RNA-Seq Analysis of Periocular Neural Crest and Embryonic Corneal Transcriptomes

Purpose: The goal of this study was to evaluate changes in the transcriptome profile during periocular neural crest differentiation into corneal endothelium and keratocytes. Methods: RNA profiles of chick embryonic day (E3) periocular neural crest, E5 corneal endothelium, and E7 corneal endothelium plus keratocytes were generated in triplicate by deep sequencing using Illumina HS4000. Bowtie2 and HISAT were used to map clean reads to reference gene and genome, respectively. An average mapping ratio of 76.26% to the reference gene and 93.10% to the genome were generated with Galgal5 reference assembly.Transcripts were normalized and presented as Fragments Per Kilobase Million (FPKM). Differentially expressed genes between pNCvsEn and pNCvsKEn were examined. Expression of some candidate genes was validated by in situ hybridization. Results: 790 transcripts were enriched between pNC and En, and 865 transcripts were enriched between pNC and KEn. Enriched transcripts correspond with KEGG pathways involved in cell proliferation, synthesis of extracellular matrix, focal adhesion, metabolism, and cancer. The RNA-Seq data serves as platform for further analyses of the molecular networks involved in NCC differentiation into corneal cells, and provides insights into genes involved in corneal dysgenesis and adult diseases. RNA profiles of chick embryonic day (E3) periocular neural crest, E5 corneal endothelium, and E7 corneal endothelium plus keratocytes were generated in triplicate by deep sequencing using Illumina HS4000.