Loss of Function Disrupts Dopaminergic Pathways in Zebrafish.

Ankyrin repeat and kinase domain containing 1 (ANKK1) is a member of the receptor-interacting protein serine/threonine kinase family, known to be involved in cell proliferation, differentiation and activation of transcription factors. Genetic variation within the locus is suggested to play a role in vulnerability to addictions. However, mechanism of action is still poorly understood. It has been suggested that may affect the development and/or functioning of dopaminergic pathways. To test this hypothesis, we generated a CRISPR-Cas9 loss of function zebrafish line causing a 27 bp insertion that disrupts the sequence introducing an early stop codon. We found that transcript levels were significantly lower in mutant ( ) fish compared to their wild type ( ) siblings. In adult zebrafish brain, ankk1 protein was detected in isocortex, hippocampus, basolateral amygdala, mesencephalon, and cerebellum, resembling the mammalian distribution pattern. In contrast, ankk1 protein was reduced in the brain of fish. Quantitative polymerase chain reaction analysis revealed an increase in expression of mRNA in at both larval and adult stages. In adult zebrafish brain, drd2 protein was detected in cerebral cortex, cerebellum, hippocampus, and caudate homolog regions, resembling the pattern in humans. In contrast, drd2 expression was reduced in cortical regions of being predominantly found in the hindbrain. No differences in the number of cell bodies or axonal projections detected by anti-tyrosine hydroxylase immunostaining on 3 days post fertilization (dpf) larvae were found. Behavioral analysis revealed altered sensitivity to effects of both amisulpride and apomorphine on locomotion and startle habituation, consistent with a broad loss of both pre and post synaptic receptors. mutants showed reduced sensitivity to the effect of the selective dopamine receptor antagonist amisulpride on locomotor responses to acoustic startle and were differentially sensitive to the effects of the non-selective dopamine agonist apomorphine on both locomotion and habituation. Taken together, our findings strengthen the hypothesis of a functional relationship between and , supporting a role for in the maintenance and/or functioning of dopaminergic pathways. Further work is needed to disentangle 's role at different developmental stages.