Carbonic anhydrase expression and CO2 excretion during early development in zebrafishDanio rerio

Abstract

SUMMARYCarbonic anhydrase (CA) is critical for CO 2 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 CO 2 production increased during early development in zebrafish (Danio rerio), CA would become necessary for effective CO 2 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 CO 2 excretion increased approximately 15-fold from 24 to 48h.p.f. whereas rates of O 2 uptake increased only 6.7-fold over the same period, indicating a relative stimulation of CO 2 excretion over O 2 uptake. Treatment of 48h.p.f. larvae with the CA inhibitor acetazolamide resulted in CO 2 excretion rates that were 52% of the value in control larvae, a significant difference that occurred in the absence of any effect on O 2 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 CO 2 excretion over O 2 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 CO 2 excretion.

Cardiac responses to hypercapnia in larval zebrafish (Danio rerio): The links between CO2 chemoreception, catecholamines and carbonic anhydrase

“…The results of a previous study (Gilmour et al, 2009) demonstrated the specificity of the zCAc knockdown and most importantly showed that zCAb was not a target for the zCAc morpholino. Thus we are confident that the similar knockdown approach used in this study also specifically targeted zCAc.…”

“…, the physiological significance of the hypercapnia-mediated tachycardia in larvae is unknown. Given that the results of a recent study (Gilmour et al, 2009) suggested that the red blood cells of larval zebrafish may be involved in CO 2 excretion, it is conceivable that internal convection might selectively promote CO 2 excretion and thereby be increased by any elevation of cardiac output associated with tachycardia. Thus the tachycardia could serve to minimize the extent of the respiratory acidosis associated with exposure to hypercapnia.…”

“…To dissolve the acetazolamide, the pH of the water was increased to ~11.0 using NaOH and then titrated back down to 7.2 using HCl (Gilmour et al, 2009).…”

“…Morpholinos (tagged with 3′-carboxyfluorescein) for zebrafish cytosolic CA (zCAc; NM_199215, also referred to as CA2-like a) (Gilmour et al, 2009), the β 1 -adrenergic receptor (β 1 -AR; AB294717) (Steele et al, 2011) and a control morpholino were purchased from Gene Tools (Philomath, OR, USA) with the following sequences, respectively: 5′-AGTGGTCAGCCAT -TCCGCCAGCTGT-3′, 5′-ACGGTAGCCCGTCTCCCATTG-3′ and 5′-CCTCTTACCTCAGTTACAATTTATA-3′. All morpholinos were prepared to a final concentration of 4 ng nl −1 in Danieau buffer (58 mmol l −1 NaCl, 0.7 mmol l −1 KCl, 0.4 mmol l −1 MgSO 4 , 0.6 mmol l −1 Ca(NO 3 ) 2 , and 5 mmol l −1 Hepes with a final pH of 7.6), and Phenol Red was used as an indicator of positive injection.…”

“…Injections into one-or two-cell embryos were performed using a Narishige IM300 microinjector (Narishige International USA Inc., Long Island, NY, USA) with needles made from 1.0 mm borosilicate glass (Sutter Instrument, Novato, CA, USA). Dosage and sequence of zCA isoforms are based on Gilmour et al (Gilmour et al, 2009) and β 1 -AR from Steele et al (Steele et al, 2011). Neither study reported adverse effects when using a dosage of 4 ng per embryo.…”