Visualization in zebrafish larvae of Na⁺uptake in mitochondria-rich cells whose differentiation is dependent onfoxi3a

Abstract

Uptake of Na(+) from the environment is an indispensable strategy for the survival of freshwater fish, as they easily lose Na(+) from the plasma to a diluted environment. Nevertheless, the location of and molecules involved in Na(+) uptake remain poorly understood. In this study, we utilized Sodium Green, a Na(+)-dependent fluorescent reagent, to provide direct evidence that Na(+) absorption takes place in a subset of the mitochondria-rich (MR) cells on the yolk sac surface of zebrafish larvae. Combined with immunohistochemistry, we revealed that the Na(+)-absorbing MR cells were exceptionally rich in vacuolar-type H(+)-ATPase (H(+)-ATPase) but moderately rich in Na(+)-K(+)-ATPase. We also addressed the function of foxi3a, a transcription factor that is specifically expressed in the H(+)-ATPase-rich MR cells. When foxi3a was depleted from zebrafish embryos by antisense morpholino oligonucleotide injection, differentiation of the MR cells was completely blocked and Na(+) influx was severely reduced, indicating that MR cells are the primary sites for Na(+) absorption. Additionally, foxi3a expression is initiated at the gastrula stage in the presumptive ectoderm; thus, we propose that foxi3a is a key gene in the control of MR cell differentiation. We also utilized a set of ion transport inhibitors to assess the molecules involved in the process and discuss the observations.

Ion uptake and acid secretion in zebrafish (Danio rerio)

“…Later, at 96 h postfertilization, both zCA15a and zCA2-like a showed decreased H + activity and increased Na + uptake, with concomitant upregulation of zNHE3b and downregulation of zATP6V1a (H + -ATPase Asubunit) expression (Lin et al, 2008), demonstrating the roles of the 2 CA isoforms in the functions of HR cells. A study by Hirose's group (Esaki et al, 2007) also supported this notion; zCA2-like mRNA was colocalized in Sodium Green-accumulating HR cells, and 10 μmol l -1 ethoxzolamide (a CA inhibitor) was found to abolish 65% of the Sodium Green accumulation in zebrafish HR cells. On the other hand, acclimation to both acidic and low-Na + FW caused upregulation of zCA15a expression but did not change the zCA2-like a mRNA level in zebrafish gills (Lin et al, 2008).…”

“…Translational knockdown of H + -ATPase in HR cells with specific A subunit (zATP6V1a) morpholinos was found to decrease the surface H + concentration in skin and whole-body Na + content in morphants, and to impair the survival of morphants in an acidic environment (Horng et al, 2007), indicating the role of HR cells in zebrafish Na + -uptake/acid-secretion mechanisms. At the same time, another molecular physiological study by Esaki and colleagues (Esaki et al, 2007) also supported this notion: Sodium Green, a fluorescence probe for Na + , was used to signal Na + uptake to demonstrate that HR cells in zebrafish embryonic skin are the exact site for absorbing Na + based on the accumulation of fluorescence signals only by HR cells.…”

“…In a study by Esaki and colleagues (Esaki et al, 2007), Na + accumulation (monitored by Sodium Green) in HR cells in the skin of zebrafish embryo was inhibited by both 10 -4 mol l -1 amiloride and 10 -5 mol l -1 EIPA, but not by a lower concentration (10 -5 mol l -1 ) of amiloride, and NHE was therefore suggested to be the major player in the Na + -uptake pathway. Subsequently, Yan and colleagues (Yan et al, 2007) provided convincing molecular physiological evidence to support the argument proposed by Boisen and colleagues (Boisen et al, 2003) and Esaki and coworkers (Esaki et al, 2007). In the zebrafish SL9 family, eight members, zNHE1, -2, -3a, -3b, -5, -6, -7 and -8, were identified, and all of them, except zNHE3a, are expressed in gills.…”

“…In zebrafish skin and gills, HR cells have been demonstrated to be responsible for acid-secretion and Na + -uptake mechanisms (Lin et al, 2006;Esaki et al, 2007;Horng et al, 2007;Hwang and Lee, 2007). So far, at least two pathways have been proposed for the apical transport of Na + in fish gill cells (Hirose et al, 2003;Perry et al, 2003;Evans et al, 2005): (1) an apical V-type H + -ATPase electrically linked to Na + absorption via the epithelial Na + channel (ENaC) and (2) an electroneutral exchange of Na + and H + via an apical Na + /H + exchanger (NHE).…”

“…In a study by Boisen and colleagues (Boisen et al, 2003), 10 -5 to 10 -4 mol l -1 of amiloride (a Na + -uptake inhibitor) did not affect the Na + uptake in zebrafish acclimated to soft fresh water (FW) ([Na + ]=0.035 mmol l -1 , [Ca 2+ ]=0.004 mmol l -1 , pH 6.0), while 5ϫ10 -5 mol l -1 EIPA (an NHE-specific inhibitor) enhanced its uptake; thus it was concluded that the pathway for Na + uptake may be distinct from ENaC. In a study by Esaki and colleagues (Esaki et al, 2007), Na + accumulation (monitored by Sodium Green) in HR cells in the skin of zebrafish embryo was inhibited by both 10 -4 mol l -1 amiloride and 10 -5 mol l -1 EIPA, but not by a lower concentration (10 -5 mol l -1 ) of amiloride, and NHE was therefore suggested to be the major player in the Na + -uptake pathway. Subsequently, Yan and colleagues (Yan et al, 2007) provided convincing molecular physiological evidence to support the argument proposed by Boisen and colleagues (Boisen et al, 2003) and Esaki and coworkers (Esaki et al, 2007).…”