Mechanisms of Autonomic Brainstem Development

描述（由申请人提供）：脑干和自主神经回路虽然在神经发育障碍中研究不足，但与病理生理学和共同发生的医学状况（如胃肠道紊乱（GID））有关。R21项目的目标是根据重要的初步数据填补这一知识空白。实验将确定分子概况和发育机制，支配自主脑干神经元亚群的发育，这些亚群由MET受体酪氨酸激酶的特异性表达界定，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、轴突导向和突触发育。MET作为脑干中的关键神经发育基因的分析将导致确定参与自主神经和内脏功能的中枢调节中断的分子网络。对于脑干自主神经核，这在很大程度上是未知的关于任何神经发育障碍（例如雷特综合征，脆性X，ASD）。一些证据表明，MET可能作为一个重要的翻译链接与自主脑干发育。我们知道MET基因的一个功能性启动子变体在患有ASD的儿童中富集，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层回路具有功能和结构影响。MET的表达在ASD和Rett受试者的颞叶新皮层中均降低，MeCP 2是MET的转录调节因子。MET是PI 3激酶/ERK信号网络的一部分，在综合征性神经发育障碍中发挥重要作用。我们提出的实验，以解决特定的假设，一个点的收敛神经发育障碍和某些共同发生的医学表型是自主和内脏脑干电路的非典型发展。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中进行这种实验努力的前景：1）产前Met/MET表达的定位揭示了Met在自主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Islet 1Cre在运动神经元中条件性缺失Met会破坏nA发育。在目的1中，将使用独特的遗传报告基因方案来差异标记，然后在nA和DMV中从MET-神经元（GFPOFF/tdTOMATOON）中分选MET+（GFPON/tdTOMATOON）运动神经元用于RNA测序。该数据将形成目标2中的分析的基础，其中在MetEGFP转基因背景上使用Islet 1Cre有条件地删除Met。将测量DMV和nA神经元的改变的组织发生、迁移和轴突模式。MET信号传导对分子分化的影响将通过分析Met基因缺失后DMV和nA中Met亚型神经元中的基因表达（Aim 1）来检查。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系统受损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.