genotyping-quantification-situ-hybridization-staining

Genotyping and Quantification of In Situ Hybridization Staining in Zebrafish

In situ hybridization (ISH) is an important technique that enables researchers to study mRNA distribution in situ and has been a critical technique in ...

University of Oxford

BHF Centre of Research Excellence

Advances in genome engineering have resulted in the generation of numerous zebrafish mutant lines. A commonly used method to assess gene expression in the mutants is  hybridisation (ISH). Because the embryos can be distinguished by genotype after ISH, comparing gene expression between wild-type and mutant siblings can be done blinded and in parallel. Such experimental design reduces the technical variation between samples and minimises the risk of bias. This approach, however, requires an efficient method of genomic DNA extraction from post-ISH fixed zebrafish samples to ascribe phenotype to genotype. Here we describe a method to obtain PCR-quality DNA from 95-100% of zebrafish embryos, suitable for genotyping after ISH. In addition, we provide an image analysis protocol for quantifying gene expression of ISH-probed embryos, adaptable for the analysis of different expression patterns. Finally, we show that intensity-based image analysis enables accurate representation of the variability of gene expression detected by ISH and that it can complement quantitative methods like qRT-PCR. By combining genotyping after ISH and computer-based image analysis, we have established a high-confidence, unbiased methodology to assign gene expression levels to specific genotypes, and applied it to the analysis of molecular phenotypes of newly generated  mutants.

An optimised pipeline for parallel image-based quantification of gene expression and genotyping after  hybridisation.

Haematopoietic stem cells are generated from the haemogenic endothelium (HE) located in the floor of the dorsal aorta (DA). Despite being integral to arteries, it is controversial whether HE and arterial endothelium share a common lineage. Here, we present a transgenic zebrafish runx1 reporter line to isolate HE and aortic roof endothelium (ARE)s, excluding non-aortic endothelium. Transcriptomic analysis of these populations identifies Runx1-regulated genes and shows that HE initially expresses arterial markers at similar levels to ARE. Furthermore, runx1 expression depends on prior arterial programming by the Notch ligand dll4. Runx1 mutants fail to downregulate arterial genes in the HE, which remains integrated within the DA, suggesting that Runx1 represses the pre-existing arterial programme in HE to allow progression towards the haematopoietic fate. These findings strongly suggest that, in zebrafish, aortic endothelium is a precursor to HE, with potential implications for pluripotent stem cell differentiation protocols for the generation of transplantable HSCs.

Monika Krecsmarik

MRC Molecular Haematology Unit,
MRC Weatherall Institute of Molecular Medicine,
John Radcliffe Hospital,
MRC Molecular Haematology Unit, MRC Weatherall Institute of Molecular Medicine, John Radcliffe Hospital

University of Oxford

Genotyping and Quantification of In Situ Hybridization Staining in Zebrafish

Monika Krecsmarik

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University of Oxford

Genotyping and Quantification of In Situ Hybridization Staining in Zebrafish

MRC Molecular Haematology Unit,
MRC Weatherall Institute of Molecular Medicine,
John Radcliffe Hospital,
MRC Molecular Haematology Unit, MRC Weatherall Institute of Molecular Medicine, John Radcliffe Hospital