Zebrafish

Zebrafish is a known model organism for almost all human diseases, such as human muscular dystrophies, neurogenesis, behavioral studies and high throughput drug testing to name a few. It is known that 82% of human disease-causing genes have at least one orthologue common in zebrafish. This and the optical transparency of zebrafish larvae makes it an attractive model to study complex mechanisms behind cardiovascular diseases and angiogenesis.

The ICG has a long expertise in identification of novel genes or loci associated with CVD using genome-wide association studies (GWAS). Ease in genetic manipulation (forward and reverse genetics), rapid breeding and short feeding time makes zebrafish an essential and crucial model organism to functionally analyze these novel GWAS hits in a short time frame.


Dr. hum. biol. Zouhair Aherrahrou

Viviana Vedder, MSc

Maren Behrensen

Projects

Krishan Vishnolia

Globally, high blood pressure (BP) is the most important risk factor for cardiovascular disease. Several genome-wide association studies (GWAS) have identified variants associated with BP traits at more than 535 chromosomal loci with genome-wide significance. The post-GWAS challenge is to annotate the most likely causal gene(s) at each locus. Chromosome 10q24.32 is a locus associated with BP that encompasses five genes: CYP17A1, BORCS7, AS3MT, CNNM2, and NT5C2 and warrants investigation to determine the specific gene or genes responsible for the phenotype.

The aim of the project is to identify the most likely causal gene(s) associated with BP at the 10q24.32 locus using zebrafish as an animal model.

We report significantly higher blood flow, increased arterial pulse, and elevated linear velocity in zebrafish larvae with cnnm2 and nt5c2 knocked down using gene-specific splice modification transcriptional morpholinos, compared with controls. No differences in blood-flow parameters were observed after as3mt, borcs7, or cyp17a1 knockdown. There was no effect on vessel diameter in animals with any of the four genes knocked down. At the molecular level, expression of hypertension markers (crp and ace) was significantly increased in cnnm2 and nt5c2 knockdown larvae. Further, the results obtained by morpholino knockdown were validated using zebrafish knockout (KO) lines with cnnm2 and nt5c2 deficiency, again resulting in higher blood flow, increased arterial pulse, and elevated linear velocity. Analysis of nt5c2a KO larvae demonstrated that lack of this gene resulted in reduced expression of cnnm2a, with reciprocal downregulation of nt5c2a in cnnm2a KO larvae. Staining of whole-blood smears from nt5c2 mutants revealed that KO of this gene might be associated with an acute lymphoblastic leukemia phenotype, consistent with literature reports. Additional experiments were designed based on previous literature on cnnm2a mutant zebrafish revealed impaired renal function, high levels of renin, and significantly increased expression of the ren gene, leading us to hypothesize that the observed elevated blood-flow parameters may be attributable to triggering of the renin–angiotensin–aldosterone signaling pathway.

Our zebrafish data establish CNNM2 and NT5C2 as the most likely causal genes at the 10q24.32 BP locus and indicate that they trigger separate downstream mechanistic pathways (Vishnolia et al. 2020).

Editorial Commentary published in Frontiers in Cardiovascular Medicine – Hypertension.

Viviana Vedder, MSc

Angiogenesis, a process that forms new blood vessels from existing ones, is associated with a variety of diseases. Irregularities in the formation of secondary blood vessels can lead to atherosclerosis, diabetes and hypertension, among others, which are considered to be major risk factors for cardiovascular diseases (CVDs), the leading cause of death worldwide.

Zebrafish provide a well-established platform for high-content screenings and can be used to investigate the effect of drugs on angiogenesis in vivo.

Previously, we established an angiogenesis assay using sunitinib malate, a known kinase inhibitor, that has been previously tested in zebrafish and which now serves as a positive control. In our phenotypical high-content screen we use a small molecule library of nearly 1300 compounds, that covers the main anatomic therapeutic chemical (ATC) groups. In order to identify and investigate drugs regulating angiogenesis and its targets for clinical applications, we examine cardiovascular phenotypes by evaluating data from fluorescent images of treated Tg(fli1a:eGFP)y1 and brightfield movies of the heart and the caudal aorta above the urogenital pore.

This data will be used to measure vessel length, thickness and lumen formation as well as pulse, heart phenotype, erythrocyte shape and count; and blood flow.

Viviana Vedder, MSc

Nadine Odenthal

Collagen VI is a component of the extracellular matrix that plays a central role in a variety of tissues, such as gastrointestinal tract, liver, eyes and heart. In addition to a biomechanical role, it has a cytoprotective function, such as counteracting apoptosis and oxidative damage, in cells such as myofibers, chondrocytes, neurons, fibroblasts and cardiomyocytes.

Mutations in genes encoding collagen VI (COL6A1-A3) are responsible for Collagen VI congenital muscular dystrophy (COL6-CMD), a spectrum disorder, that presents a wide range of mild to severe phenotypes.

In this project we seek to gain deeper understanding of the development and progression of COL6-CMD using zebrafish as a model for muscle disorders.

First, we perform a col6a2 knockdown in zebrafish larvae, which are analyzed by histology, life imaging and functional analysis to establish a suitable CMD model.

Finally, we will apply these methods to examine col6a2-Knockout zebrafish over several generations to identify potential treatment targets hidden in WT Backgrounds of certain zebrafish-lines.

Scroll to Top