Figure 2. Corneal thickness is reduced in Npnt^kd 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 Npnt^kd cornea compared with control. (B–E) Hematoxylin and eosin staining showing control (B, D) and thinner Npnt^kd corneas (C, E). Statistical analysis on measurements taken from (N = 5 control and N = 4 Npnt^kd corneas) revealed (F) significant reduction in thickness of Npnt^kd corneas. **p<0.01. ep, corneal epithelium; st, stroma; en, corneal endothelium;. Scale bars: 100 μm (B, C), 100 μm (D, E).

Figure 2—figure supplement 1. Diagram of replication-competent ASLV long terminal repeat with a splice acceptor (RCAS) vectors used for expression of either (A) GFP alone (control) or GFP together with inserts of shRNA or full-length nephronectin (Npnt).

(B) The full-length or mutated and truncated versions of Npnt driven by the viral LTR promoter and GFP driven by the IRES promoter. (C) For RT-PCR, cells plated in 30 mm dishes were homogenized in 1 mL TRIzol, and RNA was isolated following the manufacturer’s protocol. RNA samples were treated with Turbo DNA-free kit (Invitrogen) to remove residual genomic DNA, and cDNA pools were generated using SuperScript First Strand System (Invitrogen). Semi-quantitative PCR was conducted using HotStart-IT Taq polymerase (Affymetrix). Knockdown efficiency was measured on 2% agarose gel using glyceralde-hyde-3-phosphate dehydrogenase (GAPDH) as a loading control. Red rectangles indicate the constructs that were chosen for the knockdown and overexpression experiments. Primers for RT-PCR are located in Supplementary file 1.