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First published online December 1, 2006
Journal of Experimental Biology 209, 4908-4922 (2006)
Published by The Company of Biologists 2006
doi: 10.1242/jeb.02591

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The Na⁺/K⁺/2Cl^- cotransporter in the sea bass Dicentrarchus labrax during ontogeny: involvement in osmoregulation

Catherine Lorin-Nebel^*, Viviane Boulo, Charlotte Bodinier and Guy Charmantier

Equipe Adaptation Ecophysiologique et Ontogenèse, UMR 5171 UM2-CNRS-IFREMER Génome Populations Interactions Adaptation, Université Montpellier II, cc 092, Place E. Bataillon, 34095 Montpellier cedex 05, France

View larger version (71K): [in this window] [in a new window]   Fig. 1 . Amino acid alignment of the NKCC1 cotransporter from sea bass, other teleosts, Oreochromis mossambicus (NCBI accession no. AAR97731), Anguilla anguilla (NKCC1, NCBI accession no. CAD31111) and mammals, Rattus norvegicus (NCBI accession no. AAC27557), Mus musculus (NCBI accession no. AAC778332). Amino acid positions are numbered on the right. The 12 putative transmembrane TM domains are bold-underlined (Delpire et al., 1994); dashes indicate where spaces have been added to improve alignment; asterisks indicate positions where alignments show complete conservation between species; colons and semicolons indicates conserved and semi-conserved nucleotide substitution, respectively. Potential N-linked glycosylation sites (bold N, positions 494, 504, 792, 951) are indicated. The potential phosphorylation sites (underlined) were determined by NetPhos 2.0 (Blom et al., 1999). The potential site of SPAK and OSR1 interaction (Piechotta et al., 2002) is indicated (bold, italic letters).

View larger version (14K): [in this window] [in a new window]   Fig. 2. Western blot analysis of the branchial Na+/K+/2Cl- cotransporter (NKCC) in D. labrax after long-term acclimation to (A) seawater and (B) freshwater. Molecular masses in kDa are indicated on the left.

View larger version (61K): [in this window] [in a new window]   Fig. 3. Distribution of NKCC during the ontogeny of Dicentrarchus labrax. of (A) Embryo; (B) 1-day-old prelarva; (C,C',D) 3-day-old prelarvae. (A) Transverse section showing apical staining in the cells lining the digestive tract (DT) and basolateral staining in the tegumentary ionocytes (TI). (B) Transverse section of the developing branchial chamber, showing immunostained chloride cells (CC) are already present on the opercular membrane (O) and the branchial slits (BS). (C) Longitudinal and (C') transverse sections of prelarvae with intense staining of the intestinal brush-border. Note the immunopositive tegumentary ionocytes. (D) Transverse section showing weak apical staining of the renal collecting ducts (CD) and apical staining of the digestive tract. M, muscle; NC, notochord. Scale bars, 10 µm (A,C'), 20 µm (B,C,D).

View larger version (70K): [in this window] [in a new window]   Fig. 4. Distribution of NKCC during the ontogeny of Dicentrarchus labrax. (A-E) Transverse sections of 6-day-old larvae. (A) Basolateral immunostaining of the branchial and opercular chloride cells (CC). (B) Apical diffuse staining of the intestinal cells (I) and basolateral staining of the tegumentary ionocytes (TI). (C-E) Renal collecting ducts (CD) before (C), at (D) and after (E) their junction with the urinary bladder (UB); apical immunostaining is visible in the collecting duct cells (C,D) and in the cells lining the dorsal part of the urinary bladder (D,E). BA, branchial arch; CT, collecting tubule; M, muscle; O, operculum; R, rectum. Scale bars, 20 µm (A-C), 10 µm (D,E).

View larger version (68K): [in this window] [in a new window]   Fig. 5. Distribution of NKCC during the ontogeny of Dicentrarchus labrax. (A-F) Longitudinal section (A) and transverse sections (B-D) of 30-day-old larvae; (A) Predominant basolateral staining in branchial filamentary and opercular chloride cells (CC) (longitudinal section). (A') Larger magnification of a CC showing that the apical pit (arrow) presents no or weak immunostaining. (B,C,D,D',E,F) Apical immunostaining of the anterior (B) and posterior (C,D) intestine (I), of the collecting ducts (CD) and of the dorsal part of the urinary bladder (UB) (D,D',E,F). CT, collecting tubule; F, filament; L, lamellae; M, muscle; TI, tegumentary ionocyte. Scale bars, 5 µm (A'), 10 µm (A), 15 µm (D'), 20 µm (B,C,E,F), 50 µm (D).

View larger version (88K): [in this window] [in a new window]   Fig. 6. Distribution of NKCC in adult D. labrax following long-term adaptation in seawater (A,C,E,E') and freshwater (B,D,D',F,F'). (A,B) The anterior intestine (I) is stained along the brush-border membrane and the staining intensity appears stronger in the seawater-acclimated fish. (A). Note the presence of single basolaterally stained cells in both types of fish (A,B, arrows). (C,D) The renal collecting ducts (CD) have a (sub)apical staining (C,D,D') which is stronger in freshwater-acclimated fish (D,D'). Note the clearly distinguishable apical (D' arrow) and subapical (D' arrowhead) immunostaining. (E) Seawater-type chloride cells (S-CC) located on the filaments (F) are basolaterally stained (E'). (F) Freshwater-type chloride cells (F-CC) located on both the filaments and the lamellae (L), are (sub)apically stained (F'). Scale bars, 3 µm (D'), 5 µm (E',F'), 10 µm (A-F).

View larger version (8K): [in this window] [in a new window]   Fig. 7. Relative branchial NKCC abundance in D. labrax in seawater (SW; day 0) and following short-term (1 and 4 days; S) and medium-term (7, 14 and 22 days; M) freshwater (FW) acclimation. The line joins the means (open circles) of the individual measurements (black diamonds). Statistical tests were used to compare SW-acclimated sea bass (N=3) to short-term (N=4)- and medium-term (N=5)-acclimated sea bass to FW. Different letters indicate significant differences (P<0.005).

View larger version (50K): [in this window] [in a new window]   Fig. 8. Distribution of NKCC in branchial chloride cells of adult D. labrax following direct transfer from seawater to freshwater and exposure to freshwater for 1 day (A,A'), 7 days (B,B') and 14 days (C,C',C''). Note the presence of four different cell types. The SW-type chloride cells (SW-CC; A') are rich in basolateral NKCC. The intermediate cells (arrowheads, C') have moderate amounts of basolateral NKCC, the immunonegative cells (asterisks, B') lack NKCC staining and the FW-type cells (FW-CC, C'') have an apically stained cell pit. F, filament; L, lamellae. Scale bars, 20 µm (A-C), 4 µm (A',B'), 5 µm (C'), 2 µm (C'').

View larger version (8K): [in this window] [in a new window]   Fig. 9. Na+/K+/2Cl- cotransporter mRNA expression (N=10) in different organs of adult SW- and FW-acclimated Dicentrarchus labrax. (A) Gills, (B) kidney, (C) posterior intestine. Absolute expression (E-Ct) has been normalized to expression of the elongation factor (EF1). Data are expressed as the mean ± s.d. Different letters indicate significant differences (P<0.001). FW, freshwater; SW, seawater.