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First published online 15 April 2009
doi: 10.1242/dev.034082

Development 136, 1741-1750 (2009)
Published by The Company of Biologists 2009
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FGF-regulated BMP signaling is required for eyelid closure and to specify conjunctival epithelial cell fate

Jie Huang1, Lisa K. Dattilo1, Ramya Rajagopal1, Ying Liu1, Vesa Kaartinen3, Yuji Mishina4, Chu-Xia Deng5, Lieve Umans6,7, An Zwijsen6,7, Anita B. Roberts8 and David C. Beebe1,2,*

1 Department of Ophthalmology and Visual Sciences, Washington University, St Louis, MO 63130, USA.
2 Department of Cell Biology and Physiology, Washington University, St Louis, MO 63130, USA.
3 Developmental Biology Program, Childrens Hospital Los Angeles, Departments of Pathology and Surgery, Keck School of Medicine, University of Southern California, Los Angeles, CA 90027, USA.
4 Molecular Developmental Biology Group, Laboratory of Reproductive and Developmental Toxicology, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709, USA.
5 Genetics of Development and Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
6 Laboratory Molecular Biology (Celgen), Department for Molecular and Developmental Genetics, VIB, B-3000 Leuven, Belgium.
7 Laboratory Molecular Biology (Celgen), Center for Human Genetics, KU Leuven, B-3000 Leuven, Belgium.
8 Laboratory of Cell Regulation and Carcinogenesis, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.


Figure 1
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  		Fig. 1. Wild-type mouse eyelid anatomy at E15.5. The components of the normal ocular epithelia are color coded: bulbar conjunctiva, purple; palpebral conjunctiva, pink; palpebral epidermis, dark red; periderm, orange; cornea, blue.

 

Figure 2
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  		Fig. 2. Eyelid defects in mice with deficiencies in BMP signaling. (A-G) Hematoxylin and Eosin staining of frontal eye sections from wild-type (A,E), Acvr1;Bmpr1aDCKO (B), Smad4CKO (C,F), Smad1/5DCKO (D) and Tgfbr2CKO (G) mice at postnatal day 3 (P3). Wild-type eyelids are fused (arrow in A), whereas eyelids deficient in BMP signaling in the ectoderm are separate (arrowheads in B-D). (E,F) Higher magnification views of insets in A and C. (E) Eosin-stained keratin (arrow) and Hematoxylin-stained keratinocytes (arrowhead) in wild-type eyelid palpebral epidermis. (F) Ectopic Eosin-stained keratin-like protein (arrow) and ectopic Hematoxylin-stained keratinocyte-like cells (arrowhead) in Smad4CKO conjunctiva. Asterisks in E and F illustrate the hyperplasia in the Smad4CKO conjunctiva. (G) Tgfbr2CKO neonate eyelids are fused (arrow). Scale bars: 100 µm.

 

Figure 3
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  		Fig. 3. Cell proliferation in Smad4CKO eyelid epithelia at E14.5. (A-D) BrdU staining in frontal eye sections from wild-type upper eyelid (A), wild-type lower eyelid (C), Smad4CKO upper eyelid (B) and Smad4CKO lower eyelid (D). Red lines mark the approximate boundaries of the bulbar and palpebral conjunctiva and the palpebral epidermis. (E) Percentage of BrdU-labeled cells in each compartment. Compared with wild-type animals, the BrdU labeling index in Smad4CKO bulbar and palpebral conjunctival epithelia was significantly increased. The y-axes indicate the mean percentage of BrdU incorporation in each area assayed. Error bars represent the s.e.m. *P<0.05, ***P<0.001. B, bulbar conjunctiva; PC, palpebral conjunctiva; PE, palpebral epidermis. Scale bar in A: 100 µm.

 

Figure 4
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  		Fig. 4. Foxc1 and Foxc2 transcripts are greatly reduced in Fgfr2CKO and Smad4CKO eyelids. (A-C) In situ hybridization for Foxc1 mRNA on frontal eye sections from wild-type (A), Fgfr2CKO (B) and Smad4CKO (C) eyelids at E15.5. In wild-type eyelid (A), Foxc1 mRNA was detected in the palpebral epithelium, palpebral conjunctiva, bulbar conjunctiva, periderm and retina. In Fgfr2CKO (B) and Smad4CKO (C) eyelids, Foxc1 mRNA was detectable only in the retina. (D-F) In situ hybridization of Foxc2 mRNA on frontal eye sections from wild-type (D), Fgfr2CKO (E) and Smad4CKO (F) eyelids. In wild type eyelids (D), Foxc2 mRNA was detected in the palpebral conjunctiva and retina at E15.5. In Fgfr2CKO (E) and Smad4CKO (F) eyelids, Foxc2 mRNA was detectable only in the retina. Scale bars: 100 µm.

 

Figure 5
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  		Fig. 5. The nuclear localization of phosphorylated c-Jun requires Smad4. Immunostaining for phosphorylated c-Jun (p-c-Jun, green) on frontal eye sections from wild-type E15.0 (A), wild-type E15.5 (B), Smad4CKO E15.0 (C) and Smad4CKO E15.5 (D) embryos. (A) In wild-type eyelids, a small number of migrating periderm cells are present at E15.0. Staining for p-c-Jun is present in the nuclei of these cells (inset). (B) By E15.5, the number of periderm cells has increased and they have begun to migrate over the cornea. Staining for p-c-Jun is present in the nuclei of these cells (inset). (C) At E15.0, periderm cells in Smad4CKO eyelids appeared similar in number to wild type. However, p-c-Jun staining was restricted to the cytoplasm around the nuclei (inset). (D) At E15.5, fewer periderm cells were present in Smad4CKO eyelids than in wild type. Staining for p-c-Jun was weaker than in wild-type periderm cells and was still restricted to the perinuclear cytoplasm (inset). Insets show p-c-Jun staining without the nuclear counterstain (blue). Scale bar in A: 100 µm.

 

Figure 6
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  		Fig. 6. Signaling from Fgfr2 promotes Bmp4, patched 1 expression and BMP signaling in the eyelid. (A-C) In situ hybridization for Bmp4 transcripts at E15.5 in frontal eye sections from wild-type upper and lower eyelids (A) and Fgfr2CKO upper (B) and lower (C) eyelids. (A) In the wild-type eyelids, Bmp4 transcripts were present in a cluster of mesenchyme cells underlying the palpebral conjunctiva (arrows) and the palpebral epidermis (arrowhead). (B) In the upper eyelids of Fgfr2CKO mice, Bmp4 transcripts were detected in the mesenchyme underlying the palpebral conjunctiva (arrow), but not in the mesenchyme underlying the palpebral epidermis (arrowhead). (C) In the lower eyelids of Fgfr2CKO mice, Bmp4 transcripts were not detected in the mesenchyme underlying the palpebral conjunctiva (arrow). (D-F) In situ hybridization for Ptch1 transcripts in frontal eye sections from wild-type (D), Fgfr2CKO (E) and Smad4CKO (F) eyelids. In wild-type (D) and Smad4CKO eyelids (F), Ptch1 transcripts were present in clusters of mesenchyme cells and the overlying palpebral conjunctiva (arrows) and palpebral epidermis (arrowhead). (E) In the upper eyelids of Fgfr2CKO mice, Ptch1 transcripts were decreased in the mesenchyme and the overlying palpebral conjunctiva (arrow), and were barely detectable in the mesenchyme and the overlying palpebral epidermis (arrowhead). In the lower eyelids of Fgfr2CKO mice, Ptch1 transcripts were undetectable in the mesenchyme and the overlying palpebral conjunctiva (arrow). (G-J) Immunostaining for phosphorylated Smad1/5/8 (pSmad1/5/8) in frontal eye sections from wild-type upper (G), wild-type lower (H), Fgfr2CKO upper (I) and Fgfr2CKO lower (J) eyelids. (G) In the wild-type upper eyelid, nuclear pSmad1/5/8 staining was seen in the eyelid mesenchyme, the overlying palpebral conjunctiva (arrow), the palpebral epidermis (arrowhead) and the periderm (asterisk). (H) In the wild-type lower eyelid, pSmad1/5/8 staining was seen in the eyelid mesenchyme, the overlying palpebral conjunctiva (arrow) and the periderm (asterisk). (I) In the upper eyelid of Fgfr2CKO embryos, pSmad1/5/8 staining was reduced in the palpebral conjunctiva (arrow) and greatly reduced in the palpebral epidermis (arrowhead). (J) In the Fgfr2CKO lower eyelid, pSmad1/5/8 staining was greatly reduced in the palpebral conjunctiva (arrow). ul, upper eyelid; ll, lower eyelid. Scale bars: 100 µm.

 

Figure 7
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  		Fig. 7. The activity of the canonical Wnt pathway in the eyelid is modified in different ways by FGF and BMP signaling. (A-C) Whole-mount X-gal staining from the TOPGAL reporter in wild-type (A), Fgfr2CKO (B) and Smad4CKO (C) eyelids at E15.5. (D-F) Frontal sections of the eyes shown in A-C. (A) A prominent band of Wnt activity is present in the upper (black arrow) and a weaker band in the lower eyelid margins (red arrow) of wild-type eyes. Wnt activity is also present in eyelash follicles (arrowhead) and in epidermal hair follicles. (B) Wnt activity is uniformly increased in the upper and lower eyelids of Fgfr2CKO mice (black arrows) and is present in epidermal hair follicles (arrowhead). (C) Wnt activity is present in the epidermal hair follicles (arrowhead) and in ectopic patches in the upper and lower eyelids of Smad4CKO mice (black arrows). (D) Frontal sections reveal that Wnt activity is located near the anterior edge of the palpebral conjunctiva of wild-type eyelids (arrow). (E) In Fgfr2CKO eyelids, Wnt activity expands into the palpebral conjunctiva (arrows). (F) In Smad4CKO eyelids, Wnt activity is present in ectopic hair follicles (arrow), corresponding to the location of the band of cells with high Wnt activity in the wild-type palpebral conjunctiva. Scale bars: 100 µm.

 

Figure 8
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  		Fig. 8. The effects of FGF and BMP signaling on Dkk2 and Sfrp1 expression in the eyelid. (A-C) In situ hybridization for Dkk2 transcripts in frontal sections from wild-type (A), Fgfr2CKO (B) and Smad4CKO (C) embryos at E15.5. (A) Dkk2 transcript levels (blue) were strong in the eyelid epidermis, bulbar conjunctiva and corneal stroma, with weaker staining in the palpebral conjunctiva, periderm and mesenchyme in wild type. A sense probe control is shown in the inset. (B,C) No significant change in Dkk2 mRNA level was detected in Fgfr2CKO (B) and Smad4CKO (C) embryos at E15.5. (D-F) Immunostaining for DKK2 protein in frontal eye sections from wild-type (D), Fgfr2CKO (E) and Smad4CKO (F) embryos at E15.5. DKK2 protein in wild-type and CKO embryos was similar to the in situ hybridization results. (G-I) In situ hybridization for Sfrp1 transcripts in frontal sections from wild-type (G), Fgfr2CKO (H) and Smad4CKO (I) eyelids. In wild-type (G) and Smad4CKO (I) eyelids, Sfrp1 transcripts were present in the conjunctiva (asterisks), retina, retinal pigment epithelium and lens. In Fgfr2CKO embryos (H), Sfrp1 transcripts were readily detected in the retina, retinal pigment epithelium and lens, with low levels in the conjunctiva and palpebral epithelium. Scale bars: 100 µm.

 

Figure 9
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  		Fig. 9. Abnormal eyelid epithelial differentiation in Fgfr2CKO and Smad4CKO mice. (A-C) Immunostaining for keratin 14 (K14) in frontal sections from wild-type (A), Fgfr2CKO (B) and Smad4CKO (C) embryos at E15.0. In wild-type eyelids (A), K14 staining was weak in the palpebral conjunctiva and was not detected in the bulbar conjunctiva (arrows). In Fgfr2CKO (B) and Smad4CKO (C) eyelids, K14 staining was strong and uniform in the palpebral and bulbar conjunctiva (arrows). (D-F) Immunostaining for keratin 10 (K10) in frontal sections from wild type (D), Fgfr2CKO (E) and Smad4CKO (F) embryos at E17.5. In wild-type eyelids (D), K10 staining is observed only in the epidermis; it is negative in the conjunctiva (arrow). In Fgfr2CKO (E), K10 is found in epidermis and a few cells of the palpebral conjunctiva, although the majority of conjunctival cells do not express K10 (arrow). In Smad4CKO (F), K10 is detected in the epidermis and in the majority of conjunctival cells (arrow). (G-I) Immunostaining for keratin 4 (K4) in frontal sections from wild-type (G), Fgfr2CKO (H) and Smad4CKO (I) embryos at E17.5. In wild-type eyelids (G), expression of K4 is continuous in the conjunctiva (arrow). In Fgfr2CKO (H), K4 is found in most conjunctival cells (arrow). In Smad4CKO (I), K4 is only detected in a few conjunctival cells; most conjunctival cells do not express K4 (arrow). Scale bars: 100 µm.

 

Figure 10
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  		Fig. 10. Summary of the role of BMP signaling and its interaction with the other signaling pathways known or proposed to function in mouse eyelid development. (A) In eyelids that are deficient in BMP signaling, the conjunctival epithelial cells express epidermis-specific keratin 10 and form hair follicles near that lid margin. (B) The network of known or proposed signaling pathways controlling eyelid development. Observations made in this study are indicated by blue arrows, previous findings by orange arrows and findings confirmed in this study by green arrows. Our model suggests that epidermal cell fate is the default pathway and that BMP signaling is required for prospective conjunctival epithelial cells to suppress the epidermal differentiation pathway and become conjunctival cells. BMP signaling is not required to initiate the migration of periderm cells at the lid margins, but is required for the expansion of these cells across the corneal surface. During this process, BMP signaling is required for the expression of Foxc1 and Foxc2, and for the full activation (phosphorylation) of c-Jun. BMP-dependent formation of active R-Smad-Smad4 complexes is required for the translocation of p-c-Jun into the nuclei of periderm cells, where it has been reported to increase the expression of the epidermal growth factor receptor.

 

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© The Company of Biologists Ltd 2009