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Robbert De Iongh, Chaotung Ting, Jing Yee Janet Choi, Gemma Martinez; Restricted period of Smoothened requirement during lens development. Invest. Ophthalmol. Vis. Sci. 2013;54(15):3190. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Previous studies suggest that Hedgehog (Hh) signalling plays roles in ocular development. More recently, transgenic studies conditionally activating Smo in developing lens showed that Hh signals promote epithelial cell proliferation, differentiation (Foxe3, Pax6) and inhibits fibre differentiation (c-Maf, Prox1), with resultant cell death. In this study we confirmed expression of Hh pathway components in developing lens and investigated the requirement of Smo in lens development by conditional loss-of function mutations.
Hh pathway gene expression was examined by RT-PCR and immunofluorescence. Smo was deleted in embryonic mouse lenses by crossing Smofl/fl mice with LeCre and MLR10 Cre deletor mice. Lens phenotype was investigated by histology, BrdU labelling, TUNEL, immunofluorescence and RT-PCR.
Distinct expression of Ptch1 Gli2 and Gli3 were detected in developing lens epithelium from E12.5 onwards. At all stages, Ptch1 was localized to cell membranes and cytosol, whereas Gli2 was nuclear, with particularly strong staining of M-phase chromosomes. From E12.5 to E13.5 there was a shift in Gli3 localization from cytosol to nucleus, suggesting an increased role for this predominantly repressor protein. Conditional deletion of Smo in lenses, using the MLR10 Cre line (active from E13) resulted in no obvious ocular defect. However, deletion of Smo with LeCre (active from E10) resulted in anterior segment defects from E12.5 - E16.5. E14.5 and E16.5 lenses showed a defect in G2-M phase transition (phospho-histone 3 stain) of cell cycle but normal G1-S phase transition (BrdU label). Epithelial cell death (TUNEL) was detected from E14.5-E16.5. No changes in epithelial (E-cadherin, Pax6) or fibre cell markers (p57Kip2, c-maf) were detected. Analysis of corneal development showed abnormal endothelial differentiation (keratin 14) and abnormal migration of Nrp2+ mesenchymal cells in the anterior and vitreous chambers.
These data indicate that Hh pathway is required for lens epithelial cell proliferation and survival during a discrete period (E10-E13) in lens development. Moreover, defective lens development at these stages appears to have effects on semaphorin- or VEGF-responsive cells in the ocular mesenchyme.
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