Development, Maturation, and Necessity of Transcription Factors in the Mouse Suprachiasmatic Nucleus

Abstract

The suprachiasmatic nucleus (SCN) of the hypothalamus is the master mammalian circadian clock. The SCN is highly specialized as it is responsible for generating a near 24-hour rhythm, integrating external cues, and translating the rhythm throughout the body. Currently, our understanding of the developmental origin and genetic program involved in the proper specification and maturation of the SCN is limited. Herein, we provide a detailed analysis of transcription factor (TF) and developmental-gene expression in the SCN from neurogenesis to adulthood in mice (Mus musculus). TF expression within the postmitotic SCN was not static but rather showed specific temporal and spatial changes during pre- and postnatal development. In addition, we found both global and regional patterns of TF expression extending into the adult. We found the SCN is derived from a distinct region of the neuroepithelium expressing a combination of developmental genes: Six3, Six6, Fzd5, and transient Rx, allowing us to pinpoint the origin of this region within the broader developing telencephalon/diencephalon. We tested the necessity of two TFs in SCN development, RORα and Six3, which were expressed during SCN development, persisted into adulthood, and showed diurnal rhythmicity. Loss of RORα function had no effect on SCN peptide expression or localization. In marked contrast, the conditional deletion of Six3 from early neural progenitors completely eliminated the formation of the SCN. Our results provide the first description of the involvement of TFs in the specification and maturation of a neural population necessary for circadian behavior.

Ontogeny of Circadian Rhythms and Synchrony in the Suprachiasmatic Nucleus

“…The increase in synchrony in the SCN at E15.5 does coincide with Lhx1 expression across the SCN (VanDunk et al, 2011). This transcription factor is important for the expression of diverse neuropeptide coupling signals aside from VIP, such as GRP or AVP (Bedont et al, 2014).…”

“…Inclusion of these extra-SCN regions in explants could explain the discrepancy between the two studies that reported the onset of PER2 daily oscillations in hypothalamic explants at E13 or E15 (Wreschnig et al, 2014;Landgraf et al, 2015). Importantly, the correlations in the increasing fraction of rhythmic cells, increasing circadian period and increasing inter-and intracellular period precision during this time could reflect the consequences of cell-cell communication (Webb et al, 2009) and/or maturation of molecular components for circadian rhythm generation (VanDunk et al, 2011;Ono et al, 2016). Previous studies demonstrated that embryonic SCN have a strong phase-setting in response to perturbations such as surgical isolation (Wreschnig et al, 2014;Landgraf et al, 2015).…”

“…Transcription factors such as Lhx1, Shh, Six3, and Six6 participate in the embryonic specification of SCN lineages (Shimogori et al, 2010;VanDunk et al, 2011;Clark et al, 2013;Bedont et al, 2014). However, the markers of terminal differentiation and synaptogenesis peak postnatally (Moore and Bernstein, 1989;Shimogori et al, 2010).…”

Translational profiling of hypocretin neurons identifies candidate molecules for sleep regulation

“…ISH ISH was performed as described previously (VanDunk et al 2011) on coronal sections of P28 wild-type mouse brains that were immersion-fixed in 4% paraformaldehyde for 20 min and cryoprotected in sucrose before sectioning. Digoxigenin-labeled riboprobe was transcribed with T7 RNA polymerase using the templates described (Supplemental Table S3).…”

Directed Differentiation of V3 Interneurons from Mouse Embryonic Stem Cells

“…Cell cultures were fixed and stained with modifications protocol from previously described [25][26][27]. Cell cultures were fixed in 4% PFA for 10 min and washed thrice in diethylpyrocarbonate (DEPC; Sigma)-treated PBS at room temperature.…”