scholarly journals Combination of in situ hybridization and immunocytochemistry to detect messenger RNAs in identified CNS neurons and glia in tissue culture.

1991 ◽  
Vol 39 (7) ◽  
pp. 891-898 ◽  
Author(s):  
P A Trimmer ◽  
L L Phillips ◽  
O Steward
Keyword(s):  
In Situ Hybridization ◽  
Cdna Probe ◽  
Cell Types ◽  
Intermediate Filament Protein ◽  
Messenger Rnas ◽  
Cell Type ◽  
Protein Subunit ◽  
The Central Nervous System ◽  
Cell Type Specific

We have developed a technique in which immunofluorescence is combined with in situ hybridization using cDNA and RNA probes to assess the expression and distribution of messenger RNAs (mRNA) by neurons and neuroglia in tissue cultures of the rat dentate gyrus. The probes used in this study include a cDNA probe for ribosomal RNA (rRNA) and an RNA probe (cRNA) for glial fibrillary acidic protein (GEAP), an intermediate filament protein subunit expressed by astrocytes in the central nervous system. Both ubiquitous (tubulin) and cell type-specific (MAP-2 and GEAP) antibodies were used to identify neurons and neuroglia in culture. Using this procedure, the mRNA for rRNA was found in the cell bodies and large processes of MAP-2-positive neurons and throughout the cytoplasm of GEAP-positive flat astrocytes. In process-bearing astrocytes, GEAP mRNA is concentrated in the cell body, although some hybridization also occurred in astrocyte cell processes. With this combined in situ hybridization-immunofluorescence technique, the expression and distribution of an mRNA can be examined in different immunocytochemically identified cell types under identical culture and hybridization conditions. It is also possible to determine if there is a differential subcellular distribution of an mRNA in a single cell and if the distribution of the mRNA reflects the distribution of the protein itself. Finally, this technique can be utilized to verify the specificity of probes for cell type-specific mRNAs and to determine appropriate hybridization conditions to produce a specific signal.

scholarly journals Tenascin in bone morphogenesis: expression by osteoblasts and cell type-specific expression of splice variants

1992 ◽  
Vol 103 (3) ◽  
pp. 765-771 ◽  
Author(s):  
E.J. Mackie ◽  
R.P. Tucker
Keyword(s):  
In Situ Hybridization ◽  
Splice Variants ◽  
Cdna Probe ◽  
Cell Type ◽  
Specific Expression ◽  
Primary Osteoblast ◽  
Cell Type Specific ◽  
Enriched Cultures

The extracellular matrix glycoprotein, tenascin, is associated in vivo with mesenchyme undergoing osteogenesis and chondrogenesis, but is absent from mature bone and cartilage matrix. The expression of tenascin by osteoblastic cells in vitro has been investigated by immunoblotting and immunocytochemistry. Tenascin was secreted into the medium and deposited in the matrix by human and rat osteoblast-like cell lines, as well as by primary osteoblast-enriched cultures from chick embryo calvarial bones. In primary osteoblast-enriched cultures, extracellular tenascin was found only in cell aggregates expressing the osteoblast marker alkaline phosphatase. Chicken osteoblast cultures synthesized almost exclusively the largest tenascin subunit, whereas fibroblast cultures from periostea of chicken calvariae synthesized approximately equal amounts of all three subunits. In situ hybridization studies of developing chicken bones, using a cDNA probe that hybridizes to all chicken tenascin splice variants, showed specific labelling of both osteogenic and chondrogenic regions of developing endochondral bones. In contrast, a cDNA probe specific for the large tenascin splice variant showed specific hybridization in osteogenic but not chondrogenic regions. Within osteogenic regions, tenascin mRNA was expressed by osteoblasts. A comparison of in situ hybridization and immunohistochemical studies demonstrated that tenascin mRNA and protein were codistributed in osteogenic regions of endochondral and membrane bones, whereas protein was retained in regions of differentiating cartilage where mRNA was no longer detectable. The results presented here demonstrate that tenascin is synthesized by osteoblasts. Moreover, within developing bones, there are at least three different cell type-specific patterns of expression of tenascin splice variants.

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

2003 ◽  
Vol 2 (3) ◽  
pp. 627-637 ◽  
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.

scholarly journals Control of Cell Type Proportioning in Dictyostelium discoideum by Differentiation-Inducing Factor as Determined by In Situ Hybridization

2004 ◽  
Vol 3 (5) ◽  
pp. 1241-1248 ◽  
Author(s):  
Toshinari Maruo ◽  
Haruyo Sakamoto ◽  
Negin Iranfar ◽  
Danny Fuller ◽  
Takahiro Morio ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Dictyostelium Discoideum ◽  
Wild Type ◽  
Cell Type ◽  
Type Conversion ◽  
New Genes ◽  
A Cell ◽  
Cell Type Specific ◽  
Cell Type Conversion

ABSTRACT We have determined the proportions of the prespore and prestalk regions in Dictyostelium discoideum slugs by in situ hybridization with a large number of prespore- and prestalk-specific genes. Microarrays were used to discover genes expressed in a cell type-specific manner. Fifty-four prespore-specific genes were verified by in situ hybridization, including 18 that had been previously shown to be cell type specific. The 36 new genes more than doubles the number of available prespore markers. At the slug stage, the prespore genes hybridized to cells uniformly in the posterior 80% of wild-type slugs but hybridized to the posterior 90% of slugs lacking the secreted alkylphenone differentiation-inducing factor 1 (DIF-1). There was a compensatory twofold decrease in prestalk cells in DIF-less slugs. Removal of prespore cells resulted in cell type conversion in both wild-type and DIF-less anterior fragments. Thus, DIF-1 appears to act in concert with other processes to establish cell type proportions.

scholarly journals Simultaneous detection of two messenger RNAs in the central nervous system: a simple two-step in situ hybridization procedure using a combination of radioactive and non-radioactive probes.

1991 ◽  
Vol 39 (11) ◽  
pp. 1575-1578 ◽  
Author(s):  
E Normand ◽  
B Bloch
Keyword(s):  
Central Nervous System ◽  
In Situ Hybridization ◽  
Simultaneous Detection ◽  
Messenger Rnas ◽  
Tissue Sections ◽  
System A ◽  
Step Procedure ◽  
Hybridization Procedure ◽  
The Central Nervous System

We present here a method enabling the simultaneous detection of two messenger RNAs in tissue sections by use of a two-step in situ hybridization procedure. Tissue sections were hybridized with a radioactive probe and coated with emulsion. The emulsion was processed for development, fixed, and a second hybridization was performed through the emulsion with a biotinylated probe subsequently revealed with streptavidin-alkaline phosphatase. This procedure allows the detection of two mRNAs without loss of signal, removal of the emulsion, or spurious reaction. The simultaneous detection of oxytocin and vasopressin mRNAs in the hypothalamus, and of dopamine receptor and neuropeptide mRNAs in the striatum, demonstrated the efficiency of the procedure. Such a two-step procedure provides a simple and flexible way to make possible comparative analysis of the localization of two mRNAs within the same tissue section.

scholarly journals Regulation of immunoreactive GAP-43 expression in rat cortical macroglia is cell type specific.

1990 ◽  
Vol 111 (1) ◽  
pp. 209-215 ◽  
Author(s):  
A da Cunha ◽  
L Vitković
Keyword(s):  
Nervous System ◽  
Developmental Regulation ◽  
Cell Types ◽  
Type I ◽  
Cell Type ◽  
System Type ◽  
The Central Nervous System ◽  
Cell Type Specific

Growth-associated protein 43 (GAP-43) is an abundant, intensely investigated membrane phosphoprotein of the nervous system (Benowitz, L.I., and A. Routtenberg. 1987. Trends Neurosci. 10:527-532; Skene, J. H. P. 1989. Annu. Rev. Neurosci. 12:127-156), with a hitherto unknown function. We have previously demonstrated that astrocytes, brain macroglial cells, contain GAP-43 (Steisslinger, H. W., V. J. Aloyo, and L. Vitković, 1987. Brain Res. 415:375-379; Vitković, L., H. W. Steisslinger, V. J. Aloyo, and M. Mersel. 1988. Proc. Natl. Acad. Sci. USA. 85:8296-8300; Vitković L., and M. Mersel. 1989. Metab. Brain Dis. 4:47-53). Results from double immunofluorescent labeling experiments presented here show that oligodendrocytes also contain GAP-43 immunoreactivity (GAP-43ir). Thus, all three macroglial cell types of the central nervous system (type I and type 2 astrocytes and oligodendrocytes) contain GAP-43. Whereas immunoreactive GAP-43 is expressed by progenitors of all macroglial cell types, the developmental regulation of its expression is cell type specific. Immunoreactive GAP-43 is downregulated in type 1 astrocytes, and constitutively expressed in both type 2 astrocytes and oligodendrocytes. These results may be relevant to potential function(s) of GAP-43.

scholarly journals Spatiotemporal mapping of RNA editing in the developing mouse brain using in situ sequencing reveals regional and cell-type-specific regulation

BMC Biology ◽  
2020 ◽  
Vol 18 (1) ◽  
Author(s):  
Elin Lundin ◽  
Chenglin Wu ◽  
Albin Widmark ◽  
Mikaela Behm ◽  
Jens Hjerling-Leffler ◽  
...  
Keyword(s):  
Single Cell ◽  
Rna Editing ◽  
Mouse Brain ◽  
Developmental Stages ◽  
Cell Types ◽  
Specific Cell ◽  
Cell Type ◽  
Cell Type Specific ◽  
Specific Regulation

Abstract Background Adenosine-to-inosine (A-to-I) RNA editing is a process that contributes to the diversification of proteins that has been shown to be essential for neurotransmission and other neuronal functions. However, the spatiotemporal and diversification properties of RNA editing in the brain are largely unknown. Here, we applied in situ sequencing to distinguish between edited and unedited transcripts in distinct regions of the mouse brain at four developmental stages, and investigate the diversity of the RNA landscape. Results We analyzed RNA editing at codon-altering sites using in situ sequencing at single-cell resolution, in combination with the detection of individual ADAR enzymes and specific cell type marker transcripts. This approach revealed cell-type-specific regulation of RNA editing of a set of transcripts, and developmental and regional variation in editing levels for many of the targeted sites. We found increasing editing diversity throughout development, which arises through regional- and cell type-specific regulation of ADAR enzymes and target transcripts. Conclusions Our single-cell in situ sequencing method has proved useful to study the complex landscape of RNA editing and our results indicate that this complexity arises due to distinct mechanisms of regulating individual RNA editing sites, acting both regionally and in specific cell types.

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