Analysis of gene expression in zebrafish cornea during wound healing

The cornea, transparent and outermost structure of camera-type eyes, is prone to environmental challenges, but has remarkable wound healing capabilities which enables to preserve vision. The manner in which cell plasticity impacts wound healing remains to be determined. In this study, we report rapid wound closure after zebrafish corneal epithelium abrasion. Furthermore, by investigating the cellular and molecular events taking place during corneal epithelial closure, we show the induction of a bilateral response to a unilateral wound. Our transcriptomic results, together with our TGF-beta receptor inhibition experiments, demonstrate conclusively the crucial role of TGF-beta signaling in corneal wound healing. Finally, our results on Pax6 expression and bilateral wound healing, demonstrate the decisive impact of epithelial cell plasticity on the pace of healing. Altogether, our study describes terminally differentiated cell competencies in the healing of an injured cornea. These findings will enhance the translation of research on cell plasticity to organ regeneration. For RNA-sequencing, same-age fish were grouped one day prior to the experiment into four tanks (14 fish per 3 litres). Aged-matched (145, 148, 180 or 183 day-old) control and wound specimens were collected from the same tank. With respect to wounding, abrasion in both corneas of the animals was performed as described above. Control animals were anesthetized and placed onto a sponge for an equal duration as for the wounded animals. After 1.5hrs’ recovery, the fish were anesthetized again in 0.02% Tricaine and decapitated. Eyes were enucleated and cornea dissected in PBS. For one sample, 6 corneas from 3 individuals were pooled in 100µl of Tri reagent (T9424, Sigma). RNA was isolated from TRI reagent (T9424, Sigma) using Precellys beads and standard protocols (P000912-LYSK0-A.0, Bertin Technologies, Montigny-Le-Bretonneux, France). RNA was further purified using the Qiagen Rneasy MiniElute Cleanup kit (#74204, Qiagen, Hilden, Germany). Polyadenylated mRNA was purified from 50ng of Total RNA using NEBNext Poly(A) mRNA Magnetic Isolation Module (#E7490, New England Biolabs, Ipswich, Massachusetts, USA). The following steps were performed by the Biomedicum Functional Genomics Unit (FuGu) at the Helsinki Institute of Life Science and Biocenter Finland at the University of Helsinki. Library preparation was completed on purified mRNA using the NEBNext Ultra Directional RNA Library prep kit ( #E7420L, New England Biolabs) using 14 cycles of PCR amplification and indexed using single (i7) indexing. Indexed library preps from each sample were then pooled and sequenced at a pool concentration of 1.3pM on the NextSeq 500 using a NextSeq High Output 75 cycle flow cell (Illumina, San Diego, California, USA) with 75SE reads. Basecalling and demultiplexing was performed using Illumina bcl2fastq (v2.20.0.422) software. Reads were mapped to zebrafish GRCz11 genome using STAR aligner (2.6.0c). Gene counts were calculated using the featureCounts tool from the Subread package (v1.22.2) using Ensembl release 97 zebrafish gtf-files. Differential expression analysis was performed using R package DESeq2 (v1.22.2 [63]). One sample was excluded based on visual PCA inspection (Fig S2A). For downstream analyses, only genes with an entrezgene id were consider, therefore excluding predicted genes (Table S1). Gene Ontology (GO) term analysis was performed using g:Profiler (version e98_eg45_p14_ce5b097, [64]) online tool with default parameters. Volcano plots and heatmaps were generated using GraphPad Prism 8 (version 8.3.0, Graphpad Software, San Diego, California, USA).