Zfhx3-mediated genetic ablation of the SCN abolishes light entrainable circadian activity while sparing food anticipatory activity.

Circadian rhythms persist in almost all organisms and are crucial for maintaining appropriate timing in physiology and behaviour. Here, we describe a mouse mutant where the central mammalian pacemaker, the suprachiasmatic nucleus (SCN), has been genetically ablated by conditional deletion of the transcription factor Zfhx3 in the developing hypothalamus. Mutants were arrhythmic over the light-dark cycle and in constant darkness. Moreover, rhythms of metabolic parameters were ablated in vivo although molecular oscillations in the liver maintained some rhythmicity. Despite disruptions to SCN cell identity and circuitry, mutants could still anticipate food availability, yet other zeitgebers - including social cues from cage-mates - were ineffective in restoring rhythmicity although activity levels in mutants were altered. This work highlights a critical role for Zfhx3 in the development of a functional SCN, while its genetic ablation further defines the contribution of SCN circuitry in orchestrating physiological and behavioral responses to environmental signals

Circadian characterisation of the transcription factor Zfhx3 utilising a conditional mutagenesis approach

Previous work revealed an important role for the transcription factor ZFHX3 in circadian biology. A dominant missense mutation in the gene (Zfhx3Sci) results in a short circadian period in constant conditions and altered sleep homeostasis. However, constitutive knock-outs of this gene are embryonic lethal, limiting experimental characterisation options. In this thesis, the role of Zfhx3 in circadian rhythm regulation throughout the lifespan of the mouse is examined using conditional mutagenesis to produce Zfhx3 null animals. Inducible deletion of Zfhx3 in adult mice was achieved using a ubiquitously expressed inducible Cre line. These mice displayed significant circadian disruption, namely an acute shortening of circadian period and/or loss of rhythmicity in constant conditions. Following on from this, using SCN-enriched Cre lines driven by Six3 and Foxd1, Zfhx3 was deleted specifically in developing SCN. These animals showed a variety of sleep and circadian phenotypes when assessed. Homozygous mutants of the Six3-Cre line cross displayed a dramatic circadian phenotype; complete behavioural arrhythmia in all lighting conditions and an inability to entrain to a light-dark cycle. Histological examination revealed that the SCN in these mutants fails to mature, as evidenced by loss of expression of key circadian neuropeptides in this region. This work provides evidence for the sustained importance of Zfhx3 in circadian pacemaker function and highlights the use of conditional mutagenesis as an invaluable tool for the study circadian transcription factors. The data presented also suggests that Zfhx3 is crucial for development of the SCN in mice.</p

Circadian characterisation of the transcription factor Zfhx3 utilising a conditional mutagenesis approach

Previous work revealed an important role for the transcription factor ZFHX3 in circadian biology. A dominant missense mutation in the gene (Zfhx3Sci) results in a short circadian period in constant conditions and altered sleep homeostasis. However, constitutive knock-outs of this gene are embryonic lethal, limiting experimental characterisation options. In this thesis, the role of Zfhx3 in circadian rhythm regulation throughout the lifespan of the mouse is examined using conditional mutagenesis to produce Zfhx3 null animals. Inducible deletion of Zfhx3 in adult mice was achieved using a ubiquitously expressed inducible Cre line. These mice displayed significant circadian disruption, namely an acute shortening of circadian period and/or loss of rhythmicity in constant conditions. Following on from this, using SCN-enriched Cre lines driven by Six3 and Foxd1, Zfhx3 was deleted specifically in developing SCN. These animals showed a variety of sleep and circadian phenotypes when assessed. Homozygous mutants of the Six3-Cre line cross displayed a dramatic circadian phenotype; complete behavioural arrhythmia in all lighting conditions and an inability to entrain to a light-dark cycle. Histological examination revealed that the SCN in these mutants fails to mature, as evidenced by loss of expression of key circadian neuropeptides in this region. This work provides evidence for the sustained importance of Zfhx3 in circadian pacemaker function and highlights the use of conditional mutagenesis as an invaluable tool for the study circadian transcription factors. The data presented also suggests that Zfhx3 is crucial for development of the SCN in mice.</p

Inducible Knockout of Mouse Zfhx3 Emphasizes Its Key Role in Setting the Pace and Amplitude of the Adult Circadian Clock

The transcription factor zinc finger homeobox 3 (ZFHX3) plays a key role in coupling intracellular transcriptional-translational oscillations with intercellular synchrony in mouse suprachiasmatic nucleus (SCN). However, like many key players in central nervous system function, ZFHX3 serves an important role in neurulation and neuronal terminal differentiation while retaining discrete additional functions in the adult SCN. Recently, using a dominant missense mutation in mouse Zfhx3, we established that this gene can modify circadian period and sleep in adult animals. Nevertheless, we were still concerned that the neurodevelopmental consequences of ZFHX3 dysfunction in this mutant may interfere with, or confound, its critical adult-specific roles in SCN circadian function. To circumvent the developmental consequences of Zfhx3 deletion, we crossed a conditional null Zfhx3 mutant to an inducible, ubiquitously expressed Cre line (B6.Cg-Tg(UBC-cre/ERT2)1Ejb/J). This enabled us to assess circadian behavior in the same adult animals both before and after Cre-mediated excision of the critical Zfhx3 exons using tamoxifen treatment. Remarkably, we found a strong and significant alteration in circadian behavior in tamoxifen-treated homozygous animals with no phenotypic changes in heterozygous or control animals. Cre-mediated excision of Zfhx3 critical exons in adult animals resulted in shortening of the period of wheel-running in constant darkness by more than 1 h in the majority of homozygotes while, in 30% of animals, excision resulted in complete behavioral arrhythmicity. In addition, we found that homozygous animals reentrain almost immediately to 6-h phase advances in the light-dark cycle. No additional overt phenotypic changes were evident in treated homozygous animals. These findings confirm a sustained and significant role for ZFHX3 in maintaining rhythmicity in the adult mammalian circadian system. </jats:p