖细胞群体，其命运在非常早期就有明确的规定，但我们对菱形嘴唇祖细胞的细胞和分子特征几乎一无所知。在本提案的目标1中，我们将对菱形唇祖基因在野生型和Lmx1a-/-动物中的表达和细胞周期参数进行广泛的分析，以表征菱形唇细胞内的组织、发育机制和决定细胞命运的分子途径。通过对野生型和Lmx1a-/-动物的e13.5小脑菱形唇微解剖的表达谱芯片分析，我们已经鉴定了几个Lmx1a候选效应子，并将通过分析现有的小鼠突变和宫内电穿孔对基因表达的调控来表征它们在菱形嘴唇发育中的作用。我们的初步数据表明，Lmx1a还调节端脑皮质HEM的神经发生，在那里Lmx1a的丢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细胞的过度产生。在目标2中，我们提出了一系列的遗传命运图谱和体外外植体分析，以测试Lmx1a是否在皮质HEM中内在地作用于调节皮质HEM神经发生，以及邻近的端脑脉络丛(Lmx1a也表达)是否也影响HEM的发育。我们还将进行表达分析，以确定Lmx1a-下游效应器是否跨背内侧生发区保守。最后，在目标3中，我们将探讨Lmx1a和Lmx1b功能同时移除时背中线异常的基础。这些基本的神经发育分析与人类中枢神经系统结构畸形有直接的翻译相关性，并有可能揭示哺乳动物皮质大小的戏剧性进化的新见解。 与公共卫生相关：随着成像和遗传学的最新进展，人类大脑的畸形越来越多地被认识到。这些畸形会导致受影响儿童的神经发育和运动延迟，但我们对大脑发育过程中哪些过程出错或潜在的遗传原因知之甚少。这项资助使用小鼠的遗传操作来定义胚胎干细胞群体的组织原则，这些干细胞群体驻留在大脑的背侧中线，并在成人大脑中产生大量负责记忆和运动协调的神经元。当这些干细胞群体发育异常时，就会导致大脑的出生缺陷，包括小脑和大脑畸形以及脑积水。对它们基本发育生物学的更好理解最终将导致对这些毁灭性大脑疾病的诊断和最终治疗。

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.