Abstract
Various studies suggest that Hedgehog (Hh) signalling plays roles in human and zebrafish ocular development. Recent studies (Kerr et al., Invest Ophthalmol Vis Sci. 2012; 53, 3316–30) showed that conditionally activating Hh signals promotes murine lens epithelial cell proliferation and disrupts fibre differentiation. In this study we examined the expression of the Hh pathway and the requirement for the Smoothened gene in murine lens development. Expression of Hh pathway components in developing lens was examined by RT-PCR, immunofluorescence and in situ hybridisation. The requirement of Smo in lens development was determined by conditional loss-of-function mutations, using LeCre and MLR10 Cre transgenic mice. The phenotype of mutant mice was examined by immunofluorescence for various markers of cell cycle, lens and cornea differentiation. Hh pathway components (Ptch1, Smo, Gli2, Gli3) were detected in lens epithelium from E12.5. Gli2 was particularly localised to mitotic nuclei and, at E13.5, Gli3 exhibited a shift from cytosol to nucleus, suggesting distinct roles for these transcription factors. Conditional deletion of Smo, from ∼E12.5 (MLR10 Cre) did not affect ocular development, whereas deletion from ∼E9.5 (LeCre) resulted in lens and corneal defects from E14.5. Mutant lenses were smaller and showed normal expression of p57Kip2, c-Maf, E-cadherin and Pax6, reduced expression of FoxE3 and Ptch1 and decreased nuclear Hes1. There was normal G1-S phase but decreased G2-M phase transition at E16.5 and epithelial cell death from E14.5-E16.5. Mutant corneas were thicker due to aberrant migration of Nrp2+ cells from the extraocular mesenchyme, resulting in delayed corneal endothelial but normal epithelial differentiation. These results indicate the Hh pathway is required during a discrete period (E9.5–E12.5) in lens development to regulate lens epithelial cell proliferation, survival and FoxE3 expression. Defective corneal development occurs secondary to defects in lens and appears to be due to defective migration of peri-ocular Nrp2+ neural crest/mesenchymal cells.
Highlights
Vertebrate eye development involves co-ordinated interactions and signalling mechanisms between neural and surface ectoderm and periocular mesenchyme, which is derived from the neural crest (NC) and paraxial mesoderm
Ptch-1 was detected as cytoplasmic reactivity in the epithelium and fibre cells at all ages and appeared to be most strongly expressed in the equatorial region by E15.5 (Fig. 2D)
To confirm that loss of Smoothened 7-pass transmembrane protein (Smo) affected the canonical Hh signalling pathway, we examined the expression of Ptch1, which is a direct target of the Hh pathway, in LeSmox lenses
Summary
Vertebrate eye development involves co-ordinated interactions and signalling mechanisms between neural and surface ectoderm and periocular mesenchyme, which is derived from the neural crest (NC) and paraxial mesoderm. Upon contact with the surface ectoderm, the optic vesicle thickens to form the retinal disc and the pre-lens ectoderm thickens to form the lens placode. Both the lens placode and optic vesicle invaginate to form respectively the lens vesicle and the optic cup, which later gives rise to retina, iris and ciliary body. The lens continues to interact with the overlying ectoderm, which subsequently differentiates to form the corneal epithelium. The corneal stroma and endothelium arise from the migration of cranial NC and mesodermal cells into the stroma that lies between the lens and overlying presumptive corneal epithelium [2,3]. Signals from the lens have been implicated in regulating the migration of peri-ocular NC cells [4], with TGFb2 proposed to be chemo-attractive [5,6], while Sema3A was proposed as a chemo-repellant, based on studies in chick embryos [7]
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