A versatile Immuno-LCM/RNA-Seq method uncovers the rodent gonadotropin-releasing hormone neuron transcriptome

We developed and validated an Immuno-LCM/RNA-Seq method for transcriptome profiling of immunohistochemically labeled neurons. Immuno-LCM/RNA-Seq allows cell type-specific and high-throughput spatial transcriptomic studies without species limitations to genetically-modified mice. Innovative RNA-preserving strategies we introduced efficiently maintain RNA integrity in floated sections of formaldehyde-fixed rodent brains during both long-term tissue storage and immunohistochemical processing. Gonadotropin-releasing hormone (GnRH) neurons regulating reproduction were immunostained and harvested with laser-capture microdissection (LCM) for RNA isolation, random primer-based cDNA library preparation and bulk sequencing. The 13,000-14,000 transcripts we detected in GnRH neurons of rats and mice included 28 mRNAs implicated previously in human infertility. Interestingly, GnRH neurons exclusively expressed estrogen receptor alpha in mice and estrogen receptor beta in rats, among other unexpected species differences. Immuno-LCM/RNA-Seq is a versatile, sensitive and accurate bulk sequencing approach to characterize the transcriptome landscape of GnRH neurons and other neuronal phenotypes identified with immunohistochemistry in formaldehyde-fixed tissue sections of any species.

本研究开发并验证了一种可用于免疫组织化学标记神经元转录组分析的免疫激光捕获显微切割/RNA测序（Immuno-LCM/RNA-Seq）方法。该方法可实现细胞类型特异性且高通量的空间转录组学研究，无需受限于基因工程改造小鼠，可跨物种开展相关实验。本研究引入的创新性RNA保存策略，可在长期组织储存与免疫组织化学处理流程中，有效维持甲醛固定啮齿类动物脑组织漂浮切片的RNA完整性。针对调控生殖过程的促性腺激素释放激素（Gonadotropin-releasing hormone, GnRH）神经元，本研究通过免疫染色结合激光捕获显微切割（laser-capture microdissection, LCM）技术完成样本获取，随后开展RNA提取、基于随机引物的cDNA文库构建及批量测序。本研究在大小鼠的GnRH神经元中检测到13000至14000个转录本，其中包含28种此前被认为与人类不孕症相关的mRNA。值得注意的是，在诸多意外的物种差异中，小鼠的GnRH神经元仅表达雌激素受体α，而大鼠的GnRH神经元仅表达雌激素受体β。免疫激光捕获显微切割/RNA测序是一种通用、灵敏且精准的批量测序方法，可用于解析任意物种甲醛固定组织切片中经免疫组织化学鉴定的GnRH神经元及其他神经元表型的转录组全貌。