Pax6 mRNA expressed pattern during eyelid development.

<p>(A) Semi-quantitative PCR for total expression levels of Pax6 alone and Pax6 and Pax6(5a) using eyelid RNA from E14.5 to E16.5. Pax6 and Pax6(5a) are amplified by the same pair of primers. (B, C) The relative fold expressions of total Pax6 and Pax6(5a) only by real-time PCR, respectively. The reference sample is the cDNA from wild-type eyelids at E14.5. GAPDH is a reference gene. The significantly different data groups at different time point are analysed by Student’s t-test. One asterisk stands for p<0.05, two asterisks mean p<0.01, and no asterisk means no significant difference.</p

The examination of the EGFR-ERK pathway and Smad3 activation in eyelid epidermis at E15.5.

<p>(A–D) The activation state of EGFR and its downstream signalling molecules in the epidermis of wild-type and EOB-5 eyelids. Phosphorylated EGFR and ERK were predominantly stained in wild-type epidermis (A, C), while this staining was weak in the mutant eyelid (B, D). (E, F) The activation state of Smad3. Phosphorylated Smad3 was strongly expressed in the mesenchyme underlying the epidermis of wild-type eyelids (E) and was weakly expressed in the mesenchyme of EOB-5 eyelids (F). (G, H) The examination of CTCF expression. A positive signal (green) was shown in the epidermis of the wild-type eyelid (G), while no positive staining was shown in the mutant eyelid (H). (A’–H’) Negative controls (no primary antibody). Key: ep, epidermis; c, conjunctiva; m, mesenchyme; p, periderm. Bar, 100 µm; arrows indicate positive staining.</p

Cell migration in wild-type and mutant eyelids from E14.5 to E16.5.

<p>F-actin polymerisation is shown at the margin of the eyelid epithelium in both wild-type (A) and EOB-5 (D) embryos at E14.5. An intense positive staining is shown in the epithelial layer of wild-type (B) and EOB-5 (E) eyelids at E15.5. F-actin polymerisation was reduced when eyelid fusion was completed. More intense staining of polymerised F-actin was detected in the wild-type (C) embryo at E16.5 and in the un-fused eyelid of EOB-5 (F) embryo at E16.5. (A’–F’) Negative controls without phalloidin. Rhodamine-phalloidin was used to visualise F-actin (red). Bar, 100 µm.</p

Distributions of total Pax6 and the ratio of Pax6 and Pax6(5a) in the eyelid.

<p>The ratio of Pax6/Pax6(5a) was measured by fluorescence intensity. The significant differences between the values at E14.5 and those at E15.5 and between the values at E15.5 and at E16.5 were analysed in the same sample to illustrate the trend of the ratios during eyelid development (P* = 0.022<0.05, P** = 0.002<0.01).</p

Cell proliferation assays in wild-type and mutant embryos.

<p>(A–C and A’–C’) Detection of proliferating cells by PCNA staining. Epidermal cell proliferation presents in wild-type eyelids from E14.5 to E16.5 (A–C) but is almost absent in EOB-5 eyelids (A’–C’). The proliferating epidermal cells were restricted to the basal layer of epidermis in normal eyelid at E16.5 (C). The arrows indicate proliferating cells in the eyelid epidermis. Bar, 50 µm. (D, D’, E, E’) The examination of cell proliferation by Brdu staining in wild-type and mutant embryos at E15.5. Bar, 100 µm. Key: ep, epidermis; ul, upper eyelid; ll, lower eyelid. Bar, 100 µm.</p

Immunohistochemistry on eyelid sections from E14.5 to E16.5 with anti-PAX6 antibody.

<p>(A–C) Pax6 staining in wild-type sections. There is weak but clear staining shown in the conjunctiva and mesenchyme at E14.5 (A); little positive staining is shown in the mesenchymal cells, none is in the epidermis at E15.5 (B), and Pax6 is only in peridermal cells at E16.5. (D–F) Pax6 staining of EOB-5 eyelid sections. There is intense staining in epidermis and mesenchyme at all three days. (A’–F’) Negative controls without primary antibody. Key: ep, epidermis; c, conjunctiva; m, mesenchyme; p, periderm. Bar, 100 µm; arrows indicate positive staining.</p