Davidson Ann E, Straquadine Nora R W, Cook Sara A, Liu Christina G, Nie Chuhao, Spaulding Matthew C, Ganz Julia
Department of Integrative Biology, Michigan State University, East Lansing, Michigan, USA.
Concordia University, St. Paul, Minnesota, USA.
Neurogastroenterol Motil. 2025 May;37(5):e70009. doi: 10.1111/nmo.70009. Epub 2025 Apr 6.
The neural crest-derived enteric nervous system (ENS) provides the intrinsic innervation of the gut with diverse neuronal subtypes and glial cells. The ENS regulates all essential gut functions, such as motility, nutrient uptake, immune response, and microbiota colonization. Deficits in ENS neuron numbers and composition cause debilitating gut dysfunction. Yet, few studies have identified genes that control neuronal differentiation and the generation of the diverse neuronal subtypes in the ENS.
Utilizing existing CRISPR/Cas9 genome editing technology in zebrafish, we have developed a rapid and scalable screening approach for identifying genes that regulate ENS neurogenesis.
As a proof-of-concept, F0 guide RNA-injected larvae (F0 crispants) targeting the known ENS regulator genes sox10, ret, or phox2bb phenocopied known ENS phenotypes with high efficiency. We evaluated 10 transcription factor candidate genes as regulators of ENS neurogenesis and function. F0 crispants for five of the tested genes have fewer ENS neurons. Secondary assays in F0 crispants for a subset of the genes that had fewer neurons reveal no effect on enteric progenitor cell migration but differential changes in gut motility.
Our multistep, yet straightforward CRISPR screening approach in zebrafish tests the genetic basis of ENS developmental and disease gene functions that will facilitate the high-throughput evaluation of candidate genes from transcriptomic, genome-wide association, or other ENS-omics studies. Such in vivo ENS F0 crispant screens will contribute to a better understanding of ENS neuronal development regulation in vertebrates and what goes awry in ENS disorders.
神经嵴衍生的肠神经系统（ENS）为肠道提供具有多种神经元亚型和神经胶质细胞的内在神经支配。ENS调节所有基本的肠道功能，如蠕动、营养吸收、免疫反应和微生物群定植。ENS神经元数量和组成的缺陷会导致使人衰弱的肠道功能障碍。然而，很少有研究确定控制神经元分化和ENS中多种神经元亚型生成的基因。
利用斑马鱼中现有的CRISPR/Cas9基因组编辑技术，我们开发了一种快速且可扩展的筛选方法，用于鉴定调节ENS神经发生的基因。
作为概念验证，靶向已知ENS调节基因sox10、ret或phox2bb的F0引导RNA注射幼虫（F0敲减体）高效模拟了已知的ENS表型。我们评估了10个转录因子候选基因作为ENS神经发生和功能的调节因子。针对五个测试基因的F0敲减体的ENS神经元较少。对神经元较少的部分基因的F0敲减体进行的二次分析显示，对肠祖细胞迁移没有影响，但对肠道蠕动有不同的变化。
我们在斑马鱼中采用的多步骤但直接的CRISPR筛选方法测试了ENS发育和疾病基因功能的遗传基础，这将有助于对来自转录组学、全基因组关联或其他ENS组学研究的候选基因进行高通量评估。这种体内ENS F0敲减体筛选将有助于更好地理解脊椎动物中ENS神经元发育的调控以及ENS疾病中出现的问题。
