Zebrafish foxP2 zinc finger nuclease mutant has normal axon pathfinding.
Zebrafish foxP2 zinc finger nuclease mutant has normal axon pathfinding.
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DOI：
10.1371/journal.pone.0043968
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发表时间：
2012
期刊：
影响因子：
3.7
通讯作者：
Bonkowsky JL
中科院分区：
文献类型：
--
作者：
Xing L;Hoshijima K;Grunwald DJ;Fujimoto E;Quist TS;Sneddon J;Chien CB;Stevenson TJ;Bonkowsky JL
foxP2是一种叉头结构域转录因子，对人类的言语和语言发育至关重要，但其在中枢神经系统连接建立中的作用尚不清楚。虽然体外研究已经确定轴突引导分子是foxP2调节的靶点，并且细胞培养分析表明foxP2在神经突生长中起作用，但体内研究缺乏foxP2在轴突寻路中的作用。我们使用改良的锌指核酸酶方法在斑马鱼foxP2基因中产生突变。使用基于pcr的高分辨率熔融曲线分析（HRMA），我们筛选并鉴定了3个在第2编码外显子上携带无义突变的突变体：一个17碱基对（bp）缺失，一个8bp缺失和一个4bp插入。cDNA序列分析证实了这些是移码突变，预测了早期蛋白质截断。纯合子突变体鱼存活且可育，体形态不变，胚胎期中枢神经系统凋亡、增殖或模式无明显差异。已知foxP2靶基因cntnap2的表达减少，该基因通过注射野生型foxP2转录物获救。当我们使用泛轴突标记或转基因系（包括foxp2神经元特异性增强子）检测轴突寻路时，我们没有观察到任何轴突引导错误。我们的研究结果表明foxP2在发育过程中并不是轴突寻路所必需的。
foxP2, a forkhead-domain transcription factor, is critical for speech and language development in humans, but its role in the establishment of CNS connectivity is unclear. While in vitro studies have identified axon guidance molecules as targets of foxP2 regulation, and cell culture assays suggest a role for foxP2 in neurite outgrowth, in vivo studies have been lacking regarding a role for foxP2 in axon pathfinding. We used a modified zinc finger nuclease methodology to generate mutations in the zebrafish foxP2 gene. Using PCR-based high resolution melt curve analysis (HRMA) of G0 founder animals, we screened and identified three mutants carrying nonsense mutations in the 2nd coding exon: a 17 base-pair (bp) deletion, an 8bp deletion, and a 4bp insertion. Sequence analysis of cDNA confirmed that these were frameshift mutations with predicted early protein truncations. Homozygous mutant fish were viable and fertile, with unchanged body morphology, and no apparent differences in CNS apoptosis, proliferation, or patterning at embryonic stages. There was a reduction in expression of the known foxP2 target gene cntnap2 that was rescued by injection of wild-type foxP2 transcript. When we examined axon pathfinding using a pan-axonal marker or transgenic lines, including a foxP2-neuron-specific enhancer, we did not observe any axon guidance errors. Our findings suggest that foxP2 is not necessary for axon pathfinding during development.
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9.8
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