The Transcription Factor Ventral Anterior Homeobox 1 Modulates Circadian Time-Keeping and Fertility Through Direct Regulation of Vasoactive Intestinal Polypeptide Expression in the Suprachiasmatic Nucleus

Abstract

Light provides the primary timing signal that enables fine-tuned behavioral and hormonal entrainment of circadian rhythms to the environment. Light is transmitted from the eye to the brain through the retinohypothalamic tract, where one target is the hypothalamic suprachiasmatic nucleus (SCN), which generates self-sustained circadian rhythms. The vasoactive intestinal polypeptide (VIP) expressing neurons of the SCN relay light information to peripheral cells and tissues through control of hormonal and nervous signals, allowing synchronization of molecular clocks located in individual cells throughout the body. Non-natural light cycles, ie shiftwork, and weakened SCN function through genetic manipulation, disrupt the body’s circadian rhythms, causing deregulated hormone release, metabolic disorders, and negative effects on reproductive systems such as irregular menstrual cycles and decreased sperm count. To further our understanding of how the SCN translates light information into neuroendocrine control of fertility, we conditionally deleted the SCN enriched transcription factor Ventral anterior homeobox 1 (Vax1) in post-developmental VIP neurons, generating Vax1-flox/flox:Vip-Cre+ (cKO) mice. To determine if the SCN timekeeping function was impacted in cKO mice, we single housed males and females with running wheels to examine activity during both 12-hour light/dark cycles and in constant darkness. Wheel-running behavior in constant darkness revealed a shortening of the endogenous free-running period (Tau) of the SCN. Aside from Tau, wheel running behaviors were comparable to controls. Weakened SCN output can negatively impact fertility. While on 12-hour light/dark cycles, we found a modest, but significant change in follicle stimulating hormone and estrogen in cKO females and a reduced sensitivity of GnRH neurons to kisspeptin in males. The changes in hormone release were associated with a slightly lengthened estrous cycle in cKO females and reduced sperm quality in cKO males. To identify the molecular origin of the shortened SCN period, we used immunohistochemistry and RNAscope to examine expression of Vip. We found that diestrus cKO females had a significant reduction in Vip expression at ZT16 and preliminary data suggest a reduction in the circadian clock gene Bmal1. Together, these studies identify a novel role of VAX1 in VIP neurons where VAX1 is required for VIP expression and circadian timekeeping. Loss of VAX1 in VIP neurons weakens SCN output, deregulating reproductive hormone release and modestly reducing reproductive function in both males and females.