Analysis of the Dopaminergic Systems Development and Function by Targeted Genome Editing
Keywords: Dopamine, tyrosine hydroxylase, TALENs, CRISPRs, neurogenesis, prepulse inhibition
Categories: Life Sciences
Dopamine is a prominent modulatory neurotransmitter affecting a vast array of neural circuits, however, dopamine has also been shown to be an important regulator of neurogenesis both in the developing embryo and in the adult mammalian brain. A more thorough understanding of dopamine and its role in neurogenesis will be pivotal to exploit endogenously produced neural precursors for cell replacement in neurodegenerative disorders such as Parkinson’s Disease. Two genes important for dopaminergic function are tyrosine hydroxylase (th), the rate limiting enzyme in the synthesis of catecholamines, and the re-uptake carrier dopamine transporter (dat / slc6a3). Using TALENs technology, mutations were induced in the th and dat genes of zebrafish embryos. A knockout mutation in the th gene is expected to cause the complete lack of dopamine and noradrenalin production in all catecholaminergic cells, whereas a knockout of the dat gene is expected to cause the accumulation of dopamine in the synaptic cleft. TALENs targeting exon one of th induced mutant alleles with a 7 bp (th m1403 ) deletion, an 11 bp (th m1404 ) deletion and a 1 bp (th m1405 ) insertion close to the translation start, causing truncated proteins devoid of their native functions thus resulting in the knockout of the gene. TALENs targeting the dat gene induced a mutant allele with a 10 bp deletion which, however, was not expected to cause the knockout of the gene since the native start ATG was deleted and a second in frame ATG is present 20 codons downstream of the start codon. The CRISPR/Cas9 system was used to induce a mutation at a different position of exon one of the dat gene and genotyping of the F 1 generation will determine if this has lead to the knockout of the gene. Homozygous th mutants were analyzed for catecholaminergic neuron development in the brain and motor neuron development in the spine. No major differences were found in neither motor neuron cell numbers in the spine nor in catecholamine cell number in the brain when comparing th -/- and wildtype larvae at 3 days post fertilization. These results indicate that dopamine does not play a pivotal role in the generation of these neurons during embryogenesis, and question previous publications reporting changes in motor neuron number following pharmacological or antisense morpholino based manipulation of dopaminergic function. th -/- zebrafish larvae were additionally analyzed for prepulse inhibition responses, and preliminary data was consistent with previous publications suggesting that dopamine may modulate this behaviour. Genome editing techniques were in addition employed to enable the targeted insertion of transgenes. The CRISPR/Cas9 system was used to establish the protocol and its functionality was demonstrated by the insertion of a gal4 transgene at the eGFP locus of Tg(NeuroD:eGFP) x Tg(UAS:RFP, cry:eGFP) zebrafish embryos, driving red fluorescent expression in 1 of 6 of injected embryos. In the future this system can be used to establish reliable transgenic lines with a gal4 at the th and dat loci driving expression of active proteins that may control axonogenesis of dopaminergic neurons, an interesting aspect useful for potential new regeneration therapies, or driving expression of neuroprotective proteins in order to attenuate neurodegenerative processes in zebrafish models of Parkinson’s disease.