Mila Ljujić*, Jelena Kušić Tišma, Bojan Ilić, and Aleksandra Divac Rankov
Institute of Molecular Genetics and Genetic Engineering, University of Belgrade, Belgrade, Serbia
milaljujic [at] imgge.bg.ac.rs
Abstract
Zebrafish (Danio rerio) have a remarkable regenerative capacity of various tissues and organs, making them a widely used model organism for studying tissue regeneration and therapeutic development through pharmacological screens. The zebrafish larval fin consists of a two-layered, infolded epithelium and its regeneration after amputation is completed within just 3 days. We utilized this model to study the effect of human protein alpha-1 antitrypsin (AAT) on wound healing. This study aimed to identify the effect of AAT on transcriptional profiles during zebrafish larvae fin regeneration at 24h post amputation. Caudal fin was amputated at 48h post fertilization and larvae were treated with 2mg/ml AAT for 24h.
Pools of 24 larvae were collected at 24h post amputation and total RNAs were extracted using TRIzol Reagent. RNAseq was performed using Illumina Novaseq6000. Adapter trimming and low-quality reads were removed from raw data with fastp and reads were aligned to Danio rerio genome assembly GRCz11 by STAR aligner. Counts of differentially expressed transcripts were calculated at gene level with FeatureCount. Differential expression was analysed by DESeq2 package after correction for batch effect by ComBat-seq from sva bioconductor package. The threshold for significantly differential expression was set as the adjusted p-value < 0.01.
At 24hpa there were 185 differentially expressed genes (89 upregulated and 96 downregulated) in treated larvae compared to the untreated. Mgst2, Apobb.2, and Prss59.2 were in the top 10 downregulated genes while Col9a1b, Col8a1a, and Matn3a were in the top 10 upregulated genes. Enrichment analysis by Enrichr showed that key gene onthologies in AAT treated zebrafish larvae were intermediate filament, collagen containing extracellular matrix, cellular response to glucocorticoid and endoplasmic reticulum lumen among others. Our data support further evaluation of AAT as a potential promoter of wound healing.
Keywords: RNA-sequencing, zebrafish, regeneration, wound healing
Acknowledgement: This work was funded by the Ministry of Science, Technological Development and Innovation of the Republic of Serbia (Contract No. 451-03-66/2024-03/200042)