gdnf affects early diencephalic dopaminergic neuron development through regulation of differentiation‐associated transcription factors in zebrafish,Journal of Neurochemistry

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gdnf affects early diencephalic dopaminergic neuron development through regulation of differentiation‐associated transcription factors in zebrafish
Journal of Neurochemistry ( IF 4.2 ) Pub Date : 2020-06-24 , DOI: 10.1111/jnc.15108
Chee Ern David Wong _{1,

2} , Khang Hua ₂ , Simon Monis ₂ , Vishal Saxena ₂ , Anwar Norazit ₁ , Suzita Mohd Noor ₁ , Marc Ekker ₂

Affiliation

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.

中文翻译：

gdnf通过调节斑马鱼的分化相关转录因子来影响早期多脑多巴胺能神经元的发育

据报道，胶质细胞源性神经营养因子（GDNF）可以增强多巴胺能神经元的体内外存活和分化，尽管这些结果仍在争论中。胶质细胞源性神经营养因子（gdnf）在斑马鱼中高度保守，并在肠神经系统功能中发挥作用。但是，关于硬骨脑中的gdnf功能知之甚少。在这里，我们采用簇状规则间隔的短回文重复/ CRISPR相关蛋白9来阻止gdnf在维持多巴胺能神经元发育中的功能。gdnf酥油的基因分型显示成功删除了具有各种突变带大小的编码区，并下调了gdnf受精后第1、3和7天的成绩单。值得注意的是，在缺乏gdnf的脆片中，第8和第13簇的腹侧二脑多巴胺能神经元数量减少了约20％。此外，gdnf的消耗导致多巴胺能神经发生的适度减少，这是通过5-乙炔基-2'-脱氧尿苷脉冲追逐测定法确定的。这些有害影响可以部分归因于多巴胺能神经元命运规范相关的转录因子（放松管制OTP，LMX1B，shha，并ngn1两个crispants），并建立了整装原位杂交纯合突变体（ WISH）对GDNF突变体显示腹侧间脑中otpb和lmx1b.1表达降低。有趣的是，脆片的运动功能仅在7 dpf时受到影响，而未受影响。最后，正如预期的那样，gdnf缺乏症使脆片更容易受到1-甲基-4-苯基吡啶鎓中毒的伤害。我们的结果表明，哺乳动物的硬骨鱼gdnf脑功能得以保护，并揭示了多巴胺能神经元分化中gdnf与转录因子之间的相互作用。

更新日期：2020-06-24

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