Loss of Msx2 Function Down-Regulates the Foxe3 Expression and Results in Anterior Segment Dysgenesis Resembling Peters Anomaly

The American Journal of Pathology, Vol. 180, No. 6, June 2012 Copyright © 2012 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ajpath.2012.02.017 Biomarkers, Genomics, Proteomics, and Gene Regulation

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

Jiangyue Zhao,*† Kirio Kawai,† Hongyan Wang,‡ involvement of MSX2 in Peters anomaly and the distinct Di Wu,* Mingwu Wang,§ Zhicao Yue,¶ function of MSX2 in regulating the growth and develop- Jinsong Zhang,* and Yi-Hsin Liu† ment of lens vesicles. (Am J Pathol 2012, 180:2230–2239; http://dx.doi.org/10.1016/j.ajpath.2012.02.017) From the Eye Hospital of China Medical University and the Department of Ophthalmology,* the Fourth Affiliated Hospital of China Medical University, Provincial Key Laboratory of Lens Anterior segment dysgenesis (ASD) is a developmental Research, Liaoning, China; the Department of Ophthalmology failure of the anterior segment tissues of the eye and an and Doheny Eye Institute,† Keck School of Medicine, University of important cause of severe visual impairment in infants Southern California, Los Angeles, California; the Department of and young children. ASD has been classified into differ- General Surgery,‡ People’s Hospital of Liaoning Province, ent subtypes based on its specific clinical phenotypes, Liaoning, China; the Department of Ophthalmology and Vision including Peters anomaly, aniridia, and Axenfeld-Rieger Science,§ The University of Arizona College of Medicine, Tucson, syndrome or malformation.1,2 Peters anomaly is due to Arizona; and the Institute of Life Sciences,¶ Fuzhou University, the incomplete and delayed detachment of the lens ves- Fujian, China icle 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, Complex molecular interactions dictate the develop- Peters anomaly is associated with iridiocorneal adhe- mental steps that lead to a mature and functional sions, iris hyperplasia, and other developmental eye dis- cornea and lens. Peters anomaly is one subtype of orders, such as microphthalmia.3–5 anterior segment dysgenesis especially due to abnor- Lens development is a complicated process because mal development of the cornea and lens. MSX2 was recently implicated as a potential gene that is critical the formation of lens placode derives from the surface ec- for anterior segment development. However, the role toderm. It has been proposed that abnormalities seen in MSX2 Peters anomaly may also result from primary defects in the of within the complex mechanisms of eye de- 6 velopment remains elusive. Our present study ob- lens. In human embryos, formation of lens placode takes served the morphologic changes in conventional place on approximately day 33 of gestation and in mice on 7,8 Msx2 knockout (KO) mice and found phenotypes day 9.5 of embryonic development (E9.5). Coordinated consistent with Peters anomaly and microphthalmia invagination of the lens ectoderm and the optic vesicle seen in humans. The role of Msx2 in cornea and lens gives rise to the lens pit and the optic cup and subsequently development was further investigated using IHC, in a spherical lens vesicle that remains attached to the surface situ hybridization, and quantification of proliferative ectoderm via a transient lens stalk. Detachment of the lens and apoptotic lens cells. Loss of Msx2 down-regulated vesicle from the overlying surface ectoderm and disappear- FoxE3 expression and up-regulated Prox1 and crystallin expression in the lens. The FoxE3 and Prox1 mal- Supported by NIH grant EY015417 (Y.H.L.) and National Natural Science Foun- function and precocious Prox1 and crystallin expres- dation of China grants NSFC30800651 (J.Y.Z.) and NSFC30872836 (J.S.Z.). sion contribute to a disturbed lens cell cycle in lens Accepted for publication February 13, 2012. vesicles and eventually to cornea-lentoid adhesions and Supplemental material for this article can be found at http://ajp. microphthalmia in Msx2 KO mice. The observed amjpathol.org or at http://dx.doi.org/10.1016/j.ajpath.2012.02.017. changes in the expression of FoxE3 suggest that Msx2 is Address reprint requests to Jiangyue Zhao, M.D., Ph.D., Xinhua Road, an important contributor in controlling transcription of #11, Department of Ophthalmology, the Fourth Affiliated Hospital of China target genes critical for early eye development. These Medical University, Shenyang, Liaoning Province, China 110005. E-mail: results provide the first direct genetic evidence of the [email protected]. 2230 Loss of Msx2 Results in Peters Anomaly 2231 AJP June 2012, Vol. 180, No. 6 ance of the lens stalk are essential for the subsequent mozygous Msx2-deficient (Msx2Ϫ/Ϫ) and wild-type (WT) growth and differentiation of the lens vesicle and the forma- mouse embryos. The pregnant females were sacrificed at tion of the cornea. At the anterior pole of the lens vesicle, various time points after conception. One hour before proliferating lens epithelial cells continue to divide to pro- sacrificing, females were injected intraperitoneally with duce progenitor cells that migrate to the equatorial region of 100 ␮g of BrdU per gram of body weight. Animals were the lens, where they exit cell cycle, differentiate into sec- sacrificed and embryos were dissected and isolated in ondary fiber cells, and elongate to fill the cavity of the ves- ice-cold PBS. A piece of tail tissue was taken from each icle. The secondary fibers engulf the primary fiber cells, embryo for DNA extraction and identification of the ge- which now become denucleated to form the lens nucleus. notype. Then the embryos were fixed in 4% paraformal- Proliferation, migration, and differentiation of the anterior dehyde (PFA) in PBS or in Methyl-Carnoy fixative (60% lens epithelial cells continue throughout the lifespan.9,10 methanol, 30% chloroform, and 10% glacial acid) over- Several genes that are involved in the morphogenetic night at 4°C. For histologic analysis, embryos were further processes of anterior eye development have been found to dehydrated through graded alcohols, cleared in xylene, be associated with ASD, including PAX6, PITX2, PITX3, and embedded in paraffin. Then 5-␮m sections were cut FOXC1, FOXE3, SIX3, SOX11, SOX2, and MAF.5,11–17 Stud- for immunohistochemistry (IHC) and H&E staining. For in ies have shown that complex molecular interactions dictate situ hybridization analysis, the fixed embryos were rinsed in steps of development responsible for mature and functional PBS for 10 minutes then cryoprotected in 30% sucrose cornea and lens.18–20 Among these molecular complexes overnight. Embryos were then oriented in OCT compound are members of the Bmp signaling pathway21–24 and its down- (Sakura Finetek, Torrance, CA) and rapidly frozen. Then stream nuclear effectors, the MSH homobox genes.25–27 12-␮m sections were cut and mounted on Superfrost Plus They encode a group of highly conserved homeodomain glass slides (VWR, Brisbane, CA) for future experiments. proteins that mainly function as transcriptional repres- 28–33 sors. Developmental anomalies as a result of muta- Immunohistochemistry tions in MSX1 and MSX2 have clearly demonstrated the importance of these homeodomain transcriptional factors in Fixed sections were rehydrated. Endogenous peroxi- controlling the development of the skull, hair follicles, teeth, dases were blocked with 3% H2O2. Epitope retrieval was heart, and brain.34–40 Consistent with its role in controlling performed in 0.1M sodium citrate buffer (pH 6.8) at 100°C eye development is our previous finding that overexpres- for 10 minutes before adding blocking reagents. After the sion of the Msx2 gene in transgenic animals resulted in addition of primary antibodies, sections were incubated microphthalmia.41 In the present study, we provide evi- in a humidified chamber at 4°C overnight. The following dence that the Msx2 gene is critically involved in anterior primary antibodies were used: mouse monoclonal anti- segment development of the eye. Deletion of Msx2 in mice Ap2␣ (Santa Cruz Biotechnology, Santa Cruz, CA), can lead to persistent lens stalk with lens and cornea de- mouse monoclonal anti-Pax6 (Developmental Studies formities that resemble Peters anomaly. Hybridoma Bank, Iowa City, IA), mouse anti-BrdU (Sigma, St Louis, MO), and rabbit polyclonal anti–␣-crystallin and anti–␤-crystallin antibodies (generously pro- Materials and Methods vided by Dr. Samuel Zigler, John Hopkins University, Experimental Mouse Breeding and Genotyping Baltimore, MD). Fluorophore-labeled anti-mouse IgG antibody (Invitrogen, Carlsbad, CA) was used for signal All animal experiments followed the guidelines of the Asso- acquisition or diaminobenzidine (Zymed, San Francisco, ciation for Research in Vision and Ophthalmology State- CA) compound for signal amplification. ment for the Use of Animals in Ophthalmic and Vision Re- search. Experimental mice used in this study are homozygous Msx2-deficient mice (Msx2Ϫ/Ϫ)36 and trans- TUNEL Assay 42 43 44 genic mouse strains R26R, Wnt1-Cre, and Le-Cre. Apoptotic cells were detected by using the fluorescein In Le-Cre transgenic mice were obtained from David Beebe situ Cell Death Detection Kit (Roche Applied Science, (Washington University, St Louis, MO) with permission from Indianapolis, IN). Briefly, 4% PFA-fixed tissue sections Peter Gruss (Max-Planck Institute for Biophysical Chemis- were boiled for 10 minutes. Fragmented DNA was la- try, Gottingen, Germany) and Ruth Ashery-Padan (Tel Aviv beled with fluorescein-dUTP using terminal transferase. University, Ramat Aviv, Israel). The R26R transgenic strain Fluorescence and bright-field image of sections were was purchased from the Jackson Laboratory (Bar Harbor, taken using an Olympus microscope (BX51, Olympus, ME). The Wnt1-Cre transgenic strain was obtained from Center Valley, PA) with a SPOT camera. 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.45 -Galactosidase Staining The Wnt1-Cre mouse line has a ␤-galactosidase reporter BrdU Labeling, Isolation, and Preparation of gene in the Rosa26 knock-in allele mediated by the Cre Mouse Embryos recombinase, the expression of which is controlled by the Wnt-1 promoter.43 R26R;Wnt1-Cre mice were crossed Timed matings were performed between Msx2 heterozy- with Msx2Ϫ/Ϫ homozygous mice to generate Msx2Ϫ/Ϫ; gous (Msx2ϩ/Ϫ) male and female mice to generate ho- R26R;Wnt1-Cre and Msx2ϩ/Ϫ;R26R;Wnt1-Cre genotypes to 2232 Zhao et al AJP June 2012, Vol. 180, No. 6

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 man- ufacturer’s recommendations (Roche Applied Science). The reaction was revealed by immunocytochemistry using an antidigoxigenin alkaline phosphatase, Fab frag- ment 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 opac- ity and edema with microphthalmia compared with that of Figure 1. Homozygous Msx2-deficient mice (Msx2Ϫ/Ϫ) show Peters anom- the wild-type littermates. Enucleated eyes of Msx2-defi- aly with microphthalmia compared with WT littermates. Enucleated eyes of cient mice at 3 postnatal months were consistently mi- Msx2-deficient mice (arrowhead) at 3 months old were consistently mi- Ͼ crophthalmic and Ͼ50% smaller in size than eyes of WT littermates (arrow) crophthalmic and 50% smaller in size than eyes of WT (A and B). Smaller palpebral fissure, smaller eyeball, and cornea with cornea littermates (Figure 1, A and B). Overall, smaller palpebral edema were detected in the Msx2-deficient mice (arrow; D) compared with fissure, smaller eyeball, and cornea with cornea edema the WT mice (C). Histologic analysis of the eyes of WT (E) and Msx2- deficient littermates (F). Thickened cornea, iridiocorneal adhesion, iris hy- were detected in the Msx2-deficient mice. Such pheno- perplasia, and smaller lens were observed in the anterior segment of Msx2- types became more apparent after the eyelids opened deficient mice compared with WT mice. The cornea thickness was increased (Figure 1, C and D). Histologic analysis of the eyes at 3 mainly in stromal layers. The lens was small and pushed toward the cornea by overproliferated retina and mesenchyme tissues. All of the phenotypes are postnatal weeks disclosed thickened cornea, iridiocor- consistent with Peters anomaly in the human. Co, cornea; Le, lens; Ret, retnia. neal adhesion, iris hyperplasia, and smaller lens in the anterior segment compared with that of the WT litter- facilitate the observation of neural crest by X-gal staining. At mates (Figure 1, E and F). The corneal thickness was E15.5, embryos were collected and fixed for 30 minutes in 4% PFA, then snap-frozen in OCT freezing media, sec- tioned, and stained as previously described.35

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 en- coding the nls-Cre, followed by internal ribosome binding sites and green fluorescent protein (GFP).44 Le-Cre mice were crossed with Msx2Ϫ/Ϫ homozygous mice to generate LeCre;Msx2Ϫ/Ϫ and LeCre;Msx2ϩ/Ϫ genotypes so that observation of the lens development would be facil- itated 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 ␮m) were cut for viewing. Fluorescent images were cap- tured using a fluorescence microscope with a SPOT Figure 2. Expression profile of Msx2 during eye development. A: At E9.5, camera. 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 In Situ Hybridization 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 In situ hybridization was performed as previously de- zone and a moderate Msx2 expression is found throughout primary lens fiber scribed.46 The Msx2, Sox2, Mip, Prox1, and Pax6 cDNA cells but absent from the lens epithelium. Loss of Msx2 Results in Peters Anomaly 2233 AJP June 2012, Vol. 180, No. 6 increased mainly in the stromal layer. The lens was inating lens placode and a weak signal in the retina pushed toward the cornea by overproliferated retina and (Figure 2B). In the lens vesicle stage (E11.5), Msx2 tran- mesenchyme tissues. Ciliary body and iris could not be scripts were present in low level in the epithelial cells and distinguished clearly because of overproliferated pig- moderate level in the posterior portion of the optic vesicle ment tissue (Figure 1F). The phenotypes observed are (Figure 2C). When primary lens fiber cells began to dif- consistent with Peters anomaly in human. ferentiate and elongate (E12.5), a moderate Msx2 ex- Examination of eye specimens of the Msx2-deficient pression was found throughout primary lens fiber cells. mice from E14 to 3 weeks (n ϭ 24) revealed that, besides Such expression was intense in the transition zone but abnormalities consistent with Peters anomaly with mi- absent in the proliferating lens epithelium (Figure 2D). crophthalmia, folding of the retina and presence of ecto- This early-stage dynamic expression pattern suggests pic pigmented tissue in the vitreous were found in all that Msx2 expression is strictly regulated during eye de- eyes. The severity of lens and cornea defects in the eyes velopment, especially in induction, invagination, and dif- of Msx2-deficient mice varied among animals and even ferentiation of the lens vesicle. between eyes of the same animal (data not shown). Cornea and Lens Development Are Severely Normal Dynamic Expression of Msx2 during Affected as a Consequence of Targeted Early-Stage Development of the Eye Deletion of Msx2 Msx2 transcripts were detected in the optic vesicle (Figure On gross examination, subtle changes in the eyes of the 2A) as early as the lens placode stage (E9.5). At E10.5, a Msx2-deficient mice were first detected as early as E10.5 hybridization signal for Msx2 can be found in the invag- in the optic vesicle, compared with that of the littermate

Figure 3. Msx2 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. 2234 Zhao et al AJP June 2012, Vol. 180, No. 6 controls (Figure 3, A and C). Examination of histologic (Figure 4, E and F). The development of both the corneal sections revealed that the optic vesicle of the Msx2-defi- stroma and endothelial cells were interrupted. By E14.5 cient mice appeared to be larger and the lens vesicle days, the lens stalk was still present, adhering to cornea smaller and incompletely developed (Figure 3, B and D). and lens and further hindering the development of the By E11.5, abnormal development of the mutant eyes cornea. Lens vesicle was smaller than normal and lens could be easily recognized by the anterior expansion of fiber cells distributed in disarray in the lens vesicle. In the retinal pigmented epithelium (RPE) (Figure 3E). Such addition, the typically single-layered anterior lens epi- anterior movement of the RPE produced an iris that took thelium frequently became multilayered in Msx2-defi- on a bowtie-like appearance. Lens vesicle was com- cient mice and some lens fiber cells failed to be denu- pressed by the invading periocular mesenchyme (Figure cleated. Abnormal mesenchyme filled in vitreous 3F). Introduction of GFP under the control of Pax6 pro- cavity. The retina displayed abnormal proliferation and moter aided early-stage visualization of lens and lens folding that lead to shrinkage of the eye (Figure 3,M stalk (Figure 4, A–D). At E12.5 days, in eyes of the Msx2- versus O). The characteristic bowtie region formed by deficient mice, cornea and lens were not separated, the the lens nuclei that was present in the WT lens (Figure neural crest cells migrated into the vitreous cavity and 3P) was either less pronounced or completely absent pushed the lens toward the cornea (Figure 3, I and J) (Figure 3N). 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 Reduction of FoxE3 Expression in the Lens of periocular mesenchyme (Figure 3, K and L). Apparently, Msx2-Deficient Mice this migratory blockade impeded the development of the cornea in the mutant animals. Eyes of the Msx2-deficient In the lens vesicles of Msx2-deficient mice, the expres- mice had only a single layer of cornea epithelium cell. sion levels of both Ap2␣ and Pax6 were not altered (Fig- The persistent adherence of the lens vesicle to the cor- ure 5, A–F). This finding suggests that Msx2 may function neal ectoderm hindered the migration of neural crest in parallel to or downstream of the Ap2␣ and Pax6 genes. cells across the stromal space between the surface ec- Nonradioactive RNA in situ hybridization was then per- toderm and endothelium as shown by X-gal staining of formed to detect FoxE3 and major intrinsic protein (Mip) Msx2Ϫ/Ϫ;R26R;Wnt1-Cre mouse embryonic sections transcripts. At E10.5 and E11.5, FoxE3 expression was

Figure 4. Smaller 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;Msx2Ϫ/Ϫ 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 Msx2ϩ/Ϫ;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 Msx2Ϫ/Ϫ;R26R;Wnt1-Cre embryonic eye, persistent adhesion of the lens (Le) to the overlying ectoderm hinders the morphogenesis of the cornea (F). Neural crest–de- rived 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 des- tination in the vitreous (black arrow) along with some retinal neurons (F). Re, retina; Le, lens. Loss of Msx2 Results in Peters Anomaly 2235 AJP June 2012, Vol. 180, No. 6

ure 6F). No change was seen in the expression of Mip in lens fiber cells (Figure 6, I and J). The expression of FoxE3 was studied in ␣-TN4 and C2C12 cell lines that were transfected with pEGFP-Msx2 as described.33 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).

Figure 5. Loss of Msx2 activity does not alter Pax6 and Ap2␣ 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-␣ (E –F), and crystallin on E11.5 (G and H) and E12.5 (I and Figure 6. FoxE3 and Prox1 expression in the developing lens vesicle are J). At the lens vesicle stage of eye development, the intensity and the spatial 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 domain of immune-reactivity are not altered significantly. Immunofluores- (C and D), and E12.5 (E and F), Prox1 at E12.5 (G and H), and MiP at E12.5 cence of WT and Msx2-deficient embryonic eyes at E11.5 and E12.5 show ␣ ␤ (I and J). A: At E10.5, FoxE3 transcripts are present in the invaginated lens significant overexpression of - and -crystallin in the Msx2-deficient lens vesicle but absent from the anterior region of the fusing vesicle in the WT ␮ ␮ vesicle (G–J). Scale bars: 50 m(A and B), 100 m(C–J). Re, retina; Le, lens; lens vesicle. B: Similar expression pattern is detected in the Msx2-deficient OV, optic vesicle. 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 significantly reduced in the invaginating lens vesicles of detectable in the anterior half of the lens (D). At E12.5, FoxE3 expression is Msx2-deficient mice (Figure 6, A–D). At E12.5, FoxE3 was restricted to the primary lens epithelium in the WT embryo (E) but undetectable in the Msx2-deficient lens (arrows)(F). The expression of only expressed in the anterior lens epithelial cells and not Prox1 is elevated in the Msx2-deficient mouse lens vesicle at E12.5 (G) in the differentiated lens fiber cells in WT mice (Figure compared with WT mice (H). The expression level of Mip, a lens differentiation marker, does not differ between WT (I) and Msx2-deficient (J) 6E), whereas in the Msx2-deficient mice, FoxE3 tran- mouse lens vesicle at E12.5. Scale bars: 50 ␮m(A and B), 75 ␮m(C and D), 100 scripts were completely absent in the lens vesicles (Fig- ␮m(E–J). Re, retina. 2236 Zhao et al AJP June 2012, Vol. 180, No. 6

Table 1. Primers Used in Quantitative Real-Time RT-PCR We further examined programmed cell death by per- forming in situ TUNEL assay and detected significantly Primer Sequence higher level of apoptotic events in lenses of the Msx2 FoxE3 5=-TCTAACGCTGGCAGCCATCTAC deficient mice at E11.5 (Figure 8, A–C, P Ͻ 0.01). ACGAAACAGTCGTTGAGGGTGAG-3= ␤-Actin 5=-CATCCGTAAAGACCTCTATGCCAAC ATGGAGCCACCGATCCACA-3 = Discussion

In this study, using conventional Msx2 KO mice, we demonstrated that Msx2 is a significant contributor to eye Increase of Prox1 and Crystalline Expression in development. Without the Msx2 gene, mice are born with 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 ␣- and ␤-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 ␣- and ␤-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 ϭ 4, P Ͼ 0.05). 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 modi- fied from previous studies.47 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 Figure 7. Proliferation rate changes in the lens after loss of Msx2 expression, sector, cells begin to withdraw from the cell cycle in as assessed by BrdU incorporation in the lens vesicles. Comparable numbers preparation for fiber cell terminal differentiation. The BrdU of lens cells are undergoing cell division in WT and Msx2-deficient embryos, labeling rate was found to be significantly increased in 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 central lens epithelial cells (60° to 90° sectors) at E14.5 significant between Msx2-deficient mice and WT mice at E10.5 and E11.5, but and E15.5 in the Msx2-deficient mice compared with the BrdU-labeled lens cells are less at E11.5 in Msx2-deficient mice compared WT mice (P Ͻ 0.01). However, in the 15° to 45° sectors 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 BrdU-labeling rates were significantly lower in the Msx2- central lens epithelial cells in the Msx2-deficient mice at E14.5 and E15.5, and deficient mice at E15.5 (P Ͻ 0.01), whereas no difference 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 Ͻ 0.01. was found at E14.5 in this sector compared with the WT Scale bars: 50 ␮m(A–D), 75 ␮m(C and D), 100 ␮m(E and G), 150 ␮m(F and mice (Figure 7, I and J). H). Re, retina; Le, lens. Loss of Msx2 Results in Peters Anomaly 2237 AJP June 2012, Vol. 180, No. 6

Figure 8. Loss 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 ϭ 50 ␮m. Re, retina; Le, lens.

phenotypes consistent with Peters anomaly, a congenital To gain understanding at the molecular level of the per- developmental disease in human. Both the Le-Cre and sistent corneal-lentoid adhesion phenotype in the Msx2- Wntl-Cre transgenic mouse lines were used in this study deficient animals, we investigated the expression of to better reveal the persistent lens stalk and address the Pax6, Ap2␣, and FoxE3. We found FoxE3 expression was relationship between the lens and cornea in early stages significantly reduced in the lens vesicles in the Msx2- of eye development. The use of Le-Cre mice helped us to deficient mice. Because elevated expression of FoxE3 demonstrate persistent lens stalk through GFP expres- was seen in the cell lines overexpressing Msx2, we con- sion, which is under the control of the lens ectoderm clude that Msx2 can up-regulate the expression of FoxE3 44 promoter of Pax6 in such mice. Using Wnt1-Cre mice, in in the early stage of lens development. One explanation which ␤-galactosidase reporter gene expression in the could be that such adhesion is caused by a dysregula- Rosa26 knock-in allele is mediated by the Wnt-1 pro- tion of genes responsible for apoptosis within the corneal moter controlled Cre recombinase, we demonstrated that stalk, which is thought to degrade during lens morpho- cornea development was hindered by persistent lens genesis as a result of apoptosis. An alternative explana- stalk. Our findings suggest that Msx2 is embedded in the tion could be that down-regulation of FoxE3 may cause a transcriptional network, which governs the morphoge- dysregulation of cadherin proteins, such as E-cadherin, netic processes of early eye development in vertebrates. which is present in the corneal stalk.49 Proper development of the eye relies on coordinated To learn more about the signaling pathways in which interactions among the surface ectoderm, lens, optic Msx2 influences ocular development, we investigated the cup, and periocular neural crest mesenchyme. Signals expression of Prox1, which was found to affect the termi- from the lens have been shown to be critical for early eye nal differentiation and elongation of lens fiber cell through development.48 In addition, the lens itself plays a major role regulating the Cdkn1c which controlled the cell cycle.60 during innervation of the cornea.48 It is known that Msx2 Given that Prox1 was shown to function downstream of controls target gene transcription by forming activating or FoxE3 and its expression was suppressed by FoxE3,51 it suppressing protein-protein complexes.8,25,35,36,41 These was not surprising to see that Prox1 expression was Msx2-interacting partners are the likely contributors to phe- up-regulated in the lens vesicles of the Msx2-deficient notypic variability in the Msx2-deficient animals. Loss of mice due to reduced FoxE3 expression. Nevertheless, function of Msx2 led to a range of eye defects, with a Msx2 Prox1 minuscule lens vesicle and persistent lens stalk the most the possibility that may directly suppress severe phenotypes, as observed in our study. In the most transcription cannot be ruled out. The mechanism as to severe cases, retina exhibited folding due to reduced how Msx2 achieves its regulatory activity on FoxE3 ocular volume, and laminar structure of the retina was and/or Prox1 awaits further investigation. Ectopic expres- compromised. sion of Prox1 in the mouse retina was found to force 61 Previous studies have established a provisional ge- retinal progenitor cells to exit the cell cycle. Further- netic pathway that controls this morphologic process, more, results from several other studies supported the leading to apoptosis and elimination of the lens stalk after role of Prox1 in promoting neuronal differentiation and lens vesicle closure.49,50 Central to this genetic regula- cell cycle withdraw. This prodifferentiation function of tory pathway is the FoxE3 gene.51 FoxE3 mutation causes Prox1 appears to be evolutionarily conserved among fish, 62,63 ASD, with ocular phenotypes ranging from microcornea chicks, and mice. Thus, it is reasonable to hypothe- to Peters anomaly in human.52 Mutant mice with consid- size that the high level of Prox1 expression found in the erable reduction in FoxE3 expression showed severely lens of the Msx2-deficient mice may lead to premature malformed lens with persisting lens stalk during early eye cell cycle withdrawal and thus promote cell differentia- development, and the lens epithelium remained in con- tion. Because of FoxE3 and Prox1 malfunction in the tact with the corneal epithelium.48 Several transcriptional Msx2-deficient mice, precocious crystallin expression factors have been shown to regulate FoxE3 expression, occurred. Disturbed lens cell cycle in the lens vesicles and among these are Pax6, Mab21L1, Zeb2 (former might explain the microphthalmia seen in the Msx2-deficient name Sip-1), AP-2␣, Six3, Pitx3, Otx2, and KLF4.50,53–59 mice. Furthermore, the failure of the lens in detaching from 2238 Zhao et al AJP June 2012, Vol. 180, No. 6

the surface ectoderm in these mice may have also contrib- 3. Verma AS, Fitzpatrick DR: Anophthalmia and microphthalmia. Orpha- uted to the disruption to further growth of the lens. net J Rare Dis 2007, 2:47 Because 100% of the Msx2-deficient mice showed 4. Harissi-Dagher M, Colby K: Anterior segment dysgenesis: peters anomaly and sclerocornea. Int Ophthalmol Clin 2008, 48:35–42 persistence of the lens vesicles, we looked at cell prolif- 5. Wurm A, Sock E, Fuchshofer R, Wegner M, Tamm ER: Anterior seg- eration and apoptosis in the early stage of lens development dysgenesis in the eyes of mice deficient for the high-mobility- ment. We did not observe changes in the proliferation group transcription factor Sox11. Exp Eye Res 2008, 86:895–907 rate, and there was a high level of cell death in the central 6. Gould DB, John SW: Anterior segment dysgenesis and the developmental glaucomas are complex traits. Hum Mol Genet 2002, 11:1185– lens epithelium and in the Msx2-deficient mice at an early 1193 stage. Our previous studies found that overexpressed 7. Bailey AP, Bhattacharyya S, Bronner-Fraser M, Streit A: Lens speci- Msx2 could result in microphthalmia due to inhibited pro- fication is the ground state of all sensory placodes, from which FGF liferation in lens vesicles.41 It was suggested that the promotes olfactory identity. Dev Cell 2006, 11:505–517 overlapping of Msx2 malfunction and FoxE3 reduction 8. Sjodal M, Edlund T, Gunhaga L: Time of exposure to BMP signals Ϫ/Ϫ plays a key role in the specification of the olfactory and lens placodes resulted in the normal proliferation rate in the Msx2 ex vivo. Dev Cell 2007, 13:141–149 mice at an early stage. Thus, the smaller lens vesicle 9. 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