gdnf affects early diencephalic dopaminergic neuron development through regulation of differentiation-associated transcription factors in zebrafish

• Main Authors: Wong, Chee Ern David, Hua, Khang, Monis, Simon, Saxena, Vishal, Norazit, Anwar, Mohd Noor, Suzita, Ekker, Marc
• Format: Article
• Published: Wiley 2021
• Subjects: QD Chemistry; QH301 Biology; RC Internal medicine
• Online Access: http://eprints.um.edu.my/28765/

Glial cell line-derived neurotrophic factor (GDNF) has been reported to enhance dopaminergic neuron survival and differentiation in vitro and in vivo, although those results are still being debated. Glial cell line-derived neurotrophic factor (gdnf) is highly conserved in zebrafish and plays a role in enteric nervous system function. However, little is known about gdnf function in the teleost brain. Here, we employed clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 to impede gdnf function in the maintenance of dopaminergic neuron development. Genotyping of gdnf crispants revealed successful deletions of the coding region with various mutant band sizes and down-regulation of gdnf transcripts at 1, 3 and 7 day(s) post fertilization. Notably, similar to 20% reduction in ventral diencephalic dopaminergic neuron numbers in clusters 8 and 13 was observed in the gdnf-deficient crispants. In addition,gdnf depletion caused a modest reduction in dopaminergic neurogenesis as determined by 5-ethynyl-2'-deoxyuridine pulse chase assay. These deleterious effects could be partly attributed to deregulation of dopaminergic neuron fate specification-related transcription factors (otp, lmx1b, shha, and ngn1) in both crispants and established homozygous mutants with whole mount in-situ hybridization(WISH) on gdnf mutants showing reduced otpb and lmx1b.1 expression in the ventral diencephalon. Interestingly, locomotor function of crispants was only impacted at 7 dpf, but not earlier. Lastly, as expected, gdnf deficiency heightened crispants vulnerability to 1-methyl-4-phenylpyridinium toxic insult. Our results suggest conservation of teleost gdnf brain function with mammals and revealed the interactions between gdnf and transcription factors in dopaminergic neuron differentiation.