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First published online 1 September 2005
doi: 10.1242/dev.02030

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HB-EGF promotes epithelial cell migration in eyelid development

Department of Cell Biology, Research Institute for Microbial Diseases, Osaka University, 3-1, Yamadaoka, Suita, Osaka 565-0871, Japan

View larger version (77K): [in a new window]   Fig. 1. HB-EGF expression during eyelid development. (A-E) Whole-mount lacZ staining of HBdel/+ eyelids (black arrow) at (A) E14.5, (B) E15.0, (C) E15.5, (D) E16.0 and (E) E16.5. The densely blue zone (asterisk) is the iris. (F-J) lacZ and Hematoxylin/Eosin stained sections of HBdel/+ eyelids at (F) E14.5, (G) E15.0, (H) E15.5, (I) E16.0 and (J) E16.5. (K-O) Schematic illustration of eyelid development. (K) At E14.5, leading edge formation has still not occurred. (L) At E15.0, the leading edge starts to extend from the eyelid root on both sides. (M) At E15.5, extension of both the leading edge and the root region continues (arrowheads). (N) At E16.0, the leading edges from both sides contact each other. (O) At E16.5, the root regions migrate and contact each other. (G-J) The expression of HB-EGF is restricted to the tip of the leading edge during this process. Scale bars: 500 µm for A-E; 100 µm for F-J.

View larger version (31K): [in a new window]   Fig. 2. Both HB-EGF null embryos and embryos with an uncleavable mutant form of HB-EGF show delays in eyelid closure. (A,B) Representative whole-mount lacZ stained (black arrow) HBdel/+ eyelids (A) and HBdel/del eyelids (B) at E15.5. Scale bar: 500 µm for A and B. (C) Schematic diagram showing how the progression of eyelid closure was measured. (D) Comparison of the progression of eyelid closure along the long axis (left panel) and the short axis (right panel) between HBdel/+ (blue dots, n=19) and HBdel/del (orange dots, n=20) embryos. (E) Comparison of the progression of eyelid closure along the long axis (left panel) and the short axis (right panel) between HBuc/+ (blue dots, n=18) and HBuc/uc (yellow dots, n=16) embryos. Horizontal bars in the graphs indicate the mean value (D,E).

View larger version (56K): [in a new window]   Fig. 3. HB-EGF is not required for cell proliferation in eyelid closure. (A,B) BrdU incorporation in eyelids of HBdel/+ (A) and HBdel/del (B) embryos at E15.5. (C,D) High magnification of A and B, focusing on the leading edge. BrdU-positive cells (brown nuclei) are mostly in the dermis but not in the epidermis. Scale bars: 100 µm for A and B; 50 µm for C and D. (E) Scoring of BrdU-positive cells based on the data shown in A and B. Data represent the mean value±s.e.m. of the results obtained in three individual embryos. N.S, not significant.

View larger version (40K): [in a new window]   Fig. 4. HB-EGF contributes to the epithelial sheet migration in leading edge formation. (A) Schematic diagram showing how the progression of eyelid formation was measured. Contribution of leading edge formation and root formation to total eyelid formation was estimated as shown. (B) Progression of total eyelid formation (left panel), leading edge formation (middle panel) and root formation (right panel) in HBdel/+ (blue dots, n=19) and HBdel/del (orange dots, n=20) animals. Horizontal bar in the graph indicates the mean value. (C) HB-EGF promotes F-actin reorganization in the developing eyelid epithelium. Whole-mount staining of HBdel/+ (a-d) and HBdel/del (e-h) embryos at E15.0 (a,c,e,g) and E15.5 (b,d,f,h) was performed using FITC-phalloidin and propidium iodide to visualize F-actin and DNA, respectively. High-magnification views of boxed areas in a,b,e,f are shown in c,d,g,h, respectively. Both actin cable formation (white arrowheads) and radial F-actin fiber (black arrowheads) can be detected more clearly in HBdel/+ eyelids than in HBdel/del eyelids. Scale bars: 500 µm for a,b,e,f; 200 µm for c,d,g,h.

View larger version (79K): [in a new window]   Fig. 5. sHB-EGF activates EGFR-ERK signaling but not JNK signaling in the extending leading edge. (A,B) lacZ expression at the tip of the leading edge of HBdel/+ (A) and HBdel/del (B) eyelids. (C,D) Detection of sHB-EGF protein in the leading edge of HBdel/+ (C) and HBdel/del (D) eyelids. (E,F) High-magnification views of boxed areas in C and D. (G-N) State of activation of EGFR and downstream signaling molecules in the leading edge of HBdel/+ (G,I,K,M) and HBdel/del (H,J,L,N) eyelids. (G,H) Phosphorylated EGFR. (I,J) Total EGFR protein. (K,L) Phosphorylated ERK. (M,N) Phosphorylated JNK. Scale bars: 100 µm for A-D,G-N; 20 µm for E,F.

View larger version (51K): [in a new window]   Fig. 6. EGFR contributes to HB-EGF-dependent eyelid closure process. (A,B) Neonatal eyelid of a wild-type (A) and a double mutant mouse carrying the HB-EGF null allele and the waved 2 EGFR allele, showing an EOB phenotype (B). (C) Penetrance of the EOB phenotype in compound mutants carrying the HB-EGF null (`d') and waved 2 (`wa2') alleles. Blue bars indicate pups with normal closed eyes; red bars indicate pups with EOB.

View larger version (30K): [in a new window]   Fig. 7. TGF functions equally and synergistically with HB-EGF in eyelid closure. (A,B) BrdU incorporation in eyelids of Tgfa+/- (A) and Tgfa-/- (B) E15.5 embryos. Scale bar: 100 µm for A,B. (C) Scoring of BrdU-positive cells shown in A and B. Data represent the mean value±s.e.m. of the results obtained from three individual embryos. N.S, not significant. (D) Progression of total eyelid formation (left panel), leading edge formation (middle panel) and root formation (right panel) in Tgfa+/- (blue dots, n=14) and Tgfa-/- (orange dots, n=12) eyelids. Horizontal bar in the graphs indicates the mean value. (E-G) TGF functions synergistically with HB-EGF in eyelid closure. Neonatal eyelid of wild-type animal (E) and double homozygote lacking both HB-EGF and TGF with an EOB phenotype (F). (G) Penetrance of EOB phenotype in compound mutants carrying HB-EGF null and TGF null alleles. Blue bars indicate pups with normal closed eyes; red bars indicate pups with EOB.

View larger version (25K): [in a new window]   Fig. 8. Proposed model for the function of HB-EGF in leading edge formation during eyelid closure. (A) Before E15.0, the leading edge is still not formed, and HB-EGF expression is not detectable. (B) At E15.0, the leading edge starts to extend, and HB-EGF expression appears at the tip of the leading edge. (C) From E15.0 to E16.0, HB-EGF is constitutively expressed at the tip of the extending leading edge. On the surface of the cells located in this region, ectodomain shedding of proHB-EGF may be mediated by ADAM17 (TACE), allowing sHB-EGF to diffuse throughout the leading edge region. sHB-EGF binds to and activates EGFR and the ERK pathway, resulting in F-actin polymerization and promotion of epithelial sheet migration.