Single cell RNA-Seq identifies mechanisms controlling hypothalamic patterning and differentiation

The hypothalamus is a central regulator of many innate behaviors that are essential for survival, but the molecular mechanisms controlling hypothalamic patterning and cell fate specification remain poorly understood. To identify genes that control hypothalamic development, we have used single-cell RNA sequencing (scRNA-Seq) to profile mouse hypothalamic gene expression across 11 developmental time points between embryonic day 10 and postnatal day 45. This identified genes that delineated clear developmental trajectories for all major hypothalamic cell types and readily distinguished major regional subdivisions of the developing hypothalamus. We show that this approach can rapidly and comprehensively characterize mutants that control hypothalamic patterning and, in doing so, identify multiple genes that simultaneously repress posterior hypothalamic identity while promoting prethalamic identity. This argues in favor of a modified columnar organization of hypothalamus and prethalamus. These data serve as a resource for further studies of hypothalamic development, physiology, and dysfunction. Dissection and cell dissociation - Embryos or postnatal mice were collected and dissociated following previously published protocol. Embryos were collected using Hibernate-E media (Thermo Fisher Scientific) with 2% B-27 supplement (Thermo Fisher Scientific) and GlutaMAX supplement (0.5 mM final, Thermo Fisher Scientific). A small incision was made dorsal to the lower jaw to expose the ventral portion of the brain. For samples collected between E10 and E16, tissue residing posterior to the medial ganglionic eminence and anterior to the midbrain and sensory thalamus was dissected to collect developing prethalamus and hypothalamus. Prethalamus was excluded from samples aged E18 and older, with only hypothalamus collected, as previously described (2). Collection of medial ganglionic eminence and posterior structure to the supramammillary nucleus were variable between dissections. Between 8 and 12 embryos of either sex were collected for each embryonic time point. Postnatal mice were collected using Hibernate-A media with 2% B-27 and GlutaMAX (0.5 mM final), and the tissue posterior to the optic chiasm and anterior to the posterior hypothalamus were collected. Eight pups (4 male and 4 female) were collected for P4, P8, and P14 dataset, and 3 male mice were pooled for P45 dataset. E10, E12, and E15; E11 and E13; E14, E16, and P45; E18, P4, and P14 were each generated on the same day.For single-cell phenotyping studies, E12.5 time-mated mice were collected and placed in buffer mentioned above on ice. Tail-tips were collected and rapidly genotyped using GeneAmp Fast PCR mastermix (Thermo Fisher Scientific). Both control and mutant groups were collected on the same day, and E12.5 embryos were pooled from 3 different dams. Following dissection, tissues were dissociated in papain (Worthington Biochemical) as previously described in calcium-free Hibernate media. Tissue debris were removed using OptiPrep density gradient media (Sigma-Aldrich) in postnatal mice following cell dissociation. Number of viable cells was counted manually haemocytometer with Trypan Blue staining, and cell concentration was adjusted following manufacturer's protocol of 10x Genomics. 10x library generation and data processing - Suspended cells were loaded into 10x Genomics Chromium Single Cell System (10X Genomics), and libraries were generated using v1 (1 library) and V2 chemistry with manufacturer's instructions. Libraries were sequenced on Illumina MiSeq (1 library) and NextSeq500. Sequencing data were pre-processed through the Cell Ranger pipeline (10x Genomics) with default parameters, aligned to mm10 genome, and matrix files were used for bioinformatic analysis.下丘脑（hypothalamus）是诸多生存必需的先天行为的中枢调控枢纽，但调控下丘脑模式形成与细胞命运特化的分子机制仍不甚明晰。为鉴定调控下丘脑发育的基因，我们采用单细胞RNA测序（single-cell RNA sequencing, scRNA-Seq）对胚胎第10天至出生后第45天之间的11个发育时间点的小鼠下丘脑基因表达谱进行了分析。本研究鉴定出可清晰勾勒所有主要下丘脑细胞类型发育轨迹的基因，且可轻松区分发育中下丘脑的主要区域亚分区。我们证实，该方法可快速且全面地表征调控下丘脑模式形成的突变体，并借此鉴定出多个可同时抑制下丘脑后部细胞身份、同时促进丘脑前体（prethalamus）细胞身份的基因。上述发现支持一种经过修正的下丘脑与丘脑前体的柱状组织结构假说。本数据集可作为下丘脑发育、生理功能及功能异常相关后续研究的宝贵资源。 解剖与细胞解离——按照已发表的方案收集胚胎或出生后小鼠并进行细胞解离。胚胎收集采用含2% B-27添加剂（Thermo Fisher Scientific）与GlutaMAX添加剂（终浓度0.5 mM，Thermo Fisher Scientific）的Hibernate-E培养基（Thermo Fisher Scientific）。于下颌背侧做小切口以暴露脑的腹侧部分。对于E10至E16阶段采集的样本，我们解剖内侧神经节隆起（medial ganglionic eminence）后方、中脑与感觉丘脑前方的组织，以收集发育中的丘脑前体与下丘脑。对于E18及更晚阶段的样本，我们排除丘脑前体，仅收集下丘脑，如既往研究所述（2）。内侧神经节隆起与乳头体上核后方结构的采集在不同解剖操作间存在差异。每个胚胎时间点收集8~12只不限性别的胚胎。出生后小鼠采用含2% B-27与GlutaMAX（终浓度0.5 mM）的Hibernate-A培养基收集，采集视交叉后方、下丘脑前部前方的组织。对于P4、P8与P14数据集，各收集8只幼鼠（4雄4雌）；P45数据集则合并3只雄性小鼠的组织。E10、E12与E15；E11与E13；E14、E16与P45；E18、P4与P14分别于同一天完成实验。 对于单细胞表型分析研究，收集妊娠至E12.5的孕鼠并置于上述冰上缓冲液中。截取尾尖并使用GeneAmp Fast PCR预混液（Thermo Fisher Scientific）快速进行基因分型。对照组与突变体组均于同一天收集，E12.5胚胎取自3只不同孕鼠并合并。解剖完成后，按照既往研究方法，在无钙Hibernate培养基中用木瓜蛋白酶（Worthington Biochemical）解离组织。出生后小鼠的细胞解离后，使用OptiPrep密度梯度培养基（Sigma-Aldrich）去除组织碎片。采用台盼蓝染色与血球计数板手动计数活细胞数量，并按照10x Genomics的官方方案调整细胞浓度。 10x基因组文库构建与数据处理——将重悬后的细胞加载至10x Genomics Chromium单细胞系统（10x Genomics），并按照制造商说明书使用v1（1个文库）与V2化学试剂构建文库。文库在Illumina MiSeq（1个文库）与NextSeq500平台上进行测序。测序数据通过Cell Ranger流程（10x Genomics）以默认参数进行预处理，比对至mm10基因组，所得矩阵文件用于生物信息学分析。