Homeobox genes generally encode transcription factors involved in regulating developmental processes. In the pineal gland, a brain structure devoted to nocturnal melatonin synthesis, a number of homeobox genes are also expressed postnatally; among these is the LIM homeobox 4 gene (Lhx4). We here report that Lhx4 is specifically expressed in the postnatal pineal gland of rats and humans. Circadian analyses revealed a fourfold rhythm in Lhx4 expression in the rat pineal gland, with rhythmic expression detectable from postnatal day 10. Pineal Lhx4 expression was confirmed to be positively driven by adrenergic signaling, as evidenced by in vivo modulation of Lhx4 expression by pharmacological (isoprenaline injection) and surgical (superior cervical ganglionectomy) interventions. In cultured pinealocytes, Lhx4 expression was upregulated by cyclic AMP, a second messenger of norepinephrine. By use of RNAscope technology, Lhx4 transcripts were found to be exclusively localized in melatonin-synthesizing pinealocytes. This prompted us to investigate the possible role of Lhx4 in regulation of melatonin-producing enzymes. By use of siRNA technology, we knocked down Lhx4 by 95% in cultured pinealocytes; this caused a reduction in transcripts encoding the melatonin-producing enzyme arylalkylamine N-acetyl transferase (Aanat). Screening the transcriptome of siRNA-treated pinealocytes by RNAseq revealed a significant impact of Lhx4 on the phototransduction pathway and on transcripts involved in development of the nervous system and photoreceptors. These data suggest that rhythmic expression of Lhx4 in the pineal gland is controlled via an adrenergic-cyclic AMP mechanism and that Lhx4 acts to promote nocturnal melatonin synthesis. K E Y W O R D Shomeobox, melatonin, pineal gland, RNA sequencing, RNAscope, siRNA 2 of 12 | HERTZ ET al.

The pineal gland is a neuroendocrine organ responsible for production of the nocturnal hormone melatonin. A specific set of homeobox gene-encoded transcription factors govern pineal development, and some are expressed in adulthood. The brainspecific homeobox gene (Bsx) falls into both categories. We here examined regulation and function of Bsx in the mature pineal gland of the rat. We report that Bsx is expressed from prenatal stages into adulthood, where Bsx transcripts are localized in the melatonin-synthesizing pinealocytes, as revealed by RNAscope in situ hybridization. Bsx transcripts were also detected in the adult human pineal gland. In the rat pineal gland, Bsx was found to exhibit a 10-fold circadian rhythm with a peak at night.By combining in vivo adrenergic stimulation and surgical denervation of the gland in the rat with in vitro stimulation and transcriptional inhibition in cultured pinealocytes, we show that rhythmic expression of Bsx is controlled at the transcriptional level by the sympathetic neural input to the gland acting via adrenergic stimulation with cyclic AMP as a second messenger. siRNA-mediated knockdown (>80% reduction) in pinealocyte cultures revealed Bsx to be a negative regulator of other pineal homeobox genes, including paired box 4 (Pax4), but no effect on genes encoding melatonin-synthesizing enzymes was detected. RNA sequencing analysis performed on siRNA-treated pinealocytes further revealed that downstream target genes of Bsx are mainly involved in developmental processes. Thus, rhythmic Bsx expression seems to govern other developmental regulators in the mature pineal gland. K E Y W O R D SBsx, circadian, homeobox, human, pineal gland, rat, RNA sequencing, RNAscope, siRNA

A molecular circadian oscillator resides in neurons of the cerebral cortex, but its role is unknown. Using the Cre-LoxP method, we have here abolished the core clock gene Arntl in those neurons. This mouse represents the first model carrying a deletion of a circadian clock component specifically in an extrahypothalamic cell type of the brain. Molecular analyses of clock gene expression in the cerebral cortex of the Arntl conditional knockout mouse revealed disrupted circadian expression profiles, whereas clock gene expression in the suprachiasmatic nucleus was still rhythmic, thus showing that Arntl is required for normal function of the cortical circadian oscillator. Daily rhythms in running activity and temperature were not influenced, whereas the resynchronization response to experimental jet-lag exhibited minor though significant differences between genotypes. The tail-suspension test revealed significantly prolonged immobility periods in the knockout mouse indicative of a depressive-like behavioral state. This phenotype was accompanied by reduced norepinephrine levels in the cerebral cortex. Our data show that Arntl is required for normal cortical clock function and further give reason to suspect that the circadian oscillator of the cerebral cortex is involved in regulating both circadian biology and mood-related behavior and biochemistry.

The suprachiasmatic nucleus houses the central circadian clock and is characterized by the timely regulated expression of clock genes. However, neurons of the cerebellar cortex also contain a circadian oscillator with circadian expression of clock genes being controlled by the suprachiasmatic nucleus. It has been suggested that the cerebellar circadian oscillator is involved in food anticipation, but direct molecular evidence of the role of the circadian oscillator of the cerebellar cortex is currently unavailable. To investigate the hypothesis that the circadian oscillator of the cerebellum is involved in circadian physiology and food anticipation, we therefore by use of Cre-LoxP technology generated a conditional knockout mouse with the core clock gene Arntl deleted specifically in granule cells of the cerebellum, since expression of clock genes in the cerebellar cortex is mainly located in this cell type. We here report that deletion of Arntl heavily influences the molecular clock of the cerebellar cortex with significantly altered and arrhythmic expression of other central clock and clock-controlled genes. On the other hand, daily expression of clock genes in the suprachiasmatic nucleus was unaffected. Telemetric registrations in different light regimes did not detect significant differences in circadian rhythms of running activity and body temperature between Arntl conditional knockout mice and controls. Furthermore, food anticipatory behavior did not differ between genotypes. These data suggest that Arntl is an essential part of the cerebellar oscillator; however, the oscillator of the granular layer of the cerebellar cortex does not control traditional circadian parameters or food anticipation.

The pineal gland is a neuroendocrine structure in the brain, which produces and secretes the hormone melatonin at nighttime and is considered a key element in the circadian clock system. Early morphogenesis of the gland is controlled by a number of transcription factors, some of which remain active in adult life. One of these is the brain-specific homeobox (Bsx), a highly conserved homeodomain transcription factor with a developmental role in the pineal gland of several species, including zebrafish, and regulatory roles in mature pinealocytes of the rat. To determine the role of Bsx in circadian