The role of two-pore channel type 2 (TPC2, encoded by tcpn2)-mediated Ca²⁺ release was recently characterized in zebrafish during establishment of the early spinal circuitry, one of the key events in the coordination of neuromuscular activity. Here, we extend our study to investigate the in vivo role of TPC2 in the regulation of caudal primary motor neuron (CaP) axon extension. We used a combination of TPC2 knockdown with a translation-blocking morpholino antisense oligonucleotide (MO), TPC2 knockout via the generation of a tpcn2^dhkz1a mutant line of zebrafish using CRISPR/Cas9 gene-editing and pharmacological inhibition of TPC2 via incubation with bafilomycin A1 (an H⁺-ATPase inhibitor) or trans-ned-19 (an NAADP receptor antagonist), and showed that these treatments attenuated CaP Ca²⁺ signaling and inhibited axon extension. We also characterized the expression of an arc1-like transcript in CaPs grown in primary culture. MO-mediated knockdown of ARC1-like in vivo led to attenuation of the Ca²⁺ transients in the CaP growth cones and an inhibition of axon extension. Together, our new data suggest a link between ARC1-like, TPC2 and Ca²⁺ signaling during axon extension in zebrafish.

最近，斑马鱼在早期脊髓回路建立过程中表征了2 型双孔通道（TPC2，由tcpn2编码）介导的 Ca ^{2+释放的作用，这是协调神经肌肉活动的关键事件之一。}在这里，我们扩展了我们的研究，以调查TPC2 在调节尾部初级运动神经元 (CaP) 轴突延伸中的体内作用。我们将 TPC2 敲低与翻译阻断吗啉代反义寡核苷酸 (MO) 相结合，通过使用 CRISPR/Cas9 基因编辑生成斑马鱼tpcn2 ^dhkz1a突变系来敲除 TPC2，并通过与巴弗洛霉素 A1 一起孵育来对 TPC2 进行药理抑制（ H ⁺ -ATP酶抑制剂）或trans -ned-19（NAADP受体拮抗剂），并表明这些治疗减弱了CaP Ca ²⁺信号传导并抑制轴突延伸。我们还对原代培养的 CaP 中arc1 样转录本的表达进行了表征。MO介导的体内ARC1样敲除导致CaP生长锥中Ca ^{2+瞬变的减弱和轴突延伸的抑制。}总之，我们的新数据表明斑马鱼轴突延伸过程中ARC1 样、TPC2 和 Ca ^{2+信号传导之间存在联系。}