Transcriptomic analysis of differential gene expression during chick periocular neural crest differentiation into corneal cells

Abstract

Background: Multipotent neural crest cells (NCC) contribute to the corneal endothelium and keratocytes during ocular development, but the molecular mechanisms that underlie this process remain poorly understood. We performed RNA-Seq analysis on periocular neural crest (pNC), corneal endothelium, and keratocytes and validated expression of candidate genes by in situ hybridization. Results: RNA-Seq profiling revealed enrichment of genes between pNC and neural crest-derived corneal cells, which correspond to pathways involved in focal adhesion, ECM-receptor interaction, cell adhesion, melanogenesis, and MAPK signaling. Comparisons of candidate NCC genes to ocular gene expression revealed that majority of the NCC genes are expressed in the pNC, but they are either differentially expressed or maintained during corneal development. Several genes involved in RA, TGFβ, and Wnt signaling pathways and their modulators are also differentially expressed. We identified differentially expressed transcription factors as potential downstream candidates that may instruct expression of genes involved in establishing corneal endothelium and keratocyte identities. Conclusion: Combined, our data reveal novel changes in gene expression profiles as pNC differentiate into highly specialized corneal endothelial cells and keratocytes. These data serve as platform for further analyses of the molecular networks involved in NCC differentiation into corneal cells, and provide insights into genes involved in corneal dysgenesis and adult diseases.

A mouse model of brittle cornea syndrome caused by mutation in Zfp469

“…Zfp469 remains expressed at E16.5, by which point the presumptive stroma is wellestablished. This expression pattern agrees with that reported for ZNF469 during development of the cornea in the chick (Bi and Lwigale, 2019) and coincides with the induction of temporally regulated signalling pathways important to corneal development, and with increasingly high expression levels of Col1a1, Col1a2 and other ECM components important in the stroma (Bard and Kratochwil, 1987;Ma and Lwigale, 2019). As the cornea matures, up to 8 weeks of age in the mouse, or 3 years of age in humans, collagen fibrils are further organised into additional layers of the established lamella.…”

Nephronectin-Integrin α8 signaling is required for proper migration of periocular neural crest cells during chick corneal development

“…To define the expression of Npnt mRNA and protein distribution during this process, we performed section in situ hybridization and immunohistology, respectively. Consistent with our RNA-seq data (Bi & Lwigale, 2019), we found that Npnt is undetectable in the pNC by embryonic day (E) 4 and in the presumptive corneal endothelium at E5, but it is primarily expressed in the retinal pigment epithelium (RPE) and presumptive lens fiber cells at these time points (Fig. 1A, B).…”

“…Very little is known about the function of the corneal ECM during early development. Here, we focus on Npnt, which we recently found in our RNA-seq study (Bi & Lwigale, 2019) to be upregulated during early development of the chick cornea. Specifically, we have identified novel expression of Npnt and its Itga8 receptor at critical timepoints of corneal development.…”

Cell-independent matrix configuration in early corneal development

“…Type II and type IX collagens are replaced by type I and type V as principal components of the secondary stroma. Multipotent periocular cells, originating in the neural crest, undergo distinctive alterations in gene expression as they migrate to form corneal endothelium and keratocytes (Bi and Lwigale, 2019). The latter synthesise the mature stroma, and there is strong evidence that cellular mechanisms exert a profound influence upon the organisation and orientation of matrix they deposit (Birk and Trelstad, 1984; Young et al, 2014; Koudouna et al, 2018b).…”