ABSTRACT
Variants of UNC13A, a critical gene for synapse function, increase the risk of amyotrophic lateral sclerosis and frontotemporal dementia1-3, two related neurodegenerative diseases defined by mislocalization of the RNA-binding protein TDP-434,5. Here we show that TDP-43 depletion induces robust inclusion of a cryptic exon in UNC13A, resulting in nonsense-mediated decay and loss of UNC13A protein. Two common intronic UNC13A polymorphisms strongly associated with amyotrophic lateral sclerosis and frontotemporal dementia risk overlap with TDP-43 binding sites. These polymorphisms potentiate cryptic exon inclusion, both in cultured cells and in brains and spinal cords from patients with these conditions. Our findings, which demonstrate a genetic link between loss of nuclear TDP-43 function and disease, reveal the mechanism by which UNC13A variants exacerbate the effects of decreased TDP-43 function. They further provide a promising therapeutic target for TDP-43 proteinopathies.
Subject(s)
Amyotrophic Lateral Sclerosis , Frontotemporal Dementia , TDP-43 Proteinopathies , Alternative Splicing , Amyotrophic Lateral Sclerosis/genetics , Amyotrophic Lateral Sclerosis/metabolism , Codon, Nonsense , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Frontotemporal Dementia/genetics , Frontotemporal Dementia/metabolism , Humans , Nerve Tissue Proteins , Polymorphism, Single Nucleotide/geneticsABSTRACT
La-related proteins (LARPs) comprise a family of RNA-binding proteins involved in a wide range of posttranscriptional regulatory activities. LARPs share a unique tandem of two RNA-binding domains, La motif (LaM) and RNA recognition motif (RRM), together referred to as a La-module, but vary in member-specific regions. Prior structural studies of La-modules reveal they are pliable platforms for RNA recognition in diverse contexts. Here, we characterize the La-module of LARP1, which plays an important role in regulating synthesis of ribosomal proteins in response to mTOR signaling and mRNA stabilization. LARP1 has been well characterized functionally but no structural information exists for its La-module. We show that unlike other LARPs, the La-module in LARP1 does not contain an RRM domain. The LaM alone is sufficient for binding poly(A) RNA with submicromolar affinity and specificity. Multiple high-resolution crystal structures of the LARP1 LaM domain in complex with poly(A) show that it is highly specific for the RNA 3'-end, and identify LaM residues Q333, Y336 and F348 as the most critical for binding. Use of a quantitative mRNA stabilization assay and poly(A) tail-sequencing demonstrate functional relevance of LARP1 RNA binding in cells and provide novel insight into its poly(A) 3' protection activity.
Subject(s)
Autoantigens , Ribonucleoproteins , Ribonucleoproteins/metabolism , Autoantigens/genetics , RNA, Messenger/metabolism , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Poly A/metabolism , RNA/genetics , RNA/metabolism , Protein BindingABSTRACT
The mammalian pituitary gland is a complex organ consisting of hormone-producing cells, anterior lobe folliculostellate cells (FSCs), posterior lobe pituicytes, vascular pericytes and endothelial cells, and Sox2-expressing stem cells. We present single-cell RNA sequencing and immunohistofluorescence analyses of pituitary cells of adult female rats with a focus on the transcriptomic profiles of nonhormonal cell types. Samples obtained from whole pituitaries and separated anterior and posterior lobe cells contained all expected pituitary resident cell types and lobe-specific vascular cell subpopulations. FSCs and pituicytes expressed S100B, ALDOC, EAAT1, ALDH1A1, and VIM genes and proteins, as well as other astroglial marker genes, some common and some cell type-specific. We also found that the SOX2 gene and protein were expressed in ~15% of pituitary cells, including FSCs, pituicytes, and a fraction of hormone-producing cells, arguing against its stem cell specificity. FSCs comprised two Sox2-expressing subclusters; FS1 contained more cells but lower genetic diversity, while FS2 contained proliferative cells, shared genes with hormone-producing cells, and expressed genes consistent with stem cell niche formation, regulation of cell proliferation and stem cell pluripotency, including the Hippo and Wnt pathways. FS1 cells were randomly distributed in the anterior and intermediate lobes, while FS2 cells were localized exclusively in the marginal zone between the anterior and intermediate lobes. These data indicate the identity of the FSCs as anterior pituitary-specific astroglia, with FS1 cells representing differentiated cells equipped for classical FSC roles and FS2 cells exhibiting additional stem cell-like features.
Subject(s)
Pituitary Gland, Anterior , Rats , Female , Animals , Pituitary Gland, Anterior/metabolism , Astrocytes , Endothelial Cells , Stem Cells , Hormones/metabolism , MammalsABSTRACT
Infantile neuronal ceroid lipofuscinosis (INCL), also known as CLN1-disease, is a devastating neurodegenerative lysosomal storage disorder (LSD), caused by inactivating mutations in the CLN1 gene. The Cln1-/- mice, which mimic INCL, manifest progressive neuroinflammation contributing to neurodegeneration. However, the underlying mechanism of neuroinflammation in INCL and in Cln1-/- mice has remained elusive. Previously, it has been reported that microRNA-155 (miR-155) regulates inflammation and miR profiling in Cln1-/- mouse brain showed that the level of miR-155 was upregulated. Thus, we sought to determine whether ablation of miR-155 in Cln1-/- mice may suppress neuroinflammation in these mice. Towards this goal, we generated Cln1-/-/miR-155-/- double-knockout mice and evaluated the inflammatory signatures in the brain. We found that the brains of double-KO mice manifest progressive neuroinflammatory changes virtually identical to those found in Cln1-/- mice. We conclude that ablation of miR-155 in Cln1-/- mice does not alter the neuroinflammatory trajectory in INCL mouse model.
Subject(s)
Disease Models, Animal , Inflammation/metabolism , MicroRNAs/metabolism , Thiolester Hydrolases/metabolism , Animals , Inflammation/genetics , Mice , Mice, Knockout , MicroRNAs/genetics , Thiolester Hydrolases/deficiency , Thiolester Hydrolases/geneticsABSTRACT
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.
Subject(s)
Circadian Rhythm/physiology , Gene Expression Regulation, Developmental/physiology , Melatonin/metabolism , Nerve Tissue Proteins/biosynthesis , Pineal Gland/metabolism , Transcription Factors/biosynthesis , Animals , Female , Male , Pineal Gland/cytology , Rats, Sprague-DawleyABSTRACT
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.
Subject(s)
LIM-Homeodomain Proteins , Melatonin/metabolism , Pineal Gland , Transcription Factors , Transcriptome/genetics , Adult , Animals , Arylalkylamine N-Acetyltransferase/genetics , Arylalkylamine N-Acetyltransferase/metabolism , Circadian Rhythm/genetics , Cyclic AMP/metabolism , Female , Humans , LIM-Homeodomain Proteins/genetics , LIM-Homeodomain Proteins/metabolism , Male , Melatonin/genetics , Middle Aged , Norepinephrine/metabolism , Pineal Gland/chemistry , Pineal Gland/cytology , Pineal Gland/growth & development , Pineal Gland/metabolism , RNA, Small Interfering/genetics , Rats , Rats, Sprague-Dawley , Transcription Factors/genetics , Transcription Factors/metabolism , Young AdultABSTRACT
The website and database https://snengs.nichd.nih.gov provides RNA sequencing data from multi-species analysis of the pineal glands from zebrafish (Danio rerio), chicken (White Leghorn), rat (Rattus novegicus), mouse (Mus musculus), rhesus macaque (Macaca mulatta), and human (Homo sapiens); in most cases, retinal data are also included along with results of the analysis of a mixture of RNA from tissues. Studies cover day and night conditions; in addition, a time series over multiple hours, a developmental time series and pharmacological experiments on rats are included. The data have been uniformly re-processed using the latest methods and assemblies to allow for comparisons between experiments and to reduce processing differences. The website presents search functionality, graphical representations, Excel tables, and track hubs of all data for detailed visualization in the UCSC Genome Browser. As more data are collected from investigators and improved genomes become available in the future, the website will be updated. This database is in the public domain and elements can be reproduced by citing the URL and this report. This effort makes the results of 21st century transcriptome profiling widely available in a user-friendly format that is expected to broadly influence pineal research.
Subject(s)
Databases, Nucleic Acid , Gene Expression Regulation , Internet , Pineal Gland/metabolism , Retina/metabolism , Animals , Chickens , Humans , Macaca mulatta , Mice , Rats , ZebrafishABSTRACT
Melatonin (N-acetyl-5-methoxytrypamine) is the vertebrate hormone of the night: circulating levels at night are markedly higher than day levels. This increase is driven by precisely regulated increases in acetylation of serotonin in the pineal gland by arylalkylamine N-acetyltransferase (AANAT), the penultimate enzyme in the synthesis of melatonin. This unique essential role of AANAT in vertebrate timekeeping is recognized by the moniker the timezyme. AANAT is also found in the retina, where melatonin is thought to play a paracrine role. Here, we focused on the evolution of AANAT in early vertebrates. AANATs from Agnathans (lamprey) and Chondrichthyes (catshark and elephant shark) were cloned, and it was found that pineal glands and retinas from these groups express a form of AANAT that is compositionally, biochemically, and kinetically similar to AANATs found in bony vertebrates (VT-AANAT). Examination of the available genomes indicates that VT-AANAT is absent from other forms of life, including the Cephalochordate amphioxus. Phylogenetic analysis and evolutionary rate estimation indicate that VT-AANAT evolved from the nonvertebrate form of AANAT after the Cephalochordate-Vertebrate split over one-half billion years ago. The emergence of VT-AANAT apparently involved a dramatic acceleration of evolution that accompanied neofunctionalization after a duplication of the nonvertebrate AANAT gene. This scenario is consistent with the hypotheses that the advent of VT-AANAT contributed to the evolution of the pineal gland and lateral eyes from a common ancestral photodetector and that it was not a posthoc recruitment.
Subject(s)
Arylalkylamine N-Acetyltransferase/genetics , Evolution, Molecular , Gene Expression Regulation, Enzymologic , Melatonin/chemistry , Amino Acid Sequence , Animals , Gene Library , Humans , Lampreys , Likelihood Functions , Molecular Sequence Data , Photoreceptor Cells, Vertebrate/physiology , Phylogeny , Pineal Gland/physiology , Protein Conformation , Retina/physiology , Sequence Homology, Amino Acid , Sharks , Sheep , Time Factors , VertebratesABSTRACT
Melatonin is a multifunctional bioactive molecule that plays comprehensive physiological roles in all living organisms. N-acetylserotonin methyltransferase (ASMT, also known as hydroxyindole O-methyltransferase or HIOMT) is the final enzyme for biosynthesis of melatonin. Here, we performed a comparative genomic and transcriptomic survey to explore the ASMT family in fish. Two ASMT isotypes (ASMT1 and ASMT2) and a new ASMT-like (ASMTL) are all extracted from teleost genomes on the basis of phylogenetic and synteny analyses. We confirmed that C-terminal of the ASMTL proteins (ASMTL-ASMT) is homology to the full length of ASMT1 and ASMT2. Our results also demonstrate that the two ASMT isotypes and their distribution in teleosts seem to be the result of combinations of whole-genome duplication (WGD) and gene loss. Differences were also observed in tissue distribution and relative transcript abundances of ASMT1, ASMT2 and ASMTL through transcriptomic analysis. Protein sequence alignment and 3D structure prediction of ASMTs and ASMTL suggest differential roles for these ASMT genes. In summary, our current work provides novel insights into the ASMT genes in fish by combination of genomic and transcriptomic data.
Subject(s)
Acetylserotonin O-Methyltransferase , Fishes , Genomics , Transcriptome , Acetylserotonin O-Methyltransferase/genetics , Acetylserotonin O-Methyltransferase/metabolism , Animals , Melatonin/biosynthesis , Melatonin/geneticsABSTRACT
A wide variety of biochemical, physiological, and molecular processes are known to have daily rhythms driven by an endogenous circadian clock. While extensive research has greatly improved our understanding of the molecular mechanisms that constitute the circadian clock, the links between this clock and dependent processes have remained elusive. To address this gap in our knowledge, we have used RNA sequencing (RNA-seq) and DNA microarrays to systematically identify clock-controlled genes in the zebrafish pineal gland. In addition to a comprehensive view of the expression pattern of known clock components within this master clock tissue, this approach has revealed novel potential elements of the circadian timing system. We have implicated one rhythmically expressed gene, camk1gb, in connecting the clock with downstream physiology of the pineal gland. Remarkably, knockdown of camk1gb disrupts locomotor activity in the whole larva, even though it is predominantly expressed within the pineal gland. Therefore, it appears that camk1gb plays a role in linking the pineal master clock with the periphery.
Subject(s)
Circadian Clocks , Circadian Rhythm/genetics , Pineal Gland , Zebrafish Proteins , Animals , Circadian Clocks/genetics , Circadian Clocks/physiology , Circadian Rhythm/physiology , Gene Expression Regulation , Gene Knockdown Techniques , Larva/genetics , Larva/growth & development , Oligonucleotide Array Sequence Analysis , Pineal Gland/growth & development , Pineal Gland/metabolism , Pineal Gland/physiology , Sequence Analysis, RNA , Zebrafish/genetics , Zebrafish/growth & development , Zebrafish Proteins/genetics , Zebrafish Proteins/metabolism , Zebrafish Proteins/physiologyABSTRACT
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.
Subject(s)
Circadian Rhythm/genetics , Pineal Gland/metabolism , RNA, Untranslated/genetics , Animals , Bucladesine/pharmacology , Circadian Rhythm/drug effects , Computational Biology , Gene Expression Regulation/drug effects , Nervous System/drug effects , Nervous System/metabolism , Norepinephrine/pharmacology , Pineal Gland/drug effects , RNA, Untranslated/metabolism , Rats , Rats, Sprague-Dawley , Synapses/drug effects , Synapses/metabolismABSTRACT
All living organisms synchronize biological functions with environmental changes; melatonin plays a vital role in regulating daily and seasonal variations. Due to rhythmic activity of the timezyme aralkylamine N-acetyltransferase (AANAT), the blood level of melatonin increases at night and decreases during daytime. Whereas other vertebrates have a single form of AANAT, bony fishes possess various isoforms of aanat genes, though the reasons are still unclear. Here, we have taken advantage of multiple unpublished teleost aanat sequences to explore and expand our understanding of the molecular evolution of aanat in fish. Our results confirm that two rounds of whole-genome duplication (WGD) led to the existence of three fish isoforms of aanat, i.e., aanat1a, aanat1b, and aanat2; in addition, gene loss led to the absence of some forms from certain special fish species. Furthermore, we suggest the different roles of two aanat1s in amphibious mudskippers, and speculate that the loss of aanat1a, may be related to terrestrial vision change. Several important sites of AANAT proteins and regulatory elements of aanat genes were analyzed for structural comparison and functional forecasting, respectively, which provides insights into the molecular evolution of the differences between AANAT1 and AANAT2.
Subject(s)
Arylalkylamine N-Acetyltransferase/genetics , Evolution, Molecular , Fish Proteins/genetics , Fishes/genetics , Amino Acid Sequence , Animals , Arylalkylamine N-Acetyltransferase/chemistry , Fish Proteins/chemistry , Gene Dosage , Genome , Molecular Sequence Data , Phylogeny , Protein Isoforms/chemistry , Protein Isoforms/genetics , Sequence AlignmentABSTRACT
MicroRNAs (miRNAs) play a broad range of roles in biological regulation. In this study, rat pineal miRNAs were profiled for the first time, and their importance was evaluated by focusing on the main function of the pineal gland, melatonin synthesis. Massively parallel sequencing and related methods revealed the miRNA population is dominated by a small group of miRNAs as follows: ~75% is accounted for by 15 miRNAs; miR-182 represents 28%. In addition to miR-182, miR-183 and miR-96 are also highly enriched in the pineal gland, a distinctive pattern also found in the retina. This effort also identified previously unrecognized miRNAs and other small noncoding RNAs. Pineal miRNAs do not exhibit a marked night/day difference in abundance with few exceptions (e.g. 2-fold night/day differences in the abundance of miR-96 and miR-182); this contrasts sharply with the dynamic 24-h pattern that characterizes the pineal transcriptome. During development, the abundance of most pineal gland-enriched miRNAs increases; however, there is a marked decrease in at least one, miR-483. miR-483 is a likely regulator of melatonin synthesis, based on the following. It inhibits melatonin synthesis by pinealocytes in culture; it acts via predicted binding sites in the 3"-UTR of arylalkylamine N-acetyltransferase (Aanat) mRNA, the penultimate enzyme in melatonin synthesis, and it exhibits a developmental profile opposite to that of Aanat transcripts. Additionally, a miR-483 targeted antagonist increased melatonin synthesis in neonatal pinealocytes. These observations support the hypothesis that miR-483 suppresses Aanat mRNA levels during development and that the developmental decrease in miR-483 abundance promotes melatonin synthesis.
Subject(s)
3' Untranslated Regions/physiology , Arylalkylamine N-Acetyltransferase/biosynthesis , Melatonin/biosynthesis , MicroRNAs/metabolism , Pineal Gland/metabolism , Animals , Cells, Cultured , HEK293 Cells , Humans , Organ Specificity/physiology , Pineal Gland/cytology , Pineal Gland/growth & development , Rats , Rats, Sprague-DawleyABSTRACT
Introduction: Pediatric obesity has steadily increased in recent decades. Large-scale genome-wide association studies (GWAS) conducted primarily in Eurocentric adult populations have identified approximately 100 loci that predispose to obesity and type II diabetes. GWAS in children and individuals of non-European descent, both disproportionately affected by obesity, are fewer. Rare syndromic and monogenic obesities account for only a small portion of childhood obesity, so understanding the role of other genetic variants and their combinations in heritable obesities is key to developing targeted and personalized therapies. Tight and responsive regulation of the cAMP-dependent protein kinase (PKA) signaling pathway is crucial to maintaining healthy energy metabolism, and mutations in PKA-linked genes represent the most common cause of monogenic obesity. Methods: For this study, we performed targeted exome sequencing of 53 PKA signaling-related genes to identify variants in genomic DNA from a large, ethnically diverse cohort of obese or metabolically challenged youth. Results: We confirmed 49 high-frequency variants, including a novel variant in the PDE11A gene (c.152C>T). Several other variants were associated with metabolic characteristics within ethnic groups. Discussion: We conclude that a PKA pathway-specific variant search led to the identification of several new genetic associations with obesity in an ethnically diverse population.
Subject(s)
Diabetes Mellitus, Type 2 , Pediatric Obesity , Adult , Adolescent , Humans , Child , Pediatric Obesity/genetics , Genome-Wide Association Study , MutationABSTRACT
NeuroD1 encodes a basic helix-loop-helix transcription factor involved in the development of neural and endocrine structures, including the retina and pineal gland. To determine the effect of NeuroD1 knockout in these tissues, a Cre/loxP recombination strategy was used to target a NeuroD1 floxed gene and generate NeuroD1 conditional knockout (cKO) mice. Tissue specificity was conferred using Cre recombinase expressed under the control of the promoter of Crx, which is selectively expressed in the pineal gland and retina. At 2 months of age, NeuroD1 cKO retinas have a dramatic reduction in rod- and cone-driven electroretinograms and contain shortened and disorganized outer segments; by 4 months, NeuroD1 cKO retinas are devoid of photoreceptors. In contrast, the NeuroD1 cKO pineal gland appears histologically normal. Microarray analysis of 2-month-old NeuroD1 cKO retina and pineal gland identified a subset of genes that were affected 2-100-fold; in addition, a small group of genes exhibit altered differential night/day expression. Included in the down-regulated genes are Aipl1, which is necessary to prevent retinal degeneration, and Ankrd33, whose protein product is selectively expressed in the outer segments. These findings suggest that NeuroD1 may act through Aipl1 and other genes to maintain photoreceptor homeostasis.
Subject(s)
Basic Helix-Loop-Helix Transcription Factors/deficiency , Basic Helix-Loop-Helix Transcription Factors/metabolism , Gene Expression Regulation/genetics , Photoreceptor Cells, Vertebrate/metabolism , Retina/cytology , Adaptor Proteins, Signal Transducing/metabolism , Analysis of Variance , Animals , Basic Helix-Loop-Helix Transcription Factors/genetics , Bromodeoxyuridine , Cell Survival/genetics , Electroretinography , Mice , Mice, Inbred C57BL , Mice, Knockout , Microarray Analysis , Microscopy, Electron, Transmission , Mucoproteins/deficiency , Mucoproteins/genetics , Oncogene Proteins , Opsins/genetics , Opsins/metabolism , Photoreceptor Cells, Vertebrate/ultrastructure , Pineal Gland/cytology , Pineal Gland/metabolism , Pineal Gland/ultrastructure , RNA, Messenger/metabolism , Retinal Degeneration/pathology , Transcription Factors/metabolismABSTRACT
The isolation of single cells from the pineal gland plays an essential role in understanding the complex nature of such processes as differentiation, metabolism, and cell-cell communication within the pineal gland. This procedure is the portal to single-cell RNA sequencing, which produces the transcriptome of individual cells. As such, single-cell RNA sequencing is critical to the continued development of knowledge of the pineal cell physiology. This chapter describes a simple procedure for isolating individual cells. Starting with the incubation of whole tissue in an enzyme preparation, which dissociates the pineal gland into small pieces, it continues with gentle trituration and then isolation of single cells through filtration. The procedure takes less than 2 h.
Subject(s)
Pineal Gland , Astrocytes , Pineal Gland/metabolism , TranscriptomeABSTRACT
The pineal gland presents a powerful genetic tool to study a broad range of physiological processes. It has been instrumental as a model in understanding transduction processes and daily changes in gene expression and holds great promise in understanding development. Currently, the field is at an exciting point, with methods available for the isolation of individual cells and, as presented here, the preparation of these single cells for sequencing. The resulting cellular transcriptomes have played a role in categorizing cells in the pineal gland, with current estimates including two types of pinealocytes, three types of astrocytes, two types of microglia, and two types of endothelial cells, including the poorly understood vascular and meningeal cell. The methods described in this chapter will serve to support and advance cellular studies of the pineal gland in the twenty-first century.
Subject(s)
Pineal Gland , Astrocytes/metabolism , Endothelial Cells , Microglia/metabolism , Pineal Gland/metabolism , Sequence Analysis, RNAABSTRACT
The pineal transcriptome webpage is described, which provides access to the transcript expression profile of the vertebrate pineal gland and, in many cases, the retina. Experimental material was obtained during the day and night, providing an opportunity to examine rhythmicity. The vertebrates represented include human, rhesus, rat, mouse, chicken, and zebrafish. In addition, data on the effects of surgical denervation and pharmacological treatments of the rat are included. Data are freely available to users.