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

Abstract

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.

Keywords: RNAseq; bone; connective tissue; epithelium; fin; fish skin; gene expression; histology; mesenchyme; spatial transcriptomics.

Fig. 1.

Tissue sampling and optimization. a) The 4 tissue sampling sites are marked by circles, with schematic illustrations of the main tissue types present in the operculum, skin at the lateral line, pectoral fin, and dorsal fin. b) The samples (maximum size 5 × 5 mm) were held in an upright position for 5 s on a frozen metal plate, the ROI facing the metal plate. The specimens were embedded in O.C.T. and held on dry ice until fully frozen and transferred to appropriate tubes. For processing of the samples, the O.C.T.-embedded tissue was mounted on to the cryostat sample holder with the flat surface and ROI facing the operator. Skin and fins were cut into 10-μm-thick cryo sections and mounted on expression slides. Tissues were scanned with Aperio CS2 (Leica, USA).

Fig. 2.

Tissue sections and permeabilization time. a–c) Frozen tissue sections, 10 µm, of operculum, skin, and fin samples were sectioned onto Visium expression slides and stained with H&E. Abbreviations: Epithelium (Epi), loose connective tissue (Lct), dense connective tissue (Dct), mucous cell (Mc), bone (Bo), and mesenchyme (Mes). Capture spot diameter and center-to-center distance are indicated in a. d) Fluorescent cDNA print of pectoral fin (10 min optimization time). Insert with higher magnification shows epithelial tissue with mature mucous cells displayed as circles with low fluorescent signal. e and f) Similar to d with 20 and 25 min permeabilization time. The intensity of the fluorescent signal indicates cDNA/mRNA yield. At all timepoints, the intensity of the fluorescent signal was higher in the epithelial layer when compared with the dermal layer.

Fig. 3.

Normalized gene counts for Fish I and Fish II. Median-normalized average gene counts (X and Y axes) for Fish I and Fish II for skin tissue samples originating from the same position. a) Caudal fin, b) skin, c) operculum, and d) pectoral fin.

Fig. 4.

UMI counts in tissue from Fish I. a) Skin, b) caudal fin, c) operculum, and d) pectoral fin. For each sample, UMI counts are given as spots on top of the tissue section in the left panel, and the t-SNE projection with UMI counts is in the right panel. Epithelial tissue (Epi), muscle tissue (Mu), loose connective tissue (Lct), dense connective tissue (Dct), bone (Bo), and mesenchyme (Mes) are indicated in the plots.

Fig. 5.

Illustrations of the graph-based clustering for Fish I. a) Skin, b) operculum, c) caudal fin, and d) pectoral fin. In each sample, the left picture displays the complete tissue section within 1 capture frame. Additionally, magnified areas are indicated by circles, showing detailed views of the complete section on the right side. The rightmost frame depicts a capture spot that encompasses multiple tissues. Clusters are represented by spots with similar colors, where each spot corresponds to a barcoded probe on the Visium expression slide. The tissue clusters were named based on the main tissue type present. Abbreviations: loose connective tissue (Lct), dense connective tissue (Dct), bone (Bo), and epithelium (Epi).

Fig. 6.

Chord diagram displaying the interrelationships between the transcriptional profiles within each cluster. The color and the thickness of the nodes visualize the relationships between the clusters. For operculum, “loose connective tissue, sub” refers to the loose connective tissue under the opercular bone.

Fig. 7.

Expression of collagen types 1, 5, 7, and 10 in the skin, operculum, caudal fin, and pectoral fin. For each sample, the left picture shows the complete tissue section within the capture frame; magnified areas are indicated by black circles, and detailed views of the complete section are given on the right side. The figure illustrates the average gene expression for collagens annotated with the same names, as listed in Supplementary File 1, and the color of the capture spots indicate the Log2 expression. Abbreviations: epithelium (Epi), loose connective tissue (Lct), dense connective tissue (Dct), bone (Bo), scale (Sc), and mesenchyme (Mes). ENS ID of genes is given in Supplementary File 1.

Fig. 8.

Expression of interfilamentous proteins in the skin, operculum, caudal fin and pectoral fin. For each sample, the left picture shows the complete tissue section within the capture frame; magnified areas are indicated by black circles, and detailed views of the complete section are given to the right. Keratins had the overall highest expression rates in the fin and operculum. In the skin, they were primarily expressed in the epithelial layer. Vimentin (ENS0000073113) was expressed around scale pockets in the skin, in a fold in the operculum, and primarily in the mesenchyme of the fins. Desmin (ENS00000101128 and ENS0000040563) was transcribed in skeletal muscle tissue attached to the skin sample and in the opercular fold, with limited expression in fins. The figure displays keratin genes as the average gene expression of “keratins” (as listed in Supplementary File 1), and the color of the spots indicates Log2 expression.

Fig. 9.

Expression of marker genes in the epithelium, bone, and mesenchyme. a) Pectoral fin, epithelial gene markers; b) pectoral fin, bone gene markers; and c) pectoral fin, mesenchymal markers. For each tissue section, the AVG Log2 expression is marked by colored spots on top of the tissue section. The marker genes behind the avg. expression ratio are listed in Supplementary File 1.

Fig. 10.

Distribution of gene markers and their transcripts in the major tissues and organs of Atlantic salmon. Data are Log2 AVG fold change of tissue to the mean of all tissues according to Nofima’s microarray database STARS (Krasnov et al. 2011). ENS ID of genes is given in Supplementary File 1.

Fig. 11.

Expression of epithelial gene markers in the skin. a) Avg. Log2 expression of all epithelial gene markers listed in Supplementary File 1, for the skin, operculum, caudal fin, and pectoral fin. Magnified areas of each section are in the upper right corner, and area of magnification is marked by circles on the main slide. b) Expression of epithelial gene markers in skin epithelium. c and d) Expression of cldni, ass1, and apnl in the epithelium of the skin, operculum, caudal fin, and pectoral fin. Note that the complete slides with AVG expression ratios are depicted in a, while b–e only display magnified areas of the original slide, and each picture represents 1 marker gene.