Characterization of mWake expression in the murine brain

Abstract

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.

An Amygdalar Oscillator Coordinates Cellular and Behavioral Rhythms

“…We hypothesized that mWAKE labels brain subregions that represent brain oscillators and focused on the lateral nucleus of the amygdala (LA), because of its importance in emotional processing 22,23 and the relatively strong mWAKE expression in this region 20 . Neuronal tracing experiments and lesion studies suggest the presence of distinct dorsal and ventral LA subregions [24][25][26][27] .…”

“…This categorization was recently refined by a single cell RNA-Seq (scRNA-seq) analysis of the basolateral amygdala (BLA), which defined two major cell groups within the LA: a cluster labeled by Myl4 in the anterior/dorsal subregion (adLA) and a cluster marked by Otof in the posterior/ventral subregion (pvLA) 28 . To characterize mWAKE expression in these LA subregions, we used two transgenic mouse lines 20,29 . In mWake Cre mice, exon 5 of mWake is replaced by a tdTomato-P2A-Cre cassette, which simultaneously labels mWake-positive cells and generates a loss-of-function allele (Figure 1A).…”

“…Wild-type C57BL/6J mice were obtained from the Jackson Laboratory (JAX, 000664), and all mouse strains were backcrossed to this background at least seven times before use in behavioral experiments. The following previously described mouse strains were used in this study: an mWake null allele (mWake - ) 29 , an mWake conditional allele ( mWake flox ) 29 , an mWake allele with a tdTomato-P2A-Cre cassette integrated into exon 5 ( mWake Cre ) 29 , and a tagged mWake allele ( mWake V 5 ) 20 . VGLUT1 Cre mice were obtained from Jackson Laboratory (JAX, 037512).…”

“…We previously identified the clock output molecule WIDE AWAKE (WAKE) in Drosophila and characterized its role in clock neurons, where it upregulates ion channels/pumps at night to promote rhythmic excitability 18,19 . A single homolog of WAKE is present in mice (mWAKE/ANKFN1), which is expressed in the SCN and a distributed set of brain regions, many of which exhibit cycling clock gene expression 5,14,20,21 .…”

“…We previously identified the clock output molecule WIDE AWAKE (WAKE) in Drosophila and characterized its role in clock neurons, where it upregulates ion channels/pumps at night to promote rhythmic excitability 18,19 . A single homolog of WAKE is present in mice (mWAKE/ANKFN1), which is expressed in the SCN and a distributed set of brain regions, many of which exhibit cycling clock gene expression 5,14,20,21 . Because daily rhythms of electrical activity and clock gene expression are thought to be requisite features of extra-SCN oscillators 5,14,15 , we hypothesized that mWAKE defines neural circuits that function as circadian oscillators in the mouse brain.…”