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，但涉及病理生理学和共同发生的医疗状况，如胃肠道紊乱（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。实验将确定控制自主脑干神经元亚群发育的分子特征和发育机制，这些神经元由MET受体酪氨酸激酶的特定表达划分，酪氨酸激酶是一种多向信号受体，可以调节神经元迁移、轴突引导和突触发育。将MET作为脑干中重要的神经发育基因进行分析，将有助于确定参与自主神经和内脏功能中枢调控中断的分子网络。对于脑干自主神经核，对于任何神经发育障碍（如Rett综合征，脆性X染色体，ASD），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重要翻译环节。我们知道MET基因的功能性启动子变体在同时患有孤独症的ASD儿童中富集。我们也知道，降低MET基因表达的启动子变异对人类皮层回路具有功能和结构上的影响。在ASD和Rett受试者中，颞叶新皮层中MET表达减少，MeCP2是MET的转录调节因子。MET是PI3激酶/ERK信号网络的一部分，在综合征性神经发育障碍中起重要作用。我们提出实验来解决特定的假设，即神经发育障碍和某些共同发生的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将启动一个新的研究项目来验证这一假设。初步研究支持了该实验在小鼠中的前景：1)产前Met/ Met表达图谱揭示了自主脑干神经元中Met的高度选择性发育表达模式，包括背运动迷走核（DMV）和歧见核（nA）中的亚群；2)使用Islet1Cre在运动神经元中条件删除Met会破坏nA的发育。在Aim 1中，将使用一种独特的遗传报告程序对nA和DMV中的MET+ (GFPON/tdTOMATOON)神经元和MET- (GFPOFF/tdTOMATOON)神经元进行区分标记，然后进行rna测序。该数据将构成Aim 2分析的基础，在Aim 2中，Met在MetEGFP转基因背景下使用Islet1Cre有条件地删除。将测量DMV和nA神经元的组织发生、迁移和轴突模式的改变。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亚型神经元的基因表达（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.