Wnt/β-catenin signaling controls mouse eyelid growth by mediating epithelial-mesenchymal interactions

Abstract

Purpose: To investigate the role of Wnt/β-catenin signaling in mouse eyelid development.

Methods: Wnt/β-catenin signaling was disrupted by deleting supraorbital mesenchymal β-catenin or epithelial Wls. p63 was removed to determine whether the expression of Wnts is affected. The eyelid morphology was examined at different stages. Proliferation, apoptosis, and expression of Wnt ligands and their target genes were analyzed via immunofluorescence staining, TUNEL assay, and in situ hybridization.

Results: Deletion of β-catenin in supraorbital mesenchyme abolishes eyelid growth by causing decreased proliferation in supraorbital epithelium and underlying mesenchyme. Inhibition of Wnt secretion by deleting Wls in supraorbital epithelium results in failure of eyelid development, similar to the effects of deleting mesenchymal β-catenin. Knockout of p63 results in formation of hypoplastic eyelids and reduced expression of several Wnt ligands in eyelid epithelium.

Conclusions: Epithelial Wnt ligands activate mesenchymal Wnt/β-catenin signaling to control eyelid growth and their expression is partially regulated by p63.

Keywords: Epithelial-mesenchymal interactions; Eyelid development; Fgf10; Proliferation; Signaling pathway; Wls; Wnt; p63; β-catenin.

Figure 1.. Deletion of β-catenin in supraorbital mesenchyme abolishes upper eyelid growth.

(A) The eyelids of control embryos are fused at E18.5. (B) The eyes of Prx1-Cre β-catenin^fl/fl embryos are open at E18.5. (C) H&E staining of tissue sections shows that eyelids are fused in control embryos at E18.5. (D) H&E staining of tissue sections indicates that the upper eyelid is almost absent in Prx1-Cre β-catenin^fl/fl embryos, except for some epidermal thickening (black arrow). The lower eyelid is still present in these mutants. (E) H&E staining of tissue sections shows that control eyelids are well-formed at E14.5. (F) H&E staining of tissue sections indicates that there is no discernable upper eyelid in Prx1-Cre β-catenin^fl/fl embryos at E14.5 (black arrow). (G) H&E staining shows that at E12.5, control embryos have morphologically distinguishable eyelid primordia. (H) H&E staining shows that in Prx1-Cre β-catenin^fl/fl embryos, no significant upper eyelid primordia have formed at E12.5, but lower eyelid primordia are present. Samples in (A) and (B) were photographed at the same magnification. Scale bars: 150μm. Abbreviations: ul, upper eyelid; ll, lower eyelid.

Figure 2.. Deletion of β-catenin in supraorbital mesenchyme results in reduced canonical Wnt activity and decreased cell proliferation.

(A) IF shows that β-catenin is expressed in the supraorbital epithelium and mesenchyme of control embryos at E11.5. (B) IF shows that β-catenin is deleted in the supraorbital mesenchyme (yellow arrows), but patches of undeleted cells can also be identified (green arrow) in Prx1-Cre β-catenin^fl/fl embryos at E11.5. (C) Lef1 expression is detected in supraorbital mesenchyme in control embryos at E11.5. (D) Lef1 expression is reduced or absent (yellow arrows) in the supraorbital mesenchyme of Prx1-Cre β-catenin^fl/fl embryos at E11.5. (E) Twist1 is expressed in the supraorbital mesenchyme of control embryos at E11.5. (F) Expression of Twist1 is decreased in the supraorbital mesenchyme of Prx1-Cre β-catenin^fl/fl embryos at E11.5 (yellow arrows). (G and H) Expression of epithelial Wnt3 is similar between control and Prx1-Cre β-catenin^fl/fl embryos at E11.5. (I and J) Epithelial Wnt4 levels are comparable between control and Prx1-Cre β-catenin^fl/fl embryos at E11.5. (K and L) TUNEL assay indicates that levels of apoptosis are similar in control and Prx1-Cre β-catenin^fl/fl embryos at E11.5. (M and N) Ki-67 staining at E11.5 shows that proliferation is reduced upon deletion of mesenchymal β-catenin. (O) Statistical analysis of Ki-67+ cells in supraorbital epithelium and underlying mesenchyme. Three pairs of control and mutant embryos were analyzed. Unpaired student t-test was used to evaluate significance. p<0.05 was considered significant. Scale bars: 50μm. The white dashed line indicates the boundary between epithelium and mesenchyme.

Figure 3.. Epithelial Wnt ligands are required to maintain eyelid growth.

(A) The eyelids of control embryos are fused at E17.5. (B) The eyes of Msx2-Cre Wls^fl/fl embryos exhibit defects in multiple ocular tissues including loss of supraorbital skin and eyelid, and malformed eye at E17.5. (C) H&E staining shows that eyelids are fused in control embryos at E17.5. (D) H&E staining indicates that the upper eyelid is lost in Msx2-Cre Wls^fl/fl embryos (black arrow). The lower eyelid is still present in these mutants. (E) H&E staining shows that control eyelids are well-formed at E14.5. (F) H&E staining indicates that the upper eyelid is lost in Msx2-Cre Wls^fl/fl embryos at E14.5 (black arrow). (G) H&E staining shows that at E12.5, control embryos have morphologically distinguishable eyelid primordia. (H) H&E staining shows that in Msx2-Cre Wls^fl/fl embryos, no upper eyelid primordia have formed at E12.5. Samples in (A) and (B) were photographed at the same magnification. Scale bars: 150μm. Abbreviations: ul, upper eyelid; ll, lower eyelid.

Figure 4.. Epithelial Wnt ligands control mesenchymal canonical Wnt signaling in the eyelid.

(A) RNAscope ISH shows that Wls is expressed in the supraorbital epithelium and mesenchyme of control embryos at E11.5. (B) RNAscope ISH shows that Wls is deleted in the supraorbital epithelium (yellow arrows) of Msx2-Cre Wls^fl/fl embryos at E11.5. (C) IF staining shows that LEF1 is expressed in the supraorbital mesenchyme of control embryos at E11.5. (D) IF staining shows that LEF1 expression is absent (yellow arrow) in the supraorbital mesenchyme of Msx2-Cre Wls^fl/fl embryos at E11.5. (E) Twist1 is expressed in the supraorbital mesenchyme of control embryos at E11.5. (F) Expression of Twist1 is lost in the supraorbital mesenchyme of Msx2-Cre Wls^fl/fl embryos at E11.5 (white arrow). (G and H) Expression of epithelial Wnt3 is similar between control and Msx2-Cre Wls^fl/fl embryos at E11.5. (I and J) Epithelial Wnt4 levels are comparable between control and Msx2-Cre Wls^fl/fl embryos at E11.5. (K and L) TUNEL assay indicates that levels of apoptosis are similar in control and Msx2-Cre Wls^fl/fl embryos at E11.5. (M and N) Ki-67 staining at E11.5 shows that proliferation is reduced upon deletion of epithelial Wls. (O) Statistical analysis of Ki-67+ cells in supraorbital epithelium and underlying mesenchyme. Three pairs of control and mutant embryos were analyzed. Unpaired student t-test was used to evaluate significance. p<0.05 was considered significant. Scale bars: 50μm. The white dashed line indicates the boundary between epithelium and mesenchyme.

Figure 5.. Expression of Fgf10 in eyelid mesenchyme is dependent on secretion of epithelial Wnt ligands.

(A) IF staining shows that LEF1 is strongly expressed in the mesenchyme of developing upper eyelid primordia of control embryos at E12.5 (yellow arrow). (B) IF staining shows that LEF1 expression is significantly reduced in the supraorbital mesenchyme of the hypoplastic upper eyelid primordia of Msx2-Cre^low Wls^fl/fl embryos at E12.5 (yellow arrow). (C) BrdU staining shows that cell proliferation is enriched in the epithelium and posterior mesenchyme of upper eyelid primordia in control embryos at E12.5 (yellow arrow). (D) BrdU staining shows that proliferation is reduced in the upper eyelid primordia of Msx2-Cre^low Wls^fl/fl embryos at E12.5 (yellow arrow). (E) Fgf10 is expressed in the developing upper eyelid primordia of control embryos at E12.5 (black arrow). (F) Fgf10 expression is undetectable in the developing upper eyelid primordia of Msx2-Cre^low Wls^fl/fl embryos at E12.5 (black arrow). (G) Statistical analysis of BrdU+ cells in supraorbital epithelium and underlying mesenchyme. Four control and three mutant embryos were analyzed. Unpaired student t-test was used to evaluate significance. p<0.05 was considered significant. Samples in (E) and (F) were photographed at the same magnification. Scale bars: 50μm. The white dashed line indicates the boundary between epithelium and mesenchyme. Abbreviations: ul, upper eyelid; ll, lower eyelid.

Figure 6.. p63 regulates expression of Wnt genes in eyelid epithelium.

(A and C) KRT5 and KRT14 are expressed in the epithelium of the upper eyelid of control embryos at E14.5 (white arrows). (B and D) KRT5 and KRT14 expression is diminished in the epithelium of the upper eyelid of E14.5 p63^−/− embryos (white arrows). (E and G) Wnt3 and Wnt4 are expressed in the epithelium of the upper eyelid of control embryos at E14.5 (white arrows). (F and H) Expression of Wnt3 and Wnt4 is significantly reduced in the epithelium of the upper eyelid of p63^−/− embryos at E14.5 (white arrows). (I) Lef1 is expressed in the epithelium (white arrow) and underlying mesenchyme (yellow arrow) of the upper eyelid of control embryos at E14.5. (J) Expression of Lef1 is lost in both epithelium (white arrow) and underlying mesenchyme (yellow arrow) of the upper eyelid of E14.5 p63^−/− embryos. (K) Twist1 is expressed in the mesenchyme of the upper eyelid of control embryos at E14.5 (white arrow). (L) Expression of Twist1 is reduced in the mesenchyme of the upper eyelid of E14.5 p63^−/− embryos. Scale bars: 50μm. The white dashed line indicates the boundary between epithelium and mesenchyme.

Figure 7.. Schematic diagram of epithelial-mesenchymal interactions mediated by Wnt/β-catenin signaling in the developing eyelid.

Our data support a model in which p63 controls the expression of epithelial Wnt proteins, which in turn activate mesenchymal β-catenin-dependent Wnt signaling resulting in increased mesenchymal proliferation and activation of downstream target genes LEF1 and TWIST1. Epithelial Wnt ligands also control mesenchymal FGF10 expression, which is required for the proliferation and migration of epithelial cells in the developing upper eyelid [11].