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Multi-target Chromogenic Whole-mount In Situ Hybridization for Comparing Gene Expression Domains in Drosophila Embryos
Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
2016 (English)In: Journal of Visualized Experiments, ISSN 1940-087X, E-ISSN 1940-087X, no 107, e53830Article in journal (Refereed) Published
Abstract [en]

To analyze gene regulatory networks active during embryonic development and organogenesis it is essential to precisely define how the different genes are expressed in spatial relation to each other in situ. Multi-target chromogenic whole-mount in situ hybridization (MC-WISH) greatly facilitates the instant comparison of gene expression patterns, as it allows distinctive visualization of different mRNA species in contrasting colors in the same sample specimen. This provides the possibility to relate gene expression domains topographically to each other with high accuracy and to define unique and overlapping expression sites. In the presented protocol, we describe a MC-WISH procedure for comparing mRNA expression patterns of different genes in Drosophila embryos. Up to three RNA probes, each specific for another gene and labeled by a different hapten, are simultaneously hybridized to the embryo samples and subsequently detected by alkaline phosphatase-based colorimetric immunohistochemistry. The described procedure is detailed here for Drosophila, but works equally well with zebrafish embryos.

Place, publisher, year, edition, pages
2016. no 107, e53830
Keyword [en]
Developmental Biology, Issue 107, Digoxigenin, biotin, fluorescein, azo dye, Fast Blue, Fast Red, INT, alkaline phosphatase substrate, WISH, fruit fly
National Category
Developmental Biology
Research subject
Molecular Biology
Identifiers
URN: urn:nbn:se:su:diva-128914DOI: 10.3791/53830OAI: oai:DiVA.org:su-128914DiVA: diva2:917730
Available from: 2016-04-07 Created: 2016-04-07 Last updated: 2016-04-08Bibliographically approved
In thesis
1. Drosophila immune responses in a model for epithelial hypertrophy
Open this publication in new window or tab >>Drosophila immune responses in a model for epithelial hypertrophy
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Apoptosis, differentiation and proliferation have to be tightly balanced and thus regulated to maintain tissue homeostasis. Stress, metabolic cues, genetic variability, infections and physiological host-commensal interactions influence this balance and thus need to be integrated. Therefore, beyond the discrimination between self and non-self (i.e., foreign) also damage inflicted on tissues under sterile conditions is perceived by the immune system due to altered tissue integrity. Growing knowledge of the interaction between the immune system and wounded or more generally altered tissues allows inferring on anti-tumorous immune responses, too. Despite the lack of adaptive immunity, Drosophila mounts solid and versatile innate immune responses that functionally and molecularly share many properties with their vertebrate counterparts. In fact, tissue overgrowth, tissue dysplasia or endogenous danger signaling activate systemic Toll-signaling in the fat body indicating a role for the Drosophila immune system in maintaining tissue homeostasis.

Here we characterize systemic and local immune responses towards altered or transformed tissues by using a Drosophila hypertrophy model, which is based on the overexpression of a dominant-active variant of the small GTPase Ras (Ras85DG12V) in salivary glands and wing discs. We characterized the strong induction of hemocyte recruitment to the glands as a consequence of JNK-dependent MMP1-expression and basal membrane degradation. Apart from this cellular immune reaction, transcriptome profiling revealed comprehensive humoral immune responses mounted by the fat body that involved signatures of Toll- and imd-activation. Moreover, a novel tissue-autonomous response that was spatially restricted to the anterior end of the RasV12-expressing salivary gland itself was identified. While multiple immune genes were found to be upregulated in the anterior compartment as detected by RNA sequencing, particular focus was given to the effector peptide Drosomycin (Drs). Overexpression of Drs with RasV12 in the entire gland similar to the inhibition of the JNK-pathway was able to selectively rescue a characteristic set of RasV12-induced phenotypes, which ultimately blocks the recruitment of hemocytes. Thereby, local immune-related responses in RasV12-expressing salivary glands are able to restrict the tissue damage induced by hypertrophic growth.

Place, publisher, year, edition, pages
Stockholm: Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, 2016. 63 p.
National Category
Immunology Genetics
Research subject
Molecular Biology
Identifiers
urn:nbn:se:su:diva-128916 (URN)978-91-7649-400-4 (ISBN)
Public defence
2016-06-01, Nordenskiöldsalen, Geovetenskapens hus, Svante Arrhenius väg 12, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 4: Manuscript. Paper 5: Manuscript.

Available from: 2016-05-09 Created: 2016-04-07 Last updated: 2016-08-17Bibliographically approved

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Hauptmann, GiselbertKrautz, RobertTheopold, Ulrich
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