The Role of Aquaporin and Tight Junction Proteins in the Regulation of Water Movement in Larval Zebrafish (Danio rerio)

Abstract

Teleost fish living in freshwater are challenged by passive water influx; however the molecular mechanisms regulating water influx in fish are not well understood. The potential involvement of aquaporins (AQP) and epithelial tight junction proteins in the regulation of transcellular and paracellular water movement was investigated in larval zebrafish (Danio rerio). We observed that the half-time for saturation of water influx (K u) was 4.3±0.9 min, and reached equilibrium at approximately 30 min. These findings suggest a high turnover rate of water between the fish and the environment. Water influx was reduced by the putative AQP inhibitor phloretin (100 or 500 μM). Immunohistochemistry and confocal microscopy revealed that AQP1a1 protein was expressed in cells on the yolk sac epithelium. A substantial number of these AQP1a1-positive cells were identified as ionocytes, either H+-ATPase-rich cells or Na+/K+-ATPase-rich cells. AQP1a1 appeared to be expressed predominantly on the basolateral membranes of ionocytes, suggesting its potential involvement in regulating ionocyte volume and/or water flux into the circulation. Additionally, translational gene knockdown of AQP1a1 protein reduced water influx by approximately 30%, further indicating a role for AQP1a1 in facilitating transcellular water uptake. On the other hand, incubation with the Ca2+-chelator EDTA or knockdown of the epithelial tight junction protein claudin-b significantly increased water influx. These findings indicate that the epithelial tight junctions normally act to restrict paracellular water influx. Together, the results of the present study provide direct in vivo evidence that water movement can occur through transcellular routes (via AQP); the paracellular routes may become significant when the paracellular permeability is increased.

The water channel aquaporin-1a1 facilitates movement of CO2 and ammonia in zebrafish (Danio rerio) larvae

“…The methods used to achieve and confirm functional knockdown of AQP1a1 were similar to those used by Kwong et al (2013). In brief, an antisense morpholino oligonucleotide (5′-AAG CCT TGC TCT TCA GCT CGT TCA T-3′; Genetools, OR, USA) designed to be complementary to the translational start site of zebrafish AQP1a1 (GenBank NM 207059.1) was prepared to a final concentration of 4 ng nl Ca(NO 3 ) 2 , 5.0 mmol l −1 HEPES, adjusted to pH 7.6] containing 0.05% (v/v) Phenol Red (as a visible indicator of successful injection).…”

“…This antibody detects a single band at ∼27 kDa in 4 dpf zebrafish larvae (Kwong et al, 2013). Membranes were washed with TBST (3×5 min) and incubated with a 1:5000 dilution of horseradish-peroxidase (HRP) goat anti-rabbit IgG (Pierce, Rockford, IL, USA) for 2 h at room temperature.…”

“…Gene-specific primers for rhcg1 (forward, 5′-TTT GGA GGA GAG GCT GAA GA-3′; reverse, 5′-CGA AAC ACA AGG CCA CAC AC-3′) were designed using Primer3 software (http://frodo.wi.mit.edu/primer3/) and the PCR product was confirmed by direct sequencing. Primers for aqp1a1 (forward, 5′-GCG ACT GTG GCC AGC GCT AT-3′; reverse, 5′-CGT CCC GCC GCC TTT TGT CT-3′) were from Kwong et al (2013) and the ribosomal 18S subunit (GenBank FJ915075.1) was used as a reference gene with primers (forward, 5′-GGC GGC GTT ATT CCC ATG ACC-3′; reverse, 5′-GGT GGT GCC CTT CCG TCA ATT C-3′) from Kumai and Perry (2011). The conditions for all experiments were 95°C for 3 min, 40 cycles of 95°C for 20 s and 60°C for 20 s, with 5 min for a final extension at 72°C.…”

“…By contrast, in situ hybridization studies demonstrated aqp1a1 expression in the vasculature, in RBCs, and in the skin covering the yolk sac of larval zebrafish, locations in which it could potentially contribute to gas transfer (Chen et al, 2010). The yolk sac epithelium contains at least two types of ion-transporting cells, or ionocytes, that express AQP1a1 as early as 4 dpf: the V-type H + -ATPase-rich (HR) cells and the Na + ,K + -ATPase-rich (NaR) cells (Kwong et al, 2013). Moreover, expression of zebrafish aqp1a1 in Xenopus oocytes established that AQP1a1 was permeable to both CO 2 and ammonia (Chen et al, 2010).…”

Vasotocin and isotocin regulate aquaporin 1 function in the sea bream

“…Not all members of the aquaporin group are represented in fish, which lack AQP2 (Cerdá and Finn, 2010;Finn and Cerdá, 2011). In turn, Aqp1 expression has been shown in water-permeable tissues such as gills, kidney or intestine (Kwong et al, 2013;Madsen et al, 2014;Martinez et al, 2005a,b;Tipsmark et al, 2010;Raldúa et al, 2008).…”