Molecular codes and in vitro generation of hypocretin and melanin concentrating hormone neurons

Abstract

Hypocretin/orexin (HCRT) and melanin concentrating hormone (MCH) neuropeptides are exclusively produced by the lateral hypothalamus and play important roles in sleep, metabolism, reward, and motivation. Loss of HCRT (ligands or receptors) causes the sleep disorder narcolepsy with cataplexy in humans and in animal models. How these neuropeptides are produced and involved in diverse functions remain unknown. Here, we developed methods to sort and purify HCRT and MCH neurons from the mouse late embryonic hypothalamus. RNA sequencing revealed key factors of fate determination for HCRT (Peg3, Ahr1, Six6, Nr2f2, and Prrx1) and MCH (Lmx1, Gbx2, and Peg3) neurons. Loss of Peg3 in mice significantly reduces HCRT and MCH cell numbers, while knock-down of a Peg3 ortholog in zebrafish completely abolishes their expression, resulting in a 2-fold increase in sleep amount. We also found that loss of HCRT neurons in Hcrt-ataxin-3 mice results in a specific 50% decrease in another orexigenic neuropeptide, QRFP, that might explain the metabolic syndrome in narcolepsy. The transcriptome results were used to develop protocols for the production of HCRT and MCH neurons from induced pluripotent stem cells and ascorbic acid was found necessary for HCRT and BMP7 for MCH cell differentiation. Our results provide a platform to understand the development and expression of HCRT and MCH and their multiple functions in health and disease.

Paternally expressed imprintedSnord116andPeg3regulate hypothalamic orexin neurons

“…The increase in Snord116 in mice with depletion of OX neurons is probably a compensatory mechanism. Our conclusions are supported by a recent study [48] that described the role of Peg3 in the development and expression of OX and MCH neurons. Our study demonstrates for the first time that Peg3 is able to bind OX promotors by increasing the expression of OX.…”

“…Peg3 binds to DNA based on its multiple zinc finger motifs and nuclear localization [27,28]. Interestingly, a recent study found an association between Peg3 and OX expression [29]. However, whether Peg3 is able to bind and regulate the expression of OX in living mice remains unclear.…”

Loss of Snord116 impacts lateral hypothalamus, sleep, and food-related behaviors

“…This altered modulation between cortical states, behavior, and subcortical neuronal activity in Snord116-deleted mice is accompanied by a loss of OX-expressing neurons in the LH. We also report, for the first time to our knowledge, a link between Snord116 and a different paternally imprinted gene, Peg3, which has been recently associated with the control of the hypothalamic OX neuromodulatory system (16).…”

“…PEG3 binds to DNA based on its multiple zinc finger motifs and nuclear localization (31,32). Interestingly, a recent study found an association between Peg3 and OX expression (16). However, whether PEG3 is able to bind and regulate the expression of OX in living mice remains unclear.…”

Systematic investigation of imprinted gene expression and enrichment in the mouse brain explored at single-cell resolution

“…Ghrh neuron clusters were isolated at the whole-brain level and whole-hypothalamic level and was identified in the POA/ARC but significantly the dopaminergic Ghrh/Gal cluster was identified in both the dopamine-wide and arcuate nucleus datasets. Finally, other neural populations were identified which have previously been associated with imprinted genes including the Hcrt population of the LHA (Seifinejad et al, 2019) identified at the whole brain and LHA level, and the Pomc neurons of the ARC (Cassidy & Charalambous, 2018) identified at the arcuate nucleus level.…”