Homeobox gene-encoded transcription factors in development and mature circadian function of the rodent pineal gland.

Homeobox genes encode transcription factors that are widely known to control developmental processes. This is also the case in the pineal gland, a neuroendocrine brain structure devoted to nighttime synthesis of the hormone melatonin. Thus, in accordance with high prenatal gene expression, knockout studies have identified a specific set of homeobox genes that are essential for development of the pineal gland. However, as a special feature of the pineal gland, homeobox gene expression persists into adulthood, and gene product abundance exhibits 24 h circadian rhythms. Recent lines of evidence show that some homeobox genes even control expression of enzymes catalyzing melatonin synthesis. We here review current knowledge of homeobox genes in the rodent pineal gland and suggest a model for dual functions of homeobox gene-encoded transcription factors in developmental and circadian mature neuroendocrine function.

Role and neural regulation of clock genes in the rat pineal gland: Clock modulates amplitude of rhythmic expression of Aanat encoding the melatonin-producing enzyme.

Circadian clock gene expression in the suprachiasmatic nucleus (SCN) controls 24 h rhythms in body functions, but clock genes are also expressed in extra-hypothalamic tissues, including the melatonin-producing pineal gland. The nocturnal increase in pineal melatonin synthesis is a hallmark in circadian biology, but the role of local clock gene oscillations in the mammalian pineal gland is unknown. The aim of this work is to determine the role of clock genes in endocrine function of the pineal gland with focus on the Aanat transcript encoding the rhythm-generating enzyme of melatonin synthesis. Using the rat as a model, we here established 24 h expression patterns of clock genes in the pineal gland in vivo. Lesion studies showed that rhythmic clock gene expression in the pineal gland to a large extent depends on the SCN; further, clock gene rhythms could be re-established in cultured pineal cells synchronized by rhythmic stimulation with norepinephrine in 12 h pulses, suggesting that pineal cells house a slave oscillator controlled by adrenergic signaling in the gland. Histological analyses showed that clock genes are expressed in pinealocytes and colocalize with Aanat transcripts, thus potentially enabling clock gene products to control cellular melatonin production. To test this, cultured pineal cells were transfected using small interfering RNA to knock down clock gene expression. While successful knockdown of Per1 had a minor effect on Aanat, Clock knockdown produced a marked overexpression of Aanat in the pinealocytes. Our study suggests that SCN-dependent rhythmic Clock gene expression in the pinealocytes regulates the daily profile of Aanat expression.

The ISL LIM-homeobox 2 transcription factor is negatively regulated by circadian adrenergic signaling to repress the expression of Aanat in pinealocytes of the rat pineal gland.

Melatonin is synthesized in the pineal gland during nighttime in response to nocturnal increase in the activity of the enzyme aralkylamine N-acetyltransferase (AANAT), the transcription of which is modulated by several homeodomain transcription factors. Recent work suggests that the homeodomain transcription factor ISL LIM homeobox 2 (ISL2) is expressed in the pineal gland, but its role is currently unknown. With the purpose of identifying the mechanisms that control pineal expression of Isl2 and the possible function of Isl2 in circadian pineal biology, we report that Isl2 is specifically expressed in the pinealocytes of the rat pineal gland. Its expression exhibits a 24 h rhythm with high transcript and protein levels during the day and a trough in the second half of the night. This rhythm persists in darkness, and lesion studies reveal that it requires intact function of the suprachiasmatic nuclei, suggesting intrinsic circadian regulation. In vivo and in vitro experiments show that pineal Isl2 expression is repressed by adrenergic signaling acting via cyclic AMP; further, Isl2 is negatively regulated by the nocturnal transcription factor cone-rod homeobox. During development, pineal Isl2 expression is detectable from embryonic day 19, preceding Aanat by several days. In vitro knockdown of Isl2 is accompanied by an increase in Aanat transcript levels suggesting that ISL2 represses its daytime expression. Thus, rhythmic expression of ISL2 in pinealocytes is under the control of the suprachiasmatic nucleus acting via adrenergic signaling in the gland to repress nocturnal expression, while ISL2 itself negatively regulates daytime pineal expression of Aanat and thereby suggestively enhances the circadian rhythm in melatonin synthesis.

An ultrastructural study of the deep pineal gland of the Sprague Dawley rat using transmission and serial block face scanning electron microscopy: cell types, barriers, and innervation.

The morphology of the deep pineal gland of the Sprague Dawley rat was investigated by serial block face scanning electron microscopy. Cells were three-dimensionally (3-D) reconstructed using the software Fiji TrackEM. The deep pineal gland consisted of 2-5 layers of electron-lucent pinealocytes, with a euchromatic nucleus, endowed with one or two processes. Laterally, the deep pineal merged with the habenula and the stria medullaris thalami, via an intermediate area containing cells with more electron-dense cytoplasm and an indented nucleus with heterochromatin. Neither nerve terminals nor capillaries were observed in the deep pineal itself but present in the intermediate parts of the gland. The deep pineal was in contact with the third ventricle via the pineal and suprahabenular recesses. The ependymal lining in these recesses was an epithelium connected by tight junctions between their lateral cell membranes. Several intraventricular nerve terminals were in contact with the ependyma. 3-D reconstructions showed the ependymal cells endowed with long slender process penetrating the underlying pineal parenchyma. Few "tanocyte-like" ependymal cells, endowed with a process, reaching the subarachnoid space on the inferior surface of the deep pineal were observed. In addition, pinealocyte and astrocyte processes, often connected by gap junctions, bordered the inferior surface. In summary, the rat deep pineal gland is a neuroendocrine structure connected to the habenula. We here report specialized ependymal cells that might transmit signals from the cerebrospinal fluid to the deep pineal parenchyma and a "trans-pineal tanocyte-like cell" that connects the ventricular system with the subarachnoid space.

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

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 brain-specific 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.

The Lhx4 homeobox transcript in the rat pineal gland: Adrenergic regulation and impact on transcripts encoding melatonin-synthesizing enzymes.

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.

Homeobox genes in melatonin-producing pinealocytes: Otx2 and Crx act to promote hormone synthesis in the mature rat pineal gland.

Homeobox genes encode transcription factors that regulate developmental processes; however, in the pineal gland, a neuroendocrine organ responsible for nocturnal melatonin synthesis, expression of the homeobox genes Otx2 (orthodenticle homeobox 2) and Crx (cone-rod homeobox) persists postnatally. We here show that OTX2 and CRX are exclusively present in melatonin-producing pinealocytes of the rat pineal gland. To understand the roles of Otx2 and Crx in the mature pineal gland, we used siRNA technology in cultured rat pinealocytes with the nocturnal situation mimicked by adding norepinephrine to the culture media. siRNA-induced knockdown of Otx2 was found to reduce expression levels of the enzymes involved in melatonin synthesis at both transcript and protein levels. Similar results were obtained when knocking down Crx. Knocking down Otx2 and Crx simultaneously produced an even larger reduction in both transcript and protein levels of the melatonin-producing enzymes and also reduced the levels of melatonin released to the culture media. These results suggest that Otx2 and Crx, both alone and in combination, act to control pineal melatonin synthesis.

The accessory magnocellular neurosecretory system of the rostral human hypothalamus.

The morphology and neurophysin expression of the magnocellular accessory neuroendocrine system located in the rostral human hypothalamus is investigated in a series of brains obtained at autopsy. The hypothalami were fixed in formalin and embedded in paraffin, or after cryoprotection, frozen for cryostat sectioning. Paraffin sections were either stained with Luxol Fast blue or immunoreacted for neurophysin I or neurophysin II, the precursor molecule for oxytocin and vasopressin. Further, 50-µm-thick serial cryostat sections were immunoreacted with the same antibodies. Both the paraventricular and supraoptic nuclei as well as the hypothalamo-hypophysial tracts exhibited strong immunoreactivity for the neurophysin antibodies. In addition, large collections of immunoreactive accessory magnocellular nuclei and single scattered neurophysin-positive neurons were located in the preoptic region between the paraventricular and supraoptic nucleus among the hypothalamo-hypophysial nerve fibers. In addition, smaller collections of neurophysin-immunoreactive neurons were located in the basal part of this region. Among the accessory magnocellular nuclei, the classical circular nucleus was identified. Accessory magnocellular neurons were often located along the blood vessels and projections of some of these neurons penetrated the vascular endothelium. The accessory magnocellular cell bodies expressed either neurophysin I or neurophysin II immunoreactivity. Summarizing, the accessory magnocellular system in the human brain is large and differs in morphology compared to the system seen in other vertebrates. The neurons of this system contain both vasopressin and oxytocin. Some neurons of the accessory neuronal systems might secrete vasopressin or oxytocin directly into the blood stream.

Circadian changes in long noncoding RNAs in the pineal gland.

Long noncoding RNAs (lncRNAs) play a broad range of biological roles, including regulation of expression of genes and chromosomes. Here, we present evidence that lncRNAs are involved in vertebrate circadian biology. Differential night/day expression of 112 lncRNAs (0.3 to >50 kb) occurs in the rat pineal gland, which is the source of melatonin, the hormone of the night. Approximately one-half of these changes reflect nocturnal increases. Studies of eight lncRNAs with 2- to >100-fold daily rhythms indicate that, in most cases, the change results from neural stimulation from the central circadian oscillator in the suprachiasmatic nucleus (doubling time = 0.5-1.3 h). Light exposure at night rapidly reverses (halving time = 9-32 min) levels of some of these lncRNAs. Organ culture studies indicate that expression of these lncRNAs is regulated by norepinephrine acting through cAMP. These findings point to a dynamic role of lncRNAs in the circadian system.

Circadian oscillators in the mouse brain: molecular clock components in the neocortex and cerebellar cortex.

The circadian timekeeper of the mammalian brain resides in the suprachiasmatic nucleus of the hypothalamus (SCN), and is characterized by rhythmic expression of a set of clock genes with specific 24-h daily profiles. An increasing amount of data suggests that additional circadian oscillators residing outside the SCN have the capacity to generate peripheral circadian rhythms. We have recently shown the presence of SCN-controlled oscillators in the neocortex and cerebellum of the rat. The function of these peripheral brain clocks is unknown, and elucidating this could involve mice with conditional cell-specific clock gene deletions. This prompted us to analyze the molecular clockwork of the mouse neocortex and cerebellum in detail. Here, by use of in situ hybridization and quantitative RT-PCR, we show that clock genes are expressed in all six layers of the neocortex and the Purkinje and granular cell layers of the cerebellar cortex of the mouse brain. Among these, Per1, Per2, Cry1, Arntl, and Nr1d1 exhibit circadian rhythms suggesting that local running circadian oscillators reside within neurons of the mouse neocortex and cerebellar cortex. The temporal expression profiles of clock genes are similar in the neocortex and cerebellum, but they are delayed by 5 h as compared to the SCN, suggestively reflecting a master-slave relationship between the SCN and extra-hypothalamic oscillators. Furthermore, ARNTL protein products are detectable in neurons of the mouse neocortex and cerebellum, as revealed by immunohistochemistry. These findings give reason to further pursue the physiological significance of circadian oscillators in the mouse neocortex and cerebellum.

Homeobox genes in the rodent pineal gland: roles in development and phenotype maintenance.

The pineal gland is a neuroendocrine gland responsible for nocturnal synthesis of melatonin. During early development of the rodent pineal gland from the roof of the diencephalon, homeobox genes of the orthodenticle homeobox (Otx)- and paired box (Pax)-families are expressed and are essential for normal pineal development consistent with the well-established role that homeobox genes play in developmental processes. However, the pineal gland appears to be unusual because strong homeobox gene expression persists in the pineal gland of the adult brain. Accordingly, in addition to developmental functions, homeobox genes appear to be key regulators in postnatal phenotype maintenance in this tissue. In this paper, we review ontogenetic and phylogenetic aspects of pineal development and recent progress in understanding the involvement of homebox genes in rodent pineal development and adult function. A working model is proposed for understanding the sequential action of homeobox genes in controlling development and mature circadian function of the mammalian pinealocyte based on knowledge from detailed developmental and daily gene expression analyses in rats, the pineal phenotypes of homebox gene-deficient mice and studies on development of the retinal photoreceptor; the pinealocyte and retinal photoreceptor share features not seen in other tissues and are likely to have evolved from the same ancestral photodetector cell.

Developmental and diurnal expression of the synaptosomal-associated protein 25 (Snap25) in the rat pineal gland.

Snap25 (synaptosomal-associated protein) is a 25 kDa protein, belonging to the SNARE-family (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) of proteins, essential for synaptic and secretory vesicle exocytosis. Snap25 has by immunohistochemistry been demonstrated in the rat pineal gland but the biological importance of this is unknown. In this study, we demonstrate a high expression of mRNA encoding Snap25 in all parts of the rat pineal complex, the superficial-, and deep-pineal gland, as well as in the pineal stalk. Snap25 showed a low pineal expression during embryonic stages with a strong increase in expression levels just after birth. The expression showed no day/night variations. Neither removal of the sympathetic input to the pineal gland by superior cervical ganglionectomy nor bilateral decentralization of the superior cervical ganglia significantly affected the expression of Snap25 in the gland. The pineal expression levels of Snap25 were not changed following intraperitoneal injection of isoproterenol. The strong expression of Snap25 in the pineal gland suggests the presence of secretory granules and microvesicles in the rat pinealocyte supporting the concept of a vesicular release. At the transcriptional level, this Snap25-based release mechanism does not exhibit any diurnal rhythmicity and is regulated independently of the sympathetic nervous input to the gland.

Rax : developmental and daily expression patterns in the rat pineal gland and retina.

Retina and anterior neural fold homeobox (Rax) gene encodes a transcription factor essential for vertebrate eye development. Recent microarray studies indicate that Rax is expressed in the adult rat pineal gland and retina. The present study reveals that Rax expression levels in the rat change significantly during retinal development with a peak occurring at embryonic day 18, whereas Rax expression in the pineal is relatively delayed and not detectable until embryonic day 20. In both tissues, Rax is expressed throughout postnatal development into adulthood. In the mature rat pineal gland, the abundance of Rax transcripts increases 2-fold during the light period with a peak occurring at dusk. These findings are consistent with the evidence that Rax is of functional importance in eye development and suggest a role of Rax in the developing pineal gland. In addition, it would appear possible that Rax contributes to phenotype maintenance in the mature retina and pineal gland and may facilitate 24-h changes in the pineal transcriptome.

Crx broadly modulates the pineal transcriptome.

Cone-rod homeobox (Crx) encodes Crx, a transcription factor expressed selectively in retinal photoreceptors and pinealocytes, the major cell type of the pineal gland. In this study, the influence of Crx on the mammalian pineal gland was studied by light and electron microscopy and by use of microarray and qRTPCR technology, thereby extending previous studies on selected genes (Furukawa et al. 1999). Deletion of Crx was not found to alter pineal morphology, but was found to broadly modulate the mouse pineal transcriptome, characterized by a>2-fold down-regulation of 543 genes and a>2-fold up-regulation of 745 genes (p<0.05). Of these, one of the most highly up-regulated (18-fold) was Hoxc4, a member of the Hox gene family, members of which are known to control gene expression cascades. During a 24-h period, a set of 51 genes exhibited differential day/night expression in pineal glands of wild-type animals; only eight of these were also day/night expressed in the Crx⁻/⁻ pineal gland. However, in the Crx⁻/⁻ pineal gland 41 genes exhibited differential night/day expression that was not seen in wild-type animals. These findings indicate that Crx broadly modulates the pineal transcriptome and also influences differential night/day gene expression in this tissue. Some effects of Crx deletion on the pineal transcriptome might be mediated by Hoxc4 up-regulation.

Global daily dynamics of the pineal transcriptome.

Transcriptome profiling of the pineal gland has revealed night/day differences in the expression of a major fraction of the genes active in this tissue, with two-thirds of these being nocturnal increases. A set of over 600 transcripts exhibit two-fold to >100-fold daily differences in abundance. These changes appear to be primarily attributable to adrenergic-cyclic-AMP-dependent mechanisms, which are controlled via a neural pathway that includes the suprachiasmatic nucleus, the master circadian oscillator. In addition to melatonin synthesis, night/day differences in gene expression impact genes associated with several specialized functions, including the immune/inflammation response, photo-transduction, and thyroid hormone/retinoic acid biology. The following nonspecialized cellular features are also affected: adhesion, cell cycle/cell death, cytoskeleton, DNA modification, endothelium, growth, RNA modification, small molecule biology, transcription factors, vesicle biology, signaling involving Ca(2+), cyclic nucleotides, phospholipids, mitogen-activated protein kinases, the Wnt signaling pathway, and protein phosphorylation.

MicroRNA expression in the adult mouse central nervous system.

MicroRNAs are approximately 22 nucleotide endogenous noncoding RNAs that post-transcriptionally repress expression of protein-coding genes by base-pairing with the 3'-untranslated regions of the target mRNAs. We present here an inventory of miRNA expression profiles from 13 neuroanatomically distinct areas of the adult mouse central nervous system (CNS). Microarray profiling in combination with real-time RT-PCR and LNA (locked nucleic acid)-based in situ hybridization uncovered 44 miRNAs displaying more than threefold enrichment in the spinal cord, cerebellum, medulla oblongata, pons, hypothalamus, hippocampus, neocortex, olfactory bulb, eye, and pituitary gland. These findings suggest that a large number of mouse CNS-expressed miRNAs may be associated with specific functions within these regions. Notably, more than 50% of the identified mouse CNS-enriched miRNAs showed different expression patterns compared to those reported in zebrafish, although the mature miRNA sequences are nearly 100% conserved between the two vertebrate species. The inventory of miRNA profiles in the adult mouse CNS presented here provides an important step toward further elucidation of miRNA function and miRNA-related gene regulatory networks in the mammalian central nervous system.

Muscleblind-like 2: circadian expression in the mammalian pineal gland is controlled by an adrenergic-cAMP mechanism.

Muscleblind-like 2 (Mbnl2) is a zinc finger protein first identified in Drosophila. It appears to be essential for photoreceptor development and to be involved in RNA splicing. Here we report that Mbnl2 is strongly expressed in the rat pineal gland. The abundance of pineal Mbnl2 transcripts follows a marked circadian rhythm with peak levels approximately sevenfold higher at night than day levels. Mbnl2 protein exhibits a similar rhythm. In vitro studies indicate that the abundance of Mbnl2 transcripts and protein are controlled by an adrenergic/cAMP mechanism.

Developmental and daily expression of the Pax4 and Pax6 homeobox genes in the rat retina: localization of Pax4 in photoreceptor cells.

Pax4 is a homeobox gene encoding Pax4, a transcription factor that is essential for embryonic development of the endocrine pancreas. In the pancreas, Pax4 counters the effects of the related transcription factor, Pax6, which is known to be essential for eye morphogenesis. In this study, we have discovered that Pax4 is strongly expressed in retinal photoreceptors of the rat. Pax4 expression is not detectable in the foetal eye; however, postnatal Pax4 transcript levels rapidly increase. In contrast, Pax6 exhibits an inverse developmental pattern of expression being more strongly expressed in the foetal eye. Histological analysis revealed that Pax4 mRNA is exclusively expressed in the retinal photoreceptors, whereas Pax6 mRNA and protein are present in the inner nuclear layer and in the ganglion cell layer of the mature retina. In the adult retina, Pax4 transcripts exhibit a diurnal rhythm with maximal levels occurring during the light period, whereas retinal Pax6 transcript levels do not change throughout the day. The daily changes in Pax4 expression may contribute to daily changes in function in the differentiated retinal photoreceptor.

Single Cell Sequencing of the Pineal Gland: The Next Chapter.

The analysis of pineal cell biology has undergone remarkable development as techniques have become available which allow for sequencing of entire transcriptomes and, most recently, the sequencing of the transcriptome of individual cells. Identification of at least nine distinct cell types in the rat pineal gland has been made possible, allowing identification of the precise cells of origin and expression of transcripts for the first time. Here the history and current state of knowledge generated by these transcriptomic efforts is reviewed, with emphasis on the insights suggested by the findings.

Localization and regulation of dopamine receptor D4 expression in the adult and developing rat retina.

Levels of dopamine and melatonin exhibit diurnal rhythms in the rat retina. Dopamine is high during daytime adapting the retina to light, whereas melatonin is high during nighttime participating in the adaptation of the retina to low light intensities. Dopamine inhibits the synthesis of melatonin in the photoreceptors via Drd4 receptors located on the cell membrane of these cells. In this study, we show by semiquantitative in situ hybridization a prominent day/night variation in Drd4 expression in the retina of the Sprague-Dawley rat with a peak during the nighttime. Drd4 expression is seen in all retinal layers but the nocturnal increase is confined to the photoreceptors. Retinal Drd4 expression is not affected by removal of the sympathetic input to the eye, but triiodothyronine treatment induces Drd4 expression in the photoreceptors. In a developmental series, we show that the expression of Drd4 is restricted to postnatal stages with a peak at postnatal day 12. The high Drd4 expression in the rat retinal photoreceptors during the night supports physiological and pharmacologic evidence that the Drd4 receptor is involved in the dopaminergic inhibition of melatonin synthesis upon light stimulation. The sharp increase of Drd4 expression at a specific postnatal time suggests that dopamine is involved in retinal development.