GSE132355
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. Overall design: 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.