Circadian regulation and molecular role of the Bsx homeobox gene in the adult pineal gland

Abstract

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

The role of homeobox gene‐encoded transcription factors in regulation of phototransduction: Implementing the primary pinealocyte culture as a photoreceptor model

“…25,26 In addition, available immortalized cell lines related to photoreceptor cells express none or very few of the genes involved in phototransduction, 27,28 making it impossible to use them as a model for determining regulation of phototransduction. Prompted by a recent large-scale analyses of the role of homeobox genes in the pinealocyte of the pineal gland, 20,29 the purpose of the current study was to identify the possible role of homeobox gene-encoded transcription factors in regulation of phototransduction by implementing primary pinealocyte cultures as a cellular photoreceptor model.…”

Genetic ablation of the Bsx homeodomain transcription factor in zebrafish: Impact on mature pineal gland morphology and circadian behavior

“…This study establishes that the Bsx homeodomain transcription factor is essential for function of the pineal gland in adult zebrafish and consequently contributes to maintaining behavioral circadian rhythms in zebrafish larvae. Bsx in the pineal gland has been previously reported in terms of its developmental role during early life of mice, Xenopus frogs, and zebrafish, and its regulatory role in adult rats 4–7 . We here expand current knowledge of Bsx by reporting a morphological absence of the pineal gland and greatly reduced levels of pineal transcripts in adult bsx −/− zebrafish mutants through multiple approaches and establish a role of bsx in circadian biology.…”

“…Unexpectedly, increased levels of the bsx transcript were detected in the pineal area of heterozygotes ( bsx +/− ) fish; this may reflect a compensatory mechanism. In the rat, limitations of a knockout approach were recently overcome by employing siRNA‐mediated knockdown in cell cultures of mature pinealocytes from fully formed glands from adults; in this setup, BSX was shown to control two other homeobox genes, Pax4 and Otx2 , as well as genes mapped to developmental processes, but not those encoding the melatonin‐synthesizing enzymes 6 . This points to an intrinsic regulation mechanism for homeobox genes in the pineal gland, further evidenced by mutual regulation of Crx and Otx2 22,41 .…”

“…Circadian rhythms are endogenous oscillations with a period of approximately 24 h which allow animals to synchronize physiology and behavior to their surroundings; in vertebrates, the neuroendocrine hormone melatonin is produced in the pineal gland during nighttime only 1 . The brain‐specific homeobox gene ( Bsx ), which encodes a conserved homeodomain transcription factor, seems to have functional implications in both early development of the pineal gland and mature phenotype maintenance 2–6 2,7 .…”

Roles of PKCdelta in Photoperiod and Circadian Regulations in Drosophila melanogaster

“…Bsh encodes a putative transcription factor, and it is needed for the specification of neural identities in the adult brain. The rat ortholog of Bsh (Bsx) is suggested to be under circadian regulations and is expressed in the mature pineal gland of rat (Carstensen et al, 2019). Dh31-R1 is Diuretic hormone 31 Receptor which encodes a protein that exhibits diuretic hormone receptor activity, and it is involved in G protein-coupled receptor signaling pathways.…”