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Comparative Study
. 2012 Jun 5;53(7):3316-30.
doi: 10.1167/iovs.12-9595.

Activation of the hedgehog signaling pathway in the developing lens stimulates ectopic FoxE3 expression and disruption in fiber cell differentiation

Christine L Kerr  1 , Jian Huang, Trevor Williams, Judith A West-Mays
Affiliations
Comparative Study

Activation of the hedgehog signaling pathway in the developing lens stimulates ectopic FoxE3 expression and disruption in fiber cell differentiation

Christine L Kerr et al. Invest Ophthalmol Vis Sci. .

Abstract

Purpose: The signaling pathways and transcriptional effectors responsible for directing mammalian lens development provide key regulatory molecules that can inform our understanding of human eye defects. The hedgehog genes encode extracellular signaling proteins responsible for patterning and tissue formation during embryogenesis. Signal transduction of this pathway is mediated through activation of the transmembrane proteins smoothened and patched, stimulating downstream signaling resulting in the activation or repression of hedgehog target genes. Hedgehog signaling is implicated in eye development, and defects in hedgehog signaling components have been shown to result in defects of the retina, iris, and lens.

Methods: We assessed the consequences of constitutive hedgehog signaling in the developing mouse lens using Cre-LoxP technology to express the conditional M2 smoothened allele in the embryonic head and lens ectoderm.

Results: Although initial lens development appeared normal, morphological defects were apparent by E12.5 and became more significant at later stages of embryogenesis. Altered lens morphology correlated with ectopic expression of FoxE3, which encodes a critical gene required for human and mouse lens development. Later, inappropriate expression of the epithelial marker Pax6, and as well as fiber cell markers c-maf and Prox1 also occurred, indicating a failure of appropriate lens fiber cell differentiation accompanied by altered lens cell proliferation and cell death.

Conclusions: Our findings demonstrate that the ectopic activation of downstream effectors of the hedgehog signaling pathway in the mouse lens disrupts normal fiber cell differentiation by a mechanism consistent with a sustained epithelial cellular developmental program driven by FoxE3.

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Conflict of interest statement

Disclosure: C.L. Kerr, None; J. Huang, None; T. Williams, None; J.A. West-Mays, None

Figures

Figure 1.
Figure 1.
Verification of constitutively active smoothened allele expression in SE and SE derivatives. Sections of WT (A–C) and Crect activated Smo mutant (D–F) mouse eyes at E12.5 (A, D), E15.5 (B, E), and E18.5 (C, F) immunostained for GFP expression. Ptch1-LacZ (G, I) and Crect Smo-Ptch1-LacZ (H, J) at E12.5 (G, H) and P0 (I, J) were stained with X-gal to examine for Ptch1 expression as indicated by LacZ staining. The blue staining seen in the WT P0 samples in the surface ectoderm overlying the eye represents expression in the hair follicles associated with the eyelids. CE, corneal epithelium; LE, lens epithelium; C, cornea; Le, lens; R, retina; SE, surface ectoderm. All scale bars represent 100 μm.
Figure 2.
Figure 2.
H&E stains of WT (A–C) and activated Smo mutant (D–F) lenses. At E12.5 (A, D) defective lens morphology in the mutant (D) was characterized by a thicker than normal lens epithelial region (black arrow) and a misshapen lens. An aberrant group of cells developed in the mutant lens vesicle lumen (pink arrow, D). By E15.5 (B, E), the WT lens displayed distinct lens epithelial and fiber cell layers, while the mutant lens was disorganized, with the lens protruding away from the optic cup. An adhesion of the lens to the overlying SE also was observed (black star, E). By E18.5 (C, F), the mutant lens was smaller than that of the WT, remained disorganized, and continued to protrude even further away from the optic cup (C, F). FC, fiber cells. All scale bars represent 100 μm.
Figure 3.
Figure 3.
Patterns of proliferation and cell death are abnormal in developing activated Smo lens. Sections of WT and Smo mutant mouse (Smo) eyes at the indicated time points immunostained for expression of phosphohistone H3 or detection of TUNEL as shown. White arrows indicate proliferating cells (A–F) or TUNEL positive cells (G–L), respectively. Slides also were stained with DAPI to highlight nuclei (blue). All scale bars represent 100 μm.
Figure 4.
Figure 4.
Cell cycle promoting and inhibiting factors cyclin D1, p27kip1, and p57kip2 are expressed ectopically in the activated Smo lens. Sections of WT (A, B, E, F, I, J) and activated Smo mutant (C, D, G, H, K, L) mouse eyes at E12.5 (A, C, E, G, I, K), E15.5 (F, H, J, L) and E16.5 (B, D) immunostained for expression of cyclin D (A–D, green), p27kip1 (E–H, green), or p57kip2 (I–L, red). Slides also were stained with DAPI to highlight nuclei (blue). White arrows (F, J) show localized expression of p27kip1 and p57kip2 within the transitional zone of the lens. White arrows (D, L) show expression of cyclin D and p57kip2 in cells within the lens epithelial region of the mutant. LE, lens epithelium; FC, fiber cells. All scale bars represent 100 μm.
Figure 5.
Figure 5.
Pax6 and FoxE3 are expressed ectopically in developing activated Smo lens. Normal sections of WT (A, B, C, G, H) and activated Smo mutant (D, E, F, I, J) mouse eyes at E12.5 (A, D, G, I), E15.5 (B, E, H, J), and E18.5 (C, F) immunostained for expression of Pax6 (A–F, red) or Foxe3 (G–J, red). Slides also were stained with DAPI to highlight nuclei (blue). White arrows (E, F, J) indicate Pax6 (E, F) and FoxE3 (J) staining within the lens epithelial cell region. All scale bars represent 100 μm.
Figure 6.
Figure 6.
γ-Crystallin and β-crystallin are expressed in an appropriate spatial pattern, though crystallin-expressing FCs fail to de-nucleate and maintain Pax6 expression. Sections of WT (A–C, G–I, M–O) and activated Smo mutant (D–F, J–L, P–R) mouse eyes at E12.5 (A, D, G, J, M, P), E15.5 (B, E, H, K, N, Q), and E18.5 (C, F, I, L, O, R) immunostained for expression of the γ-crystallin lens fiber cell marker (A–F, green), β-crystallin (G–L, red) or both Pax6 and β-crystallin (M–R, green and red, respectively). Slides also were stained with DAPI to highlight nuclei (blue). All scale bars represent 100 μm.
Figure 7.
Figure 7.
c-maf and Prox1 expression is expanded throughout the anterior and posterior lens of the activated Smo mutants. Sections of WT (A–C, G–I) and activated Smo mutant (D–F, J–L) mouse eyes at E12.5 (A, D, G, J), E15.5 (B, E, H, K), and E18.5 (C, F, I, L) immunostained for expression of c-maf (A–F, red), or Prox1 (G–L, red). Slides also were stained with DAPI to highlight nuclei (blue). White arrows show c-maf and Prox1 expression at the equatorial transitional zone. All scale bars represent 100 μm.
Figure 8.
Figure 8.
Retinal lamination is lost between E18.5 and P0. Sections of WT (A–D) and activated Smo mutant (E–H) mouse eyes at E12.5 (A, E), E15.5 (B, F), E18.5 (C, G), and P0 (D, H) stained with H&E. inbl, inner neuroblast layer; IPL, inner plexiform layer. All scale bars represent 100 μm.
Figure 9.
Figure 9.
Activated Smo mutants show disorganized retinal morphology, and expression of Pax6 and Calretinin, while exhibiting abnormal retinal cell proliferation and death by P0. Sections of WT (A–D, I–L) and activated Smo mutant (E–H, M–P) mouse eyes at E18.5 and P0. Slides were immunostained for expression of Pax6 (A, B, E, F) and Calretinin (C, D, G, H), as well as PCNA (I, J, M, N) and TUNEL (K, L, O, P). White arrows indicate irregular proliferation and cell death in the IR of the mutants. All scale bars represent 100 μm.
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