Molecular characterization and transcriptional regulation of the Na+/K+ ATPase α subunit isoforms during development and salinity challenge in a teleost fish, the Senegalese sole (Solea senegalensis) (original) (raw)
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Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology, 2015
In the present work, five genes encoding different Na + ,K + ATPase (NKA) α-isoforms in the teleost Solea senegalensis are described for the first time. Sequence analysis of predicted polypeptides revealed a high degree of conservation across teleosts and mammals. Phylogenetic analysis clustered the five genes into three main clades: α1 (designated atp1a1a and atp1a1b), α2 (designated atp1a2) and α3 (designated atp1a3a and atp1a3b) isoforms. Transcriptional analysis in larvae showed distinct expression profiles during development. In juvenile tissues, the atp1a1a gene was highly expressed in osmoregulatory organs, atp1a2 in skeletal muscle, atp1a1b in brain and heart and atp1a3a and atp1a3b mainly in brain. Quantification of mRNA abundance after a salinity challenge showed that atp1a1a transcript levels increased significantly in the gill of soles transferred to high salinity water (60 ppt). In contrast, atp1a3a transcripts increased at low salinity (5 ppt). In situ hybridization (ISH) analysis revealed that the number of ionocytes expressing atp1a1a transcripts in the primary gill filaments was higher at 35 and 60 ppt than at 5 ppt and remained undetectable or at very low levels in the lamellae at 5 and 35 ppt but increased at 60 ppt. Immunohistochemistry showed a higher number of positive cells in the lamellae. Whole-mount analysis of atp1a1a mRNA in young sole larvae revealed that it was localized in gut, pronephric tubule, gill, otic vesicle, yolk sac ionocytes and chordacentrum. Moreover, atp1a1a mRNAs increased at mouth opening (3 DPH) in larvae incubated at 36 ppt with a greater signal in gills.
Cell and Tissue Research, 2016
The euryhaline shrimp Palaemonetes argentinus exemplifies an evolutionary transition from brackish to freshwater habitats that requires adequate osmoregulatory capacities. Hyperosmoregulation is functional at hatching and it likely begins during the embryonic phase allowing this species to develop entirely in fresh water. Here, we investigated the Na + / K +-ATPase α-subunit gene (nka-α) expression using quantitative real-time PCR and localized Na + /K +-ATPase (NKA) in ion-transporting epithelia through immunofluorescence microscopy. We reared shrimps from spawning to juvenile stages at two salinities (1, 15 ‰) and maintained adults for 3 weeks at three salinity treatments (1, 15, 25 ‰). nka-α gene expression was measured in: (1) embryos at an early (SI), intermediate (SII) and late (SIII) stage of embryonic development; (2) newly hatched larvae (Zoea I, ZI); and (3) isolated gill tissue of adults. The nka-α expression was low in SI and SII embryos and reached maximum levels prior to hatching (SIII), which were similar to expression levels detected in the ZI. The nka-α expression in SIII and ZI was highest at 15 ‰, whereas salinity did not affect expression in earlier embryos. In SIII, in ZI and in a later zoeal stage ZIV, NKA was localized in epithelial cells of pleurae, in the inner-side epithelium of branchiostegite and in the antennal glands. Gills appeared in the ZIV but NKA immunolabeling of the cells of the gill shaft occurred in a subsequent developmental larval stage, the decapodid. Extrabranchial organs constitute the main site of osmoregulation in early ontogenetic stages of this freshwater shrimp.
Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology, 2012
In the present work, five genes encoding different Na + ,K + ATPase (NKA) α-isoforms in the teleost Solea senegalensis are described for the first time. Sequence analysis of predicted polypeptides revealed a high degree of conservation across teleosts and mammals. Phylogenetic analysis clustered the five genes into three main clades: α1 (designated atp1a1a and atp1a1b), α2 (designated atp1a2) and α3 (designated atp1a3a and atp1a3b) isoforms. Transcriptional analysis in larvae showed distinct expression profiles during development. In juvenile tissues, the atp1a1a gene was highly expressed in osmoregulatory organs, atp1a2 in skeletal muscle, atp1a1b in brain and heart and atp1a3a and atp1a3b mainly in brain. Quantification of mRNA abundance after a salinity challenge showed that atp1a1a transcript levels increased significantly in the gill of soles transferred to high salinity water (60 ppt). In contrast, atp1a3a transcripts increased at low salinity (5 ppt). In situ hybridization (ISH) analysis revealed that the number of ionocytes expressing atp1a1a transcripts in the primary gill filaments was higher at 35 and 60 ppt than at 5 ppt and remained undetectable or at very low levels in the lamellae at 5 and 35 ppt but increased at 60 ppt. Immunohistochemistry showed a higher number of positive cells in the lamellae. Whole-mount analysis of atp1a1a mRNA in young sole larvae revealed that it was localized in gut, pronephric tubule, gill, otic vesicle, yolk sac ionocytes and chordacentrum. Moreover, atp1a1a mRNAs increased at mouth opening (3 DPH) in larvae incubated at 36 ppt with a greater signal in gills.
Journal of Crustacean Biology, 2006
Immunolocalization and ontogenetical changes in Na þ ,K þ-ATPase were investigated in the antennal glands, intestine, and branchial cavity in developing Homarus gammarus. The antennal glands lined by undifferentiated cells are detectable in embryos at stage EI 150 lm (EI: eye index; 640 lm at hatching). From a mesodermic sac and an ectodermic tubular epithelium (EI 225 lm), they develop into coelomosac, labyrinth, and bladder up to larval stage II. In larval stage III, the end parts of the labyrinth fold inward. In the first post-larvae, the coelomosac is completely enclosed in the folded labyrinth, there is no nephridial tubule. The definitive organization of the antennal gland found in adults is achieved in post-larval stage V. At EI 425 lm, the intestine epithelium is composed of cuboidal cells, and the branchial cavity is already formed at EI 325 lm. The presence of Na þ ,K þ-ATPase was detected on the basolateral side of ionocytes lining the epithelium sac of the antennal gland starting at EI 425 lm, in the intestine at EI 625 lm, and in the epipodites of the branchial cavity at EI 525 lm. In post-larvae (stage V), a strong fluorescence was also found on the inner-side of the branchiostegite epithelium. Thus the ontogeny of the osmoregulatory epithelia is completed only after metamorphosis and it conditions the occurrence of the adult pattern of osmoregulation.
Cell and Tissue Research, 2005
The ontogeny of osmoregulation was examined in the branchial cavity of embryonic and early post-embryonic stages of the crayfish Astacus leptodactylus maintained in freshwater, at the sub-cellular level through the detection of the sodium–potassium adenosine triphosphatase (Na+,K+-ATPase). The embryonic rate of development was calculated according to the eye index (EI) which was 430–450 μm at hatching. The distribution of the enzyme was identified by immunofluorescence microscopy using a monoclonal antibody IgGα5 raised against the avian α-subunit of the Na+,K+-ATPase. Immunoreactivity staining, indicating the presence of Na+, K+-ATPase appeared in the gills of late embryos (EI≥400 μm), i.e. a few days before hatching time, and steadily increased throughout the late embryonic and early post-embryonic development. The appearance of the enzyme correlates with the ability to osmoregulate which also occurs late in the embryonic development at EI 410–420 μm and with tissue differentiation within the gill filaments. These observations indicate that the physiological shift from osmoconforming embryos to hyper-regulating late embryos and post-hatching stages in freshwater must originate partly from the differentiation in the gill epithelia of ionocytes which are the site of ion pumping, as suggested by the location of Na+,K+-ATPase. Only the gills were immunostained and a lack of specific staining was noted in the lamina and the branchiostegites. Therefore, osmoregulation through Na+active uptake is likely achieved in embryos at the gill level; all the newly formed gills in embryos function in ion regulation; other parts of the branchial chamber such as the branchiostegites and lamina do not appear to be involved in osmoregulation.
2008
Embryos, larvae, and adults of Palaemonetes argentinus tolerate a wide range of salinities (1 to 25 ‰). While osmoregulatory capacities have previously been demonstrated in all postembryonic stages, little is known about the occurrence of osmoregulation during the embryonic phase. We examined ontogenetic and salinity-induced changes in the activity of a key enzyme involved in osmoregulation, Na + ,K + -ATPase. Its activity was studied in: (1) eggs at an early (SI), an intermediate (SII), and a late stage of embryonic development (SIII); (2) in newly hatched larvae (Zoea-I, ZI); and in homogenates of (3) whole adults and (4) isolated gill tissue. All stages were directly exposed to 1, 15, or 25 ‰, and Na + ,K + -ATPase activity was chemically determined 24 h (embryos, larvae) or 48 h later (adults). Enzyme activity was detected in all developmental stages, being low in SI and SII, maximum in SIII, and intermediate in ZI and adults. Maximum salinityinduced activity changes prior to hatching (SIII) suggest that hyper-osmoregulatory functions are expressed by the end of the embryonic phase. The ontogenetic activity maximum at this stage, however, may also be related to the hatching process. Comparing different salinities, Na + ,K + -ATPase activity in SIII was always highest at 15 ‰, whereas the activity in gills was higher at both 15 and 25 ‰ than at 1 ‰. While gills are absent in the embryonic and early larval stages, ion-transporting cells must be located elsewhere during these early ontogenetic stages, probably in the brachiostegites.
Journal of Experimental Biology, 2001
SUMMARYMany studies have shown that hyperosmoregulation in euryhaline crabs is accompanied by enhanced Na++K+-ATPase activity in the posterior gills, but it remains unclear whether the response is due to regulation of pre-existing enzyme or to increased gene transcription and mRNA translation. To address this question, the complete open reading frame and 3′ and 5′ untranslated regions of the mRNA coding for the α-subunit of Na++K+-ATPase from the blue crab Callinectes sapidus were amplified by reverse transcriptase/polymerase chain reaction (RT-PCR) and sequenced. The resulting 3828-nucleotide cDNA encodes a putative 1039-amino-acid protein with a predicted molecular mass of 115.6 kDa. Hydrophobicity analysis of the amino acid sequence indicated eight membrane-spanning regions, in agreement with previously suggested topologies. The α-subunit amino acid sequence is highly conserved among species, with the blue crab sequence showing 81–83 % identity to those of other arthropods and 74...
Journal of Experimental Biology, 2008
SUMMARYThe ability to reverse the net direction of gill ion transport in response to a salinity change is critical for euryhaline teleosts and involves a complex cellular and molecular remodelling of the gill epithelium. The present study aimed to clarify the cellular localisation and exact quantitative inter-relationship of Na+,K+–ATPase α- andβ-subunit transcripts in Atlantic salmon gill during salinity change. The combined expression level of all α-isoforms in the gill increased by 100% after freshwater (FW) to seawater (SW) transfer. The α1aand α1b isoforms were both in the range 1–6 amol 20 ng–1 total RNA; α1a decreased andα 1b increased after SW-transfer, their ratio changing from 5:1 in FW to 0.26:1 in SW. The α1c and α3levels were 10- and 100-fold lower, respectively. Theβ 1-subunit mRNA level was 0.1–0.3 amol 20 ng–1 total RNA, thus much lower than the sum ofα-subunits. Even though increasing 3-fold after SW-transfer,β-subunit availability may still limit functional pump sy...
Journal of Experimental Biology, 2009
SUMMARY The ability to reverse the net direction of gill ion transport in response to a salinity change is critical for euryhaline teleosts and involves a complex cellular and molecular remodelling of the gill epithelium. The present study aimed to clarify the cellular localisation and exact quantitative inter-relationship of Na+,K+–ATPase α- andβ-subunit transcripts in Atlantic salmon gill during salinity change. The combined expression level of all α-isoforms in the gill increased by 100% after freshwater (FW) to seawater (SW) transfer. The α1aand α1b isoforms were both in the range 1–6 amol 20 ng–1 total RNA; α1a decreased andα 1b increased after SW-transfer, their ratio changing from 5:1 in FW to 0.26:1 in SW. The α1c and α3levels were 10- and 100-fold lower, respectively. Theβ 1-subunit mRNA level was 0.1–0.3 amol 20 ng–1 total RNA, thus much lower than the sum ofα-subunits. Even though increasing 3-fold after SW-transfer,β-subunit availability may still limit functional pump s...