scholarly journals Transcriptional Profiling of Identified Neurons in Leech

2020 ◽  
Author(s):  
Elizabeth Heath-Heckman ◽  
Shinja Yoo ◽  
Christopher Winchell ◽  
Maurizio Pellegrino ◽  
James Angstadt ◽  
...  
Keyword(s):  
Nervous System ◽  
Transient Receptor Potential ◽  
Transcriptional Profiling ◽  
Expression Patterns ◽  
Gene Families ◽  
Cell Types ◽  
Sensory Transduction ◽  
System Structure ◽  
Identified Neurons ◽  
Hcn Channels

ABSTRACTWhile leeches in the genus Hirudo have long been models for neurobiology, the molecular underpinnings of nervous system structure and function in this group remain largely unknown. To begin to bridge this gap, we performed RNASeq on pools of identified neurons of the central nervous system (CNS): sensory T (touch), P (pressure) and N (nociception) neurons; neurosecretory Retzius cells; and ganglia from which these four cell types had been removed. Bioinformatic analyses identified 2,812 putative genes whose expression differed significantly among the samples. These genes clustered into 7 groups which could be associated with one or more of the identified cell types. We verified predicted expression patterns through in situ hybridization on whole CNS ganglia, and found that orthologous genes were for the most part similarly expressed in a divergent leech genus, suggesting evolutionarily conserved roles for these genes. Transcriptional profiling allowed us to identify candidate phenotype-defining genes from expanded gene families. Thus, we identified one of eight hyperpolarization-activated cyclic-nucleotide gated (HCN) channels as a candidate for mediating the prominent sag current in P neurons, and found that one of five inositol triphosphate receptors (IP3Rs), representing a sub-family of IP3Rs absent from vertebrate genomes, is expressed with high specificity in T cells. We also identified one of two piezo genes, two of ~65 deg/enac genes, and one of at least 16 transient receptor potential (trp) genes as prime candidates for involvement in sensory transduction in the three distinct classes of leech mechanosensory neurons.

scholarly journals Transcriptional profiling of identified neurons in leech

BMC Genomics ◽  
2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Elizabeth Heath-Heckman ◽  
Shinja Yoo ◽  
Christopher Winchell ◽  
Maurizio Pellegrino ◽  
James Angstadt ◽  
...  
Keyword(s):  
Nervous System ◽  
Transient Receptor Potential ◽  
Transcriptional Profiling ◽  
Expression Patterns ◽  
Gene Families ◽  
Cell Types ◽  
Sensory Transduction ◽  
System Structure ◽  
Identified Neurons ◽  
Hcn Channels

Abstract Background While leeches in the genus Hirudo have long been models for neurobiology, the molecular underpinnings of nervous system structure and function in this group remain largely unknown. To begin to bridge this gap, we performed RNASeq on pools of identified neurons of the central nervous system (CNS): sensory T (touch), P (pressure) and N (nociception) neurons; neurosecretory Retzius cells; and ganglia from which these four cell types had been removed. Results Bioinformatic analyses identified 3565 putative genes whose expression differed significantly among the samples. These genes clustered into 9 groups which could be associated with one or more of the identified cell types. We verified predicted expression patterns through in situ hybridization on whole CNS ganglia, and found that orthologous genes were for the most part similarly expressed in a divergent leech genus, suggesting evolutionarily conserved roles for these genes. Transcriptional profiling allowed us to identify candidate phenotype-defining genes from expanded gene families. Thus, we identified one of eight hyperpolarization-activated cyclic-nucleotide gated (HCN) channels as a candidate for mediating the prominent sag current in P neurons, and found that one of five inositol triphosphate receptors (IP3Rs), representing a sub-family of IP3Rs absent from vertebrate genomes, is expressed with high specificity in T cells. We also identified one of two piezo genes, two of ~ 65 deg/enac genes, and one of at least 16 transient receptor potential (trp) genes as prime candidates for involvement in sensory transduction in the three distinct classes of leech mechanosensory neurons. Conclusions Our study defines distinct transcriptional profiles for four different neuronal types within the leech CNS, in addition to providing a second ganglionic transcriptome for the species. From these data we identified five gene families that may facilitate the sensory capabilities of these neurons, thus laying the basis for future work leveraging the strengths of the leech system to investigate the molecular processes underlying and linking mechanosensation, cell type specification, and behavior.

Protein expression patterns of identified neurons and of sprouting cells from the leech central nervous system

2000 ◽  
Vol 44 (3) ◽  
pp. 320-332 ◽  
Author(s):  
Xueqing Wu ◽  
Barbara Ritter ◽  
Jan Henrik Schlattjan ◽  
Volkmar Lessmann ◽  
Rolf Heumann ◽  
...  
Keyword(s):  
Central Nervous System ◽  
Nervous System ◽  
Protein Expression ◽  
Expression Patterns ◽  
Identified Neurons

scholarly journals Genetic reagents for making split-GAL4 lines in Drosophila

2017 ◽  
Author(s):  
Heather Dionne ◽  
Karen L. Hibbard ◽  
Amanda Cavallaro ◽  
Jui-Chun Kao ◽  
Gerald M. Rubin
Keyword(s):  
Nervous System ◽  
Genomic Dna ◽  
Expression Patterns ◽  
Cell Types ◽  
Biological Processes ◽  
Single Segment ◽  
Distinct Cell ◽  
Experimental Tool ◽  
Transgenic Lines

AbstractThe ability to reproducibly target expression of transgenes to small, defined subsets of cells is a key experimental tool for understanding many biological processes. The Drosophila nervous system contains thousands of distinct cell types and it has generally not been possible to limit expression to one or a few cell types when using a single segment of genomic DNA as an enhancer to drive expression. Intersectional methods, in which expression of the transgene only occurs where two different enhancers overlap in their expression patterns, can be used to achieve the desired specificity. This report describes a set of over 2,800 transgenic lines for use with the split-GAL4 intersectional method.

scholarly journals FIN-Seq: transcriptional profiling of specific cell types from frozen archived tissue of the human central nervous system

2019 ◽  
Author(s):  
Ryoji Amamoto ◽  
Emanuela Zuccaro ◽  
Nathan C Curry ◽  
Sonia Khurana ◽  
Hsu-Hsin Chen ◽  
...  
Keyword(s):  
Central Nervous System ◽  
Nervous System ◽  
Rna Sequencing ◽  
Human Tissue ◽  
Transcriptional Profiling ◽  
Cell Types ◽  
Specific Cell ◽  
Human Central Nervous System ◽  
Tissue Samples ◽  
Rna Profiling

Abstract Thousands of frozen, archived tissue samples from the human central nervous system (CNS) are currently available in brain banks. As recent developments in RNA sequencing technologies are beginning to elucidate the cellular diversity present within the human CNS, it is becoming clear that an understanding of this diversity would greatly benefit from deeper transcriptional analyses. Single cell and single nucleus RNA profiling provide one avenue to decipher this heterogeneity. An alternative, complementary approach is to profile isolated, pre-defined cell types and use methods that can be applied to many archived human tissue samples that have been stored long-term. Here, we developed FIN-Seq (Frozen Immunolabeled Nuclei Sequencing), a method that accomplishes these goals. FIN-Seq uses immunohistochemical isolation of nuclei of specific cell types from frozen human tissue, followed by bulk RNA-Sequencing. We applied this method to frozen postmortem samples of human cerebral cortex and retina and were able to identify transcripts, including low abundance transcripts, in specific cell types.

Immunolocalization of TRPC channel subunits 1 and 4 in the chicken retina

2003 ◽  
Vol 20 (4) ◽  
pp. 453-463 ◽  
Author(s):  
SCOTT CROUSILLAC ◽  
MICHELLE LEROUGE ◽  
MICHELE RANKIN ◽  
EVANNA GLEASON
Keyword(s):  
Transient Receptor Potential ◽  
Trp Channels ◽  
Expression Patterns ◽  
Plexiform Layer ◽  
Cell Types ◽  
Retinal Cell ◽  
Inner Plexiform Layer ◽  
Double Labeling ◽  
Adult Chicken ◽  
Chicken Retina

In the vertebrate retina, multiple cell types express G protein-coupled receptors linked to the IP3 signaling pathway. The signaling engendered by activation of this pathway can involve activation of calcium permeable transient receptor potential (TRP) channels. To begin to understand the role of these channels in the retina, we undertake an immunocytochemical localization of two TRP channel subunits. Polyclonal antibodies raised against mammalian TRPC1 and TRPC4 are used to localize the expression of these proteins in sections of the adult chicken retina. Western blot analysis indicates that these antibodies recognize avian TRPC1 and TRPC4. TRPC1 labeling is almost completely confined to the inner plexiform layer (IPL) where it labels a subset of processes that ramify in three broad stripes. Occasionally, cell bodies are labeled. These can be found in the inner nuclear layer (INL) proximal to the IPL, the IPL, and the ganglion cell layer (GCL). Double-labeling experiments using a polyclonal antibody that recognizes brain nitric oxide synthase (bNOS) in the chicken indicate that many of the TRPC1-positive processes and cell bodies also express bNOS. Labeling with the TRPC4 antibody was much more widespread with some degree of labeling found in all layers of the retina. TRPC4 immunoreactivity was found in the photoreceptor layer, in the outer plexiform layer (OPL), in radially oriented cells in the INL, diffusely in the IPL, and in vertically oriented elements below the GCL. Double-labeling experiments with a monoclonal antibody raised against vimentin indicate that the TRPC4-positive structures in the INL and below the GCL are Müller cells. Thus, TRPC1 and TRPC4 subunits have unique expression patterns in the adult chicken retina. The distributions of these two subunits indicate that different retinal cell types express TRP channels containing different subunits.

scholarly journals Lamprey Lecticans Link New Vertebrate Genes to the Origin and Elaboration of Vertebrate Tissues

2020 ◽  
Author(s):  
Zachary D. Root ◽  
David Jandzik ◽  
Cara Allen ◽  
Margaux Brewer ◽  
Marek Romášek ◽  
...  
Keyword(s):  
Gene Family ◽  
Expression Patterns ◽  
Gene Families ◽  
Cell Types ◽  
Specific Cell ◽  
Skeletal Tissue ◽  
Genomic Arrangement ◽  
New Gene ◽  
Morphological Novelties ◽  
Head Skeleton

ABSTRACTThe evolution of vertebrates from an invertebrate chordate ancestor involved the evolution of new organs, tissues, and cell types. It was also marked by the origin and duplication of new gene families. If, and how, these morphological and genetic innovations are related is an unresolved question in vertebrate evolution. Hyaluronan is an extracellular matrix (ECM) polysaccharide important for water homeostasis and tissue structure. Vertebrates possess a novel family of hyaluronan binding proteins called Lecticans, and studies in jawed vertebrates (gnathostomes) have shown they function in many of the cells and tissues that are unique to vertebrates. This raises the possibility that the origin and/or expansion of this gene family helped drive the evolution of these vertebrate novelties. In order to better understand the evolution of the lectican gene family, and its role in the evolution of vertebrate morphological novelties, we investigated the phylogeny, genomic arrangement, and expression patterns of all lecticans in the sea lamprey (Petromyzon marinus), a jawless vertebrate. Though both P. marinus and gnathostomes have four lecticans, our phylogenetic and syntenic analyses suggest lamprey lecticans are the result of one or more cyclostome-specific duplications. Despite the independent expansion of the lamprey and gnathostome lectican families, we find highly conserved expression of lecticans in vertebrate-specific and mesenchyme-derived tissues. We also find that, unlike gnathostomes, lamprey expresses its lectican paralogs in distinct subpopulations of head skeleton precursors, potentially reflecting an ancestral diversity of skeletal tissue types. Together, these observations suggest that the ancestral pre-duplication lectican had a complex expression pattern, functioned to support mesenchymal histology, and likely played a role in the evolution of vertebrate-specific cell and tissue types.

Hox-7 expression during murine craniofacial development

Development ◽  
1991 ◽  
Vol 113 (2) ◽  
pp. 601-611 ◽  
Author(s):  
A. MacKenzie ◽  
M.W. Ferguson ◽  
P.T. Sharpe
Keyword(s):  
Nervous System ◽  
Choroid Plexus ◽  
Expression Pattern ◽  
Neural Tube ◽  
Anterior Pituitary ◽  
Expression Patterns ◽  
In Situ Hybridisation ◽  
Cell Types ◽  
Subsequent Formation ◽  
Dorsal Midline

We have used in situ hybridisation to establish the temporal and spatial expression patterns of the mouse homeobox-containing gene; Hox-7, in the developing embryonic cranium and nervous system of the mouse between embryonic days 9.5 (E9.5) and E15.5. Hox-7 has previously been associated with areas of mesenchymal-epithelial interaction and cell migration especially in neural crest ectomesenchymal cells. Aside from the expression patterns seen in the facial anlage at E9.5, Hox-7 transcripts were also detected in the neuroepithelium including cells of the dorsal midline of the neural tube. This expression pattern persisted throughout the embryonic time span studied. At E11.5, expression of Hox-7 became obvious in the neuroepithelium of the forming tela choroida and the telencephelii in areas destined to form the choroid plexus before any atrophy of the neuroepithelium took place. High expression of Hox-7 was also present in the mesenchyme cells invading the pouch formed by the involuting choroid plexus neuroepithelium. A second major site where Hox-7 was expressed was the anlage of the anterior pituitary; the Rathke's pouch. Expression became obvious at E10.5 throughout the pouch but by E12.5 became more regionalised in areas of the pouch destined to form the pars distalis. Hox-7 was also expressed in the forming meninges and skull bone precursors from E10.5 onwards. Expression of the Hox-7 gene is also seen in the external ear, the forming eye, the nasal pits and forming Jacobson's organs. When these expression patterns are considered together with characterised human and mouse retinoic acid embryopathies and the congenital malformations seen in human children associated with deletion of chromosome 4p16.1 (Wolf-Hirschhorn syndrome), Hox-7 may be a good candidate as one of the genes involved in the initiation of the choroid plexus phenotype and its subsequent formation, the formation of the outer ear, formation of the dentition and the differentiation of the cell types of the anterior pituitary. The expression pattern of Hox-7 in the dorsal midline of the neural tube further suggests that it may also be involved in the specification of the dorsal-ventral axis of the developing nervous system.

scholarly journals Molecular Profiling to Infer Neuronal Cell Identity: Lessons from small ganglia of the Crab Cancer borealis

2019 ◽  
Author(s):  
Adam J. Northcutt ◽  
Daniel R. Kick ◽  
Adriane G. Otopalik ◽  
Benjamin M. Goetz ◽  
Rayna M. Harris ◽  
...  
Keyword(s):  
Gene Expression ◽  
Expression Profile ◽  
Transcriptional Profiling ◽  
Cell Types ◽  
Identified Neurons ◽  
Cell Type ◽  
Cell Identity ◽  
Cell Identification ◽  
Neuronal Identity ◽  
Type Classification

ABSTRACTUnderstanding circuit organization depends on identification of cell types. Recent advances in transcriptional profiling methods have enabled classification of cell types by their gene expression. While exceptionally powerful and high throughput, the ground-truth validation of these methods is difficult: if cell type is unknown, how does one assess whether a given analysis accurately captures neuronal identity? To shed light on the capabilities and limitations of solely using transcriptional profiling for cell type classification, we performed two forms of transcriptional profiling – RNA-seq and quantitative RT-PCR, in single, unambiguously identified neurons from two small crustacean networks: the stomatogastric and cardiac ganglia. We then combined our knowledge of cell type with unbiased clustering analyses and supervised machine learning to determine how accurately functionally-defined neuron types can be classified by expression profile alone. Our results demonstrate that expression profile is able to capture neuronal identity most accurately when combined with multimodal information that allows for post-hoc grouping so analysis can proceed from a supervised perspective. Solely unsupervised clustering can lead to misidentification and an inability to distinguish between two or more cell types. Therefore, our study supports the general utility of cell identification by transcriptional profiling, but adds a caution: it is difficult or impossible to know under what conditions transcriptional profiling alone is capable of assigning cell identity. Only by combining multiple modalities of information such as physiology, morphology or innervation target can neuronal identity be unambiguously determined.SIGNIFICANCE STATEMENTSingle cell transcriptional profiling has become a widespread tool in cell identification, particularly in the nervous system, based on the notion that genomic information determines cell identity. However, many cell type classification studies are unconstrained by other cellular attributes (e.g., morphology, physiology). Here, we systematically test how accurately transcriptional profiling can assign cell identity to well-studied anatomically- and functionally-identified neurons in two small neuronal networks. While these neurons clearly possess distinct patterns of gene expression across cell types, their expression profiles are not sufficient to unambiguously confirm their identity. We suggest that true cell identity can only be determined by combining gene expression data with other cellular attributes such as innervation pattern, morphology, or physiology.

scholarly journals Localization of TRP channels in healthy oral mucosa from human donors

2021 ◽  
Author(s):  
Yalda Moayedi ◽  
Stephanie Michlig ◽  
Mark Park ◽  
Alia Koch ◽  
Ellen Annette Lumpkin
Keyword(s):  
Epithelial Cells ◽  
Oral Cavity ◽  
Transient Receptor Potential ◽  
Trp Channels ◽  
Thermal Sensation ◽  
Expression Patterns ◽  
Nerve Fibers ◽  
Confocal Imaging ◽  
Sensory Transduction ◽  
Human Tongue

The oral cavity is exposed to a remarkable range of noxious and innocuous conditions, including temperature fluctuations, mechanical forces, inflammation and environmental and endogenous chemicals. How such changes in the oral environment are sensed by oral cells and tissues is not completely understood. Transient receptor potential (TRP) ion channels are a diverse family of molecular receptors that are activated by chemicals, temperature changes, and tissue damage. In non-neuronal cells, TRP channels play roles in inflammation, as well as tissue development and maintenance. In somatosensory neurons, TRP channels mediate nociception, thermosensation and chemosensation. To assess whether TRP channels might be involved in environmental sensing in the human oral cavity, we investigated the distribution of TRP channels in human tongue and hard palate. Oral biopsies were collected from volunteers and underwent fluorescent immunohistochemistry followed by confocal imaging. We analyzed immunoreactivity of TRP channels in human oral epithelia including TRPV3, TRPV4, TRPV1, TRPM8, and TRPA1. TRPV3 and TRPV4 were expressed in epithelial cells with inverse expression patterns where they are likely to contribute to epithelial development and integrity. TRPA1 immunoreactivity was found in fibroblasts, subsets immune cells, and neurons, consistent with known roles of TRPA1 in sensory transduction, as well as in response to damage and inflammation. TRPM8 immunoreactivity was found in lamina propria cells and some neuronal subpopulations including some neurons within the end bulbs of Krause, consistent with a role in thermal sensation. TRPV1 immunoreactivity was identified in intraepithelial nerve fibers, in some end bulbs of Krause, and in epithelial cells, consistent with roles in nociception and thermosensation. Immunoreactivity of TRPM8 and TRPV1 in end bulbs of Krause suggest that these structures contain a variety of neuronal afferents, including those that mediate nociception, thermosensation and mechanotransduction. Collectively, these studies support the role of TRP channels in oral environmental surveillance and response.

scholarly journals The VT GAL4, LexA, and split-GAL4 driver line collections for targeted expression in the Drosophila nervous system

2017 ◽  
Author(s):  
Laszlo Tirian ◽  
Barry J. Dickson
Keyword(s):  
Nervous System ◽  
Transgene Expression ◽  
Expression Patterns ◽  
Neuronal Cell ◽  
Cell Types ◽  
Specific Cell ◽  
Cellular Interactions ◽  
Gal4 Driver ◽  
Targeted Expression ◽  
Driver Line

AbstractIn studying the cellular interactions within complex tissues, it is extremely valuable to be able to reproducibly and flexibly target transgene expression to restricted subsets of cells. This approach is particularly valuable in studying the nervous system, with its bewildering diversity of neuronal cell types. We report here the generation of over 18,000 driver lines (the VT collection) that exploit the GAL4, LexA, and split-GAL4 systems to express transgenes in distinct and highly specific cell types in Drosophila. We document the expression patterns of over 14,000 of these lines in the adult male brain.

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