Project description:General somatic sensation is conveyed to the central nervous system at cranial levels by the trigeminal ganglion (TG), and at spinal levels by the dorsal root ganglia (DRG). Although these ganglia have similar functions, they have distinct embryological origins, in that both contain neurons originating from the neural crest, while only the TG includes cells derived from the placodal ectoderm. Here we use microarray analysis of E13.5 embryos to demonstrate that the developing DRG and TG have very similar overall patterns of gene expression. In mice lacking the POU-domain transcription factor Brn3a the DRG and TG exhibit many common changes in downstream gene expression, but a subset of genes show increased expression only at cranial levels. Although silent in wild-type ganglia, the promoter regions of genes which are activated in the absence of Brn3a also exhibit increased histone H3-acetylation at levels similar to constitutively transcribed gene loci, and this H3-acetylation is tissue-specific for genes which are increased only in the TG. These results demonstrate that one developmental role of Brn3a is to repress potential differences in gene expression between sensory neurons generated at different axial levels, and to regulate a convergent program of developmental gene expression, in which functionally similar populations of neurons are generated from different embryological substrates. Experiment Overall Design: Microarrays used to compare the patterns of gene expression in the dorsal root ganglia and trigeminal ganglia of Brn3a knockout and wild-type mice. Embryonic day 13.5 (E13.5) was chosen because at this point in development mutant mice exhibit major defects in sensory axon growth, but have yet to undergo the period of extensive sensory neuron death associated with later stages.
Project description:General somatic sensation is conveyed to the central nervous system at cranial levels by the trigeminal ganglion (TG), and at spinal levels by the dorsal root ganglia (DRG). Although these ganglia have similar functions, they have distinct embryological origins, in that both contain neurons originating from the neural crest, while only the TG includes cells derived from the placodal ectoderm. Here we use microarray analysis of E13.5 embryos to demonstrate that the developing DRG and TG have very similar overall patterns of gene expression. In mice lacking the POU-domain transcription factor Brn3a the DRG and TG exhibit many common changes in downstream gene expression, but a subset of genes show increased expression only at cranial levels. Although silent in wild-type ganglia, the promoter regions of genes which are activated in the absence of Brn3a also exhibit increased histone H3-acetylation at levels similar to constitutively transcribed gene loci, and this H3-acetylation is tissue-specific for genes which are increased only in the TG. These results demonstrate that one developmental role of Brn3a is to repress potential differences in gene expression between sensory neurons generated at different axial levels, and to regulate a convergent program of developmental gene expression, in which functionally similar populations of neurons are generated from different embryological substrates. Keywords: Gene expression in the developing nervous system
Project description:The goal of this study was to analyze global gene expression in specific populations of nociceptor sensory neurons, the neurons that detect damaging/noxious stimuli. The dorsal root ganglia (DRG), trigeminal ganglia, and nodose ganglia are anatomically distinct peripheral sensory ganglia that contain nociceptors which innervate skin, gut, lungs, and other distinct organ tissues. We used flow cytometry to purify nociceptors from these ganglia and profiled their global gene expression signatures to compare gene expression between these different anatomically distinct nociceptors. Nav1.8-Cre were bred with Rosa26-TdTomato to generate Nav1.8-Cre/R26-TdTomato reporter progeny, where all peripheral nociceptor neurons are genetically marked with red fluroescence due to specific expression of the TTX- resistant sodium channel Nav1.8. Lumbar region dorsal root ganglia (DRG), trigeminal ganglia, and nodose ganglia were dissected from mice (3 mice were pooled/sample). Highly red fluorescent neurons were Facs purified, RNA extracted, and processed for microarray analysis.
Project description:Vagal afferent neurons are thought to convey primarily physiological information, whereas spinal afferents transmit noxious signals from the viscera to the central nervous system. In order to elucidate molecular identities for these different properties, we compared gene expression profiles of neurons located in nodose ganglia (NG) and dorsal root ganglia (DRG) in mice. Intraperitoneal administration of Alexa Fluor-488 conjugated Cholera toxin B allowed identification of neurons projecting to the viscera. Fluorescent neurons in DRG (from T10 to T13) and NG were isolated using laser capture microdissection. Gene expression profiles of visceral afferent neurons, obtained by microarray hybridization, were analysed using multivariate spectral map analysis, SAM algorithm (Significance Analysis of Microarray data) and fold-difference filtering. A total of 1996 genes were found to be differentially expressed in DRG versus NG, including 41 G-protein coupled receptors and 60 ion channels. Expression profiles obtained on laser-captured neurons were contrasted to those obtained on whole ganglia demonstrating striking differences and the need for microdissection when studying visceral sensory neurons because of dilution of the signal by somatic sensory neurons. Furthermore, a detailed catalogue of all adrenergic and cholinergic, GABA, glutamate, serotonin and dopamine receptors, voltage-gated potassium, sodium and calcium channels and transient receptor potential cation channels present in visceral afferents is provided. Our genome-wide expression profiling data provide novel insight into molecular signatures that underlie both functional differences and similarities between NG and DRG visceral sensory neurons. Moreover, these findings will offer novel insight into mode of action of pharmacologic agents modulating visceral sensation. Experiment Overall Design: Three separate experiments were performed. First, 5 whole dorsal root ganglia were compared to 7 whole nodose ganglia. Second, Laser captured visceral neurons derived from 5 dorsal root ganglia and 5 nodose ganglia were compared on MG-U74Av2. Third, Laser captured visceral neurons derived from 9 dorsal root ganglia and 11 nodose ganglia were compared on Mouse430_2.
Project description:The goal of this study was to analyze global gene expression in specific populations of somatosensory neurons in the periphery, including major, non-overlapping populations that include nociceptors, pruriceptors, and prorioceptors. The mammalian somatosensory nervous system encodes the perception of specific environmental stimuli. The dorsal root ganglion (DRG) contains distinct somatosensory neuron subtypes that innervate diverse peripheral tissues, mediating the detection of thermal, mechanical, proprioceptive, pruriceptive, and nociceptive stimuli. We purified discrete subtypes of mouse DRG somatosensory neurons by flow cytometry using fluorescently labeled mouse lines (SNS-Cre/TdTomato, Parv-Cre/TdTomato) in combination with Isolectin B4-FITC surface staining (IB4). This allowed identification of transcriptional differences between these major populations, revealing enrichment of voltage-gated ion channels, TRP channels, G-protein coupled receptors, transcription factors, and other functionally important classes of genes within specific somatosensory neuron subsets. SNS-Cre mice were bred with Rosa26-TdTomato mice to generate SNS-Cre/TdTomato reporter mice. Parv-Cre mice were bred with Rosa26-TdTomato mice to generate Parv-Cre/TdTomato mice. Isolectin B4-FITC was used to stain the surface of SNS-Cre/TdTomato reporter mice. We used these strategies of fluorescent labeling to purify distinct murine sensory neuron subsets from the dorsal root ganglia (DRG) by fluorescence activated cell sorting (FACS). Neurons were sorted directly in Qiazol for total RNA extraction and microarray analysis. Whole DRG tissue was also included for transcriptome analysis to compare with purified neuronal populations.
Project description:The goal of this study was to analyze global gene expression in specific populations of nociceptor sensory neurons, the neurons that detect damaging/noxious stimuli. The dorsal root ganglia (DRG), trigeminal ganglia, and nodose ganglia are anatomically distinct peripheral sensory ganglia that contain nociceptors which innervate skin, gut, lungs, and other distinct organ tissues. We used flow cytometry to purify nociceptors from these ganglia and profiled their global gene expression signatures to compare gene expression between these different anatomically distinct nociceptors.
Project description:The vast majority of cold sensitive DRG neurons from mice do not express the voltage-gated sodium channel NaV1.8. Therefore, we aimed to compare the molecular profiles of NaV1.8 and non-NaV1.8-expressing neurons using microarray analysis. Fluorescent activated cell sorting was performed at 4 degrees centigrade on acutely dissociated DRG neurons from mice expressing NaV1.8-Cre, Pirt-GCaMP3 and a Cre-dependent global reporter (td tomato). NaV1.8-expressing neurons were sorted based on their reporter fluorescence (td tomato; red) and putative cold sensing neurons were sorted based on their GCaMP3 fluorescence at 4 degrees centigrade and absence of Cre-dependent reporter fluorescence. A total of three mice were used (samples one, two and three) with GCaMP3 only and NaV1.8-expressing neurons forming two relative populations within each sample (eg. GC3 one is the experimental counterpart of Tom one).
Project description:Nociceptors play an essential role in both acute pain and chronic pain conditions. In this study, we examined the proteome of mouse dorsal root ganglia and compared NaV1.8Cre+/-; ROSA26-flox-stop-flox-DTA (Diphtheria toxin fragment A) mutant mice (NaV1.8Cre-DTA), in which NaV1.8-positive neurons (mainly nociceptors) in dorsal root ganglia (DRG) were ablated, with respective littermate wildtype controls.
Project description:Unbiased 'omics' techniques, such as next generation RNA-sequencing, can provide entirely novel insights into biological systems. However, cellular heterogeneity presents a significant barrier to analysis and interpretation of these datasets. The neurons of the dorsal root ganglia (DRG) are an important model for studies of neuronal injury, regeneration and pain. The majority of investigators utilize a dissociated preparation of whole ganglia when studying cellular and molecular function. We demonstrate that the standard methods for producing these preparations gives a 10%-neuronal mixture of cells, with the remainder of cells constituting satellite glia and other non-neuronal cell types. Using a novel application of magnetic purification, we consistently obtain over 95% pure, viable neurons from adult tissue, significantly enriched for small diameter nociceptors expressing the voltage gated ion channel Nav1.8. Using genome-wide RNA-sequencing we compare the currently used (10% neuronal) and pure (95% nociceptor) preparations and find 920 genes enriched. This gives an unprecedented insight into the molecular composition of small nociceptive neurons in the DRG, potentially altering the interpretation of previous studies performed at the tissue level, and indicating a number of novel markers of this widely-studied population of cells. We anticipate that the ease of use, affordability and speed of this technique will see it become widely adopted, delivering a greatly improved capacity to study the roles of nociceptors in health and disease. RNA-Seq was performed for 4 biological replicates from three different groups: intact DRG, acutely dissociated DRG and magnetically-purified DRG neurons. Differential expression was analyzed between acutely dissociated and MACS-dissociated samples to define the 'nociceptor transcriptome'.