The transcription factors SIX3 and VAX1 are required for suprachiasmatic nucleus circadian output and fertility in female mice

Abstract

The homeodomain transcription factors sine oculis homeobox 3 (Six3) and ventral anterior homeobox 1 (Vax1) are required for brain development. Their expression in specific brain areas is maintained in adulthood, where their functions are poorly understood. To identify the roles of Six3 and Vax1 in neurons, we conditionally deleted each gene using Synapsin cre , a promoter targeting maturing neurons, and generated

Deletion of Six3 in post-proliferative neurons produces weakened SCN circadian output, improved metabolic function, and dwarfism in male mice

“…Indeed, as can be observed on the individual actigraphy data from 6 controls and 8 Six3 syn males ( Supplementary Figure 1 ), the activity onset of the controls during DD was relatively variable, suggesting the Six3 fl/fl allele by itself impacts SCN function. Interestingly, as we had previously observed for Six3 syn females [ 25 ], 6/8 Six3 syn males on DD had very variable activity periods, where they initially trended to have a period shorter than 24 h, but after variable times on DD, suddenly switched to a longer Tau. Owing to the low activity profile of some of the Six3 syn males ( Supplementary Figure 1 , Six3 syn ), we were unable to analyze the data from some of the mice.…”

“…We recently showed that the Synapsin cre -allele recombines the Six3 flox -allele throughout the brain including the SCN, impairing SCN circadian output in Six3 syn females [ 25 ]. To determine whether circadian function was also weakened in Six3 syn males, we placed Six3 fl/fl and Six3 syn males on running-wheels ( Figure 1 A).…”

“…To determine whether circadian function was also weakened in Six3 syn males, we placed Six3 fl/fl and Six3 syn males on running-wheels ( Figure 1 A). To limit the number of mice in the male wheel-running study, as it was an extension of our previous study in Six3 syn females, we decided to estimate the number of mice required to reach the lower power of 60% based on chi square amplitude (Qp) values of our previous publication studying wheel-running of Six3 syn females [ 25 ]. Our ad-hoc power analysis indicated that a total of 16 mice would allow us to achieve more than 60% of power.…”

“…To determine whether the reduced SCN circadian precision was associated with changes in the molecular clock within SCN neurons, we analyzed the capacity of SIX3 to modulate the molecular clock genes Per2 and Bmal1 through in vitro transient transfection studies. We recently showed that SIX3 overexpression in vitro increased Per2 - luciferase expression through ATTA-sites in the Per2 regulatory region [ 25 ]. To determine whether SIX3 also regulates Bmal1 expression, we analyzed the Bmal1 regulatory region for potential SIX3 binding sites (ATTA-like sites) and identified five potential SIX3 binding sites ( Supplementary Table 2 ).…”

“…SIX3 is a homeodomain transcription factor required for retinal, forebrain, and pituitary development [ [16] , [17] , [18] , [19] , [20] , [21] , [22] , [23] , [24] ]. After embryonic development, Six3 retains a broad expression pattern in the brain [ 25 ], where it regulates radial glia maturation into ependymal cells [ 26 ], medium spiny neuron plasticity and striatal function [ [27] , [28] , [29] ], hypothalamic kisspeptin neuron function [ 30 ], as well as SCN function [ 25 ]. However, the potential role of Six3 in metabolism remains unexplored.…”