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• W2037810765 abstract "Complex molecular interactions dictate the developmental steps that lead to a mature and functional cornea and lens. Peters anomaly is one subtype of anterior segment dysgenesis especially due to abnormal development of the cornea and lens. MSX2 was recently implicated as a potential gene that is critical for anterior segment development. However, the role of MSX2 within the complex mechanisms of eye development remains elusive. Our present study observed the morphologic changes in conventional Msx2 knockout (KO) mice and found phenotypes consistent with Peters anomaly and microphthalmia seen in humans. The role of Msx2 in cornea and lens development was further investigated using IHC, in situ hybridization, and quantification of proliferative and apoptotic lens cells. Loss of Msx2 down-regulated FoxE3 expression and up-regulated Prox1 and crystallin expression in the lens. The FoxE3 and Prox1 malfunction and precocious Prox1 and crystallin expression contribute to a disturbed lens cell cycle in lens vesicles and eventually to cornea-lentoid adhesions and microphthalmia in Msx2 KO mice. The observed changes in the expression of FoxE3 suggest that Msx2 is an important contributor in controlling transcription of target genes critical for early eye development. These results provide the first direct genetic evidence of the involvement of MSX2 in Peters anomaly and the distinct function of MSX2 in regulating the growth and development of lens vesicles. Complex molecular interactions dictate the developmental steps that lead to a mature and functional cornea and lens. Peters anomaly is one subtype of anterior segment dysgenesis especially due to abnormal development of the cornea and lens. MSX2 was recently implicated as a potential gene that is critical for anterior segment development. However, the role of MSX2 within the complex mechanisms of eye development remains elusive. Our present study observed the morphologic changes in conventional Msx2 knockout (KO) mice and found phenotypes consistent with Peters anomaly and microphthalmia seen in humans. The role of Msx2 in cornea and lens development was further investigated using IHC, in situ hybridization, and quantification of proliferative and apoptotic lens cells. Loss of Msx2 down-regulated FoxE3 expression and up-regulated Prox1 and crystallin expression in the lens. The FoxE3 and Prox1 malfunction and precocious Prox1 and crystallin expression contribute to a disturbed lens cell cycle in lens vesicles and eventually to cornea-lentoid adhesions and microphthalmia in Msx2 KO mice. The observed changes in the expression of FoxE3 suggest that Msx2 is an important contributor in controlling transcription of target genes critical for early eye development. These results provide the first direct genetic evidence of the involvement of MSX2 in Peters anomaly and the distinct function of MSX2 in regulating the growth and development of lens vesicles. Anterior segment dysgenesis (ASD) is a developmental failure of the anterior segment tissues of the eye and an important cause of severe visual impairment in infants and young children. ASD has been classified into different subtypes based on its specific clinical phenotypes, including Peters anomaly, aniridia, and Axenfeld-Rieger syndrome or malformation.1Sowden J.C. Molecular and developmental mechanisms of anterior segment dysgenesis.Eye (Lond). 2007; 21: 1310-1318Crossref PubMed Scopus (111) Google Scholar, 2Idrees F. Vaideanu D. Fraser S.G. Sowden J.C. Khaw P.T. A review of anterior segment dysgeneses.Surv Ophthalmol. 2006; 51: 213-231Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar Peters anomaly is due to the incomplete and delayed detachment of the lens vesicle from the surface ectoderm and the persistence of the lens stalk. The lens adheres to the posterior cornea, leading to central corneal opacities in deep stromal layers and local absence of corneal endothelium. In addition, Peters anomaly is associated with iridiocorneal adhesions, iris hyperplasia, and other developmental eye disorders, such as microphthalmia.3Verma A.S. Fitzpatrick D.R. Anophthalmia and microphthalmia.Orphanet J Rare Dis. 2007; 2: 47Crossref PubMed Scopus (267) Google Scholar, 4Harissi-Dagher M. Colby K. Anterior segment dysgenesis: peters anomaly and sclerocornea.Int Ophthalmol Clin. 2008; 48: 35-42Crossref PubMed Scopus (39) Google Scholar, 5Wurm A. Sock E. Fuchshofer R. Wegner M. Tamm E.R. Anterior segment dysgenesis in the eyes of mice deficient for the high-mobility-group transcription factor Sox11.Exp Eye Res. 2008; 86: 895-907Crossref PubMed Scopus (47) Google Scholar Lens development is a complicated process because the formation of lens placode derives from the surface ectoderm. It has been proposed that abnormalities seen in Peters anomaly may also result from primary defects in the lens.6Gould D.B. John S.W. Anterior segment dysgenesis and the developmental glaucomas are complex traits.Hum Mol Genet. 2002; 11: 1185-1193Crossref PubMed Scopus (130) Google Scholar In human embryos, formation of lens placode takes place on approximately day 33 of gestation and in mice on day 9.5 of embryonic development (E9.5).7Bailey A.P. Bhattacharyya S. Bronner-Fraser M. Streit A. Lens specification is the ground state of all sensory placodes, from which FGF promotes olfactory identity.Dev Cell. 2006; 11: 505-517Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar, 8Sjodal M. Edlund T. Gunhaga L. Time of exposure to BMP signals plays a key role in the specification of the olfactory and lens placodes ex vivo.Dev Cell. 2007; 13: 141-149Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar Coordinated invagination of the lens ectoderm and the optic vesicle gives rise to the lens pit and the optic cup and subsequently a spherical lens vesicle that remains attached to the surface ectoderm via a transient lens stalk. Detachment of the lens vesicle from the overlying surface ectoderm and disappearance of the lens stalk are essential for the subsequent growth and differentiation of the lens vesicle and the formation of the cornea. At the anterior pole of the lens vesicle, proliferating lens epithelial cells continue to divide to produce progenitor cells that migrate to the equatorial region of the lens, where they exit cell cycle, differentiate into secondary fiber cells, and elongate to fill the cavity of the vesicle. The secondary fibers engulf the primary fiber cells, which now become denucleated to form the lens nucleus. Proliferation, migration, and differentiation of the anterior lens epithelial cells continue throughout the lifespan.9Song N. Schwab K.R. Patterson L.T. Yamaguchi T. Lin X. Potter S.S. Lang R.A. pygopus 2 has a crucial, Wnt pathway-independent function in lens induction.Development. 2007; 134: 1873-1885Crossref PubMed Scopus (64) Google Scholar, 10Kreslova J. Machon O. Ruzickova J. Lachova J. Wawrousek E.F. Kemler R. Krauss S. Piatigorsky J. Kozmik Z. Abnormal lens morphogenesis and ectopic lens formation in the absence of beta-catenin function.Genesis. 2007; 45: 157-168Crossref PubMed Scopus (56) Google Scholar Several genes that are involved in the morphogenetic processes of anterior eye development have been found to be associated with ASD, including PAX6, PITX2, PITX3, FOXC1, FOXE3, SIX3, SOX11, SOX2, and MAF.5Wurm A. Sock E. Fuchshofer R. Wegner M. Tamm E.R. Anterior segment dysgenesis in the eyes of mice deficient for the high-mobility-group transcription factor Sox11.Exp Eye Res. 2008; 86: 895-907Crossref PubMed Scopus (47) Google Scholar, 11Ali M. Buentello-Volante B. McKibbin M. Rocha-Medina J.A. Fernandez-Fuentes N. Koga-Nakamura W. Ashiq A. Khan K. Booth A.P. Williams G. Raashid Y. Jafri H. Rice A. Inglehearn C.F. Zenteno J.C. Homozygous FOXE3 mutations cause non-syndromic, bilateral, total sclerocornea, aphakia, microphthalmia and optic disc coloboma.Mol Vis. 2010; 16: 1162-1168PubMed Google Scholar, 12Reis L.M. Tyler R.C. Schneider A. Bardakjian T. Semina E.V. Examination of SOX2 in variable ocular conditions identifies a recurrent deletion in microphthalmia and lack of mutations in other phenotypes.Mol Vis. 2010; 16: 768-773PubMed Google Scholar, 13Jia X. Guo X. Xiao X. Li S. Zhang Q. A novel mutation of PAX6 in Chinese patients with new clinical features of Peters' anomaly.Mol Vis. 2010; 16: 676-681PubMed Google Scholar, 14Strungaru M.H. Dinu I. Walter M.A. Genotype-phenotype correlations in Axenfeld-Rieger malformation and glaucoma patients with FOXC1 and PITX2 mutations.Invest Ophthalmol Vis Sci. 2007; 48: 228-237Crossref PubMed Scopus (125) Google Scholar, 15Summers K.M. Withers S.J. Gole G.A. Piras S. Taylor P.J. Anterior segment mesenchymal dysgenesis in a large Australian family is associated with the recurrent 17 bp duplication in PITX3.Mol Vis. 2008; 14: 2010-2015PubMed Google Scholar, 16Hsieh Y.W. Zhang X.M. Lin E. Oliver G. Yang X.J. The homeobox gene Six3 is a potential regulator of anterior segment formation in the chick eye.Dev Biol. 2002; 248: 265-280Crossref PubMed Scopus (23) Google Scholar, 17Jamieson R.V. Perveen R. Kerr B. Carette M. Yardley J. Heon E. Wirth M.G. van Heyningen V. Donnai D. Munier F. Black G.C. Domain disruption and mutation of the bZIP transcription factor, MAF, associated with cataract, ocular anterior segment dysgenesis and coloboma.Hum Mol Genet. 2002; 11: 33-42Crossref PubMed Scopus (227) Google Scholar Studies have shown that complex molecular interactions dictate steps of development responsible for mature and functional cornea and lens.18Lang R.A. Pathways regulating lens induction in the mouse.Int J Dev Biol. 2004; 48: 783-791Crossref PubMed Scopus (146) Google Scholar, 19Donner A.L. Lachke S.A. Maas R.L. Lens induction in vertebrates: variations on a conserved theme of signaling events.Semin Cell Dev Biol. 2006; 17: 676-685Crossref PubMed Scopus (56) Google Scholar, 20Cvekl A. Duncan M.K. Genetic and epigenetic mechanisms of gene regulation during lens development.Prog Retin Eye Res. 2007; 26: 555-597Crossref PubMed Scopus (142) Google Scholar Among these molecular complexes are members of the Bmp signaling pathway21Wawersik S. Purcell P. Rauchman M. Dudley A.T. Robertson E.J. Maas R. BMP7 acts in murine lens placode development.Dev Biol. 1999; 207: 176-188Crossref PubMed Scopus (226) Google Scholar, 22Belecky-Adams T.L. Adler R. Beebe D.C. Bone morphogenetic protein signaling and the initiation of lens fiber cell differentiation.Development. 2002; 129: 3795-3802Crossref PubMed Google Scholar, 23Grogg M.W. Call M.K. Okamoto M. Vergara M.N. Del Rio-Tsonis K. Tsonis P.A. BMP inhibition-driven regulation of six-3 underlies induction of newt lens regeneration.Nature. 2005; 438: 858-862Crossref PubMed Scopus (105) Google Scholar, 24Haynes T. Gutierrez C. Aycinena J.C. Tsonis P.A. Del Rio-Tsonis K. BMP signaling mediates stem/progenitor cell-induced retina regeneration.Proc Natl Acad Sci U S A. 2007; 104: 20380-20385Crossref PubMed Scopus (72) Google Scholar and its downstream nuclear effectors, the MSH homobox genes.25Tribulo C. Aybar M.J. Nguyen V.H. Mullins M.C. Mayor R. Regulation of Msx genes by a Bmp gradient is essential for neural crest specification.Development. 2003; 130: 6441-6452Crossref PubMed Scopus (246) Google Scholar, 26Hussein S.M. Duff E.K. Sirard C. Smad4 and beta-catenin co-activators functionally interact with lymphoid-enhancing factor to regulate graded expression of Msx2.J Biol Chem. 2003; 278: 48805-48814Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar, 27Trousse F. Esteve P. Bovolenta P. Bmp4 mediates apoptotic cell death in the developing chick eye.J Neurosci. 2001; 21: 1292-1301Crossref PubMed Google Scholar They encode a group of highly conserved homeodomain proteins that mainly function as transcriptional repressors.28Newberry E.P. Latifi T. Battaile J.T. Towler D.A. Structure-function analysis of Msx2-mediated transcriptional suppression.Biochemistry. 1997; 36: 10451-10462Crossref PubMed Scopus (70) Google Scholar, 29Bendall A.J. Abate-Shen C. Roles for Msx and Dlx homeoproteins in vertebrate development.Gene. 2000; 247: 17-31Crossref PubMed Scopus (227) Google Scholar, 30Shirakabe K. Terasawa K. Miyama K. Shibuya H. Nishida E. Regulation of the activity of the transcription factor Runx2 by two homeobox proteins, Msx2 and Dlx5.Genes Cells. 2001; 6: 851-856Crossref PubMed Scopus (159) Google Scholar, 31Ichida F. Nishimura R. Hata K. Matsubara T. Ikeda F. Hisada K. Yatani H. Cao X. Komori T. Yamaguchi A. Yoneda T. Reciprocal roles of MSX2 in regulation of osteoblast and adipocyte differentiation.J Biol Chem. 2004; 279: 34015-34022Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar, 32Park K. Kim K. Rho S.B. Choi K. Kim D. Oh S.H. Park J. Lee S.H. Lee J.H. Homeobox Msx1 interacts with p53 tumor suppressor and inhibits tumor growth by inducing apoptosis.Cancer Res. 2005; 65: 749-757Crossref PubMed Google Scholar, 33Zhuang F. Nguyen M.P. Shuler C. Liu Y.H. Analysis of Msx1 and Msx2 transactivation function in the context of the heat shock 70 (Hspa1b) gene promoter.Biochem Biophys Res Commun. 2009; 381: 241-246Crossref PubMed Scopus (11) Google Scholar Developmental anomalies as a result of mutations in MSX1 and MSX2 have clearly demonstrated the importance of these homeodomain transcriptional factors in controlling the development of the skull, hair follicles, teeth, heart, and brain.34Liu Y.H. Kundu R. Wu L. Luo W. Ignelzi Jr., M.A. Snead M.L. Maxson Jr., R.E. Premature suture closure and ectopic cranial bone in mice expressing Msx2 transgenes in the developing skull.Proc Natl Acad Sci U S A. 1995; 92: 6137-6141Crossref PubMed Scopus (204) Google Scholar, 35Liu Y.H. Tang Z. Kundu R.K. Wu L. Luo W. Zhu D. Sangiorgi F. Snead M.L. Maxson R.E. Msx2 gene dosage influences the number of proliferative osteogenic cells in growth centers of the developing murine skull: a possible mechanism for MSX2-mediated craniosynostosis in humans.Dev Biol. 1999; 205: 260-274Crossref PubMed Scopus (190) Google Scholar, 36Satokata I. Ma L. Ohshima H. Bei M. Woo I. Nishizawa K. Maeda T. Takano Y. Uchiyama M. Heaney S. Peters H. Tang Z. Maxson R. Maas R. Msx2 deficiency in mice causes pleiotropic defects in bone growth and ectodermal organ formation.Nat Genet. 2000; 24: 391-395Crossref PubMed Scopus (628) Google Scholar, 37Ma L. Liu J. Wu T. Plikus M. Jiang T.X. Bi Q. Liu Y.H. Muller-Rover S. Peters H. Sundberg J.P. Maxson R. Maas R.L. Chuong C.M. ‘Cyclic alopecia’ in Msx2 mutants: defects in hair cycling and hair shaft differentiation.Development. 2003; 130: 379-389Crossref PubMed Scopus (136) Google Scholar, 38Chen Y.H. Ishii M. Sun J. Sucov H.M. Maxson Jr., R.E. Msx1 and Msx2 regulate survival of secondary heart field precursors and post-migratory proliferation of cardiac neural crest in the outflow tract.Dev Biol. 2007; 308: 421-437Crossref PubMed Scopus (72) Google Scholar, 39Ramos C. Robert B. msh/Msx gene family in neural development.Trends Genet. 2005; 21: 624-632Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 40Cheng S.L. Shao J.S. Cai J. Sierra O.L. Towler D.A. Msx2 exerts bone anabolism via canonical Wnt signaling.J Biol Chem. 2008; 283: 20505-20522Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar Consistent with its role in controlling eye development is our previous finding that overexpression of the Msx2 gene in transgenic animals resulted in microphthalmia.41Wu L.Y. Li M. Hinton D.R. Guo L. Jiang S. Wang J.T. Zeng A. Xie J.B. Snead M. Shuler C. Maxson Jr., R.E. Liu Y.H. Microphthalmia resulting from MSX2-induced apoptosis in the optic vesicle.Invest Ophthalmol Vis Sci. 2003; 44: 2404-2412Crossref PubMed Scopus (44) Google Scholar In the present study, we provide evidence that the Msx2 gene is critically involved in anterior segment development of the eye. Deletion of Msx2 in mice can lead to persistent lens stalk with lens and cornea deformities that resemble Peters anomaly. All animal experiments followed the guidelines of the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research. Experimental mice used in this study are homozygous Msx2-deficient mice (Msx2−/−)36Satokata I. Ma L. Ohshima H. Bei M. Woo I. Nishizawa K. Maeda T. Takano Y. Uchiyama M. Heaney S. Peters H. Tang Z. Maxson R. Maas R. Msx2 deficiency in mice causes pleiotropic defects in bone growth and ectodermal organ formation.Nat Genet. 2000; 24: 391-395Crossref PubMed Scopus (628) Google Scholar and transgenic mouse strains R26R,42Soriano P. Generalized lacZ expression with the ROSA26 Cre reporter strain.Nat Genet. 1999; 21: 70-71Crossref PubMed Scopus (4174) Google Scholar Wnt1-Cre,43Danielian P.S. Muccino D. Rowitch D.H. Michael S.K. McMahon A.P. Modification of gene activity in mouse embryos in utero by a tamoxifen-inducible form of Cre recombinase.Curr Biol. 1998; 8: 1323-1326Abstract Full Text Full Text PDF PubMed Google Scholar and Le-Cre.44Ashery-Padan R. Marquardt T. Zhou X. Gruss P. Pax6 activity in the lens primordium is required for lens formation and for correct placement of a single retina in the eye.Genes Dev. 2000; 14: 2701-2711Crossref PubMed Scopus (460) Google Scholar Le-Cre transgenic mice were obtained from David Beebe (Washington University, St Louis, MO) with permission from Peter Gruss (Max-Planck Institute for Biophysical Chemistry, Gottingen, Germany) and Ruth Ashery-Padan (Tel Aviv University, Ramat Aviv, Israel). The R26R transgenic strain was purchased from the Jackson Laboratory (Bar Harbor, ME). The Wnt1-Cre transgenic strain was obtained from Andrew McMahon (Harvard University, Boston, MA). Mouse genomic DNA was extracted from the embryonic tail tissue using the hot sodium hydroxide and Tris (HotSHOT) method.45Truett G.E. Heeger P. Mynatt R.L. Truett A.A. Walker J.A. Warman M.L. Preparation of PCR-quality mouse genomic DNA with hot sodium hydroxide and tris (HotSHOT).Biotechniques. 2000; 29: 52-54Crossref PubMed Scopus (1091) Google Scholar Timed matings were performed between Msx2 heterozygous (Msx2+/−) male and female mice to generate homozygous Msx2-deficient (Msx2−/−) and wild-type (WT) mouse embryos. The pregnant females were sacrificed at various time points after conception. One hour before sacrificing, females were injected intraperitoneally with 100 μg of BrdU per gram of body weight. Animals were sacrificed and embryos were dissected and isolated in ice-cold PBS. A piece of tail tissue was taken from each embryo for DNA extraction and identification of the genotype. Then the embryos were fixed in 4% paraformaldehyde (PFA) in PBS or in Methyl-Carnoy fixative (60% methanol, 30% chloroform, and 10% glacial acid) overnight at 4°C. For histologic analysis, embryos were further dehydrated through graded alcohols, cleared in xylene, and embedded in paraffin. Then 5-μm sections were cut for immunohistochemistry (IHC) and H&E staining. For in situ hybridization analysis, the fixed embryos were rinsed in PBS for 10 minutes then cryoprotected in 30% sucrose overnight. Embryos were then oriented in OCT compound (Sakura Finetek, Torrance, CA) and rapidly frozen. Then 12-μm sections were cut and mounted on Superfrost Plus glass slides (VWR, Brisbane, CA) for future experiments. Fixed sections were rehydrated. Endogenous peroxidases were blocked with 3% H2O2. Epitope retrieval was performed in 0.1M sodium citrate buffer (pH 6.8) at 100°C for 10 minutes before adding blocking reagents. After the addition of primary antibodies, sections were incubated in a humidified chamber at 4°C overnight. The following primary antibodies were used: mouse monoclonal anti-Ap2α (Santa Cruz Biotechnology, Santa Cruz, CA), mouse monoclonal anti-Pax6 (Developmental Studies Hybridoma Bank, Iowa City, IA), mouse anti-BrdU (Sigma, St Louis, MO), and rabbit polyclonal anti–α-crystallin and anti–β-crystallin antibodies (generously provided by Dr. Samuel Zigler, John Hopkins University, Baltimore, MD). Fluorophore-labeled anti-mouse IgG antibody (Invitrogen, Carlsbad, CA) was used for signal acquisition or diaminobenzidine (Zymed, San Francisco, CA) compound for signal amplification. Apoptotic cells were detected by using the fluorescein In situ Cell Death Detection Kit (Roche Applied Science, Indianapolis, IN). Briefly, 4% PFA-fixed tissue sections were boiled for 10 minutes. Fragmented DNA was labeled with fluorescein-dUTP using terminal transferase. Fluorescence and bright-field image of sections were taken using an Olympus microscope (BX51, Olympus, Center Valley, PA) with a SPOT camera. The Wnt1-Cre mouse line has a β-galactosidase reporter gene in the Rosa26 knock-in allele mediated by the Cre recombinase, the expression of which is controlled by the Wnt-1 promoter.43Danielian P.S. Muccino D. Rowitch D.H. Michael S.K. McMahon A.P. Modification of gene activity in mouse embryos in utero by a tamoxifen-inducible form of Cre recombinase.Curr Biol. 1998; 8: 1323-1326Abstract Full Text Full Text PDF PubMed Google Scholar R26R;Wnt1-Cre mice were crossed with Msx2−/− homozygous mice to generate Msx2−/−; R26R;Wnt1-Cre and Msx2+/−;R26R;Wnt1-Cre genotypes to facilitate the observation of neural crest by X-gal staining. At E15.5, embryos were collected and fixed for 30 minutes in 4% PFA, then snap-frozen in OCT freezing media, sectioned, and stained as previously described.35Liu Y.H. Tang Z. Kundu R.K. Wu L. Luo W. Zhu D. Sangiorgi F. Snead M.L. Maxson R.E. Msx2 gene dosage influences the number of proliferative osteogenic cells in growth centers of the developing murine skull: a possible mechanism for MSX2-mediated craniosynostosis in humans.Dev Biol. 1999; 205: 260-274Crossref PubMed Scopus (190) Google Scholar The Le-Cre mouse line has a 6.5-kb SacII/XmnI genomic region, including the upstream regulatory sequences and the first Pax6 promoter (P0) upstream of sequences encoding the nls-Cre, followed by internal ribosome binding sites and green fluorescent protein (GFP).44Ashery-Padan R. Marquardt T. Zhou X. Gruss P. Pax6 activity in the lens primordium is required for lens formation and for correct placement of a single retina in the eye.Genes Dev. 2000; 14: 2701-2711Crossref PubMed Scopus (460) Google Scholar Le-Cre mice were crossed with Msx2−/− homozygous mice to generate LeCre;Msx2−/− and LeCre;Msx2+/− genotypes so that observation of the lens development would be facilitated by GFP fluorescence. The pregnant females were sacrificed at E9.5 to E12.5 after conception. Embryos were fixed overnight in 4% PFA, embedded in OCT compound, and rapidly frozen in liquid nitrogen. Sections (10 μm) were cut for viewing. Fluorescent images were captured using a fluorescence microscope with a SPOT camera. In situ hybridization was performed as previously described.46Li M. Wu X. Zhuang F. Jiang S. Jiang M. Liu Y.H. Expression of murine ELL-associated factor 2 (Eaf2) is developmentally regulated.Dev Dyn. 2003; 228: 273-280Crossref PubMed Scopus (19) Google Scholar The Msx2, Sox2, Mip, Prox1, and Pax6 cDNA plasmids were part of the Mammalian Gene Collection clones purchased from ATCC (Manassas, VA) or Open Biosystems (Huntsville, AL). The FoxE3 cDNA plasmid was kindly provided by Peter Carlsson (University of Gothenburg, Gothenburg, Sweden). All RNA probes were labeled with digoxigenin-UTP according to the manufacturer's recommendations (Roche Applied Science). The reaction was revealed by immunocytochemistry using an antidigoxigenin alkaline phosphatase, Fab fragment antibody (Roche Applied Science). The sections were photographed using Olympus microscope (BX51, Olympus) with a SPOT camera. When applicable, the unpaired t-test was used for quantitative analysis using SPSS software version 13.0 (SPSS Inc, Chicago, IL). Homozygous Msx2-deficient mice showed cornea opacity and edema with microphthalmia compared with that of the wild-type littermates. Enucleated eyes of Msx2-deficient mice at 3 postnatal months were consistently microphthalmic and >50% smaller in size than eyes of WT littermates (Figure 1, A and B). Overall, smaller palpebral fissure, smaller eyeball, and cornea with cornea edema were detected in the Msx2-deficient mice. Such phenotypes became more apparent after the eyelids opened (Figure 1, C and D). Histologic analysis of the eyes at 3 postnatal weeks disclosed thickened cornea, iridiocorneal adhesion, iris hyperplasia, and smaller lens in the anterior segment compared with that of the WT littermates (Figure 1, E and F). The corneal thickness was increased mainly in the stromal layer. The lens was pushed toward the cornea by overproliferated retina and mesenchyme tissues. Ciliary body and iris could not be distinguished clearly because of overproliferated pigment tissue (Figure 1F). The phenotypes observed are consistent with Peters anomaly in human. Examination of eye specimens of the Msx2-deficient mice from E14 to 3 weeks (n = 24) revealed that, besides abnormalities consistent with Peters anomaly with microphthalmia, folding of the retina and presence of ectopic pigmented tissue in the vitreous were found in all eyes. The severity of lens and cornea defects in the eyes of Msx2-deficient mice varied among animals and even between eyes of the same animal (data not shown). Msx2 transcripts were detected in the optic vesicle (Figure 2A) as early as the lens placode stage (E9.5). At E10.5, a hybridization signal for Msx2 can be found in the invaginating lens placode and a weak signal in the retina (Figure 2B). In the lens vesicle stage (E11.5), Msx2 transcripts were present in low level in the epithelial cells and moderate level in the posterior portion of the optic vesicle (Figure 2C). When primary lens fiber cells began to differentiate and elongate (E12.5), a moderate Msx2 expression was found throughout primary lens fiber cells. Such expression was intense in the transition zone but absent in the proliferating lens epithelium (Figure 2D). This early-stage dynamic expression pattern suggests that Msx2 expression is strictly regulated during eye development, especially in induction, invagination, and differentiation of the lens vesicle. On gross examination, subtle changes in the eyes of the Msx2-deficient mice were first detected as early as E10.5 in the optic vesicle, compared with that of the littermate controls (Figure 3, A and C). Examination of histologic sections revealed that the optic vesicle of the Msx2-deficient mice appeared to be larger and the lens vesicle smaller and incompletely developed (Figure 3, B and D). By E11.5, abnormal development of the mutant eyes could be easily recognized by the anterior expansion of the retinal pigmented epithelium (RPE) (Figure 3E). Such anterior movement of the RPE produced an iris that took on a bowtie-like appearance. Lens vesicle was compressed by the invading periocular mesenchyme (Figure 3F). Introduction of GFP under the control of Pax6 promoter aided early-stage visualization of lens and lens stalk (Figure 4, A–D). At E12.5 days, in eyes of the Msx2-deficient mice, cornea and lens were not separated, the neural crest cells migrated into the vitreous cavity and pushed the lens toward the cornea (Figure 3, I and J) whereas in WT littermates, the lens vesicle and the surface ectoderm were already completely separated and the space between was filled with invading cells from the periocular mesenchyme (Figure 3, K and L). Apparently, this migratory blockade impeded the development of the cornea in the mutant animals. Eyes of the Msx2-deficient mice had only a single layer of cornea epithelium cell. The persistent adherence of the lens vesicle to the corneal ectoderm hindered the migration of neural crest cells across the stromal space between the surface ectoderm and endothelium as shown by X-gal staining of Msx2−/−;R26R;Wnt1-Cre mouse embryonic sections (Figure 4, E and F). The development of both the corneal stroma and endothelial cells were interrupted. By E14.5 days, the lens stalk was still present, adhering to cornea and lens and further hindering the development of the cornea. Lens vesicle was smaller than normal and lens fiber cells distributed in disarray in the lens vesicle. In addition, the typically single-layered anterior lens epithelium frequently became multilayered in Msx2-deficient mice and some lens fiber cells failed to be denucleated. Abnormal mesenchyme filled in vitreous cavity. The retina displayed abnormal proliferation and folding that lead to shrinkage of the eye (Figure 3, M versus O). The characteristic bowtie region formed by the lens nuclei that was present in the WT lens (Figure 3P) was either less pronounced or completely absent (Figure 3N).Figure 4Smaller lens and persistent lens stalk in eyes of the Msx2-deficient mice. Micrographs of the whole mount (A and B) or cryosections (C and D) of the eyes from embryos at E11.5. GFP under the control of the Pax6 promoter helps to illuminate the lens (white arrowhead). In the LeCre;Msx2−/− embryo, GFP illuminated the lens (B) and the lens stalk (white arrow) (D). WT mice are shown in A and C. A section stained with X-gal (blue stained) demonstrates contribution of neural crest to the stroma and endothelium of the normal cornea in this eye from a Msx2+/−;R26R;Wnt1-Cre embryo at E15.5 (E). Corneal stroma is populated by cells of neural crest origin as shown here by cells stained blue. In the Msx2−/−;R26R;Wnt1-Cre embryonic eye, persistent adhesion of the lens (Le) to the overlying ectoderm hinders the morphogenesis of the cornea (F). Neural crest–derived corneal stroma is prevented by the lens from migrating across the midsection of the cornea (black arrowheads). A population of cells originated from the neural crest found their destination in the vitreous (black arrow) along with some retinal neurons (F). Re, retina; Le, lens.View Large" @default.
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• W2037810765 date "2012-06-01" @default.
• W2037810765 modified "2023-10-07" @default.
• W2037810765 title "Loss of Msx2 Function Down-Regulates the FoxE3 Expression and Results in Anterior Segment Dysgenesis Resembling Peters Anomaly" @default.
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