Decoding the origin of neuronal diversity in hypothalamus by single-cell RNA-seq

The hypothalamus is one of the most complex brain structures whose development involves a plastic process of neuronal fate specification. Progress has been made to decipher the gene regulatory programs that are responsible for hypothalamus development; however, the molecular developmetal trajectory of hyothalamus is largely unknown. To understand how pre- and postmitotic transcriptional programs interact and coordinate to endow neuronal cell subtypes with their characteristic properties during hypothalamic development, we performed single-cell RNA sequencing (scRNA-seq) on single cells derived from Rax+ hypothalamic neuroepithelium at four critical developmental points during hypothalamic development. Our single-cell analysis provides a developmental landscape of mouse hypothalamus. We show that while the fate of radial glial cells (RGCs) is predetermined before differentiation but lack spatial code to distinguish from each other, different clusters of intermediate progenitors (IPCs) emerge to display diversifying fates and subdivide hypothalamic primordium into distinct spatially-restricted progenitor domains. We further characterize the maturation dynamics of hypothalamic neurons and suggest that immature neurons could evolve into multiple peptidergic neuronal subtypes. Finally, we identify sets of transcription factors (TFs) serving as regulons to determine the fate of diverse GABAergic and Glutamatergic neurons in hypothalamus. Together, our study offers a single-cell transcriptional framework for the hypothalamus developmental trajectory and propose a cascade diversifying model to deconstruct the origin of neuronal diversity in hypothalamus. Overall design: To infer the lineage progression underlying hypothalamus development, we first labeled hypothalamic neuroepithelium by applying a single dose of tamoxifen in Rax-CreERT2::Ai14 mice at embryonic day 9 (E9) and performed lineage tracing for cell collection. Next, we microdissected hypothalamus from induced Rax-CreERT2::Ai14 mice across 4 different time points that cover the process of neurogenesis and early maturation for hypothalamic neurons [E11, E14, postnatal days 0 (P0) and P7], and collected live tdTomato+ cells by fluorescent-activated cell sorting for single-cell transcriptomic sequencing