Methods to Enhance Signal Using Isotopic In Situ Hybridization

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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Cell-specific metallothionein gene expression in mouse decidua and placentae

Development ◽

10.1242/dev.107.3.611 ◽

1989 ◽

Vol 107 (3) ◽

pp. 611-621 ◽

Cited By ~ 2

Author(s):

S.K. De ◽

M.T. McMaster ◽

S.K. Dey ◽

G.K. Andrews

Keyword(s):

Gene Expression ◽

In Situ Hybridization ◽

Embryo Implantation ◽

Normal Pregnancy ◽

Mrna Levels ◽

Metallothionein Gene ◽

Visceral Yolk Sac ◽

Rna In Situ Hybridization ◽

Low Levels

Oligodeoxyribonucleotide excess solution hybridization, Northern blot and in situ hybridization were used to analyze metallothionein gene expression in mouse decidua and placentae during gestation. Metallothionein (MT) -I and -II mRNA levels were constitutively elevated, 11- and 13-fold, respectively, relative to the adult liver, in the deciduum (D8), and decreased coordinately about 6-fold during the period of development when the deciduum is replaced by the developing placenta (D10-16). Coincident with this decline, levels of MT mRNA increased dramatically in the visceral yolk sac endoderm. In situ hybridization established that MT-I mRNA was present at low levels in the uterine luminal epithelium (D4), but was elevated at the site of embryo implantation exclusively in the primary decidual zone by D5, and then in the secondary decidual zone (D6-8). Although low levels of MT mRNA were detected in total placental RNA, in situ hybridization revealed constitutively high levels in the outer placental spongiotrophoblasts. Analysis of pulse-labeled proteins from decidua and placentae established that these tissues are active in the synthesis of MT. The constitutively high levels of MT mRNA in decidua were only slightly elevated following injection of cadmium (Cd) and/or zinc (Zn), whereas in placentae they increased several-fold. MT mRNA levels were equally high in decidua and experimentally induced deciduomata (D8) which establishes that decidual MT gene expression is not dependent on the presence of the embryo or some embryo-derived factor. Although the functional role of MT during development is speculative, these results establish the concept that, from the time of implantation to late in gestation, the mouse embryo is surrounded by cells, interposed between the maternal and embryonic environments, which actively express the MT genes. This suggests that MT plays an important role in the establishment and maintenance of normal pregnancy.

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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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Kisspeptin Cells in the Ewe Brain Respond to Leptin and Communicate with Neuropeptide Y and Proopiomelanocortin Cells

Endocrinology ◽

10.1210/en.2009-1190 ◽

2010 ◽

Vol 151 (5) ◽

pp. 2233-2243 ◽

Cited By ~ 164

Author(s):

Kathryn Backholer ◽

Jeremy T. Smith ◽

Alix Rao ◽

Alda Pereira ◽

Javed Iqbal ◽

...

Keyword(s):

Gene Expression ◽

In Situ Hybridization ◽

Neuropeptide Y ◽

Preoptic Area ◽

Cell Types ◽

Normal Weight ◽

Target Cells ◽

Reciprocal Connections ◽

Fluorescent Immunohistochemistry

Kisspeptin stimulates reproduction, and kisspeptin cells in the arcuate nucleus (ARC) express Ob-Rb in the mouse. Herein we report studies in ewes to determine whether kisspeptin cells express Ob-Rb and respond to leptin and whether reciprocal connections exist between kisspeptin cells and proopiomelanocortin (POMC) or neuropeptide Y (NPY) cells to modulate reproduction and metabolic function. Kiss1 mRNA was measured by in situ hybridization in ovariectomized ewes that were normal body weight, lean, or lean with leptin treatment by intracerebroventricular (icv) infusion (4 μg/h, 3 d). Kiss1 expression in the ARC and the preoptic area was lower in hypogonadotropic lean animals than animals of normal weight, and icv infusion of leptin partially restored Kiss1 expression in lean animals. Single-cell laser capture microdissection coupled with real-time PCR showed that Kiss1 cells in the preoptic area and ARC express Ob-Rb. Double-label fluorescent immunohistochemistry showed that reciprocal connections exist between kisspeptin cells and NPY and POMC cells. Accordingly, we treated ovariectomized ewes with kisspeptin (5 μg/h, icv) or vehicle for 20 h and examined POMC and NPY gene expression by in situ hybridization. Kisspeptin treatment reduced POMC and increased NPY gene expression. Thus, kisspeptin neurons respond to leptin and expression of Kiss1 mRNA is affected by leptin status. Kisspeptin cells communicate with NPY and POMC cells, altering expression of the relevant genes in the target cells; reciprocal connections also exist. This network between the three cell types could coordinate brain control of reproduction and metabolic homeostatic systems.

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Changing Patterns of Gene Expression in Dictyostelium Prestalk Cell Subtypes Recognized by In Situ Hybridization with Genes from Microarray Analyses

Eukaryotic Cell ◽

10.1128/ec.2.3.627-637.2003 ◽

2003 ◽

Vol 2 (3) ◽

pp. 627-637 ◽

Cited By ~ 57

Author(s):

Mineko Maeda ◽

Haruyo Sakamoto ◽

Negin Iranfar ◽

Danny Fuller ◽

Toshinari Maruo ◽

...

Keyword(s):

Gene Expression ◽

In Situ Hybridization ◽

Cell Types ◽

Cell Type ◽

Developmentally Regulated ◽

Specific Expression ◽

Cell Type Specific Expression ◽

Changing Patterns ◽

Cell Type Specific

ABSTRACT We used microarrays carrying most of the genes that are developmentally regulated in Dictyostelium to discover those that are preferentially expressed in prestalk cells. Prestalk cells are localized at the front of slugs and play crucial roles in morphogenesis and slug migration. Using whole-mount in situ hybridization, we were able to verify 104 prestalk genes. Three of these were found to be expressed only in cells at the very front of slugs, the PstA cell type. Another 10 genes were found to be expressed in the small number of cells that form a central core at the anterior, the PstAB cell type. The rest of the prestalk-specific genes are expressed in PstO cells, which are found immediately posterior to PstA cells but anterior to 80% of the slug that consists of prespore cells. Half of these are also expressed in PstA cells. At later stages of development, the patterns of expression of a considerable number of these prestalk genes changes significantly, allowing us to further subdivide them. Some are expressed at much higher levels during culmination, while others are repressed. These results demonstrate the extremely dynamic nature of cell-type-specific expression in Dictyostelium and further define the changing physiology of the cell types. One of the signals that affect gene expression in PstO cells is the hexaphenone DIF-1. We found that expression of about half of the PstO-specific genes were affected in a mutant that is unable to synthesize DIF-1, while the rest appeared to be DIF independent. These results indicate that differentiation of some aspects of PstO cells can occur in the absence of DIF-1.

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Pro-opiomelanocortin (POMC) gene expression, as identified by in situ hybridization, in purified populations of interstitial macrophages and Leydig cells of the adult rat testis

Reproduction Fertility and Development ◽

10.1071/rd9930545 ◽

1993 ◽

Vol 5 (5) ◽

pp. 545 ◽

Cited By ~ 3

Author(s):

H Li ◽

GP Risbridger ◽

JA Clements

Keyword(s):

Gene Expression ◽

In Situ Hybridization ◽

Leydig Cells ◽

Cell Types ◽

Rat Testis ◽

Adult Rat ◽

Pomc Gene ◽

Rna Probe ◽

Silver Grains

The presence of testicular pro-opiomelanocortin (POMC) mRNA and POMC-derived peptides has recently been demonstrated in purified preparations of interstitial macrophages and in Leydig cells of the adult rat testis by Northern blot analysis and immunocytochemistry. In the present study, in situ hybridization provided further evidence that the POMC gene is expressed by both purified interstitial macrophages and Leydig cells. The cellular localization of the POMC transcripts was similar for both cell types, silver grains being predominantly located in the cytoplasm. The specificity of the labelling was demonstrated by the lack of silver grains in the preparations pretreated with RNAase or hybridized with an insulin cDNA probe, a gene known not to be expressed in these cell types. An additional control was provided by hybridization with a sense POMC RNA probe, which gave a less intense signal when compared with the antisense RNA probe under the same experimental conditions. The results confirm POMC gene expression in both macrophages and Leydig cells in the adult rat testis.

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RNA In Situ Hybridization for Detecting Gene Expression Patterns in the Abdomens and Wings of Drosophila Species

Methods and Protocols ◽

10.3390/mps4010020 ◽

2021 ◽

Vol 4 (1) ◽

pp. 20

Author(s):

Mujeeb Shittu ◽

Tessa Steenwinkel ◽

William Dion ◽

Nathan Ostlund ◽

Komal Raja ◽

...

Keyword(s):

Gene Expression ◽

In Situ Hybridization ◽

Expression Patterns ◽

Drosophila Species ◽

Gene Expression Patterns ◽

Probe Design ◽

Tissue Preparation ◽

Design And Synthesis ◽

Rna In Situ Hybridization

RNA in situ hybridization (ISH) is used to visualize spatio-temporal gene expression patterns with broad applications in biology and biomedicine. Here we provide a protocol for mRNA ISH in developing pupal wings and abdomens for model and non-model Drosophila species. We describe best practices in pupal staging, tissue preparation, probe design and synthesis, imaging of gene expression patterns, and image-editing techniques. This protocol has been successfully used to investigate the roles of genes underlying the evolution of novel color patterns in non-model Drosophila species.

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Analysis of the transcriptome of bovine endometrial cells isolated by laser micro-dissection (1): specific signatures of stromal, glandular and luminal epithelial cells

BMC Genomics ◽

10.1186/s12864-021-07712-0 ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Wiruntita Chankeaw ◽

Sandra Lignier ◽

Christophe Richard ◽

Theodoros Ntallaris ◽

Mariam Raliou ◽

...

Keyword(s):

Gene Expression ◽

Protein Localization ◽

Expression Profiles ◽

Cell Types ◽

Molecular Signature ◽

Receptor Protein ◽

Specific Gene ◽

Specific Cell ◽

Biological Processes ◽

Endometrial Cells

Abstract Background A number of studies have examined mRNA expression profiles of bovine endometrium at estrus and around the peri-implantation period of pregnancy. However, to date, these studies have been performed on the whole endometrium which is a complex tissue. Consequently, the knowledge of cell-specific gene expression, when analysis performed with whole endometrium, is still weak and obviously limits the relevance of the results of gene expression studies. Thus, the aim of this study was to characterize specific transcriptome of the three main cell-types of the bovine endometrium at day-15 of the estrus cycle. Results In the RNA-Seq analysis, the number of expressed genes detected over 10 transcripts per million was 6622, 7814 and 8242 for LE, GE and ST respectively. ST expressed exclusively 1236 genes while only 551 transcripts were specific to the GE and 330 specific to LE. For ST, over-represented biological processes included many regulation processes and response to stimulus, cell communication and cell adhesion, extracellular matrix organization as well as developmental process. For GE, cilium organization, cilium movement, protein localization to cilium and microtubule-based process were the only four main biological processes enriched. For LE, over-represented biological processes were enzyme linked receptor protein signaling pathway, cell-substrate adhesion and circulatory system process. Conclusion The data show that each endometrial cell-type has a distinct molecular signature and provide a significantly improved overview on the biological process supported by specific cell-types. The most interesting result is that stromal cells express more genes than the two epithelial types and are associated with a greater number of pathways and ontology terms.

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Pituitary adenoma producing growth hormone and adrenocorticotropin: a histological, immunocytochemical, electron microscopic, and in situ hybridization study

Journal of Neurosurgery ◽

10.3171/jns.1998.88.6.1111 ◽

1998 ◽

Vol 88 (6) ◽

pp. 1111-1115 ◽

Cited By ~ 27

Author(s):

Kalman Kovacs ◽

Eva Horvath ◽

Lucia Stefaneanu ◽

Juan Bilbao ◽

William Singer ◽

...

Keyword(s):

Growth Hormone ◽

In Situ Hybridization ◽

Pituitary Adenoma ◽

Immunoelectron Microscopy ◽

Secretory Granules ◽

Cell Types ◽

Content Type ◽

Separate Cell ◽

Fine Print

✓ The authors report on the morphological features of a pituitary adenoma that produced growth hormone (GH) and adrenocorticotropic hormone (ACTH). This hormone combination produced by a single adenoma is extremely rare; a review of the available literature showed that only one previous case has been published. The tumor, which was removed from a 62-year-old man with acromegaly, was studied by histological and immunocytochemical analyses, transmission electron microscopy, immunoelectron microscopy, and in situ hybridization. When the authors used light microscopy, the tumor appeared to be a bimorphous mixed pituitary adenoma composed of two separate cell types: one cell population synthesized GH and the other ACTH. The cytogenesis of pituitary adenomas that produce more than one hormone is obscure. It may be that two separate cells—one somatotroph and one corticotroph—transformed into neoplastic cells, or that the adenoma arose in a common stem cell that differentiated into two separate cell types. In this case immunoelectron microscopy conclusively demonstrated ACTH in the secretory granules of several somatotrophs. This was associated with a change in the morphological characteristics of secretory granules. Thus it is possible that the tumor was originally a somatotropic adenoma that began to produce ACTH as a result of mutations that occurred during tumor progression.

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