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Bonkhofer, F.

Publications and source records attributed to Bonkhofer, F..

2 recordsLinked to original sources

Runx1 promotes scar deposition and inhibits myocardial proliferation and survival during zebrafish heart regeneration

Runx1 is a transcription factor that plays a key role in determining the proliferative and differential state of multiple cell-types, during both development and adulthood. Here, we report how runx1 is specifically upregulated at the injury site during zebrafish heart regeneration, but unexpectedly, absence of runx1 results in enhanced regeneration. Using single cell sequencing, we found that the wild-type injury site consists of Runx1-positive endocardial cells and thrombocytes that induce expression of smooth muscle and collagen genes without differentiating into myofibroblasts. Both these populations are absent in runx1 mutants, resulting in a less collagenous and fibrinous scar. The reduction in fibrin in the mutant is further explained by reduced myofibroblast formation and by upregulation of components of the fibrin degradation pathway, including plasminogen receptor Annexin 2A as well as downregulation of plasminogen activator inhibitor serpine1 in myocardium and endocardium, resulting in increased levels of Plasminogen. In addition, we find enhanced myocardial proliferation as well as increased myocardial survival in the mutant. Our findings suggest that Runx1 controls the regenerative response of multiple cardiac cell-types and that targeting Runx1 is a novel therapeutic strategy to induce endogenous heart repair.

developmental biology↗

An optimized pipeline for parallel image-based quantification of gene expression and genotyping after in situ hybridization

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 in situ hybridization (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 lmo4a mutants.\n\nSUMMARY STATEMENTOur optimized protocol to genotype zebrafish mutant embryos after in situ hybridization and digitally quantify the in situ signal will help to standardize existing experimental designs and methods of analysis.

developmental biology↗