Mechanisms of Autonomic Brainstem Development

描述(申请人提供)：脑干和自主神经回路，虽然在神经发育障碍方面研究不多，但与病理生理学和共同发生的医学状况有关，如胃肠功能障碍(GID)。这个R21项目的目标是基于重要的初步数据来填补这一知识空白。实验将确定支配自主脑干神经元亚群发育的分子图谱和发育机制，这些亚群由MET受体酪氨酸激酶的特定表达所界定，MET受体酪氨酸激酶是一种多效性信号受体，可以调节神经元迁移、轴突引导和突触发育。对脑干中关键的神经发育基因MET的分析将导致确定参与自主神经和内脏功能中枢调节紊乱的分子网络。对于脑干自主神经核团，对于任何神经发育障碍(如Rett综合征、脆性X综合征、ASD)来说，这在很大程度上是未知的。一些证据表明，MET可能是自主脑干发育的一个重要的翻译环节。我们知道，MET基因的一个功能性启动子变体在患有自闭症的儿童中丰富，这些儿童也患有GID。我们还知道，减少MET基因表达的启动子变体对人类皮质回路有功能和结构上的影响。在ASD和Rett受试者中，MET在颞叶新皮质中的表达均减少，而MeCP2是MET的转录调节因子。MET是PI3Kinase/ERK信号网络的一部分，在综合征性神经发育障碍中发挥着重要作用。我们提出实验来解决这一特定假设，即神经发育障碍和某些共生医学表型的一个趋同点是自主神经和内脏脑干回路的非典型发育。R21将启动一项新的研究计划来验证这一假设。初步研究支持在小鼠上进行此实验：1)产前定位Met/Met表达图谱显示Met在自主神经脑干神经元中的高度选择性发育表达模式，包括迷走神经背核(DMV)和疑核(NA)中的亚群；2)使用Islet1Cre有条件地删除运动神经元中的Met可干扰NA的发育。在目标1中，将使用一种独特的遗传报告方案来对NA和DMV中的MET(GFPON/tdTOMATOON)运动神经元进行差异标记和排序，以用于RNA测序。这些数据将成为目标2分析的基础，在目标2中，在MetEGFP转基因背景下使用Islet1Cre有条件地删除Met。将测量DMV和NA神经元改变的组织发生、迁移和轴突模式。Met信号对分子分化的影响将通过对Met基因缺失后DMV和NA中Met亚型神经元的基因表达分析(Aim 1)来检验。这些数据将为未来的功能研究奠定基础，以了解由于中枢自主神经回路中断而导致的与临床表型相关的遗传风险。

DESCRIPTION (provided by applicant): Brainstem and autonomic circuitry, though understudied in neurodevelopmental disorders, are implicated in pathophysiology and co-occurring medical conditions, such as gastrointestinal disturbances (GID). The goal of this R21 project is to fill this knowledge gap, based on significant preliminary data. Experiments will determine the molecular profiles and developmental mechanisms that govern the development of subpopulations of autonomic brainstem neurons that are delimited by specific expression of the MET receptor tyrosine kinase, a pleiotropic signaling receptor that can regulate neuronal migration, axon guidance and synapse development. The analysis of MET as a critical neurodevelopmental gene in the brainstem will lead to determining the molecular networks that are involved in disrupted central regulation of autonomic and visceral functions. For brainstem autonomic nuclei, this is largely unknown with regard to any neurodevelopmental disorder (e.g. Rett Syndrome, Fragile X, ASD). Several lines of evidence suggest that MET may serve as an important translational link with autonomic brainstem development. We know that a functional promoter variant of the MET gene is enriched in children with ASD who also have GIDs. We also know that the promoter variant, which reduced MET gene expression, has functional and structural impact on human cortical circuits. MET expression is reduced in the temporal neocortex in both ASD and Rett subjects, and MeCP2 is a transcriptional regulator of MET. MET is part of PI3 Kinase/ERK signaling networks that play a significant role in syndromic neurodevelopmental disorders. We propose experiments to address the specific hypothesis that one point of convergence for neurodevelopmental disorders and certain co-occurring medical phenotypes is atypical development of autonomic and visceral brainstem circuits. The R21 will launch a new research program for testing this hypothesis. Preliminary studies support the promise of this experimental effort in mice: 1) Mapping of Met/MET expression prenatally reveals a highly selective developmental expression pattern of Met in autonomic brainstem neurons, including subpopulations in dorsal motor vagal nucleus (DMV) and nucleus Ambiguus (nA); and 2) conditional deletion of Met in motor neurons using Islet1Cre disrupts nA development. In Aim 1, a unique genetic reporter scheme will be used to label differentially and then sort MET+ (GFPON/tdTOMATOON) motor neurons from MET- neurons (GFPOFF/tdTOMATOON) in nA and DMV for RNA-sequencing. This data will form the basis for the analysis in Aim 2, in which Met is deleted conditionally using Islet1Cre on a MetEGFP transgenic background. Altered histogenesis, migration and axon patterning of DMV and nA neurons will be measured. The influence of MET signaling on molecular differentiation will be examined by analysis of gene expression (Aim 1) in Met-subytpe neurons in DMV and nA following genetic deletion of Met. These data will establish a basis for future functional studies o understand genetic risk related to clinical phenotypes due to disrupted central autonomic circuitry.