In fishes, branchial cytosolic carbonic anhydrase (CA) plays an important role in ion and acid-base regulation. The Ca17a isoform in zebrafish (Danio rerio) is expressed abundantly in Na⁺-absorbing/H⁺-secreting H⁺-ATPase-rich (HR) cells. The present study aimed to identify the role of Ca17a in ion and acid-base regulation across life stages using CRISPR/Cas9 gene editing. However, in preliminary experiments, we established that ca17a knockout is lethal with ca17a^-/-mutants exhibiting a significant decrease in survival beginning at approximately 12 days post-fertilization (dpf) and with no individuals surviving past 19 dpf. Based on these findings, we hypothesized that ca17a^-/- mutants would display alterations in ion and acid-base balance and that these physiological disturbances might underlie their early demise. Na⁺ uptake rates were significantly increased by up to 300% in homozygous mutants compared to wild-type individuals at 4 and 9 dpf, however whole-body Na⁺ content remained constant. In contrast, Cl^- uptake was significantly reduced in ca17a^-/- mutants, while Cl^- content also was unaffected. Reduction of CA activity by Ca17a morpholino knockdown or ethoxzolamide treatments similarly reduced Cl^- uptake, implicating Ca17a in the mechanism of Cl^- uptake by larval zebrafish. H⁺ secretion, O₂ consumption, CO₂ excretion, and ammonia excretion were generally unaltered in ca17a^-/- mutants. In conclusion, while loss of Ca17a caused marked changes in ion uptake rates, providing strong evidence for a Ca17a-depedent Cl^- uptake mechanism, the underlying causes of the lethality of this mutation in zebrafish remain unclear.

中文翻译：

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使用碳酸酐酶ca17a敲除研究斑马鱼（Danio rerio）中离子吸收的机制

在鱼类中，分支胞质碳酸酐酶（CA）在离子和酸碱调节中起重要作用。斑马鱼（Danio rerio）中的Ca17a亚型在富Na ^+-吸收/ H ^+-分泌H ⁺ -ATPase的（HR）细胞中大量表达。本研究旨在使用CRISPR / Cas9基因编辑来确定Ca17a在生命各个阶段对离子和酸碱调节中的作用。但是，在初步实验中，我们确定ca17a敲除对ca17a具有致命性^-/-突变体在受精后约12天（dpf）开始表现出明显的存活率下降，并且没有个体存活超过19 dpf。基于这些发现，我们假设ca17a ^-/-突变体将显示离子和酸碱平衡的变化，并且这些生理紊乱可能是其早期灭亡的基础。与野生型个体相比，在4和9 dpf时，纯合突变体中Na ^+的吸收率显着提高了300％，但是全身Na ⁺含量保持恒定。与此相反，氯^-摄取显著在ca17a降低^{- / -}突变体，而氯^-内容也不受影响。通过Ca17a CA活性的降低吗啉代或敲低治疗ethoxzolamide类似地减少氯^-摄取，以Cl机制牵连Ca17a ^-摄取幼虫斑马鱼。在ca17a ^-/-突变体中，H ⁺分泌，O ₂消耗，CO ₂排泄和氨排泄通常保持不变。总之，尽管Ca17a的流失造成标志着离子的吸收率的变化，对于Ca17a-depedent氯提供了强有力的证据^-吸收机制，这种突变在斑马鱼的杀伤力的根本原因仍不清楚。