BACKGROUND Signaling cascades, such as the extracellular signal-regulated kinase (ERK) pathway, play vital roles in early vertebrate development. Signals through these pathways are initiated by a growth factor or hormone, are transduced through a kinase cascade, and result in the expression of specific downstream genes that promote cellular proliferation, growth, or differentiation. Tight regulation of these signals is provided by positive or negative modulators at varying levels in the pathway, and is required for proper development and function. Two members of the dual-specificity phosphatase (Dusp) family, dusp6 and dusp2, are believed to be negative regulators of the ERK pathway and are expressed in both embryonic and adult zebrafish, but their specific roles in embryogenesis remain to be fully understood. RESULTS Using CRISPR/Cas9 genome editing technology, we generated zebrafish lines harboring germ line deletions in dusp6 and dusp2. We do not detect any overt defects in dusp2 mutants, but we find that approximately 50% of offspring from homozygous dusp6 mutants do not proceed through embryonic development. These embryos are fertilized, but are unable to proceed past the first zygotic mitosis and stall at the 1-cell stage for several hours before dying by 10 h post fertilization. We demonstrate that dusp6 is expressed in gonads of both male and female zebrafish, suggesting that loss of dusp6 causes defects in germ cell production. Notably, the 50% of homozygous dusp6 mutants that complete the first cell division appear to progress through embryogenesis normally and give rise to fertile adults. CONCLUSIONS The fact that offspring of homozygous dusp6 mutants stall prior to activation of the zygotic genome, suggests that loss of dusp6 affects gametogenesis and/or parentally-directed early development. Further, since only approximately 50% of homozygous dusp6 mutants are affected, we postulate that ERK signaling is tightly regulated and that dusp6 is required to keep ERK signaling within a range that is permissive for proper embryogenesis. Lastly, since dusp6 is expressed throughout zebrafish embryogenesis, but dusp6 mutants do not exhibit defects after the first cell division, it is possible that other regulators of the ERK pathway compensate for loss of dusp6 at later stages.

中文翻译：

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斑马鱼对双特异性磷酸酶6的父母要求。

背景技术信号级联反应，例如细胞外信号调节激酶（ERK）途径，在早期脊椎动物发育中起重要作用。通过这些途径的信号由生长因子或激素引发，通过激酶级联转导，并导致特定下游基因的表达，从而促进细胞增殖，生长或分化。这些信号的严格调节是由正调节剂或负调节剂在通路中的不同水平提供的，是适当发育和功能所必需的。双特异性磷酸酶（Dusp）家族的两个成员dusp6和dusp2被认为是ERK途径的负调控因子，并且在胚胎和成年斑马鱼中都有表达，但是它们在胚胎发生中的特定作用尚待充分了解。结果使用CRISPR / Cas9基因组编辑技术，我们生成了在dusp6和dusp2中带有种系缺失的斑马鱼系。我们没有在dusp2突变体中发现任何明显的缺陷，但是我们发现纯合dusp6突变体的约50％的后代不会通过胚胎发育进行。这些胚胎受精，但无法继续经过第一个合子有丝分裂，并在1细胞阶段停滞数小时，直到受精后10小时死亡。我们证明了dusp6在雄性和雌性斑马鱼的性腺中都有表达，这表明dusp6的缺失会导致生殖细胞生产中的缺陷。值得注意的是，完成第一次细胞分裂的纯合dusp6突变体的50％似乎正常地通过胚胎发生进展，并产生可育的成年。结论纯合的dusp6突变体的后代在合子​​基因组激活之前停滞的事实表明，dusp6的缺失会影响配子发生和/或父母主导的早期发育。此外，由于仅约50％的纯合dusp6突变体受到影响，因此我们假设ERK信号传导受到严格调节，并且要求dusp6将ERK信号传导保持在允许正常胚胎发生的范围内。最后，由于dusp6在整个斑马鱼的胚胎发生中表达，但是dusp6突变体在第一次细胞分裂后没有表现出缺陷，因此ERK途径的其他调节子有可能在以后的阶段补偿dusp6的损失。由于仅约50％的纯合dusp6突变体受到影响，因此我们假设ERK信号传导受到严格调节，并且要求dusp6将ERK信号传导保持在适当的胚胎发生所允许的范围内。最后，由于dusp6在整个斑马鱼的胚胎发生中表达，但是dusp6突变体在第一次细胞分裂后没有表现出缺陷，因此ERK途径的其他调节子有可能在以后的阶段补偿dusp6的损失。由于仅约50％的纯合dusp6突变体受到影响，因此我们假设ERK信号传导受到严格调节，并且要求dusp6将ERK信号传导保持在适当的胚胎发生所允许的范围内。最后，由于dusp6在整个斑马鱼的胚胎发生中表达，但是dusp6突变体在第一次细胞分裂后没有表现出缺陷，因此ERK途径的其他调节子有可能在以后的阶段补偿dusp6的损失。