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The sensing of environmental fluctuations and initiation of appropriate physiological responses is crucial to homeostasis. Neuroepithelial cells (NECs) in fishes are putative chemoreceptors, resembling mammalian Type I (glomus) cells, that respond in vitro to changes in O 2 , CO 2 , NH 3 , and pH. Cytosolic carbonic anhydrase (Ca17a) is thought to be involved in CO 2 sensing owing to its presence in NECs. Zebrafish ( Danio rerio ) lacking functional Ca17a were generated via CRISPR/Cas9 technology and used to assess the role of Ca17a in initiating the cardiorespiratory responses to elevated CO 2 (hypercapnia). Unfortunately, the homozygous knockout mutants ( ca17a -/- ) did not survive more than ∼12-14 days postfertilization (dpf), restricting experiments to early developmental stages (4-8 dpf). Changes in ventilation ( f V ) and cardiac ( f H ) frequency in response to hypercapnia (1% CO 2 ) in wild-type ( ca17a +/+ ), heterozygous ( ca17a +/- ) and ca17a -/- fish were used to investigate Ca17a-dependent CO 2 sensing and downstream signaling. Wild-type fish exhibited hyperventilation during hypercapnia as indicated by an increase in f V . In the ca17a -/- fish, the hyperventilatory response was attenuated markedly but only at 8 dpf. Hypercapnic tachycardia was observed for all genotypes and did not appear to be influenced by the absence of Ca17a. Interestingly, ca17a -/- fish exhibited a significantly lower resting f H that became more pronounced as the fish aged. The decrease in resting f H was prevented ("rescued") when ca17a -/- embryos were injected with ca17a mRNA. Collectively, the results of this study support a role for Ca17a in promoting hyperventilation during hypercapnia in larval zebrafish and suggest a previously unrecognized role for Ca17a in determining resting heart rate.

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 ∼12 days postfertilization (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 with wild-type individuals at 4 and 9 dpf; however, whole body Na + content remained constant. While Cl - uptake was significantly reduced in ca17a -/- mutants, Cl - content 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, O 2 consumption, CO 2 excretion, and ammonia excretion were generally unaltered in ca17a -/- mutants. In conclusion, while the loss of Ca17a caused marked changes in ion uptake rates, providing strong evidence for a Ca17a-dependent Cl - uptake mechanism, the underlying causes of the lethality of this mutation in zebrafish remain unclear.