Gene expression during Drosophila melanogaster oogenesis: Analysis by in situ hybridization to tissue sections

Abstract Multiplexed RNA in situ hybridization for the analysis of gene expression patterns plays an important role in investigating development and disease. Here, we present a method for multiplexed RNA-ISH to detect spatial tumor heterogeneity in tissue sections. We made use of a microfluidic chip to deliver ISH-probes locally to regions of a few hundred micrometers over time periods of tens of minutes. This spatial multiplexing method can be combined with ISH-approaches based on signal amplification, with bright field detection and with the commonly used format of formalin-fixed paraffin-embedded tissue sections. By using this method, we analyzed the expression of HER2 with internal positive and negative controls (ActB, dapB) as well as predictive biomarker panels (ER, PgR, HER2) in a spatially multiplexed manner on single mammary carcinoma sections. We further demonstrated the applicability of the technique for subtype differentiation in breast cancer. Local analysis of HER2 revealed medium to high spatial heterogeneity of gene expression (Cohen effect size r = 0.4) in equivocally tested tumor tissues. Thereby, we exemplify the importance of using such a complementary approach for the analysis of spatial heterogeneity, in particular for equivocally tested tumor samples. As the method is compatible with a range of ISH approaches and tissue samples, it has the potential to find broad applicability in the context of molecular analysis of human diseases.

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The random primer labeling technique applied to in situ hybridization on tissue sections.

Journal of Histochemistry & Cytochemistry ◽

10.1177/36.10.3138309 ◽

1988 ◽

Vol 36 (10) ◽

pp. 1335-1340 ◽

Cited By ~ 13

Author(s):

M Thomas-Cavallin ◽

O Aït-Ahmed

Keyword(s):

Drosophila Melanogaster ◽

In Situ Hybridization ◽

Specific Activity ◽

High Specific Activity ◽

High Sensitivity ◽

Random Primer ◽

Tissue Sections ◽

In Situ Hybridizations ◽

Labeling Method

We report an application of the random primer labeling technique to in situ hybridizations on tissue sections. The ease of the method and the high specific activity achieved make it valuable when a large number of probes must be analyzed and high sensitivity is needed. We have applied this technique to study the spatial expression of a cluster of maternally acting genes (the yema gene region of Drosophila melanogaster which encodes eleven transcripts, some of them having a very low level of expression) (Aït-Ahmed et al., 1978: Dev Biol 122:153; Aït-Ahmed et al., unpublished results). The results reported here concern one of the transcripts of the yema region, which displays a peculiar anterior localization in the oocyte. We demonstrate that the "oligo-labeling" method allows a far better level of detection of the transcript of interest.

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The in situ localization of Adh transcripts in Drosophila species reveals evolved regulatory differences in spatially restricted expression

Genome ◽

10.1139/g94-139 ◽

1994 ◽

Vol 37 (6) ◽

pp. 984-991

Author(s):

Gogineni Ranganayakulu

Keyword(s):

Gene Expression ◽

Drosophila Melanogaster ◽

In Situ Hybridization ◽

Transcriptional Activity ◽

Fat Body ◽

Interspecific Variation ◽

Specific Expression ◽

Temporal Aspects ◽

Drosophila Adh

Spatial and temporal aspects of Adh expression were examined during oogenesis and embryogenesis of Drosophila melanogaster, D. simulans, and D. virilis by in situ hybridization. In stage 14 and 15 embryos, differences in zygotic expression of Adh in the primordia of the gastric caecae of D. simulans and in the fat body of D. virilis were observed. These zygotic differences appear to be transient because Adh expression is seen in the gastric caecae of stage 16 embryos of D. simulans and in the fat body of stage 17 embryos of D. virilis. Analysis of D. melanogaster × D. simulans hybrids revealed that the parental difference for transcriptional activity of Adh in the primordia of the gastric caecae is under dominant control. These results provide the basis for exploring evolved regulatory differences in Adh expression during oogenesis and embryogenesis of Drosophila, which are until now unexplored. The potential of in situ hybridization in analyzing evolved regulatory differences in gene expression is briefly discussed.Key words: Drosophila, Adh, tissue-specific expression, interspecific variation, in situ hybridization.

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Expression of human mucous proteinase inhibitor in respiratory tract: a study by in situ hybridization.

Journal of Histochemistry & Cytochemistry ◽

10.1177/43.6.7769236 ◽

1995 ◽

Vol 43 (6) ◽

pp. 645-648 ◽

Cited By ~ 14

Author(s):

I Van Seuningen ◽

J P Audie ◽

B Gosselin ◽

J J Lafitte ◽

M Davril

Keyword(s):

Gene Expression ◽

In Situ Hybridization ◽

Proteinase Inhibitor ◽

Tissue Level ◽

Leukocyte Elastase ◽

Tissue Sections ◽

Submucosal Glands ◽

Large Airways ◽

Immunohistochemical Studies

Human mucous proteinase inhibitor (MPI) is present in bronchial secretions, where it participates in the protection of lung structures against degradation by leukocyte elastase. The protein has been localized by immunohistochemical studies and immunogold labeling essentially in the serous cells of the submucosal glands and also in the surface epithelial cells of central and peripheral airways. However, until now no gene expression study has been performed at the tissue level. In this study, in situ hybridization was used to precisely study MPI mRNA expression in bronchial tissue sections with a specific radiolabeled oligonucleotide probe. By light microscopy, MPI gene expression was localized exclusively in the serous and seromucinous acini of the submucosal glands of large airways. The MPI gene was expressed in submucosal glands of normal and carcinomatous tissue sections, whereas it was not expressed in bronchial and bronchiolar surface epithelia.

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Radioactive In Situ Hybridization to Detect Gene Expression in Skeletal Tissue Sections

Methods in Molecular Biology - Skeletal Development and Repair ◽

10.1007/978-1-62703-989-5_16 ◽

2014 ◽

pp. 217-232 ◽

Cited By ~ 3

Author(s):

Kyu Sang Joeng ◽

Jenna Regan ◽

Fanxin Long

Keyword(s):

Gene Expression ◽

In Situ Hybridization ◽

Skeletal Tissue ◽

Tissue Sections ◽

Detect Gene Expression

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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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