Jelena Kusic-Tisma*, Nevena Vezmar, Igor Davidović, Mateja Ilic, Mila Ljujić and Aleksandra Divac Rankov
Institute of Molecular Genetics and Genetic Engineering, University of Belgrade, Serbia
jelena.kusic [at] imgge.bg.ac.rs
Abstract
In order to better characterise expression profile of 3-day old zebrafish larvae, we performed spatial transcriptomic analysis using STomics stereoseq technology. Analyses were performed on 4 sagittal cryosections from 2 different larvae (2 sections per sample, ~60 μm apart). Stereoseq is a grid-based technology that aggregates a number of DNA nanoball spots (DNB) and clusters their spatial profile into substructures. Here, we used grid size of 10 μm (bin 20, 20 × 20 DNB), which is equivalent to approximately one cell diameter. The number of transcripts and genes detected among different sections were comparable, with an average of 1064 unique molecular identifiers (UMIs) and 535 genes per unit. Unsupervised clustering was performed by spatially aware clustering algorithm BANKSY using lambda parameter 0.2. Number of clusters varied between sections from 10 to 13, which was in good correlation with expected number of organs/tissues per section of 3-day old zebrafish larvae. Analyses of spatially variable genes (SVG) detected 95 genes with spatial pattern having Global Moran’s I coefficient greater than 0.4. In order to identify co-varying genes showing similar spatial distribution, we applied Hotspot algorithm. Ten spatial modules per section were recognised. Gene ontology (GO) enrichment analysis on the spatially correlated genes of each module indicated the identity of these spatial modules (e.g. heart, epidermis, cartilage, eye, blood, forebrain, notochord, liver). Spatial visualization of modules confirmed that the distribution of detected modules matched their region-specific biological functions. Taken together, our analyses contribute to the gene expression landscape of zebrafish at the 3-day old developmental stage.
Keywords: spatial transcriptomics, stereoseq, zebrafish
Acknowledgement: This study is a part of the Enhancing Non-Communicable Disease Research Excellence Through Zebrafish Capacity Building (ZeNCure) project, supported by the European Union, under Horizon Europe programme Widening Participation and Spreading Excellence, HORIZON-WIDERA-2023-ACCESS-02, Grant Agreement number 101160259.