Abstract
During development, cells aggregate at tissue boundaries to form normal tissue architecture of organs. However, how cells are segregated into tissue precursors remains largely unknown. Cornea development is a perfect example of this process whereby neural crest cells aggregate in the periocular region prior to their migration and differentiation into corneal cells. Our recent RNA-seq analysis identified upregulation of nephronectin (Npnt) transcripts during early stages of corneal development where its function has not been investigated. We found that Npnt mRNA and protein are expressed by various ocular tissues, including the migratory periocular neural crest (pNC), which also express the integrin alpha 8 (Itgα8) receptor. Knockdown of either Npnt or Itgα8 attenuated cornea development, whereas overexpression of Npnt resulted in cornea thickening. Moreover, overexpression of Npnt variants lacking RGD-binding sites did not affect corneal thickness. Neither the knockdown nor augmentation of Npnt caused significant changes in cell proliferation, suggesting that Npnt directs pNC migration into the cornea. In vitro analyses showed that Npnt promotes pNC migration from explanted periocular mesenchyme, which requires Itgα8, focal adhesion kinase, and Rho kinase. Combined, these data suggest that Npnt augments cell migration into the presumptive cornea extracellular matrix by functioning as a substrate for Itgα8-positive pNC cells.
Highlights
In vertebrates, the cornea is comprised of three cellular layers: the epithelium, stroma and endothelium
Consistent with our RNA-seq data (Bi & Lwigale, 2019), we found that Npnt is undetectable in the periocular neural crest (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 (Figure 1A, B)
In contrast, we observed that treatment with the 8 1 inhibitor significantly decreased the density of migratory cells from the explant (Figure 3F, 3G; Figure 3-Video 2). These results indicate that 8 1 functions as a receptor for Npnt signaling in the presumptive corneal pNC, and that it is required for their migration. 196 Knockdown of integrin alpha 8 (Itg 8) disrupts corneal thickness during development Given that Itg 8-expressing pNC appear to be in direct contact with Npnt secreted into the primary stroma, we wanted to characterize the role of Itg 8 during pNC migration
Summary
The cornea is comprised of three cellular layers: the epithelium, stroma and endothelium. The primary stroma serves an important role as a scaffold for pNC migration throughout the process of corneal development (Hay and Revel, 1969; Bard and Hay, 1975; Quantock and Young, 2008) It consists of multiple ECM proteins including hyaluronan (Toole and Trelstad, 1971), collagen type I and II (Hayashi et al, 1988; Fitch et al, 1988), laminin (Doane et al, 1996), and fibronectin (Fn) (Fitch et al, 1991). The role of collagens and proteoglycans have been the focus of several investigations due to their indispensable functions in corneal transparency Most of these studies were conducted in rodents, in which early corneal development does not involve the primary stroma (Pei and Rhodin, 1970; Haustein, 1983; Feneck et al, 2019). Our findings show that Npnt secreted into the ECM of the nascent cornea provides a substrate that promotes migration of Itg 8-positive pNC during development
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