Martin F. Rath

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.

Night/Day Changes in Pineal Expression of >600 Genes CENTRAL ROLE OF ADRENERGIC/cAMP SIGNALING

The pineal gland plays an essential role in vertebrate chronobiology by converting time into a hormonal signal, melatonin, which is always elevated at night. Here we have analyzed the rodent pineal transcriptome using Affymetrix GeneChip® technology to obtain a more complete description of pineal cell biology. The effort revealed that 604 genes (1,268 probe sets) with Entrez Gene identifiers are differentially expressed greater than 2-fold between midnight and mid-day (false discovery rate <0.20). Expression is greater at night in ∼70%. These findings were supported by the results of radiochemical in situ hybridization histology and quantitative real time-PCR studies. We also found that the regulatory mechanism controlling the night/day changes in the expression of most genes involves norepinephrine-cyclic AMP signaling. Comparison of the pineal gene expression profile with that in other tissues identified 334 genes (496 probe sets) that are expressed greater than 8-fold higher in the pineal gland relative to other tissues. Of these genes, 17% are expressed at similar levels in the retina, consistent with a common evolutionary origin of these tissues. Functional categorization of the highly expressed and/or night/day differentially expressed genes identified clusters that are markers of specialized functions, including the immune/inflammation response, melatonin synthesis, photodetection, thyroid hormone signaling, and diverse aspects of cellular signaling and cell biology. These studies produce a paradigm shift in our understanding of the 24-h dynamics of the pineal gland from one focused on melatonin synthesis to one including many cellular processes.

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.

Expression of the Otx2 homeobox gene in the developing mammalian brain : embryonic and adult expression in the pineal gland

Otx2 is a vertebrate homeobox gene, which has been found to be essential for the development of rostral brain regions and appears to play a role in the development of retinal photoreceptor cells and pinealocytes. In this study, the temporal expression pattern of Otx2 was revealed in the rat brain, with special emphasis on the pineal gland throughout late embryonic and postnatal stages. Widespread high expression of Otx2 in the embryonic brain becomes progressively restricted in the adult to the pineal gland. Crx (cone–rod homeobox), a downstream target gene of Otx2, showed a pineal expression pattern similar to that of Otx2, although there was a distinct lag in time of onset. Otx2 protein was identified in pineal extracts and found to be localized in pinealocytes. Total pineal Otx2 mRNA did not show day–night variation, nor was it influenced by removal of the sympathetic input, indicating that the level of Otx2 mRNA appears to be independent of the photoneural input to the gland. Our results are consistent with the view that pineal expression of Otx2 is required for development and we hypothesize that it plays a role in the adult in controlling the expression of the cluster of genes associated with phototransduction and melatonin synthesis.

Pineal function: Impact of microarray analysis

Microarray analysis has provided a new understanding of pineal function by identifying genes that are highly expressed in this tissue relative to other tissues and also by identifying over 600 genes that are expressed on a 24-h schedule. This effort has highlighted surprising similarity to the retina and has provided reason to explore new avenues of study including intracellular signaling, signal transduction, transcriptional cascades, thyroid/retinoic acid hormone signaling, metal biology, RNA splicing, and the role the pineal gland plays in the immune/inflammation response. The new foundation that microarray analysis has provided will broadly support future research on pineal function.

Developmental and diurnal dynamics of Pax4 expression in the mammalian pineal gland: nocturnal down-regulation is mediated by adrenergic-cyclic adenosine 3',5'-monophosphate signaling.

Pax4 is a homeobox gene that is known to be involved in embryonic development of the endocrine pancreas. In this tissue, Pax4 counters the effects of the related protein, Pax6. Pax6 is essential for development of the pineal gland. In this study we report that Pax4 is strongly expressed in the pineal gland and retina of the rat. Pineal Pax4 transcripts are low in the fetus and increase postnatally; Pax6 exhibits an inverse pattern of expression, being more strongly expressed in the fetus. In the adult the abundance of Pax4 mRNA exhibits a diurnal rhythm in the pineal gland with maximal levels occurring late during the light period. Sympathetic denervation of the pineal gland by superior cervical ganglionectomy prevents the nocturnal decrease in pineal Pax4 mRNA. At night the pineal gland is adrenergically stimulated by release of norepinephrine from the sympathetic innervation; here, we found that treatment with adrenergic agonists suppresses pineal Pax4 expression in vivo and in vitro. This suppression appears to be mediated by cAMP, a second messenger of norepinephrine in the pineal gland, based on the observation that treatment with a cAMP mimic reduces pineal Pax4 mRNA levels. These findings suggest that the nocturnal decrease in pineal Pax4 mRNA is controlled by the sympathetic neural pathway that controls pineal function acting via an adrenergic-cAMP mechanism. The daily changes in Pax4 expression may influence gene expression in the pineal gland.

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.

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.

Expression of the homeobox genes PAX6, OTX2, and OTX1 in the early human fetal retina.

We studied the spatial and temporal expression of the homeobox genes PAX6, OTX2, and OTX1 using a developmental series of human fetal eyes aged from 6 to 10 weeks post‐conception. Previous animal studies have shown that PAX6 may regulate progenitor cell proliferation, timing of differentiation and neural retina cell fate determination. OTX2 may play a role in development of the retinal pigment epithelium and photoreceptor differentiation, whereas OTX1 may be important in ciliary body development.

A standardized surgical technique for rat superior cervical ganglionectomy

Superior cervical ganglionectomy (SCGx) is a valuable microsurgical model to study the role of the sympathetic nervous system in a vast array of physiological and pathological processes, including homeostatic regulation, circadian biology and the dynamics of neuronal dysfunction and recovery after injury. Despite having several experimental applications in the rat, a thorough description of a standardized procedure has never been published. Here, we provide a brief review of the principal features and experimental uses of the SCGx, the surgical anatomy of the neck and sympathetic cervical chain, and a step-by-step description of how to consistently remove the superior cervical ganglia through the omohyoid muscle or the carotid triangle. Furthermore, we suggest procedures and precautions to be taken during and after surgery to optimize results and describe tools to validate surgical success. We expect that the following standardized and optimized protocol will allow researchers to organize knowledge into a cohesive framework in those areas where the SCGx is applied.