Transcriptomic landscape of Atlantic salmon (Salmo salar L.) skin.

In this study, we present the first spatial transcriptomic atlas of Atlantic salmon skin using the Visium Spatial Gene Expression protocol. We utilized frozen skin tissue from 4 distinct sites, namely the operculum, pectoral and caudal fins, and scaly skin at the flank of the fish close to the lateral line, obtained from 2 Atlantic salmon (150 g). High-quality frozen tissue sections were obtained by embedding tissue in optimal cutting temperature media prior to freezing and sectioning. Further, we generated libraries and spatial transcriptomic maps, achieving a minimum of 80 million reads per sample with mapping efficiencies ranging from 79.3 to 89.4%. Our analysis revealed the detection of over 80,000 transcripts and nearly 30,000 genes in each sample. Among the tissue types observed in the skin, the epithelial tissues exhibited the highest number of transcripts (unique molecular identifier counts), followed by muscle tissue, loose and fibrous connective tissue, and bone. Notably, the widest nodes in the transcriptome network were shared among the epithelial clusters, while dermal tissues showed less consistency, which is likely attributable to the presence of multiple cell types at different body locations. Additionally, we identified collagen type 1 as the most prominent gene family in the skin, while keratins were found to be abundant in the epithelial tissue. Furthermore, we successfully identified gene markers specific to epithelial tissue, bone, and mesenchyme. To validate their expression patterns, we conducted a meta-analysis of the microarray database, which confirmed high expression levels of these markers in mucosal organs, skin, gills, and the olfactory rosette.

Roles of aquaporin-4 isoforms and amino acids in square array assembly.

Aquaporin-4 (AQP4) is a water channel found at high concentrations around blood vessels in the brain and is organized into elaborate assemblies called square arrays. The natural functions of AQP4 and the square arrays remain unknown, but under pathophysiological conditions, AQP4 has been shown to influence brain edema, synapse function, and cellular migration. AQP4 was recently found to have six isoforms, where AQP4a (also known as M1), AQP4c (also known as M23), and AQP4e are functional water transport channels. Furthermore, by two-dimensional blue native polyacrylamide gel electrophoresis (BN-PAGE) analysis of the internal composition of square arrays, three distinct isoforms were visualized. Here we combine these advances in technique with mutational analysis to test a series of current hypotheses about AQP4 functional structure. We find that the square array destabilizing N-terminus of AQP4a is partly functional through the C13 and C17 amino acids, and not through R8 and R9. We find a discrepancy between our data and the proposed tetramer-tetramer binding site based on the in vitro AQP4 two-dimensional crystal structure. On the other hand, we find that isoforms AQP4a and AQP4e, while not being able to form square arrays alone, are able to interact with AQP4c and be incorporated into higher-order structures. Our results with the novel BN-PAGE analysis technique point toward a model in which the presence of accessory isoforms (AQP4a and AQP4e) regulates the square array assembly process of the main isoform, AQP4c.