Molecular codes defining lateral hypothalamic sleep regulating neurons

Hypothalamic hypocretin (HCRT) and melanin concentrating hormone (MCH) have multiple functions including sleep and metabolism. How these neuropeptides are produced and involved in divers functions remain unknown. We developed methods to sort and purify HCRT and MCH neurons from mouse hypothalamus. RNA-sequencing revealed key factors of fate determination for HCRT (Peg3, Ahr1, Six6, Nr2f2 and Prrx1) and MCH (Lmx1, Gbx2 and Peg3) neurons. Amongst these, loss of Peg3 in mice significantly reduces HCRT and MCH cell numbers while knock-down of Peg3 ortholog in zebrafish completely abolishes their expression resulting in a two fold increase in sleep. The transcriptome results were used to produce HCRT and MCH neurons from induced pluripotent stem cells (iPSCs) 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. Overall design: Sorted cells mCherry positive (reporter in HCRT cells) versus mCherry negative; and sorted cells EGFP positive (reporter in MCH cells) versus EGFP negative.