Shifts in the relationship between mRNA and protein abundance of gill ion-transporters during smolt development and seawater acclimation in Atlantic salmon (Salmo salar).

Smolting Atlantic salmon exhibit a seasonal increase in seawater tolerance that is associated with changes in the abundance of major gill ion-transporter transcripts and proteins. In the present study, we investigate how the transcript and protein abundance of specific ion-transporter isoforms relate to each other during smolt development and seawater acclimation, and how each correlates to seawater tolerance. We show that during smolt development both mRNA and protein abundance of gill Na+/K+-ATPase α1a subunit (NKAα1a) decreased but the decrease in the mRNA was five-times greater than that of the protein. Gill NKAα1b mRNA levels increased only slightly (1.5-fold) throughout development whereas protein abundance increased 30-fold at its peak. Gill Na+/K+/2Cl- co-transporter 1 (NKCC1) increased at the mRNA and protein level (5- and 12-fold) in smolts. The abundance of a gill ion-transporter's mRNA and protein changed in the same direction through development and after seawater transfer, but the changes were not always strongly correlated: NKAα1a (r = 0.768), NKAα1b (r = 0.40), and NKCC1 (r = 0.898). The maintenance of plasma chloride concentration correlated most strongly with the abundance of NKAα1a mRNA, and the ratio of NKAα1b to NKAα1a mRNA and protein. Growth performance after seawater transfer correlated most strongly with the abundance of NKAα1b protein and the ratio of NKAα1b to NKAα1a protein. Our results indicate that the abundance of ion-transporter mRNA and protein do not always correlate well and a decrease in the abundance of gill NKAα1a mRNA and increase in NKAα1b protein are strong predictors of seawater tolerance and growth performance after seawater transfer.

Differential regulation of sodium-potassium pump isoforms during smolt development and seawater exposure of Atlantic salmon.

Freshwater and seawater isoforms of the alpha subunit of Na(+)/K(+)-ATPase (NKA) have previously been identified in gill ionocytes of Atlantic salmon (Salmo salar). In the present study we examine the abundance and cellular localization of these isoforms during the parr-smolt transformation, a developmental process that is preparatory for seawater entry. The abundance of NKAα1a was lower in smolts than in parr, remained relatively constant during spring and decreased in summer. NKAα1b increased tenfold in smolts during spring, peaking in late April, coincident with downstream migration and increased salinity tolerance. NKAα1b increased a further twofold after seawater exposure of smolts, whereas NKAα1a decreased by 98%. The abundance of NKAα1b-positive, and NKAα1b and NKAα1a co-labeled ionocytes increased during smolt development, whereas the number of NKAα1a cells decreased. After seawater exposure of smolts, NKAα1b-positive ionocytes increased, NKAα1a-positive cells decreased, and co-labeled cells disappeared. Plasma growth hormone and cortisol increased during spring in smolts, but not in parr, peaking just prior to the highest levels of NKAα1b. The results indicate that the increase in the abundance of NKAα1b during smolt development is directly linked to the increase in salinity tolerance that occurs at this stage, but that significant changes also occur after seawater exposure. Spring increases in circulating levels of growth hormone and cortisol indicate that these hormones may be instrumental in upregulating NKAα1b during smolt development.

Tissue-specific requirements for specific domains in the FERM protein Moe/Epb4.1l5 during early zebrafish development.

BACKGROUND: The FERM domain containing protein Mosaic Eyes (Moe) interacts with Crumbs proteins, which are important regulators of apical identity and size. In zebrafish, loss-of-function mutations in moe result in defects in brain ventricle formation, retinal pigmented epithelium and neural retinal development, pericardial edema, and tail curvature. In humans and mice, there are two major alternately spliced isoforms of the Moe orthologue, Erythrocyte Protein Band 4.1-Like 5 (Epb4.1l5), which we have named Epb4.1l5long and Epb4.1l5short, that differ after the FERM domain. Interestingly, Moe and both Epb4.1l5 isoforms have a putative C' terminal Type-I PDZ-Binding Domain (PBD). We previously showed that the N' terminal FERM domain in Moe directly mediates interactions with Crumbs proteins and Nagie oko (Nok) in zebrafish, but the function of the C'terminal half of Moe/Epb4.1l5 has not yet been examined. RESULTS: To define functionally important domains in zebrafish Moe and murine Epb4.1l5, we tested whether injection of mRNAs encoding these proteins could rescue defects in zebrafish moe- embryos. Injection of either moe or epb4.1l5long mRNA, but not epb4.1l5short mRNA, could rescue moe- embryonic defects. We also tested whether mRNA encoding C' terminal truncations of Epb4.1l5long or chimeric constructs with reciprocal swaps of the isoform-specific PBDs could rescue moe- defects. We found that injection of the Epb4.1l5short chimera (Epb4.1l5short+long_PBD), containing the PBD from Epb4.1l5long, could rescue retinal and RPE defects in moe- mutants, but not brain ventricle formation. Injection of the Epb4.1l5long chimera (Epb4.1l5long+short_PBD), containing the PBD from Epb4.1l5short, rescued retinal defects, and to a large extent rescued RPE integrity. The only construct that caused a dominant phenotype in wild-type embryos, was Epb4.1l5long+short_PBD, which caused brain ventricle defects and edema that were similar to those observed in moe- mutants. Lastly, the morphology of rod photoreceptors in moe- mutants where embryonic defects were rescued by moe or epb4.1l5long mRNA injection is abnormal and their outer segments are larger than normal. CONCLUSION: Taken together, the data reveal tissue specificity for the function of the PBD in Epb4.1l5long, and suggest that additional C' terminal sequences are important for zebrafish retinal development. Additionally, our data provide further evidence that Moe is a negative regulator of rod outer segment size.

Mosaic Eyes is a novel component of the Crumbs complex and negatively regulates photoreceptor apical size.

Establishment of apical-basal cell polarity has emerged as an important process during development, and the Crumbs complex is a major component of this process in Drosophila. By comparison, little is known about the role of Crumbs (Crb) proteins in vertebrate development. We show that the FERM protein Mosaic Eyes (Moe) is a novel regulatory component of the Crumbs complex. Moe coimmunoprecipitates with Ome/Crb2a and Nok (Pals1) from adult eye and in vitro interaction experiments suggest these interactions are direct. Morpholino knockdown of ome/crb2a phenocopies the moe mutations. Moe and Crumbs proteins colocalize apically and this apical localization requires reciprocal protein function. By performing genetic mosaic analyses, we show that moe- rod photoreceptors have greatly expanded apical structures, suggesting that Moe is a negative regulator of Crumbs protein function in photoreceptors. We propose that Moe is a crucial regulator of Crumbs protein cell-surface abundance and localization in embryos.