Characterization of mWake expression in the murine brain

• Published in: Journal of comparative neurology (1911) Vol. 529; no. 8; pp. 1954 - 1987
• Main Authors: Bell, Benjamin J., Wang, Annette A., Kim, Dong Won, Xiong, Jiali, Blackshaw, Seth, Wu, Mark N.
• Format: Journal Article
• Language: English
• Published: Hoboken, USA John Wiley & Sons, Inc 01.06.2021; Wiley Subscription Services, Inc
• Subjects: Animals; Arousal; Brain - metabolism; Brain stem; circadian; Circadian rhythms; Ependymal cells; Functional anatomy; Gene expression; Hypothalamus; Hypothalamus (dorsomedial); Hypothalamus (lateral); Information processing; Limbic system; Male; Mice; Mice, Inbred C57BL; mRNA; mWAKE; Nerve Tissue Proteins - metabolism; Physiology; Sensory integration; Sleep; Structure-function relationships; Suprachiasmatic nucleus; Tuberomammillary nucleus; WAKE; mWAKE; circadian; WAKE; suprachiasmatic nucleus; hypothalamus
• ISSN: 0021-9967; 1096-9861
• DOI: 10.1002/cne.25066

Structure–function analyses of the mammalian brain have historically relied on anatomically‐based approaches. In these investigations, physical, chemical, or electrolytic lesions of anatomical structures are applied, and the resulting behavioral or physiological responses assayed. An alternative approach is to focus on the expression pattern of a molecule whose function has been characterized and then use genetic intersectional methods to optogenetically or chemogenetically manipulate distinct circuits. We previously identified WIDE AWAKE (WAKE) in Drosophila, a clock output molecule that mediates the temporal regulation of sleep onset and sleep maintenance. More recently, we have studied the mouse homolog, mWAKE/ANKFN1, and our data suggest that its basic role in the circadian regulation of arousal is conserved. Here, we perform a systematic analysis of the expression pattern of mWake mRNA, protein, and cells throughout the adult mouse brain. We find that mWAKE labels neurons in a restricted, but distributed manner, in multiple regions of the hypothalamus (including the suprachiasmatic nucleus, dorsomedial hypothalamus, and tuberomammillary nucleus region), the limbic system, sensory processing nuclei, and additional specific brainstem, subcortical, and cortical areas. Interestingly, mWAKE is also observed in non‐neuronal ependymal cells. In addition, to describe the molecular identities and clustering of mWake+ cells, we provide detailed analyses of single cell RNA sequencing data from the hypothalamus, a region with particularly significant mWAKE expression. These findings lay the groundwork for future studies into the potential role of mWAKE+ cells in the rhythmic control of diverse behaviors and physiological processes. WIDE AWAKE (WAKE) was first identified in Drosophila as a clock‐output molecule that regulates the timing of sleep and arousal. There is a single homolog of WAKE (mWAKE/ANKFN1/NMF9) in mice, which has previously shown to be enriched in the suprachiasmatic nucleus, the master circadian pacemaker. This present work surveys the brain expression pattern of mWAKE using multiple approaches. First, mWake mRNA expression is examined using RNAscope in situ hybridization. Second, mWAKE protein expression is investigated using two different transgenic animal models: an mWakeV5 mouse where a V5 tag is fused directly to the end of the protein and an mWaketdTomato‐2A‐Cre mouse where tdTomato is inserted in‐frame within the gene. Third, we analyze scRNA‐Seq data to characterize the molecular identity of mWake neurons in the hypothalamus. These data reveal that mWAKE labels a distributed subset of GABAergic and glutamatergic neurons across the brainstem, subcortical regions, and cortex, with significant expression in the hypothalamus. This study may provide a potential roadmap for future studies of the circadian regulation of distinct circuits and behaviors.