A missense mutation in zinc finger homeobox-3 (ZFHX3) impedes growth and alters metabolism and hypothalamic gene expression in mice.

A protein altering variant in the gene encoding zinc finger homeobox-3 (ZFHX3) has recently been associated with lower BMI in a human genome-wide association study. We investigated metabolic parameters in mice harboring a missense mutation in Zfhx3 (Zfhx3Sci/+ ) and looked for altered in situ expression of transcripts that are associated with energy balance in the hypothalamus to understand how ZFHX3 may influence growth and metabolic effects. One-year-old male and female Zfhx3Sci/+ mice weighed less, had shorter body length, lower fat mass, smaller mesenteric fat depots, and lower circulating insulin, leptin, and insulin-like growth factor-1 (IGF1) concentrations than Zfhx3+/+ littermates. In a second cohort of 9-20-week-old males and females, Zfhx3Sci/+ mice ate less than wildtype controls, in proportion to body weight. In a third cohort of female-only Zfhx3Sci/+ and Zfhx3+/+ mice that underwent metabolic phenotyping from 6 to 14 weeks old, Zfhx3Sci/+ mice weighed less and had lower lean mass and energy expenditure, but fat mass did not differ. We detected increased expression of somatostatin and decreased expression of growth hormone-releasing hormone and growth hormone-receptor mRNAs in the arcuate nucleus (ARC). Similarly, ARC expression of orexigenic neuropeptide Y was decreased and ventricular ependymal expression of orphan G protein-coupled receptor Gpr50 was decreased. We demonstrate for the first time an energy balance effect of the Zfhx3Sci mutation, likely by altering expression of key ARC neuropeptides to alter growth, food intake, and energy expenditure.

Zfhx3 modulates retinal sensitivity and circadian responses to light.

Mutations in transcription factors often exhibit pleiotropic effects related to their complex expression patterns and multiple regulatory targets. One such mutation in the zinc finger homeobox 3 (ZFHX3) transcription factor, short circuit (Sci, Zfhx3Sci/+ ), is associated with significant circadian deficits in mice. However, given evidence of its retinal expression, we set out to establish the effects of the mutation on retinal function using molecular, cellular, behavioral and electrophysiological measures. Immunohistochemistry confirms the expression of ZFHX3 in multiple retinal cell types, including GABAergic amacrine cells and retinal ganglion cells including intrinsically photosensitive retinal ganglion cells (ipRGCs). Zfhx3Sci/+ mutants display reduced light responsiveness in locomotor activity and circadian entrainment, relatively normal electroretinogram and optomotor responses but exhibit an unexpected pupillary reflex phenotype with markedly increased sensitivity. Furthermore, multiple electrode array recordings of Zfhx3Sci/+ retina show an increased sensitivity of ipRGC light responses.

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.

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.