The pioneer/terminal nerve neurons of rodents control GnRH-1 neuronal migration and olfactory bulb formation.

During embryonic development, the olfactory placode (OP) gives rise to various populations of neurons; these include putative olfactory pioneer neurons, different neurons of unknown identity and function, cells of the terminal nerve, and the Gonadotropin-releasing hormone-1 (GnRH-1) neurons. In mice, the GnRH-1 neurons are first detectable in the developing olfactory system around mid-gestation. From here, the GnRH-1 neurons migrate, along the axons of the terminal nerve (TN), to various regions of the developing brain. Once in the brain, the GnRH-1 neurons play a central role in controlling the hypothalamic-pituitary-gonadal (HPG) axis. Early migratory neurons forming from the olfactory placode have been proposed to play a vital role in inducing olfactory bulb morphogenesis. Kallmann syndrome is a condition characterized by defective development of the olfactory system and infertility. Murine studies have demonstrated the critical role of the Prokineticin 2-Prokinteicin Receptor 2 pathway in olfactory bulb morphogenesis and GnRH-1 neuronal migration. Loss-of-function Prokr2 mutations cause both Kallmann syndrome associated with bulb agenesis and normosmic idiopathic hypogonadotropic hypogonadism (nIHH). Following Prokr2 expression and lineage tracing, we found that Prokr2 is not expressed by the cells of the developing olfactory bulb but by migratory putative pioneer/terminal nerve neurons. Performing single-cell-RNA-sequencing, we identified genes enriched in the migratory cells of the putative terminal nerve. By integrating genetic lineage tracing and single-cell transcriptomics, we identified previously undescribed populations of migratory neurons that appear to be enriched in the expression of several genes related to olfactory defects, GnRH migratory deficiencies and infertility. Whole embryonic noses were dissected from E14.5 wild-type mice, cells were isolated then analyzed using scRNAseq. ProKR2iCre/R26tdTomato E14.5 mice had noses dissected, then cells were isolated using Flourescence-activated cell sorting (FACS), then scRNAseq was performed.在胚胎发育过程中，嗅基板（olfactory placode）可产生多种神经元群，包括推定的嗅觉先锋神经元、多种身份与功能尚不明确的神经元、终神经（terminal nerve, TN）细胞，以及促性腺激素释放激素1（Gonadotropin-releasing hormone-1, GnRH-1）神经元。在小鼠体内，GnRH-1神经元最早可在妊娠中期的发育中嗅觉系统内被检测到。自此之后，GnRH-1神经元会沿着终神经的轴突迁移至发育中大脑的多个区域。一旦进入大脑，GnRH-1神经元便在调控下丘脑-垂体-性腺（hypothalamic-pituitary-gonadal, HPG）轴的过程中发挥核心作用。有研究提出，源自嗅基板的早期迁移神经元在诱导嗅球形态发生过程中起到关键作用。卡尔曼综合征（Kallmann syndrome）是一类以嗅觉系统发育缺陷与不育为特征的疾病。小鼠实验已证实，促动力素2-促动力素受体2（Prokineticin 2-Prokineticin Receptor 2）通路在嗅球形态发生以及GnRH-1神经元迁移过程中发挥着至关重要的作用。功能缺失型Prokr2突变可引发伴有嗅球发育不全的卡尔曼综合征，以及嗅觉正常的特发性低促性腺激素性性腺功能减退症（normosmic idiopathic hypogonadotropic hypogonadism, nIHH）。通过对Prokr2的表达进行检测并开展谱系示踪，我们发现Prokr2并非在发育中的嗅球细胞中表达，而是在迁移中的推定先锋神经元/终神经神经元中表达。借助单细胞RNA测序（single-cell RNA sequencing, scRNAseq），我们在推定终神经的迁移细胞中鉴定出了富集表达的基因。通过整合遗传谱系示踪与单细胞转录组学分析，我们发现了此前未被描述的迁移神经元群，这类神经元群似乎富集了多种与嗅觉缺陷、GnRH迁移缺陷以及不育相关的基因。我们从胚胎发育第14.5天（E14.5）的野生型小鼠体内分离出全胚胎鼻部组织，对细胞进行解离后采用scRNAseq进行分析。同时，我们对ProKR2iCre/R26tdTomato基因型的E14.5小鼠进行鼻部组织解剖，通过荧光激活细胞分选（Fluorescence-activated cell sorting, FACS）分离细胞后开展scRNAseq实验。