Dorsal midline patterning in the vertebrate CNS

描述（由申请人提供）：本提案的目标是使用Lmx 1a作为分子切入点，定义背内侧生发区组织和调节的一般原则。背侧脑生发区，如小脑菱形唇和端脑皮质边缘，对中枢神经系统的神经元多样性有很大贡献，但驱动其神经发生的机制尚不明确。使用遗传命运作图和突变分析，我们已经证明，在小脑发育过程中，LIM同源结构域转录因子Lmx 1a 1）分离的顶板谱系神经元菱形唇衍生物，2）是需要维持整个晚期胚胎菱形唇和3）赋予后蚓身份的菱形唇细胞的一个子集。这些实验表明，小脑菱形唇是一个异质的祖细胞群体的命运在非常早期的阶段，但我们几乎一无所知的菱形唇祖细胞的细胞和分子特征。在本提案的目标1中，我们将对野生型和Lmx 1a-/-动物中的菱形唇祖基因表达和细胞周期参数进行广泛分析，以表征菱形唇内赋予细胞命运的组织、发育机制和分子途径。使用表达微阵列分析的显微切割e13.5小脑菱形唇野生型和Lmx 1a-/-动物，我们已经确定了几个Lmx 1a-候选效应，我们将通过分析现存的小鼠突变体和基因表达的操纵在子宫内小鼠电穿孔菱形唇发育中的作用。我们的初步数据表明，Lmx 1a还调节端脑皮质hem中的神经发生，其中Lmx 1a的缺失导致皮质选择基因Lhx 2的异常表达，这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映射和体外外植体分析，以测试是否Lmx 1a的行为本质上在皮质出血调节皮质出血神经发生，如果相邻的端脑脉络丛，Lmx 1a也表达，也影响出血的发展。我们还将进行表达分析，以确定Lmx 1a下游效应子是否在背内侧胚层区保守。最后，在目标3中，我们将探索当Lmx 1a和Lmx 1b功能被去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演变的新见解。

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.