title: A role for smoothened during murine lens and cornea development.
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.
authors.affiliation: Ocular Development Laboratory, Anatomy & Neuroscience, University of Melbourne, Parkville, Australia.
authors.forename: Janet J Y
authors.initials: JJ
authors.lastname: Choi
authors.affiliation: Ocular Development Laboratory, Anatomy & Neuroscience, University of Melbourne, Parkville, Australia.
authors.forename: Chao-Tung
authors.initials: CT
authors.lastname: Ting
authors.affiliation: Ocular Development Laboratory, Anatomy & Neuroscience, University of Melbourne, Parkville, Australia.
authors.forename: Lidia
authors.initials: L
authors.lastname: Trogrlic
authors.affiliation: Ocular Development Laboratory, Anatomy & Neuroscience, University of Melbourne, Parkville, Australia.
authors.forename: Stefan V
authors.initials: SV
authors.lastname: Milevski
authors.affiliation: Department of Zoology, University of Melbourne, Parkville, Australia.
authors.forename: Mary
authors.initials: M
authors.lastname: Familari
authors.affiliation: Ocular Development Laboratory, Anatomy & Neuroscience, University of Melbourne, Parkville, Australia.
authors.forename: Gemma
authors.initials: G
authors.lastname: Martinez
authors.affiliation: Ocular Development Laboratory, Anatomy & Neuroscience, University of Melbourne, Parkville, Australia.
authors.forename: Robb U
authors.initials: RU
authors.lastname: de Iongh
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D002453:Cell Cycle; D002465:Cell Movement; D003315:Cornea / Q000254:growth & development / Q000378:metabolism* / Q000473:pathology; D004622:Embryo, Mammalian; D042783:Endothelial Cells / Q000378:metabolism* / Q000473:pathology; D004847:Epithelial Cells / Q000378:metabolism* / Q000473:pathology; D051858:Forkhead Transcription Factors / Q000235:genetics* / Q000378:metabolism; D018507:Gene Expression Regulation, Developmental; D019426:Integrases / Q000235:genetics / Q000378:metabolism; D051741:Kruppel-Like Transcription Factors / Q000235:genetics / Q000378:metabolism; D007908:Lens, Crystalline / Q000254:growth & development / Q000378:metabolism* / Q000473:pathology; D008565:Membrane Proteins; D059630:Mesenchymal Stem Cells / Q000378:metabolism / Q000473:pathology; D051379:Mice; D008822:Mice, Transgenic; D009024:Morphogenesis; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism; D039943:Neuropilin-2 / Q000235:genetics / Q000378:metabolism; D000072079:Patched Receptors; D000072081:Patched-1 Receptor; D011956:Receptors, Cell Surface / Q000235:genetics / Q000378:metabolism; D043562:Receptors, G-Protein-Coupled / Q000235:genetics* / Q000378:metabolism; D015398:Signal Transduction; D000072086:Smoothened Receptor; D029961:Zebrafish Proteins; D000075746:Zinc Finger Protein Gli2; D000074290:Zinc Finger Protein Gli3
chemical_list: D051858:Forkhead Transcription Factors; C498612:Foxe3 protein, mouse; C000618551:Gli2 protein, mouse; C465571:Gli3 protein, mouse; D051741:Kruppel-Like Transcription Factors; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D039943:Neuropilin-2; C000606236:PTCH1 protein, human; D000072079:Patched Receptors; D000072081:Patched-1 Receptor; C000606240:Ptch1 protein, mouse; D011956:Receptors, Cell Surface; D043562:Receptors, G-Protein-Coupled; C491022:Smo protein, mouse; D000072086:Smoothened Receptor; D029961:Zebrafish Proteins; D000075746:Zinc Finger Protein Gli2; D000074290:Zinc Finger Protein Gli3; C491715:ptch1 protein, zebrafish; C045073:Cre recombinase; D019426:Integrases