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First published online 15 June 2005
doi: 10.1242/dev.01892

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A dual role of FGF10 in proliferation and coordinated migration of epithelial leading edge cells during mouse eyelid development

Hirotaka Tao^1,*, Miyuki Shimizu¹, Ryo Kusumoto¹, Katsuhiko Ono², Sumihare Noji¹ and Hideyo Ohuchi^1,

¹ Department of Biological Science and Technology, Faculty of Engineering, University of Tokushima, 2-1 Minami-Jyosanjima, Tokushima 770-8506, Japan
² Division of Neurobiology and Bioinformatics, National Institute for Physiological Sciences, Okazaki 444-8787, Japan

View larger version (131K): [in a new window]   Fig. 1. Eyelid development of the mouse at embryonic day 11.5 (E11.5) (A-A''), E13.5 (B-B''), E15 (C-C''), E16 (D,D'), and E17.5 (E,E'). The upper and lower eyelids are at the left and right, respectively. (A'-E',A''-C'') Higher magnifications of the eyelid primordia shown in A-E, respectively. (A) The ocular surface epithelium forms small grooves (arrows), and eyelid primordia (arrowheads) begin to emerge. (A',A'') The epithelial cells at the bottom of the groove have become cuboid (arrowheads). (B) The arrowheads indicate eyelid protrusions. (B',B'') The eyelid epithelium has become two-layered, with the leading edge cells yet to be formed, while the eyelid dermis starts proliferating. (C-C'') The protruding epithelial ridge has formed at the leading edge (arrow in C' and C''). (D,D') The suprabasal epithelial cells between the two lids meet and fuse to form an epithelial bridge. (E,E') After epithelial fusion, the ocular surface epithelia stratify and differentiate, while the upper and lower lid mesenchyme (m) have extended towards the junctional area and faced each other. co, cornea; ep, epidermis; hf, hair follicle; le, lens; lld, lower eyelid; p, periderm; uld, upper eyelid. Scale bars: A and B'-E', 100 µm; A',A'', 50 µm; B-E, 200 µm.

View larger version (91K): [in a new window]   Fig. 2. Expression patterns of Fgf10 (A-I,M) and Fgfr2b (J-L,M) in the developing eyelid. (A) Fgf10 is expressed in the underlying mesenchyme of the prospective eyelid region (arrowheads). (B) Higher magnification of the lower eyelid region in A. (C) Fgf10 is expressed in the mesenchyme of the developing whiskers (arrowheads), which is shown as an internal control for in situ hybridization. (D) Fgf10 is expressed in the mesenchyme underneath the upper (u) and lower (l) eyelid protrusions. The arrowhead indicates Fgf10 expression in the future corneal stroma. (E,F) Higher magnification of the eyelid protrusion in D. Fgf10 is expressed in the surrounding mesenchyme at the bottom of the eyelid groove (arrow in F) in addition to the eyelid tip. (G) Whole-mount in situ hybridization of the eye region. Fgf10 is expressed in the eyelid region and hair follicles. The Fgf10 expression is more intense in the lower eyelid. The inner canthus (towards the nose) is to the right; the outer canthus (towards the temples) is to the left. (H,I) Section in situ hybridization, showing more intense Fgf10 expression (arrowheads) in the lower eyelid mesenchyme in (I). (J-L) Fgfr2b is expressed in the eyelid epithelium. A sense probe for Fgfr2b produces no signals (L). (M) RT-PCR analysis was conducted for mRNA purified from E15 normal eyelid mesenchyme (lanes 1, 2), E18.5 wild-type keratinocytes (lanes 3, 5), E18.5 Fgf10-null keratinocytes (lanes 4, 6), and E15 normal back skin (lane 7). Fgf10 is expressed in the eyelid mesenchyme and not by keratinocytes, while Fgfr2b is expressed by keratinocytes from wild-type and Fgf10-null fetuses. The amplification reaction was performed after incubation with (lanes 2, 5, 6, 7) or without (lanes 1, 3, 4) reverse transcriptase. co, cornea; le, lens; lld, lower eyelid; re, retina; uld, upper eyelid. Scale bars: 100 µm (A,D); 50 µm (B,C,E,F); 100 µm (H-L).

View larger version (105K): [in a new window]   Fig. 3. Eyelid defects in Fgf10-null mice. (A,C) Lateral views of the face from wild-type (A) and Fgf10–/– (C) mice at birth. (B,D) Histology of the eyes of Fgf10+/– (B) and Fgf10–/– (D) mice at birth. The upper and lower eyelids are at the left and right, respectively. The Fgf10+/– neonate eyelids are fused (arrow in B), while the Fgf10-null eyelids are wide apart (arrowheads in D). The involution of the retina in D is an artifact. (E-S) HE staining of the coronal eye sections from wild-type (E,F), Fgf10+/– (I-K,O,P), and Fgf10–/– (G,H, L-N, and Q-S) embryos at E11.5 (E-H), E13.5 (I-N) and E16 (O-S). (E-H) The arrows in E and G indicate the developing eyelid groove. The epidermal cells at the bottom of the lower groove (arrowhead in F) have become cuboid by this stage in the wild-type mice, while they remain flat in the Fgf10-null mice (arrowhead in H). (I-N) The eyelid protrusion (arrowheads) is smaller, and the eyelid groove (arrows) shallower, in the Fgf10-null fetuses. (O-S) The arrowheads in O,Q indicate the eyelid leading edge. Eyelid closure is disrupted in Fgf10-null fetuses. (P) The junctional region of recently fused eyelids of the Fgf10+/– fetus consists of a loose grouping of cells overlaid by periderm cells (p), which appear to be spilling out onto both the internal and external surfaces. (R,S) The first sign of leading edge cells extending across the corneal surface can be seen in one primitive eyelid (asterisk in S) but not in the other for the Fgf10-null fetus. The arrows indicate the basal layer of the eyelid epidermis, which has spindle-shaped nuclei in P and round ones in R. m, mesenchyme. Scale bars: 250 µm (B,D); 100 µm (E and G, F and H, I and L, J,K,M and N,O and Q); 25 µm (P,R and S).

View larger version (98K): [in a new window]   Fig. 4. Cell proliferation in the developing eyelid epithelium in wild-type and Fgf10–/– mice. (A-C) BrdU analysis of the E11.5 eyelid. (A,B) Histological sections for BrdU staining. The red line underlies the epithelium where the number of BrdU-positive cells was counted (see Materials and methods for details). (C) The total number of proliferating cells from three sections was processed for quantitative histomorphometry. Compared to wild-type animals, the proliferation in Fgf10–/– mutant epithelia was significantly reduced. BrdU incorporation was particularly noticeable in the wild-type eyelid groove, as shown in A. (D-H) BrdU analysis of the E13.5 eyelid. (D-G) Histological sections for BrdU staining. The upper and lower eyelids are at the left and right, respectively. The proliferation rates were significantly reduced in the Fgf10–/– mutants. BrdU incorporation was particularly noticeable in the apex region of the wild-type eyelid, as shown in F. (I-M) BrdU analysis of the E15 eyelid. (I-L) Histological sections for BrdU staining. At E15, the Fgf10-null upper and lower eyelid epithelia showed no significant decrease in proliferation. In C, H and M the y axes indicate the cell number or mean percentage of BrdU incorporation in each area assayed. The error bars represent the s.e.m.; an asterisk denotes a significant finding (P<0.05), as compared with the wild-type value. In D,E,I,J, the dotted lines indicate the length of epithelium measured. Scale bars: 25 µm (A,B); 100 µm (D,E); 50 µm (F,G); 100 µm (I,J); 50 µm (K,L).

View larger version (162K): [in a new window]   Fig. 5. Scanning electron micrographs of E15 (A-H) and E16 (K, L) eyelids from Fgf10+/– (A,C,E,G,K) and Fgf10–/– (B,D,F,H,L) fetuses. C,E,G and D,F,H are higher magnifications of A and B, respectively. (C,D) The inner canthus region, (E,F) the lower eyelid margin, and (G,H) the outer canthus region. Clumps of rounded periderm cells are observed in the inner canthus of the Fgf10–/– mutant (D), but rarely seen in the outer canthus (H). (E) The arrowheads indicate a regular accumulation of epithelial cells, with the future periderm cells lined up on the eyelid margin of the heterozygote. (F) In the homozygote, rounded periderm cells are scattered away from the eyelid margin. (I,J) Transmission electron micrographs of the eyelid tip epithelium from wild-type (I) and Fgf10–/– (J) E15 fetuses. Section near the outer canthus region. Insets show higher magnifications of filopodia of a leading edge cell (indicated by an arrowhead). (I) Numerous filopodia are produced. (J) In the Fgf10–/– mutant leading edge, epidermal cells still produce filopodia, although these are distinctly fewer and shorter. co, cornea. (K,L) Fgf10+/–eyelids are fused, whereas Fgf10-null ones are wide open. Scale bars: 380 µm (A,B); 60 µm (C-H); 5 µm (I,J); 300 µm (K); 500 µm (L).

View larger version (88K): [in a new window]   Fig. 6. Cultures and analyses of primary keratinocytes. (A) In vitro scratch assay. Both the wild-type and mutant keratinocytes migrated into the gap within 20 hours. (B-E) Laser scanning microscopic analysis of actin organization in wild-type and mutant keratinocytes at the leading edge of a scratch assay performed as above 5 hours post-scratch. The cells were fixed and stained with phalloidin. Lamellipodia (B,C) and filopodia (D,E) are observed in both the wild-type and mutant keratinocytes, although in the mutant the lamellipodia are often not oriented toward the gap (arrows in C; arrowheads in B for comparison) and the filopodia appear thinner. Scale bars: 100 µm (A; all images are the same magnification); 50 µm (B,C); 10 µm (D,E).

View larger version (111K): [in a new window]   Fig. 7. FGF10 controls actin fiber formation (A-I), cell polarity (J-K) and expression of vimentin (L-M) in the developing eyelid epithelium. Whole-mount (A, D-G) and section (B,C,H,I) staining for F-actin of the eyes from wild-type, Fgf10+/– and Fgf10–/– fetuses at E15 (A-D,G-M) and E16 (E,F). The upper (B,H) and lower (C,I,J-M) eyelid primordia are shown. (A,D-F) The accumulation of actin fibers at the eyelid leading edge is accelerated, as eyelid closure proceeds. (B,C) Actin fibers are accumulated in the leading edge cells (arrowheads). (G-I) The Rhodamine-phalloidin binding to F-actin is much less on the eyelid margin and in the epithelial leading edge (arrowheads in H and I) of Fgf10-null fetuses. (J-K) Immunofluorescence of -tubulin. (J,K) The localization of -tubulin is indicated by the red, dotted signals. The borders of the cells are shown in green (Bodipy-ceramide). The expression of -tubulin in the inner basal layer (along the dotted line; approximately 17 cells were assessed) appears disorganized and reduced in the Fgf10–/– eyelid (K). (J',K') Higher magnifications of the eyelid tips shown in J and K, respectively. Weak expression of -tubulin is detected apically in the mutant epidermal cells (arrowhead in K'). (L,M) Immunofluorescence of vimentin. Note that high levels of vimentin protein are expressed in the eyelid mesenchyme (m). (L) In the wild type, vimentin is also localized in the leading edge epidermal cells. (M) Mutant eyelid epidermal cells express much lower levels of vimentin. (L',M') Higher magnifications of the eyelid tip shown in L and M, respectively. The arrowheads indicate the expression of vimentin. All images except A,D-G, which were obtained with a fluorescence stereomicroscope, were captured by laser scanning confocal microscopy. Scale bars: 300 µm (A,D-G); 25 µm (B,C,H,I); 25 µm (J,K); 50 µm (L,M).

View larger version (102K): [in a new window]   Fig. 8. Expression of Shh, activin ßB, and Tgfa during eyelid closure of normal and Fgf10-null mice. To better visualize the normal expression pattern, a mouse strain with no pigment in the eye was used, as shown in A-C,L,M,R,S. Whole-mount in situ hybridization of the eyes (A-O, R-U) and section in situ hybridization of the upper eyelid primordia (P,Q,V,W). Higher magnifications of the upper (H,I,N,O,T,U) and lower (J,K) eyelid margins. In A-O, R-U, the inner canthus is to the right, with the outer canthus to the left. (A-C) Shh is expressed along the upper eyelid margin and the temporal canthus at E13.5. By E15, Shh expression is also detected along the lower eyelid margin and in the prospective upper eyelash. Shortly after that, Shh expression along the eyelid margin becomes down-regulated and restricted to the eyelash anlagen. (D-G) The onset of Shh expression along the eyelid margin is delayed in Fgf10–/– mutants. The eyes shown in D,E and F,G were from the same littermates, respectively, and were processed for in situ hybridization simultaneously. In Fgf10-null eyelids, Shh expression appears down-regulated at E13.5 (E) and up-regulated at E15 (G,I,K), as compared with normal eyelids (D,F,H,J). (L,P) At E15, activin ßB is expressed by the leading edge cells (arrow in P) on the eyelid margin, the future periderm cells. (M) By E16, activin ßB is expressed by the periderm cells at the fusion line. (N-Q) The expression of activin ßB is down-regulated on the Fgf10-null eyelid margin, as compared with normal littermates. (R,V) Tgfa mRNA is concentrated in the leading edge cells (arrow in V) on the eyelid margin, the future periderm cells. (S) By E16, Tgfa is expressed by the periderm cells of the fusing eyelids. (T-W) The expression of Tgfa is down-regulated or more diffuse at the Fgf10-null eyelid margin, as compared with normal littermates. Scale bars: 50 µm (P,Q,V,W).

View larger version (79K): [in a new window]   Fig. 9. FGF10 protein induces up-regulation of activin ßB and Tgfa mRNAs and promotes eyelid closure in an explant culture. An FGF10- or PBS-soaked bead was implanted in the eyelid mesenchyme. The expression patterns of activin ßB, Tgfa and F-actin were examined 12 hours after the culture of E15 normal eyelid anlagen. (A-D) The expression domain of activin ßB (indicated by the dotted lines in A and B) was enlarged after FGF10 bead implantation (B). (C) Higher magnification of the area boxed in B. Activin ßB is expressed by subsets of cells in the thickened epidermis after FGF10 application. (D) Histology of a serial section of the explant shown in C and I. The epidermis (shown by the green line) in the vicinity of an FGF10 bead was thickened, as compared with that in F. (E) Activin ßB is not expressed in the normal epidermis abutting a PBS bead. (F) Histology of a serial section of the explant shown in E and J. Normal thickness of the epidermis is indicated by the green line. (G-J) The expression domain of Tgfa is enlarged after FGF10-bead implantation (H). The arrowhead in G and H indicates the leading edge of the eyelid. (I) Tgfa is distinctly expressed in the thickened epithelium abutting the FGF10-bead. (K,L) Phalloidin staining of the eyelid tips after FGF10-bead (K) or PBS-bead (L) implantation. The area showing accumulation of F-actin is enlarged after FGF10 application (arrowheads in K). The arrows in K and L indicate the epithelial leading edge. (M) Schematic representation of an explant culture of Fgf10-null eyelid anlagen and calculation of the percentage of eyelid closure after FGF10 application. The area of the eyelid opening was measured before and after the culture. Two beads were implanted in the eyelid mesenchyme. (N) FGF10 protein can promote eyelid closure in the Fgf10-null eyelid anlagen. ActB, activin ßB; bd, bead; co, cornea; lld, lower eyelid; uld, upper eyelid. Scale bars: 100 µm (A,B,G,H); 50 µm (C-F,I,J); 50 µm (K,L).

View larger version (22K): [in a new window]   Fig. 10. A dual role for FGF10 in controlling mouse eyelid development. (A) In the early phase of eyelid development, FGF10-FGFR2b signaling is required for cell shape changes and proliferation of the prospective eyelid epithelium, leading to coordinated growth of the eyelid anlagen. (B) In the late phase, FGF10-FGFR2b signaling is involved in up-regulation of Tgfa and activin ßB, and accumulation of F-actin in the epithelial leading edge cells, thus directing epithelial cell migration, epithelial sheet movement, and eyelid closure. The pathways indicated by the broken lines are suggested by other studies (for review, see Xia and Kao, 2004). It is not known whether FGF10 could directly regulate the accumulation of F-actin or indirectly through TGF- and/or activin pathways. This study has shown that FGF10 is necessary for proper expression of Shh in the basal layer of the eyelid tip epidermis (Motoyama et al., 1998), and for the integrity of the cell polarity of the eyelid basal epidermis. FGF10-FGFR2b signaling orchestrates these genetic and cellular activities during mouse eyelid fusion processes. These molecular interplays indeed result from combinatorial regulation of FGF10 and other extrinsic and intrinsic factors, which define the developmental context of developing eyelids. Other ligands of FGFR2b must be required for growth of the eyelid anlagen, as well.