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Loss of Msx2 Function Down-Regulates the FoxE3 Expression and Results in Anterior Segment Dysgenesis Resembling Peters Anomaly

  • Jiangyue Zhao
    Correspondence
    Address reprint requests to Jiangyue Zhao, M.D., Ph.D., Xinhua Road, #11, Department of Ophthalmology, the Fourth Affiliated Hospital of China Medical University, Shenyang, Liaoning Province, China 110005
    Affiliations
    Eye Hospital of China Medical University and the Department of Ophthalmology, the Fourth Affiliated Hospital of China Medical University, Provincial Key Laboratory of Lens Research, Liaoning, China

    Department of Ophthalmology and Doheny Eye Institute, Keck School of Medicine, University of Southern California, Los Angeles, California
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  • Kirio Kawai
    Affiliations
    Department of Ophthalmology and Doheny Eye Institute, Keck School of Medicine, University of Southern California, Los Angeles, California
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  • Hongyan Wang
    Affiliations
    Department of General Surgery, People's Hospital of Liaoning Province, Liaoning, China
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  • Di Wu
    Affiliations
    Eye Hospital of China Medical University and the Department of Ophthalmology, the Fourth Affiliated Hospital of China Medical University, Provincial Key Laboratory of Lens Research, Liaoning, China
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  • Mingwu Wang
    Affiliations
    Department of Ophthalmology and Vision Science, The University of Arizona College of Medicine, Tucson, Arizona
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  • Zhicao Yue
    Affiliations
    Institute of Life Sciences, Fuzhou University, Fujian, China
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  • Jinsong Zhang
    Affiliations
    Eye Hospital of China Medical University and the Department of Ophthalmology, the Fourth Affiliated Hospital of China Medical University, Provincial Key Laboratory of Lens Research, Liaoning, China
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  • Yi-Hsin Liu
    Affiliations
    Department of Ophthalmology and Doheny Eye Institute, Keck School of Medicine, University of Southern California, Los Angeles, California
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Open AccessPublished:April 16, 2012DOI:https://doi.org/10.1016/j.ajpath.2012.02.017
      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.
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      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.

      Materials and Methods

      Experimental Mouse Breeding and Genotyping

      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−/−)
      • Satokata 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.
      and transgenic mouse strains R26R,
      • Soriano P.
      Generalized lacZ expression with the ROSA26 Cre reporter strain.
      Wnt1-Cre,
      • Danielian 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.
      and Le-Cre.
      • Ashery-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.
      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.
      • Truett 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).

      BrdU Labeling, Isolation, and Preparation of Mouse Embryos

      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.

      Immunohistochemistry

      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.

      TUNEL Assay

      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.

      β-Galactosidase Staining

      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.
      • Danielian 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.
      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.
      • Liu 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.

      GFP Fluorescence

      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).
      • Ashery-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.
      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

      In situ hybridization was performed as previously described.
      • Li M.
      • Wu X.
      • Zhuang F.
      • Jiang S.
      • Jiang M.
      • Liu Y.H.
      Expression of murine ELL-associated factor 2 (Eaf2) is developmentally regulated.
      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).

      Results

      Eyes of Msx2-Deficient Mice Develop Peters Anomaly with Microphthalmia

      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.
      Figure thumbnail gr1
      Figure 1Homozygous Msx2-deficient mice (Msx2−/−) show Peters anomaly with microphthalmia compared with WT littermates. Enucleated eyes of Msx2-deficient mice (arrowhead) at 3 months old were consistently microphthalmic and >50% smaller in size than eyes of WT littermates (arrow) (A and B). Smaller palpebral fissure, smaller eyeball, and cornea with cornea edema were detected in the Msx2-deficient mice (arrow; D) compared with the WT mice (C). Histologic analysis of the eyes of WT (E) and Msx2-deficient littermates (F). Thickened cornea, iridiocorneal adhesion, iris hyperplasia, and smaller lens were observed in the anterior segment of Msx2-deficient mice compared with WT mice. The cornea thickness was increased mainly in stromal layers. The lens was small and pushed toward the cornea by overproliferated retina and mesenchyme tissues. All of the phenotypes are consistent with Peters anomaly in the human. Co, cornea; Le, lens; Ret, retnia.
      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).

      Normal Dynamic Expression of Msx2 during Early-Stage Development of the Eye

      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.
      Figure thumbnail gr2
      Figure 2Expression profile of Msx2 during eye development. A: At E9.5, Msx2 transcripts are present in the optic vesicle (OV) and adjacent ectoderm. B: At E10.5, a weak hybridization signal for Msx2 emerges in the invaginating lens placode (Lp), and the signal in the retina (Re) was very weak. C: At E11.5, a low level expression of Msx2 remained in the lens epithelial (Le) cells and a moderate signal was found in posterior portion of lens vesicle. D: At E12.5, an intense Msx2 hybridization signal is localized to the transition zone and a moderate Msx2 expression is found throughout primary lens fiber cells but absent from the lens epithelium.

      Cornea and Lens Development Are Severely Affected as a Consequence of Targeted Deletion of Msx2

      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 thumbnail gr3
      Figure 3Msx2 inactivation resulted in defective optic vesicle development at E10.5 (A–D), E11.5 (E–H), E12.5 (I–L), and E14.5 (M–P). Whole mount of optic vesicles from Msx2-deficient mice at E10.5 to E14.5 (A, E, I, and M, respectively) show subtle changes in the Msx2-deficient eyes. A: These changes were first detected as early as E10.5 in the optic vesicle compared with that of the littermate controls (arrows). E: The mutant eyes could be easily recognized by the anterior expansion of the RPE as early as E11.5. The anterior movement of the RPE produced an iris that took on a bowtie-like appearance. Histologic sections of Msx2-deficient mice's optic vesicles from E10.5 to E14.5 (B, F, J, and N, respectively). Optic vesicle of the Msx2-deficient mice appeared to be larger and the lens vesicle smaller and incompletely developed. The lens vesicle in the Msx2-deficient mice appears anamorphic and was compressed by the invading periocular mesenchyme. The persistent lens stalk can be seen during lens development (arrowheads). Whole mount of WT mice's optic vesicles from WT mice at E10.5 to E14.5 (C, G, K, and O, respectively). Histologic sections of optic vesicles from WT mice at E10.5 to E14.5 (D, H, L, and P, respectively). Lp, lens placode; Re, retina; Le, lens.
      Figure thumbnail gr4
      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.

      Reduction of FoxE3 Expression in the Lens of Msx2-Deficient Mice

      In the lens vesicles of Msx2-deficient mice, the expression levels of both Ap2α and Pax6 were not altered (Figure 5, A–F). This finding suggests that Msx2 may function in parallel to or downstream of the Ap2α and Pax6 genes. Nonradioactive RNA in situ hybridization was then performed to detect FoxE3 and major intrinsic protein (Mip) transcripts. At E10.5 and E11.5, FoxE3 expression was significantly reduced in the invaginating lens vesicles of Msx2-deficient mice (Figure 6, A–D). At E12.5, FoxE3 was only expressed in the anterior lens epithelial cells and not in the differentiated lens fiber cells in WT mice (Figure 6E), whereas in the Msx2-deficient mice, FoxE3 transcripts were completely absent in the lens vesicles (Figure 6F). No change was seen in the expression of Mip in lens fiber cells (Figure 6, I and J).
      Figure thumbnail gr5
      Figure 5Loss of Msx2 activity does not alter Pax6 and Ap2α expression. Protein expression in WT and Msx2-deficient embryos, respectively, was examined by immunofluorescence for Pax6 on E9.5 (A and B) and E11.5 (C and D), Ap2-α (EF), and crystallin on E11.5 (G and H) and E12.5 (I and J). At the lens vesicle stage of eye development, the intensity and the spatial domain of immune-reactivity are not altered significantly. Immunofluorescence of WT and Msx2-deficient embryonic eyes at E11.5 and E12.5 show significant overexpression of α- and β-crystallin in the Msx2-deficient lens vesicle (G–J). Scale bars: 50 μm (A and B), 100 μm (C–J). Re, retina; Le, lens; OV, optic vesicle.
      Figure thumbnail gr6
      Figure 6FoxE3 and Prox1 expression in the developing lens vesicle are altered in Msx2-deficient mice. mRNA expression in WT and Msx2-deficient embryos, respectively, was examined for FoxE3 at E10.5 (A and B), E11.5 (C and D), and E12.5 (E and F), Prox1 at E12.5 (G and H), and MiP at E12.5 (I and J). A: At E10.5, FoxE3 transcripts are present in the invaginated lens vesicle but absent from the anterior region of the fusing vesicle in the WT lens vesicle. B: Similar expression pattern is detected in the Msx2-deficient mice, although the level of expression is reduced. At E11.5, FoxE3 is expressed throughout the lens in the WT embryo (C), whereas in the Msx2-deficient mice, its expression is reduced in the posterior half and barely detectable in the anterior half of the lens (D). At E12.5, FoxE3 expression is restricted to the primary lens epithelium in the WT embryo (E) but undetectable in the Msx2-deficient lens (arrows) (F). The expression of Prox1 is elevated in the Msx2-deficient mouse lens vesicle at E12.5 (G) compared with WT mice (H). The expression level of Mip, a lens differentiation marker, does not differ between WT (I) and Msx2-deficient (J) mouse lens vesicle at E12.5. Scale bars: 50 μm (A and B), 75 μm (C and D), 100 μm (E–J). Re, retina.
      The expression of FoxE3 was studied in α-TN4 and C2C12 cell lines that were transfected with pEGFP-Msx2 as described.
      • Zhuang 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.
      Primers used in quantitative real-time RT-PCR are listed in Table 1. Expression of FoxE3 increased 28.53-fold in cell lines overexpressing Msx2, comparing with the same cell lines transfected with pEGFP-C1 plasmid (see Supplemental Figure S1 at http://ajp.amjpathol.org).
      Table 1Primers Used in Quantitative Real-Time RT-PCR
      PrimerSequence
      FoxE35′-TCTAACGCTGGCAGCCATCTAC
      ACGAAACAGTCGTTGAGGGTGAG-3′
      β-Actin5′-CATCCGTAAAGACCTCTATGCCAAC
      ATGGAGCCACCGATCCACA-3′

      Increase of Prox1 and Crystalline Expression in the Lens of the Msx2-Deficient Mice

      At E12.5, when lens begin to differentiate, Prox1 expression in the lens vesicle was found to be augmented significantly in the Msx2-deficient mice by nonradioactive RNA in situ hybridization (Figure 6, G and H). Both the α- and β-crystallin, the maker proteins for lens development, were also significantly overexpressed in the lens vesicles at E11.5 and E12.5 (Figure 5, G–J), whereas at E11.5, only very weak α- and β-crystallin expression was seen in the lens of the WT mice (Figure 5G), indicating that lens differentiation started inappropriately early in the Msx2-deficient mice.

      Msx2 Regulates Cell Proliferation and Apoptosis in the Lens Vesicle

      To examine lens epithelial cell proliferation, in vivo BrdU labeling was performed. The rate of BrdU incorporation at E10.5 was indistinguishable between control and the Msx2-deficient mice in the lens vesicles (Figure 7, A and B). At E11.5, the lens vesicles in the Msx2-deficient mice appeared conspicuously smaller in size and labeled cells disarrayed within the lens vesicle, whereas most of labeled cells were localized at the anterior portion of the lens vesicle in WT lens (Figure 7, C and D). The absolute number of lens cells that incorporated BrdU was significantly reduced in the Msx2-deficient mice compared with that of the WT (data not shown), but the ratio of BrdU-labeled cells to unlabeled cells was virtually identical between the Msx2-deficient and the WT mice (n = 4, P > 0.05).
      Figure thumbnail gr7
      Figure 7Proliferation rate changes in the lens after loss of Msx2 expression, as assessed by BrdU incorporation in the lens vesicles. Comparable numbers of lens cells are undergoing cell division in WT and Msx2-deficient embryos, respectively, at E10.5 (A and B), E11.5 (C and D), E14.5 (E and F), and E15.5 (G and H). The percentage of BrdU-labeled lens cells is not statistically significant between Msx2-deficient mice and WT mice at E10.5 and E11.5, but BrdU-labeled lens cells are less at E11.5 in Msx2-deficient mice compared with WT. I and J: Histogram showing the mean ratio of BrdU-labeled lens cells at E14.5 (I) and E15.5 (J). BrdU-labeling rate significantly increased in central lens epithelial cells in the Msx2-deficient mice at E14.5 and E15.5, and the proliferation of lens epithelial cells around the equator is disturbed in the Msx2-deficient mice at E15.5 (equators marked by black lines). *P < 0.01. Scale bars: 50 μm (A–D), 75 μm (C and D), 100 μm (E and G), 150 μm (F and H). Re, retina; Le, lens.
      We also evaluated the proliferation rate at E14.5 and E15.5, a relatively late stage of lens development. Cell counting was conducted according to a protocol modified from previous studies.
      • Rajagopal R.
      • Dattilo L.K.
      • Kaartinen V.
      • Deng C.X.
      • Umans L.
      • Zwijsen A.
      • Roberts A.B.
      • Bottinger E.P.
      • Beebe D.C.
      Functions of the type 1 BMP receptor Acvr1 (Alk2) in lens development: cell proliferation, terminal differentiation, and survival.
      We found that the BrdU labeling rate in lens epithelial cells was the lowest in the 0° to 15° sector in both the Msx2-deficient and the WT mice, and there is no significant difference between the two groups at E14.5 and E15.5 (Figure 7, E–H). In this sector, cells begin to withdraw from the cell cycle in preparation for fiber cell terminal differentiation. The BrdU labeling rate was found to be significantly increased in central lens epithelial cells (60° to 90° sectors) at E14.5 and E15.5 in the Msx2-deficient mice compared with the WT mice (P < 0.01). However, in the 15° to 45° sectors BrdU-labeling rates were significantly lower in the Msx2-deficient mice at E15.5 (P < 0.01), whereas no difference was found at E14.5 in this sector compared with the WT mice (Figure 7, I and J).
      We further examined programmed cell death by performing in situ TUNEL assay and detected significantly higher level of apoptotic events in lenses of the Msx2 deficient mice at E11.5 (Figure 8, A–C, P < 0.01).
      Figure thumbnail gr8
      Figure 8Loss of Msx2 expression influences the apoptotic response in the lens vesicle at an early stage (E11.5). A: In the WT embryo, few apoptotic cells are observed in the lens vesicle and some apoptotic cells are found in the retina. B: In the Msx2-deficient embryos, more apoptotic cells are observed in the lens vesicle. C: Histogram showing the mean number of apoptotic lens cells of serial sections. The difference between Msx2-deficient and WT embryos is statistically significant at E11.5. Scale bars = 50 μm. Re, retina; Le, lens.

      Discussion

      In this study, using conventional Msx2 KO mice, we demonstrated that Msx2 is a significant contributor to eye development. Without the Msx2 gene, mice are born with phenotypes consistent with Peters anomaly, a congenital developmental disease in human. Both the Le-Cre and Wntl-Cre transgenic mouse lines were used in this study to better reveal the persistent lens stalk and address the relationship between the lens and cornea in early stages of eye development. The use of Le-Cre mice helped us to demonstrate persistent lens stalk through GFP expression, which is under the control of the lens ectoderm promoter of Pax6 in such mice.
      • Ashery-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.
      Using Wnt1-Cre mice, in which β-galactosidase reporter gene expression in the Rosa26 knock-in allele is mediated by the Wnt-1 promoter controlled Cre recombinase, we demonstrated that cornea development was hindered by persistent lens stalk. Our findings suggest that Msx2 is embedded in the transcriptional network, which governs the morphogenetic processes of early eye development in vertebrates.
      Proper development of the eye relies on coordinated interactions among the surface ectoderm, lens, optic cup, and periocular neural crest mesenchyme. Signals from the lens have been shown to be critical for early eye development.
      • Medina-Martinez O.
      • Brownell I.
      • Amaya-Manzanares F.
      • Hu Q.
      • Behringer R.R.
      • Jamrich M.
      Severe defects in proliferation and differentiation of lens cells in Foxe3 null mice.
      In addition, the lens itself plays a major role during innervation of the cornea.
      • Medina-Martinez O.
      • Brownell I.
      • Amaya-Manzanares F.
      • Hu Q.
      • Behringer R.R.
      • Jamrich M.
      Severe defects in proliferation and differentiation of lens cells in Foxe3 null mice.
      It is known that Msx2 controls target gene transcription by forming activating or suppressing protein-protein complexes.
      • Sjodal 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.
      • Tribulo 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.
      • Liu 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.
      • Satokata 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.
      • Wu 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.
      These Msx2-interacting partners are the likely contributors to phenotypic variability in the Msx2-deficient animals. Loss of function of Msx2 led to a range of eye defects, with a minuscule lens vesicle and persistent lens stalk the most severe phenotypes, as observed in our study. In the most severe cases, retina exhibited folding due to reduced ocular volume, and laminar structure of the retina was compromised.
      Previous studies have established a provisional genetic pathway that controls this morphologic process, leading to apoptosis and elimination of the lens stalk after lens vesicle closure.
      • Medina-Martinez O.
      • Jamrich M.
      Foxe view of lens development and disease.
      • Pontoriero G.F.
      • Deschamps P.
      • Ashery-Padan R.
      • Wong R.
      • Yang Y.
      • Zavadil J.
      • Cvekl A.
      • Sullivan S.
      • Williams T.
      • West-Mays J.A.
      Cell autonomous roles for AP-2alpha in lens vesicle separation and maintenance of the lens epithelial cell phenotype.
      Central to this genetic regulatory pathway is the FoxE3 gene.
      • Blixt A.
      • Landgren H.
      • Johansson B.R.
      • Carlsson P.
      Foxe3 is required for morphogenesis and differentiation of the anterior segment of the eye and is sensitive to Pax6 gene dosage.
      FoxE3 mutation causes ASD, with ocular phenotypes ranging from microcornea to Peters anomaly in human.
      • Doucette L.
      • Green J.
      • Fernandez B.
      • Johnson G.J.
      • Parfrey P.
      • Young T.L.
      A novel, non-stop mutation in FOXE3 causes an autosomal dominant form of variable anterior segment dysgenesis including Peters anomaly.
      Mutant mice with considerable reduction in FoxE3 expression showed severely malformed lens with persisting lens stalk during early eye development, and the lens epithelium remained in contact with the corneal epithelium.
      • Medina-Martinez O.
      • Brownell I.
      • Amaya-Manzanares F.
      • Hu Q.
      • Behringer R.R.
      • Jamrich M.
      Severe defects in proliferation and differentiation of lens cells in Foxe3 null mice.
      Several transcriptional factors have been shown to regulate FoxE3 expression, and among these are Pax6, Mab21L1, Zeb2 (former name Sip-1), AP-2α, Six3, Pitx3, Otx2, and KLF4.
      • Pontoriero G.F.
      • Deschamps P.
      • Ashery-Padan R.
      • Wong R.
      • Yang Y.
      • Zavadil J.
      • Cvekl A.
      • Sullivan S.
      • Williams T.
      • West-Mays J.A.
      Cell autonomous roles for AP-2alpha in lens vesicle separation and maintenance of the lens epithelial cell phenotype.
      • Yoshimoto A.
      • Saigou Y.
      • Higashi Y.
      • Kondoh H.
      Regulation of ocular lens development by Smad-interacting protein 1 involving Foxe3 activation.
      • Brownell I.
      • Dirksen M.
      • Jamrich M.
      Forkhead Foxe3 maps to the dysgenetic lens locus and is critical in lens development and differentiation.
      • Liu W.
      • Lagutin O.V.
      • Mende M.
      • Streit A.
      • Oliver G.
      Six3 activation of Pax6 expression is essential for mammalian lens induction and specification.
      • Ogino H.
      • Fisher M.
      • Grainger R.M.
      Convergence of a head-field selector Otx2 and Notch signaling: a mechanism for lens specification.
      • Yamada R.
      • Mizutani-Koseki Y.
      • Hasegawa T.
      • Osumi N.
      • Koseki H.
      • Takahashi N.
      Cell-autonomous involvement of Mab21l1 is essential for lens placode development.
      • Swindell E.C.
      • Zilinski C.A.
      • Hashimoto R.
      • Shah R.
      • Lane M.E.
      • Jamrich M.
      Regulation and function of foxe3 during early zebrafish development.
      • Medina-Martinez O.
      • Shah R.
      • Jamrich M.
      Pitx3 controls multiple aspects of lens development.
      To gain understanding at the molecular level of the persistent corneal-lentoid adhesion phenotype in the Msx2-deficient animals, we investigated the expression of Pax6, Ap2α, and FoxE3. We found FoxE3 expression was significantly reduced in the lens vesicles in the Msx2-deficient mice. Because elevated expression of FoxE3 was seen in the cell lines overexpressing Msx2, we conclude that Msx2 can up-regulate the expression of FoxE3 in the early stage of lens development. One explanation could be that such adhesion is caused by a dysregulation of genes responsible for apoptosis within the corneal stalk, which is thought to degrade during lens morphogenesis as a result of apoptosis. An alternative explanation could be that down-regulation of FoxE3 may cause a dysregulation of cadherin proteins, such as E-cadherin, which is present in the corneal stalk.
      • Medina-Martinez O.
      • Jamrich M.
      Foxe view of lens development and disease.
      To learn more about the signaling pathways in which Msx2 influences ocular development, we investigated the expression of Prox1, which was found to affect the terminal differentiation and elongation of lens fiber cell through regulating the Cdkn1c which controlled the cell cycle.
      • Wigle J.T.
      • Chowdhury K.
      • Gruss P.
      • Oliver G.
      Prox1 function is crucial for mouse lens-fibre elongation.
      Given that Prox1 was shown to function downstream of FoxE3 and its expression was suppressed by FoxE3,
      • Blixt A.
      • Landgren H.
      • Johansson B.R.
      • Carlsson P.
      Foxe3 is required for morphogenesis and differentiation of the anterior segment of the eye and is sensitive to Pax6 gene dosage.
      it was not surprising to see that Prox1 expression was up-regulated in the lens vesicles of the Msx2-deficient mice due to reduced FoxE3 expression. Nevertheless, the possibility that Msx2 may directly suppress Prox1 transcription cannot be ruled out. The mechanism as to how Msx2 achieves its regulatory activity on FoxE3 and/or Prox1 awaits further investigation. Ectopic expression of Prox1 in the mouse retina was found to force retinal progenitor cells to exit the cell cycle.
      • Dyer M.A.
      • Livesey F.J.
      • Cepko C.L.
      • Oliver G.
      Prox1 function controls progenitor cell proliferation and horizontal cell genesis in the mammalian retina.
      Furthermore, results from several other studies supported the role of Prox1 in promoting neuronal differentiation and cell cycle withdraw. This prodifferentiation function of Prox1 appears to be evolutionarily conserved among fish, chicks, and mice.
      • Misra K.
      • Gui H.
      • Matise M.P.
      Prox1 regulates a transitory state for interneuron neurogenesis in the spinal cord.
      • Pistocchi A.
      • Gaudenzi G.
      • Carra S.
      • Bresciani E.
      • Del Giacco L.
      • Cotelli F.
      Crucial role of zebrafish prox1 in hypothalamic catecholaminergic neurons development.
      Thus, it is reasonable to hypothesize that the high level of Prox1 expression found in the lens of the Msx2-deficient mice may lead to premature cell cycle withdrawal and thus promote cell differentiation. Because of FoxE3 and Prox1 malfunction in the Msx2-deficient mice, precocious crystallin expression occurred. Disturbed lens cell cycle in the lens vesicles might explain the microphthalmia seen in the Msx2-deficient mice. Furthermore, the failure of the lens in detaching from the surface ectoderm in these mice may have also contributed to the disruption to further growth of the lens.
      Because 100% of the Msx2-deficient mice showed persistence of the lens vesicles, we looked at cell proliferation and apoptosis in the early stage of lens development. We did not observe changes in the proliferation rate, and there was a high level of cell death in the central lens epithelium and in the Msx2-deficient mice at an early stage. Our previous studies found that overexpressed Msx2 could result in microphthalmia due to inhibited proliferation in lens vesicles.
      • Wu 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.
      It was suggested that the overlapping of Msx2 malfunction and FoxE3 reduction resulted in the normal proliferation rate in the Msx2−/− mice at an early stage. Thus, the smaller lens vesicle seen can be attributed to apoptosis in the lens vesicles and inappropriate early differentiation in the lens of the Msx2-deficient mice. One previous study showed that there were severe defects in proliferation and differentiation of lens cells in FoxE3 null mice especially after E14.5.
      • Medina-Martinez O.
      • Brownell I.
      • Amaya-Manzanares F.
      • Hu Q.
      • Behringer R.R.
      • Jamrich M.
      Severe defects in proliferation and differentiation of lens cells in Foxe3 null mice.
      Therefore, we investigated lens epithelium cell proliferation change at E14.5 and E15.5. We found that the BrdU-labeling rate significantly increased in central lens epithelial cells in the Msx2-deficient mice. However, the proliferation of lens epithelial cells around the equator was disturbed, which affected the differentiation process in the Msx2-deficient mice. We hypothesized that the increase of lens epithelial cell proliferation in the central region probably was due to lack of suppression for such proliferation from the Msx2-deficient mice and the resulting persistent lens stalk beginning at a relatively later stage. The proliferation changes around the equator at E15.5 in the Msx2-deficient mice were consistent in part with a previous report that FoxE3 expression affects the lens epithelial cell proliferation at a later stage during lens development.
      In summary, our morphologic study provided the first direct genetic evidence of the role of MSX2 in Peters anomaly and the distinct function of MSX2 in regulating the growth and development of lens vesicle. The observed changes in the expression of FoxE3 and Prox1 further support the importance of MSX2 in controlling transcription of target genes critical for early eye development. Further investigation into these complex interactions among MSX2 and various transcriptional regulators and signaling molecules in directing growth and morphogenetic events in the developing eye should help clarify the pathogenesis of ASD and other congenital eye diseases. Thus far, mutations in genes such as PAX6, PITX3, and SOX1 have been identified in cases of Peters anomaly in humans.
      • Wurm 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.
      • Meyer I.
      • Rolim H.
      • Medeiros A.
      • Paiva L.
      • Galvao Filho R.
      [Peter's anomaly, clinical and therapeutic aspects: case report].
      • Shi X.
      • Bosenko D.V.
      • Zinkevich N.S.
      • Foley S.
      • Hyde D.R.
      • Semina E.V.
      • Vihtelic T.S.
      Zebrafish pitx3 is necessary for normal lens and retinal development.
      We suggest genetic testing of MSX2 mutations be included in patients with a clinical diagnosis of Peters anomaly in the future to corroborate our findings in this animal study.

      Supplementary data

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        The American Journal of PathologyVol. 181Issue 1
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          In the article entitled, “Toward Routine Use of 3D Histopathology as a Research Tool” (Volume 180, pages 1835–1842 of the May 2012 issue of The American Journal of Pathology), the support footnote should have included the following: “This work was partially funded through WELMEC, a Centre of Excellence in Medical Engineering funded by the Wellcome Trust and EPSRC (grant WT088908/Z/09/Z ).”
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