Department of Biomedical Science, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia.
Department of Biology, Faculty of Science, University of Ottawa, Ottawa, ON, Canada.
J Neurochem. 2021 Feb;156(4):481-498. doi: 10.1111/jnc.15108. Epub 2020 Jul 19.
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, ~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.
胶质细胞源性神经营养因子（GDNF）已被报道在体外和体内增强多巴胺能神经元的存活和分化，尽管这些结果仍存在争议。胶质细胞源性神经营养因子（gdnf）在斑马鱼中高度保守，在肠神经系统功能中发挥作用。然而，关于 gdnf 在硬骨鱼脑中的功能知之甚少。在这里，我们采用簇状规律间隔短回文重复序列/CRISPR 相关蛋白 9 来干扰 gdnf 在维持多巴胺能神经元发育中的功能。gdnf crispants 的基因分型显示编码区域的成功缺失，具有各种突变带大小，并在受精后 1、3 和 7 天下调 gdnf 转录本。值得注意的是，在 gdnf 缺陷 crispants 中观察到 8 号和 13 号腹侧神经节多巴胺能神经元数量减少约 20%。此外，通过 5-乙炔基-2'-脱氧尿苷脉冲追踪试验，发现 gdnf 耗竭导致多巴胺能神经发生适度减少。这些有害影响部分归因于crispants 中多巴胺能神经元命运特化相关转录因子（otp、lmx1b、shha 和 ngn1）的失调，以及全胚胎原位杂交（WISH）在 gdnf 突变体中的建立纯合突变体，显示出腹侧神经节中 otpb 和 lmx1b.1 的表达减少。有趣的是，crispants 的运动功能仅在 7 天出现影响，而不是更早。最后，正如预期的那样，gdnf 缺乏使 crispants 对 1-甲基-4-苯基吡啶毒性易感性增加。我们的结果表明硬骨鱼 gdnf 脑功能与哺乳动物的保守性，并揭示了 gdnf 与多巴胺能神经元分化中的转录因子之间的相互作用。
