Carbonic anhydrase expression and CO2 excretion during early development in zebrafish Danio rerio

Carbonic anhydrase (CA) is critical for CO2 excretion in adult fish, but little is known of the expression or function of CA during early development. The present study examined the hypothesis that, as rates of CO2 production increased during early development in zebrafish (Danio rerio), CA would become necessary for effective CO2 excretion, and that the pattern of CA expression during early development would reflect this transition. Real-time RT-PCR was used to examine the mRNA expression of the two main intracellular CA isoforms over a time course of early development ranging from 0 to 120 h post fertilization (h.p.f.). The mRNA expression of zCAb was generally higher than that of zCAc, particularly during the earliest stages of development. Rates of CO2 excretion increased approximately 15-fold from 24 to 48 h.p.f. whereas rates of O2 uptake increased only 6.7-fold over the same period, indicating a relative stimulation of CO2 excretion over O2 uptake. Treatment of 48 h.p.f. larvae with the CA inhibitor acetazolamide resulted in CO2 excretion rates that were 52% of the value in control larvae, a significant difference that occurred in the absence of any effect on O2 uptake. Antisense morpholino oligonucleotides were used to selectively knock down one or both of the main intracellular CA isoforms. Subsequent measurement of gas transfer rates at 48 h.p.f. indicated that CA knockdown caused a significant relative inhibition of CO2 excretion over O2 uptake, regardless of which cytosolic CA isoform was targeted for knockdown. These results suggest that between 24 h.p.f. and 48 h.p.f., developing zebrafish begin to rely on CA to meet requirements for increased CO2 excretion.