Fig 2: Inhibition of Rho-kinase attenuates periocular neural crest (pNC) migration on nephronectin (Npnt).(A–D) Comparisons of pNC migration from explants cultured for 24 hr on either (A) Npnt substrate alone or in the presence of (B) the ROCK inhibitor Y27632. (C, D) Quantification of cell area taken from N = 8 Npnt and N = 8 Npnt plus ROCK inhibitor showing significant reduction in (C) cell area and (D) cell migration in the presence of ROCK inhibitor. (E) Quantification of cell orientation from N = 6 Npnt and N = 6 Npnt plus ROCK inhibitor explants showing significant difference in the fraction aligned between 90° ± 30°. (F, G) Cells stained for actin (phalloidin) and focal adhesion (pY118 paxillin) showing (F) formation of actin stress fibers and focal adhesions by pNC migrating on Npnt. (G) Substantial decrease in actin stress fibers in the presence of the ROCK inhibitor. (H) Quantification taken from N = 4 Npnt and N = 4 ROCK inhibitor explants, showing significant increase in the number of pY118 paxillin-positive puncta. *p<0.05; **p<0.01. Scale bars: (A, B) 100 μm; (F, G) 50 μm.

Fig 3: Effects of overexpression of nephronectin (Npnt) during corneal development.Embryos were injected with RCAS virus expressing GFP alone (control) or GFP and the full-length Npnt protein, and corneas were analyzed at the developmental stages indicated. (A–F) Representative corneal sections from embryonic day (E)9 control (A–C) and Npntoe corneas (D–F) showing levels of Npnt transcript (A, D) and protein (B, E) expression, and hematoxylin and eosin staining indicating corneal thickness (double-sided arrows, C, F). (G, H) Measurements for corneal thickness and cell counts were taken from E7, N = 10 control, N = 10 Npntoe; E9; N = 7 control, N = 12 Npntoe; E15; N = 6 control, N = 9 Npntoe. Bar graphs show no difference at E7, but a significant increase at E9 and E15 in corneal thickness (G) and corneal cells (H). (I) Cell densities were determined from E7, N = 8 control, N = 7 Npntoe; E9; N = 6 control, N = 11 Npntoe; E15; N = 7 control, N = 6 Npntoe. Bar graph shows that there were no differences at E7 and anterior cell densities at E9 and E15, but the posterior cell densities were significantly decreased. (J, K) Bromodeoxyuridine (BrdU) analysis and quantification of cell proliferation in corneal sections taken from E7, N = 8 control, N = 7 Npntoe; E9; N = 9 control, N = 9 Npntoe; E15; N = 7 control, N = 6 Npntoe. No significant differences were observed at E7 and E15, but there was a significant reduction at E9. ns, not significant; *p<0.05; **p<0.01; ***p<0.001. ep, corneal epithelium; st, stroma; en, corneal endothelium. Scale bars: 100 μm.

Fig 4: Corneal thickness is reduced in Npntkd corneas.(A) Schematic of in ovo injection of viral constructs (green) to cover the anterior region of stage 8 chick embryo. Following 7 days of incubation, embryos were screened for GFP expression in the anterior eye region. Knockdown was verified by section in situ hybridization, which revealed reduced expression of Npnt in Npntkd cornea compared with control. (B–E) Hematoxylin and eosin staining showing control (B, D) and thinner Npntkd corneas (C, E). Statistical analysis on measurements taken from (N = 5 control and N = 4 Npntkd corneas) revealed (F) significant reduction in thickness of Npntkd corneas. **p<0.01. ep, corneal epithelium; st, stroma; en, corneal endothelium;. Scale bars: 100 μm (B, C), 100 μm (D, E).

Fig 5: Dynamics of periocular neural crest (pNC) response to the extracellular matrix (ECM) in the context of the expression of nephronectin (Npnt) and fibronectin (Fn) during corneal development.(A) Cross-section of embryonic day (E)5 eye immunostained for Npnt and Fn. Npnt appears in an increasing gradient from the periocular region into the cornea, whereas Fn stains both the periocular mesenchyme and cornea. (B) All pNC respond to Fn via expression of α5β1, but a subpopulation of pNC that reside in the region adjacent to the presumptive cornea express both α5β1 and α8β1, and become competent to also read the additional gradient of Npnt in the ECM, thus migrating into the corneal region. oc, optic cup; ps, primary stroma; en, corneal endothelium.