Samenvatting

To retrieve new mutants with a cardiac left-right patterning defect a zebrafish (Danio rerio) forward genetic screen was set-up. We were especially interested in the phenotypes that did not show a subsequently affected visceral looping, because genes affected in these mutants are specific for the left-right patterning of the heart. For this reason we looked at the cardiac and visceral looping in mutant embryos. This is visualized by expression patterns of respectively Cmlc2 and FoxA3 at 48 hours post-fertilization.

There were 99 F2 families and consequently 99 genomes available for screening of which 54 genomes were actually screened. We found four mutants with a distorted cardiac left-right patterning; HU119, HU305, HU304 and HU202. HU202 and HU304 showed in addition an affected visceral looping, thus the affected genes are not specific for cardiac left-right patterning. The HU305 mutation is specific for genes controlling left-right patterning. However, the cardiac left-right patterning defect seen in HU305 mutants was correlated with C1 dorsalization, a phenotype linked with a defect in the BMP pathway. Complementation assays with Alk8, Smad5 and BMP mutants ruled out HU305 linkage to one of these genes. The HU119 mutation was also specific for the cardiac left-right patterning and did not show any additional phenotypes. Our findings provide evidence that in addition to general mechanism left-right patterning can also be regulated by organ-specific mechanisms.

The AB and TL zebrafish lineages that were used for the set-up of the screen were polymorphic for several SSLP markers. This made it possible to link the mutations to the affected genes with the help of genomic mapping, a forward genetics technique that is based on the chance that recombination occurs between a marker and the mutation. The first steps of gene mapping has been performed on HU119 mutant embryos and showed linkage to chromosome 7. Initial research of a gene database did not result in possible candidate genes, therefore further gene mapping steps are needed.


Keywords: Zebrafish forward screen, forward genetics, cardiac jogging, left-right patterning, genomic mapping, BMP pathway.