HypoMap - a unified single cell gene expression atlas of the murine hypothalamus

The hypothalamus plays a key role in coordinating a plethora of fundamental body functions, such as energy homeostasis, social behavior and sleep. Despite recent progress in single-cell technologies, we still lack a unified catalogue and molecular characterization of the heterogeneous cell types and, specifically, neuronal subtypes in this brain region. Here, we combined 17 publicly available hypothalamic single cell sequencing datasets and in-house single-nuclear sequencing of 36,626 cells to create 'HypoMap', an integrated reference atlas of the murine hypothalamus of 384,925 cells. We have leveraged the power of this vast amount of available data by combining them in a single platform, which allows the future incorporation of additional experiments. We validated HypoMap by comparing data collected from SmartSeq2 and bulk RNA sequencing of selected neuronal cell types with different degrees of cellular heterogeneity. Finally, we identified novel classes of glucagon-like peptide-1 receptor (Glp1r)- and Prepronociceptin (Pnoc-) expressing neurons and validated them using single-molecule in situ hybridization. Collectively, HypoMap provides a unified framework for the systematic functional annotation of murine hypothalamic cell types, and will serve as an important platform for future studies to further unravel the functional organization of hypothalamic neurocircuits, as well as identifying novel druggable targets for treating metabolic disorders. We generated bacTRAP RNA-seq datasets of specific hypothalamic cell types, which have been shown to be of critical importance in control of energy and glucose homeostasis, but vary in their heterogeneity and anatomical distribution. To this end, we first crossed mice expressing Cre recombinase in either AgRP neurons, POMC neurons, Pnoc-expressing neurons or in Glp1r-expressing neurons with mice allowing for Cre-dependent expression of a fusion protein of the ribosomal protein L10a and GFP.下丘脑（hypothalamus）在协调大量基础生理功能中发挥关键作用，诸如能量稳态、社会行为与睡眠。尽管单细胞技术（single-cell technologies）近年来取得长足进展，我们仍缺乏该脑区异质性细胞类型——尤其是神经元亚型——的统一目录与分子特征解析。本研究整合了17个公开可用的下丘脑单细胞测序数据集，以及本实验室生成的36626个细胞的单细胞核测序（single-nuclear sequencing）数据，构建了名为“HypoMap”的小鼠下丘脑整合参考图谱，共涵盖384925个细胞。我们通过将这些海量数据整合至单一平台，充分发挥了其数据规模优势，该平台未来可纳入更多新增实验数据。我们通过对比SmartSeq2测序与选定神经元细胞类型的批量RNA测序（bulk RNA sequencing）数据（涵盖不同细胞异质性程度），验证了HypoMap的可靠性。最后，我们鉴定出表达胰高血糖素样肽-1受体（glucagon-like peptide-1 receptor, Glp1r）和前原孤啡肽（prepronociceptin, Pnoc）的新型神经元类群，并通过单分子原位杂交（single-molecule in situ hybridization）技术对其进行了验证。综上，HypoMap为小鼠下丘脑细胞类型的系统性功能注释提供了统一框架，将作为重要研究平台助力未来研究进一步解析下丘脑神经环路的功能组织，并为代谢紊乱的治疗发现新型可成药靶点。我们还生成了特定下丘脑细胞类型的bacTRAP RNA测序（bacTRAP RNA-seq）数据集，这些细胞类型在能量与葡萄糖稳态调控中发挥关键作用，但其异质性与解剖分布各不相同。为此，我们首先将分别在AgRP神经元、POMC神经元、表达Pnoc的神经元或表达Glp1r的神经元中表达Cre重组酶（Cre recombinase）的小鼠，与可实现Cre依赖表达核糖体蛋白L10a与绿色荧光蛋白（GFP）融合蛋白的小鼠进行杂交。