Regional encoding of enteric nervous system responses to microbiota and type 2 inflammation [10xPerturbSeq]

Enteric neurons are essential regulators of intestinal physiology, yet their responses to varying microbial and immune environments along the intestinal tract and or during challenges remain poorly understood. Here, we regionally profile enteric neurons across gnotobiotic, allergic and parasite-infected mice. We demonstrate that timing and complexity of microbial perturbations and type 2 inflammation result in motor neuron state shifts and alter multiple functionally distinct sensory neurons, including IL-13- and leukotriene-responsive Nmu-hi cells and Grp-hi neurons, which expand in germ-free colonic tissue and interact with Grpr+ interstitial cells of Cajal. Leveraging AAV-based Perturb-seq to define genetic controllers of inhibitory motor neuron state transition, we identified Edf1 and Mitf as controllers of this program and of gastrointestinal transit time, directly linking enteric neuron states to physiology. Overall design: Adeno-associated virus (AAV)-based gene delivery was used for CRISPR screening and retrograde labeling of projecting neurons. The AAV-PHP.S capsid, tailored to infect peripheral neurons, was used for in vivo Perturb-seq access to the ENS. In addition, the piggyBac transposon element was used for AAV-mediated stable transgene expression . We generated separate pools of guide RNAs (gRNA) for the different classes of genes discussed above and stably introduced them in Cas9-expressing animals by injecting AAV PHP.S vectors encoding gRNAs with a tdTomato-KASH reporter, and the piggyBac transposon system. The latter was used to sort nuclei from infected cells. Four weeks after AAV infection, we isolated nuclei from infected submucosal and myenteric plexuses, and subjected them to single nuclei RNA-seq to generate transcriptome profiles and gRNA sequences for individual cells.

肠神经元（enteric neurons）是肠道生理功能的核心调控因子，但目前对于其沿肠道梯度分布的不同微生物与免疫微环境下，以及在应激状态下的应答机制仍知之甚少。
本研究对悉生（gnotobiotic）、过敏性致敏及寄生虫感染的小鼠体内的肠神经元开展了区域层面的表征分析。研究发现，微生物扰动的时序与复杂程度、2型炎症反应，可引发运动神经元的状态转变，并重塑多种功能迥异的感觉神经元亚群——包括响应IL-13与白三烯的Nmu高表达细胞（Nmu-hi cells）以及Grp高表达神经元（Grp-hi neurons）；这类神经元在无菌结肠组织中发生扩增，并与表达Grpr的Cajal间质细胞（interstitial cells of Cajal, ICC）发生相互作用。
本研究利用基于腺相关病毒（AAV, adeno-associated virus）的Perturb-seq技术，解析调控抑制性运动神经元状态转变的遗传因子，最终鉴定出Edf1与Mitf作为该程序以及胃肠道转运时间的调控因子，直接将肠神经元状态与生理功能关联起来。

实验设计概述：
基于腺相关病毒（AAV）的基因递送系统被用于投射神经元的CRISPR筛选与逆行标记。其中，专为外周神经元感染优化的AAV-PHP.S衣壳，被用于体内开展肠神经系统（enteric nervous system, ENS）的Perturb-seq分析。此外，本研究借助piggyBac转座子元件实现AAV介导的稳定转基因表达。针对上述不同类别基因，我们分别构建了向导RNA（gRNA, guide RNA）文库，并通过注射搭载gRNA与tdTomato-KASH报告基因的AAV PHP.S载体，结合piggyBac转座子系统，将其稳定导入表达Cas9的小鼠体内。其中piggyBac转座子系统可用于分选感染细胞的细胞核。在AAV感染四周后，我们从受感染的黏膜下神经丛与肌间神经丛中分离细胞核，并对其进行单细胞核RNA测序（single nuclei RNA-seq），从而获取单个细胞的转录组谱与gRNA序列信息。