Abstract
The hypothalamus is a central regulator of physiological homeostasis. During development, multiple transcription factors coordinate the patterning and specification of hypothalamic nuclei. However, the molecular mechanisms controlling hypothalamic patterning and cell fate specification are poorly understood. To identify genes that control these processes, we have used single-cell RNA sequencing (scRNA-Seq) to profile mouse hypothalamic gene expression across multiple developmental time points. We have further utilised scRNA-Seq to phenotype mutations in genes that play major roles in early hypothalamic patterning. To first understand hypothalamic development, hypothalami were collected at both embryonic (E10-E16, E18) and postnatal (PN4, PN8, PN14, PN45) time points. At early stages, when the bulk of hypothalamic patterning occurs (E11-E13), we observe a clear separation between mitotic progenitors and postmitotic neural precursor cells. We likewise observed clean segregation among cells expressing regional hypothalamic markers identified in previous large-scale analysis of hypothalamic development. This analysis reveals new region-specific markers and identifies candidate genes for selectively regulating patterning and cell fate specification in individual hypothalamic regions. With our rich dataset of developing mouse hypothalamus, we integrated our dataset with the Allen Brain Atlas in situ data, publicly available adult hypothalamic scRNA-Seq dataset to understand hierarchy of hypothalamic cell differentiation, as well as re-defining cell types of the hypothalamus.
We next used scRNA-Seq to phenotype multiple mutant lines, including a line that has been extensively characterised as a proof of concept (Ctnnb1 overexpression), and lines that have not been characterised (Nkx2.1, Nkx2.2, Dlx1/2 deletion). We show that this approach can rapidly and comprehensively characterize mutants that have altered hypothalamic patterning, and in doing so, have identified multiple genes that simultaneously repress posterior hypothalamic identity while promoting prethalamic identity. This result supports a modified columnar model of organization for the diencephalon, where prethalamus and hypothalamus are situated in adjacent dorsal and ventral domains of the anterior diencephalon. These data serve as a resource for further studies of hypothalamic development and dysfunction, and able to delineate transcriptional regulatory networks of hypothalamic formation.
Lastly, using our mouse hypothalamus as a guideline, we are comparing dataset of developing chicken, zebrafish and human hypothalamus, to identify evolutionarily conserved and divergent region-specific gene regulatory networks. We aim to use this knowledge and information of key molecular pathways of human hypothalamic development and produce human hypothalamus organoids.
Chromatin-mediated translational control is essential for neural cell fate specification