The Transcription Factors Six3 and Six6 in Neuromedin-S Neurons Differentially Affect Circadian Rhythms

Circadian rhythms are ~24-h rhythms generated by the suprachiasmatic nucleus (SCN) in the mammalian hypothalamus. The regulation of circadian rhythms and downstream processes is highly dependent on the proper development and function of the SCN. Six3 and Six6 are homologous homeodomain transcription factors that have been shown to be required for SCN development; intriguingly, both Six3 and Six6 remain expressed in the adult SCN. To determine the role of Six3 and Six6 in the SCN after neurogenesis, we used Cre-lox to conditionally knockdown either Six3 or Six6 from cells that express neuromedin-S (NMS), a neuropeptide expressed in approximately half of SCN neurons. We found that the Nms^cre allele turns on in the SCN after embryonic Day 16.5, limiting Cre-lox-mediated loss of Six3 or Six6 to the period after SCN neurogenesis. Using this approach, we hypothesized that Six3 and Six6 in NMS neurons regulate SCN circadian output and resulting reproductive function in males and females. Loss of Six6 from NMS neurons had no impact on puberty and reproduction. While loss of Six3 from NMS neurons had no effect in females, we found significantly decreased sperm motility in males, potentially through direct effects of Six3 in the testis. Loss of Six3, but not Six6, in NMS neurons resulted in shortened wheel-running periods in constant darkness, indicating a shortening of the endogenous rhythm within the SCN. Together, these data indicate a role of Six3 in determining the circadian period, suggesting differing functions of Six3 and Six6 in the adult SCN.