Systematic analysis of expression signatures of neuronal subpopulations in the VTA

AbstractGene expression profiling across various brain areas at the single-cell resolution enables the identification of molecular markers of neuronal subpopulations and comprehensive characterization of their functional roles. Despite the scientific importance and experimental versatility, systematic methods to analyze such data have not been established yet. To this end, we developed a statistical approach based on in situ hybridization data in the Allen Brain Atlas and thereby identified specific genes for each type of neuron in the ventral tegmental area (VTA). This approach also allowed us to demarcate subregions within the VTA comprising specific neuronal subpopulations. We further identified WW domain-containing oxidoreductase as a molecular marker of a population of VTA neurons that co-express tyrosine hydroxylase and vesicular glutamate transporter 2, and confirmed their region-specific distribution by immunohistochemistry. The results demonstrate the utility of our analytical approach for uncovering expression signatures representing specific cell types and neuronal subpopulations enriched in a given brain area.

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Temporal changes in the expression of the insulin-like growth factor II gene associated with tissue maturation in the human fetus

Development ◽

10.1242/dev.106.3.543 ◽

1989 ◽

Vol 106 (3) ◽

pp. 543-554 ◽

Cited By ~ 1

Author(s):

A.L. Brice ◽

J.E. Cheetham ◽

V.N. Bolton ◽

N.C. Hill ◽

P.N. Schofield

Keyword(s):

Growth Factor ◽

Cell Types ◽

Specific Cell ◽

Insulin Like Growth Factor ◽

Vitro Fertilization ◽

Age Related ◽

Factor Ii ◽

Metanephric Blastema

The insulin-like growth factors are broadly distributed in the human conceptus and are thought to play a role in the growth and differentiation of tissues during development. Using in situ hybridization we have shown that a wide variety of specific cell types within tissues express the gene for insulin-like growth factor II at times of development from 18 days to 14 weeks of gestation. Examination of blastocysts produced by in vitro fertilization showed no expression, thus bracketing the time of first accumulation of IGF-II mRNA to between 5 and 18 days postfertilization. The pattern of IGF-II expression shows specific age-related differences in different tissues. In the kidney, for example, expression is found in the cells of the metanephric blastema which is dramatically reduced as the blastema differentiates. The reverse is also seen, and we have noted an increase in expression of IGF-II in the cytotrophoblast layer of the placenta with gestational age. The sites of expression do not correlate with areas of either high mitotic activity or specific types of differentiation, but the observed pattern of expression in the kidney, adrenal glands and liver suggests an explanation for the abnormally high IGF-II mRNA expression in developmental tumours such as Wilms' tumour.

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Methods to Enhance Signal Using Isotopic In Situ Hybridization

Journal of Histochemistry & Cytochemistry ◽

10.1177/002215540205000805 ◽

2002 ◽

Vol 50 (8) ◽

pp. 1031-1037 ◽

Cited By ~ 13

Author(s):

Betty Ky ◽

Paul J. Shughrue

Keyword(s):

Gene Expression ◽

In Situ Hybridization ◽

Cell Types ◽

Oxytocin Receptor ◽

Specific Cell ◽

Rat Hypothalamus ◽

Low Levels ◽

Tuberoinfundibular Peptide ◽

Cryostat Sections

Isotopic in situ hybridization (ISH) has been established as a uniquely powerful tool for the study of gene expression in specific cell types. This technique allows the visualization and quantification of gene expression and gene expression changes in cells. In our study of biological and molecular phenomena, we have increasingly encountered the need to detect small changes in gene expression as well as genes of low abundance, such as the oxytocin receptor (OTR) and the tuberoinfundibular peptide of 39 residues (Tip39). To increase the sensitivity of isotopic ISH for detection of rare mRNAs, we performed ISH on cryostat sections of rat hypothalamus and thalamus with 35S-labeled riboprobes and amplified the signal by hybridizing over 2 nights as well as labeling the probe with both [35S]-UTP and [35S]-ATP. These two methods of enhancement independently and in combination demonstrated a dramatic increase in signal, allowing the visualization of low levels of gene expression previously undetectable by conventional methods.

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Marine Planktonic Archaea Take Up Amino Acids

Applied and Environmental Microbiology ◽

10.1128/aem.66.11.4829-4833.2000 ◽

2000 ◽

Vol 66 (11) ◽

pp. 4829-4833 ◽

Cited By ~ 248

Author(s):

Cleber C. Ouverney ◽

Jed A. Fuhrman

Keyword(s):

Amino Acids ◽

In Situ Hybridization ◽

Cell Types ◽

Similar Proportion ◽

Specific Cell ◽

Natural Conditions ◽

The Pacific ◽

The Pacific Ocean ◽

Archaeal Communities

ABSTRACT Archaea are traditionally thought of as “extremophiles,” but recent studies have shown that marine planktonic Archaea make up a surprisingly large percentage of ocean midwater microbial communities, up to 60% of the total prokaryotes. However, the basic physiology and contribution of Archaea to community microbial activity remain unknown. We have studied Archaea from 200-m depths of the northwest Mediterranean Sea and the Pacific Ocean near California, measuring the archaeal activity under simulated natural conditions (8 to 17°C, dark and anaerobic) by means of a method called substrate tracking autoradiography fluorescence in situ hybridization (STARFISH) that simultaneously detects specific cell types by 16S rRNA probe binding and activity by microautoradiography. In the 200-m-deep Mediterranean and Pacific samples, cells binding the archaeal probes made up about 43 and 14% of the total countable cells, respectively. Our results showed that the Archaea are active in the uptake of dissolved amino acids from natural concentrations (nanomolar) with about 60% of the individuals in the archaeal communities showing measurable uptake. Bacteria showed a similar proportion of active cells. We concluded that a portion of these Archaea is heterotrophic and also appears to coexist successfully with Bacteria in the same water.

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Self-reporting transposons enable simultaneous readout of gene expression and transcription factor binding in single cells

10.1101/538553 ◽

2019 ◽

Cited By ~ 3

Author(s):

Arnav Moudgil ◽

Michael N. Wilkinson ◽

Xuhua Chen ◽

June He ◽

Alex J. Cammack ◽

...

Keyword(s):

Gene Expression ◽

Transcription Factor ◽

Single Cell ◽

Binding Sites ◽

Expression Profiles ◽

Single Cells ◽

Gene Expression Profiles ◽

Cell Types ◽

Specific Cell

AbstractIn situ measurements of transcription factor (TF) binding are confounded by cellular heterogeneity and represent averaged profiles in complex tissues. Single cell RNA-seq (scRNA-seq) is capable of resolving different cell types based on gene expression profiles, but no technology exists to directly link specific cell types to the binding pattern of TFs in those cell types. Here, we present self-reporting transposons (SRTs) and their use in single cell calling cards (scCC), a novel assay for simultaneously capturing gene expression profiles and mapping TF binding sites in single cells. First, we show how the genomic locations of SRTs can be recovered from mRNA. Next, we demonstrate that SRTs deposited by the piggyBac transposase can be used to map the genome-wide localization of the TFs SP1, through a direct fusion of the two proteins, and BRD4, through its native affinity for piggyBac. We then present the scCC method, which maps SRTs from scRNA-seq libraries, thus enabling concomitant identification of cell types and TF binding sites in those same cells. As a proof-of-concept, we show recovery of cell type-specific BRD4 and SP1 binding sites from cultured cells. Finally, we map Brd4 binding sites in the mouse cortex at single cell resolution, thus establishing a new technique for studying TF biology in situ.

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Automated Spatially Targeted Optical Micro Proteomics (AutoSTOMP) 2.0 identifies proteins enriched within inflammatory lesions in tissue sections and human clinical biopsies.

10.1101/2021.03.19.436192 ◽

2021 ◽

Author(s):

BOCHENG YIN ◽

Laura R Caggiano ◽

Rung-Chi Li ◽

Emily McGowan ◽

Jeffery W. Holmes ◽

...

Keyword(s):

Protein Expression ◽

Cell Biology ◽

Cell Types ◽

Specific Cell ◽

Fixed Tissue ◽

Tissue Samples ◽

Novel Approach ◽

Esophageal Tissue ◽

Inflammatory Proteins

Tissue microenvironment properties like blood flow, extracellular matrix or proximity to immune infiltrate are important regulators of cell biology. However, methods to study regional protein expression in context of the native tissue environment are limited. To address this need we have developed a novel approach to visualize, purify and measure proteins in situ using Automated Spatially Targeted Optical Micro Proteomics (AutoSTOMP) 2.0. We previously implemented AutoSTOMP to identify proteins localized to the vacuoles of obligate intracellular microbes at the 1-2 μm scale within infected host cells1. Here we report custom codes in SikuliX to specify regions of heterogeneity in a tissue section and then biotin tag and identify proteins belonging to specific cell types or structures within those regions. To enrich biotinylated targets from fixed tissue samples we developed a biochemical protocol compatible with LC-MS. These tools were applied to a) identify inflammatory proteins expressed by CD68+ macrophages in rat cardiac infarcts and b) characterize inflammatory proteins enriched in IgG4+ lesions in esophageal tissue. These data indicate that AutoSTOMP is a flexible approach to determine regional protein expression in situ on a range of primary tissues and clinical biopsies where current tools are limited.

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Probe-Seq enables transcriptional profiling of specific cell types from heterogeneous tissue by RNA-based isolation

10.1101/735738 ◽

2019 ◽

Author(s):

Ryoji Amamoto ◽

Mauricio D. Garcia ◽

Emma R. West ◽

Jiho Choi ◽

Sylvain W. Lapan ◽

...

Keyword(s):

Transcriptional Profiling ◽

Cell Types ◽

Cell Populations ◽

Specific Cell ◽

Rna Profiling ◽

Complementary Method ◽

Dissociated Cells ◽

Multiple Cell ◽

Heterogeneous Tissue

ABSTRACTRecent transcriptional profiling technologies are uncovering previously-undefined cell populations and molecular markers at an unprecedented pace. While single cell RNA (scRNA) sequencing is an attractive approach for unbiased transcriptional profiling of all cell types, a complementary method to isolate and sequence specific cell populations from heterogeneous tissue remains challenging. Here, we developed Probe-Seq, which allows deep transcriptional profiling of specific cell types isolated using RNA as the defining feature. Dissociated cells are labelled using fluorescent in situ hybridization (FISH) for RNA, and then isolated by fluorescent activated cell sorting (FACS). We used Probe-Seq to purify and profile specific cell types from mouse, human, and chick retinas, as well as the Drosophila midgut. Probe-Seq is compatible with frozen nuclei, making cell types within archival tissue immediately accessible. As it can be multiplexed, combinations of markers can be used to create specificity. Multiplexing also allows for the isolation of multiple cell types from one cell preparation. Probe-Seq should enable RNA profiling of specific cell types from any organism.

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IL7R⍺ is required for hematopoietic stem cell reconstitution of tissue-resident lymphoid cells

10.1101/2021.07.13.452134 ◽

2021 ◽

Author(s):

Atesh K Worthington ◽

Taylor S Cool ◽

Donna M Poscablo ◽

Adeel Hussaini ◽

Anna E Beaudin ◽

...

Keyword(s):

Cell Types ◽

Lineage Tracing ◽

Lymphoid Cells ◽

Lymphoid Tissues ◽

Hematopoietic Stem ◽

Functional Roles ◽

Reciprocal Transplants ◽

B1a Cells

Traditional, adult-derived lymphocytes that circulate provide adaptive immunity to infection and pathogens. However, subsets of lymphoid cells are also found in non-lymphoid tissues and are called tissue-resident lymphoid cells (TLCs). TLCs encompass a wide array of cell types that span the spectrum of innate-to-adaptive immune function. Unlike traditional lymphocytes that are continuously generated from hematopoietic stem cells (HSCs), many TLCs are of fetal origin and poorly generated from adult HSCs. Here, we sought to understand the development of murine TLCs across multiple tissues and therefore probed the roles of Flk2 and IL7R⍺, two cytokine receptors with known roles in traditional lymphopoiesis. Using Flk2- and Il7r-Cre lineage tracing models, we found that peritoneal B1a cells, splenic marginal zone B (MZB) cells, lung ILC2s and regulatory T cells (Tregs) were highly labeled in both models. Despite this high labeling, highly quantitative, in vivo functional approaches showed that the loss of Flk2 minimally affected the generation of these cells in situ. In contrast, the loss of IL7R⍺, or combined deletion of Flk2 and IL7R⍺, dramatically reduced the cell numbers of B1a cells, MZBs, ILC2s, and Tregs both in situ and upon transplantation, indicating an intrinsic and more essential role for IL7Rα. Surprisingly, reciprocal transplants of WT HSCs showed that an IL7Rα-/- environment selectively impaired reconstitution of TLCs when compared to TLC numbers in situ. Taken together, our data revealed functional roles of Flk2 and IL7Rα in the establishment of tissue-resident lymphoid cells.

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Expansion Sequencing: Spatially Precise In Situ Transcriptomics in Intact Biological Systems

10.1101/2020.05.13.094268 ◽

2020 ◽

Cited By ~ 4

Author(s):

Shahar Alon ◽

Daniel R Goodwin ◽

Anubhav Sinha ◽

Asmamaw T Wassie ◽

Fei Chen ◽

...

Keyword(s):

Splice Variants ◽

Metastatic Breast ◽

Cell Types ◽

Specific Cell ◽

Long Read ◽

Multiple Cell ◽

Cancer Biopsy ◽

Subcellular Resolution ◽

Mouse Visual Cortex

Abstract:Methods for highly multiplexed RNA imaging are limited in spatial resolution, and thus in their ability to localize transcripts to nanoscale and subcellular compartments. We adapt expansion microscopy, which physically expands biological specimens, for long-read untargeted and targeted in situ RNA sequencing. We applied untargeted expansion sequencing (ExSeq) to mouse brain, yielding readout of thousands of genes, including splice variants and novel transcripts. Targeted ExSeq yielded nanoscale-resolution maps of RNAs throughout dendrites and spines in neurons of the mouse hippocampus, revealing patterns across multiple cell types; layer-specific cell types across mouse visual cortex; and the organization and position-dependent states of tumor and immune cells in a human metastatic breast cancer biopsy. Thus ExSeq enables highly multiplexed mapping of RNAs, from nanoscale to system scale.One Sentence SummaryIn situ sequencing of physically expanded specimens enables multiplexed mapping of RNAs at nanoscale, subcellular resolution.

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SpaGE: Spatial Gene Enhancement using scRNA-seq

Nucleic Acids Research ◽

10.1093/nar/gkaa740 ◽

2020 ◽

Vol 48 (18) ◽

pp. e107-e107 ◽

Cited By ~ 2

Author(s):

Tamim Abdelaal ◽

Soufiane Mourragui ◽

Ahmed Mahfouz ◽

Marcel J T Reinders

Keyword(s):

Spatial Information ◽

Spatial Configuration ◽

Single Cells ◽

Large Datasets ◽

Brain Atlas ◽

Hybridization Data ◽

Transcriptome Expression ◽

Whole Transcriptome ◽

Allen Mouse Brain Atlas

Abstract Single-cell technologies are emerging fast due to their ability to unravel the heterogeneity of biological systems. While scRNA-seq is a powerful tool that measures whole-transcriptome expression of single cells, it lacks their spatial localization. Novel spatial transcriptomics methods do retain cells spatial information but some methods can only measure tens to hundreds of transcripts. To resolve this discrepancy, we developed SpaGE, a method that integrates spatial and scRNA-seq datasets to predict whole-transcriptome expressions in their spatial configuration. Using five dataset-pairs, SpaGE outperformed previously published methods and showed scalability to large datasets. Moreover, SpaGE predicted new spatial gene patterns that are confirmed independently using in situ hybridization data from the Allen Mouse Brain Atlas.

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Functional and comparative analysis of globin loci in pufferfish and humans

Blood ◽

10.1182/blood-2002-09-2850 ◽

2003 ◽

Vol 101 (7) ◽

pp. 2842-2849 ◽

Cited By ~ 43

Author(s):

Nynke Gillemans ◽

Tara McMorrow ◽

Rita Tewari ◽

Albert W. K. Wai ◽

Carola Burgtorf ◽

...

Keyword(s):

Binding Sites ◽

Globin Gene ◽

Cell Types ◽

Specific Cell ◽

Fish Analysis ◽

Globin Genes ◽

Site Mapping ◽

Physical Linkage ◽

Human And Mouse

To further our understanding of the regulation of vertebrate globin loci, we have isolated cosmids containing α- and β-globin genes from the pufferfish Fugu rubripes. By DNA fluorescence in situ hybridization (FISH) analysis, we show thatFugu contains 2 distinct hemoglobin loci situated on separate chromosomes. One locus contains only α-globin genes (α-locus), whereas the other also contains a β-globin gene (αβ-locus). This is the first poikilothermic species analyzed in which the physical linkage of the α- and β-globin genes has been uncoupled, supporting a model in which the separation of the α- and β-globin loci has occurred through duplication of a locus containing both types of genes. Surveys for transcription factor binding sites and DNaseI hypersensitive site mapping of the Fugu αβ-locus suggest that a strong distal locus control region regulating the activity of the globin genes, as found in mammalian β-globin clusters, may not be present in the Fugu αβ-locus. Searching the human and mouse genome databases with the genes surrounding the pufferfish hemoglobin loci reveals that homologues of some of these genes are proximal to cytoglobin, a recently described novel member of the globin family. This provides evidence that duplication of the globin loci has occurred several times during evolution, resulting in the 5 human globin loci known to date, each encoding proteins with specific functions in specific cell types.

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