Scrib is required for epithelial cell identity and prevents epithelial to mesenchymal transition in the mouse

Scrib is required for epithelial cell identity and prevents epithelial to mesenchymal transition in the mouse

Author's Accepted Manuscript Scrib is Required for Epithelial Cell Identity and Prevents Epithelial To Mesenchymal Transition in the Mouse Idella F. ...

1MB Sizes 3 Downloads 40 Views

Author's Accepted Manuscript

Scrib is Required for Epithelial Cell Identity and Prevents Epithelial To Mesenchymal Transition in the Mouse Idella F. Yamben, Rivka A. Rachel, Shalini Shatadal, Neal G. Copeland, Nancy A. Jenkins, Soren Warming, Anne E. Griep

www.elsevier.com/locate/developmentalbiology

PII: DOI: Reference:

S0012-1606(13)00513-7 http://dx.doi.org/10.1016/j.ydbio.2013.09.027 YDBIO6224

To appear in:

Developmental Biology

Received date: 15 April 2013 Revised date: 3 September 2013 Accepted date: 23 September 2013 Cite this article as: Idella F. Yamben, Rivka A. Rachel, Shalini Shatadal, Neal G. Copeland, Nancy A. Jenkins, Soren Warming, Anne E. Griep, Scrib is Required for Epithelial Cell Identity and Prevents Epithelial To Mesenchymal Transition in the Mouse, Developmental Biology, http://dx.doi.org/10.1016/j.ydbio.2013.09.027 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Scrib‹•‡“—‹”‡†ˆ‘”’‹–Š‡Ž‹ƒŽ‡ŽŽ †‡–‹–›ƒ†”‡˜‡–•’‹–Š‡Ž‹ƒŽ ‘‡•‡…Š›ƒŽ”ƒ•‹–‹‘‹–Š‡‘—•‡ Idella F. Yambena, Rivka A. Rachelb1, Shalini Shatadala, Neal G. Copelandb2, Nancy A. Jenkinsb2, Soren Warmingb3, Anne E. Griepa*I a

Department of Cell and Regenerative Biology, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, WI 53706 and

b

Mouse Cancer Genetics Program, Center for Cancer Research, National Cancer Institute, Frederick, MD 21702 *

Corresponding author. Tel.: +6082 62 8988; fax: 6082 62 7306.

[email protected] Abstract The integrity and function of epithelial tissues depends on the establishment and maintenance of defining characteristics of epithelial cells, cell-cell adhesion and cell polarity. Disruption of these characteristics can lead to the loss of epithelial identity through a process called epithelial to mesenchymal transition (EMT), which can contribute to pathological conditions such as tissue fibrosis and invasive cancer. In invertebrates, the epithelial polarity gene scrib plays a critical role in establishing and maintaining cell adhesion and polarity. In this study we asked if the mouse homolog, Scrib, is required for establishment and/or maintenance of epithelial identity in vivo. To do so, we conditionally deleted Scrib in the head ectoderm tissue that gives rise to both the ocular lens and the corneal epithelium. Deletion of Scrib in the lens resulted in a change in epithelial cell shape from cuboidal to flattened and elongated. Early in the process, the cell adhesion protein, E-cadherin, and apical polarity protein, ZO-1, were downregulated and the myofibroblast protein, DSMA, was upregulated, suggesting EMT was occurring in the Scrib deficient lenses. Correlating temporally with the upregulation of DSMA, Smad3 and Smad4, TGFE signaling intermediates, accumulated in the nucleus and Snail, a TGFE target and transcriptional repressor of the gene encoding E-cadherin, was  1

Present address: Neurobiology Neurodegeneration and Repair Laboratory, National Eye Institute, Bethesda, MD 20892.

2

Present address: The Methodist Cancer Research Program, The Methodist Academy, Houston, TX

77030. 3

Present address: Molecular Biology, Genentech, San Francisco, CA 94080.

upregulated. Pax6, a lens epithelial transcription factor required to maintain lens epithelial cell identity also was downregulated. Loss of Scrib in the corneal epithelium also led to molecular changes consistent with EMT, suggesting that the effect of Scrib deficiency was not unique to the lens. Together, these data indicate that mammalian Scrib is required to maintain epithelial identity and that loss of Scrib can culminate in EMT, mediated, at least in part, through TGFE signaling.

 Highlights  x x x x x

ConditionaldeletionofScribinthelensleadstoepithelialtomesenchymaltransition. ThefirstmolecularchangeobservedwasdisruptionofEcadherinfollowedbylossof ZO1andPax6. UpregulationofthemesenchymalproteinDSMAcoincidedwithdownregulationof Pax6. ActivationofTGFEsignalingcoincidedwithupregulationofDSMA. ConditionaldeletionofScribinthecornealepitheliumleadstoepithelialto mesemchymaltransition.

Keywords Scrib,PDZproteins,Lensdevelopment,Epithelialtomesenchymaltransition,Celladhesion,Cell polarity

INTRODUCTION The integrity and function of epithelial tissues depends on the establishment and maintenance of defining properties of epithelial cells, cell-cell adhesion and cell polarity, which are composed of cell junctional complexes including tight and adherens junctions. Junctional complexes, furthermore, are essential factors in organogenesis through their linkages to the cytoskeleton and the associated signaling pathways that modulate cell migration, proliferation, differentiation and 

2

death (Baum and Georgiou, 2011; Giepmans and van Ijzendoorn, 2009). Identifying the factors that are required for the establishment and maintenance of epithelial identity is crucial because of their roles in normal development and homeostasis and because loss of epithelial identity is associated with disease processes such as fibrosis and cancer (Acloque et al., 2009; Kalluri and Weinberg, 2009; Thiery et al., 2009). In Drosophila, the PDZ domain containing protein Scribble has been shown to play a role in establishing and maintaining epithelial cell adhesion, polarity and proliferation (Bilder, 2004; Bilder et al., 2000; Bilder and Perrimon, 2000). Recent studies suggest that the mouse homolog, Scrib, may perform similar and additional functions in mammalian species (Montcouquiol et al., 2003; Murdoch et al., 2003; Pearson et al., 2011; Qin et al., 2005). In this study, we address the possibility that Scrib is required for epithelial identity in vivo in the mouse. The failure to maintain epithelial cell properties can result in a change in cell identity from an epithelial cell to a mesenchymal cell through a process referred to as epithelial to mesenchymal transition (EMT). EMT is an important normal biological process contributing to embryonic development (Nakaya and Sheng, 2008), wound healing (Kalluri and Weinberg, 2009), and tissue repair (Kalluri and Weinberg, 2009). Aberrant activation of EMT can contribute to diseased states such as tissue fibrosis in the kidney, liver, lung, and ocular lens (Kalluri and Weinberg, 2009), and to malignant progression of tumors (Acloque et al., 2009; Humbert et al., 2008; Klymkowsky and Savagner, 2009; Saika et al., 2008; Tsuji et al., 2009). EMT is characterized by the disassembly of adhesion junctions, loss of apical-basal polarity, and the acquisition of migratory capacity (Kalluri and Weinberg, 2009; Tsuji et al., 2009). A number of signaling pathways, including TGFE, are involved in promoting EMT during both development and disease (Kalluri and Weinberg, 2009; Thiery et al., 2009). The phenotypic 

3

changes found in EMT are associated with molecular changes including reduced expression of epithelial proteins such as the adherens junction protein, E-cadherin, and the tight junction protein, zonula occludens 1 (ZO-1), and upregulation in the expression of mesenchymal proteins (Kalluri and Weinberg, 2009; Thiery et al., 2009), and proteins involved in remodeling of the extracellular matrix such as certain matrix metalloproteinases (Dwivedi et al., 2006). While it is known that factors such as E-cadherin and ZO-1 are essential components of cell adhesion and polarity, and their loss promotes EMT, much less is known about the upstream factors that are required to establish and maintain epithelial structure in vertebrates. In invertebrates such as Drosophila, the PDZ (PSD-95, Dlg-1, and ZO-1) domain containing protein, Scribble (Scrib), has been shown to be necessary for the establishment, positioning and maintenance of adherens junctions and apical determinants (Bilder et al., 2000; Bilder and Perrimon, 2000; Woods et al., 1996). Scrib is a leucine repeat and PDZ (LAP) protein containing 16 leucine rich-repeats and four PDZ domains (Johnson and Wodarz, 2003). The PDZ domain is a protein-protein interaction domain of 80-90 amino acids found in common amongst members of this family of proteins which assemble large macromolecular complexes involved in polarity, vesicle transport and signaling in various cell types (Harris and Lim, 2001; Humbert et al., 2003). In Drosophila, scrib mutants exhibit loss of apical-basal polarity that correlates with hyperproliferation and loss of tissue architecture. scrib has been shown to act as a neoplastic tumor suppressor (Bilder, 2004; Bilder et al., 2000; Bilder and Perrimon, 2000) and scribdeficient follicular cells exhibit mislocalization of basolateral junction proteins such as Ecadherin (Zhao et al., 2008). Scrib-depleted MDCK cells were less adherent apparently due to compromised E-cadherin function (Qin et al., 2005). Analysis of the mouse mutant, Crc, which expresses a truncated form of Scrib in which the last two PDZ domains are absent (Murdoch et



4

al., 2003), has shown that Scrib is essential for planar cell polarity (Montcouquiol et al., 2003), for maintaining epithelial cohesion during lung development (Yates et al., 2013) and for angiogenesis (Michaelis et al., 2013). Finally, loss of Scrib in conjunction with expression of an oncogenic K-ras gene in mice promotes prostate carcinogenesis (Pearson et al., 2011). Although these findings indicate roles for Scrib in epithelial adhesion and polarity, whether and through what mechanism Scrib is required specifically for establishing or maintaining the epithelial identity in vivo in mammals has yet to be elucidated. Previously, we reported that Scrib is expressed in the mouse ocular lens (Nguyen et al., 2005). The lens is an ideal in vivo model for studying the mechanisms of establishing and maintaining epithelial identity during embryogenesis. It is composed entirely of epithelial cells, facilitating biochemical analysis, and the tissue itself is dispensable for the animal’s viability, allowing one to follow the effects of loss of gene function throughout the life of the animal. Development of the mouse lens begins around embryonic day 9.5 (E9.5) when a discrete region of the head ectoderm is induced to thicken and form the lens placode, which then invaginates along with the optic cup, the future retina (Piatigorsky, 1981). The invaginating lens detaches from the overlying head ectoderm, forming the lens vesicle. Cells in the vesicle opposite the optic cup differentiate into lens epithelial cells while cells closest to the optic cup differentiate into lens fibers. By E13.5, the anterior surface of the lens is composed of a monolayered epithelium overlaying a mass of elongated cells, the primary fiber cells, that arise from differentiation of the cells in the posterior of the lens vesicle. From that point, the lens grows in size as the self-renewing epithelium gives rise to cells, which, as they undergo terminal differentiation, are added to the fiber cell compartment.



5

EMT is associated with two types of lens cataract, posterior capsular opacification (PCO) and anterior subcapsular cataract (ASC). Using murine model systems, some aspects of the mechanism through which these cataracts develop have been identified (de Iongh et al., 2005; Saika et al., 2008; Saika et al., 2009; Wormstone et al., 2009). For example, conditionally inactivating the gene encoding E-cadherin early in lens development (Pontoriero et al., 2009) or the polarity protein aPKCO (Sugiyama et al., 2009) results in EMT. Also, overexpression of self-activating form of TGFE in transgenic mouse lens (Srinivasan et al., 1998) and treatment of rat lenses ex vivo (Hales et al., 1995) and lens explants in culture with TGFE2 result in cataractous plaques expressing markers of EMT (Symonds et al., 2006). Importantly, in humans, PCO is also associated with TGFE driven EMT as treatment of human capsular bags with TGFE2 results in matrix wrinkling and expression of EMT markers (Wormstone et al., 2002). Herein, we assessed the impact on epithelial cell identity of conditionally inactivating Scrib in the precursor cells of the embryonic surface ectoderm that give rise to the corneal epithelium and the lens. We found that inactivating Scrib resulted in lens defects that were associated with the progressive downregulation of epithelial proteins and upregulation of the mesenchymal protein, DSMA.

Furthermore, we identified TGFE as a potential signaling

pathway mediating the EMT phenotype that arises in the lens as a consequence of loss of Scrib. Finally, loss of Scrib also led to changes consistent with EMT in the corneal epithelium, suggesting together with previous literature that Scrib is required to maintain epithelial identity, and the integrity of epithelial tissues, in a variety of epithelia in vivo.

MATERIALS AND METHODS 

6

Animal Maintenance and Use All experiments using mice conformed to the Public Health Service Policy on Humane and Use of Laboratory Animals and ARVO statement for the Use of Animals in Ophthalmic and Vision Research and were approved by the Institutional Animal Care and Use Committee of the University of Wisconsin-Madison School of Medicine and Public Health.

Generation of Scrib Conditional Null and Scrib Germline Null Mice To conditionally delete Scrib prior to the vesicle stage, Lens-Cre (LeCre) transgenic mice (Ashery-Padan et al., 2000) were mated to Scribf/+ mice. Progeny containing the floxed allele were identified using ScribF and Scrib1-X2-R primers, producing a wild type band of 257 bp and floxed allele product of 325 bp (Table S1). Progeny containing the Cre recombinase gene were identified using Cre-1 and Cre-3 primers, producing a single band of 420 bp (Table S1). Next, Scribfl/+;LeCre mice were mated to Scribfl/+ mice to produce litters containing Scribfl/fl;LeCre mice. The LeCre mice were maintained in the hemizygous on the FVB/N genetic background. No overt ocular abnormalities were observed in these mice. Mice carrying a germline mutation in Scrib were generated by crossing Scribfl/+ mice to ACTB-Cre mice (Lewandoski et al., 1997) and Scrib+/- progeny were intercrossed to produce Scrib-/- day E17.5 embryos. To confirm Cre-mediated deletion in the lens, genomic DNA samples isolated from P2 microdissected lenses from Scribfl/fl and Scribfl/fl;LeCre, and the eye and tail from E17.5 Scrib-/embryos were subjected to PCR amplification using the ScribF/ScribwtR and ScribF/ScribckoR primer pairs (Table S1).



7

Histological Analysis Embryos from E11.5, E13.5, heads from E15.5 and eyes from E17.5 and P10 Scribfl/fl and ScribLecre mice were dissected, fixed overnight in 4% paraformaldehyde (PFA) at 4°C and embedded in paraffin. Sections (5 Pm) were cut, rehydrated, and stained with hematoxylin and eosin and viewed by light microscopy as previously described (Rivera et al., 2009). In addition, to verify that expression of the LeCre transgene itself did not lead to lens abnormalities, eyes from stock LeCre mice and Scrib+/+;LeCre mice (generated via the intercrosses between Scribfl/+;LeCre and Scribfl/+ mice) were fixed, embedded, sectioned and stained with hematoxylin and eosin and viewed by light microscopy. No lens abnormalities were observed. Embryos were staged by designating midday on the day of the vaginal plug as embryonic day 0.5. Postnatal animals were staged by designating the day of birth as neonate (neo) and subsequent days as postnatal day 1 (P1), P2, etc.

Immunofluorescence Paraffin embedded sections were prepared as described above. To detect Pax6, Ecadherin, -catenin, ZO-1, and Smad4, and Scrib, rehydrated sections were boiled in sodium citrate buffer (0.01 M, pH 6.0) in a rice cooker for 30 minutes, cooled, washed in 1X PBS, blocked as previously described (Rivera et al., 2009), and incubated with primary antibody overnight at 40°C (see Table S2 for antibody sources and dilutions). Following incubation with primary antibody, sections were washed 1X PBS then incubated with FITC-conjugated horse anti-mouse (Vector Laboratories), AlexaFluor 568 conjugated goat anti-rabbit, or AlexaFluor 568 conjugated donkey anti-goat (Molecular Probes) antibodies for one hour at room 

8

temperature (RT) then washed and viewed by confocal microscopy. For Smad4 staining, nuclei were counterstained with To-Pro 3 (Invitrogen) and for Pax6 and ZO-1 staining, nuclei were counterstained with propidium iodide. To detect Snail or Smad3, rehydrated sections were treated for 10 min in H2O2 (0.3%) in methanol to quench endogenous peroxidases prior to antigen retrieval. Sections were blocked for one hour in 5% rabbit serum diluted in 1X PBS at RT. The sections were incubated with SNA1 primary or Smad3 antibodies (Table S2) diluted in 2% (or 5% for Smad3) blocking solution overnight at 4°C (SNA1) or one hour at RT (Smad3). Samples were washed in PBS and treated with an anti-goat biotinylated secondary antibody for one hour at 1:100 at RT (or 1:200 for Smad3) diluted in 2% blocking solution. Positive cells were identified using a Goat Elite Vectastain ABC Kit (Vector Laboratories) and DAB peroxidase substrate kit (Vector Laboratories). Nuclei were counterstained in hematoxylin. Sections were viewed by light microscopy. Smad3 and Snail positive nuclei were counted on 6-8 sections from at least 3 different E17.5 Scribfl/fl and ScribLeCre eyes and the data subjected to statistical analysis using the two-sided Wilcoxon Rank Sum test and p<0.05 was considered to be significantly different. To immunostain for SMA, cryosections were prepared from E17.5 and P10 Scribfl/fl and ScribLeCre eyes, as previously described (Rivera et al., 2009). Sections (10 Pm) were incubated in DSMA-Cy3 conjugated antibody overnight at 4°C (Table S2) washed, counterstained with ToPro 3, and viewed by confocal microscopy. To immunostain for cytokeratin 12, cryosections were incubated with primary antibody overnight at 4°C, washed and incubated with AlexaFluor 568 conjugated donkey anti-goat antibody (Molecular Probes) for one hour at RT, washed, counterstained with To-Pro3, and viewed by confocal microscopy. To immunostain the cornea for E-cadherin, cryosections from P10 Scribfl/fl and ScribLeCre were incubated with primary 

9

antibody overnight at 40°C, washed, incubated with FITC conjugated rabbit anti-rat antibody (Vector Laboratories) for one hour at RT, washed, and counterstained with propidium iodide.

Western Blotting Protein lysates of P2 whole lenses and P10 corneas from Scribfl/fl and ScribLeCre mice and protein lysates of brain tissue from E17.5 Scrib-/- embryos were prepared in 1X RIPA buffer with protease inhibitors, as described previously (Rivera et al., 2009). A total of 50-100 μg of each lysate was run on a 7.5% acrylamide gel, the proteins transferred to polyvinylidene difluoride (PVDF) membranes, and the membranes incubated with anti-Scrib, anti-Pax6, and anti-Ecadherin antibodies (Table S3). Membranes were washed in 1X PBST, incubated with goat antimouse horseradish peroxidase (HRP, Pierce) or donkey anti-goat HRP (Santa Cruz) diluted in blocking solution. After incubation with secondary antibodies, blots were washed and bands visualized using Enhanced Chemiluminescence Plus Kit (ECL, Plus, GE Healthcare) and exposed to film or scanned on a StormScanner phosphorimager. Blots were stripped and reprobed with anti-Gapdh as a loading control. Bands were quantified and subjected to statistical analysis using the two-sided Wilcoxon Rank Sum test and p<0.05 was considered to be significantly different.

RESULTS Generation of Scrib Conditional Mutants To study the role of Scrib in lens development, we generated a mouse strain carrying a conditional allele of Scrib. In brief, a targeting vector with loxP sites flanking exons 2-8 and frt sites flanking a positive neomycin (neo) marker was introduced into the mouse genome (Figure



10

1A). Mice transmitting this allele were mated with transgenic mice expressing Flippase in the germline to remove the neo marker thereby generating the conditional null allele we used in this study. These mice were mated to LeCre transgenic mice, in which the Cre recombinase enzyme is expressed beginning at day E9.0 in the surface ectoderm overlying the optic vesicle and is continually expressed in surface ectoderm-derived ocular structures such as the lens and corneal epithelium (Ashery-Padan et al., 2000). This Cre line was chosen because Scrib expression is observed in the invaginating lens pit at day E10.5 where it localizes primarily along the apical surface of the lens vesicle, and to a lesser extent along the lateral membranes (Figure 1B). The tissue specificity and timing of Cre expression in the ScribLeCre mice were confirmed by monitoring expression of a Cre-activatable GFP reporter that is incorporated into the LeCre transgene (Figure 1C). To demonstrate that Cre-mediated deletion had occurred in the lens, genomic DNA was prepared for PCR analysis from lenses of P2 Scribfl/fl and Scribfl/fl;LeCre mice (hereafter referred to as control and ScribLeCre mice, respectively), eye tissue from day E17.5 embryos Scrib-/-, and tails from Scribfl/fl, Scrib+/,and Scribfl/+mice and Scrib-/- embryos. Cre-mediated deletion of exons 2-8 was evident in DNA from the lens of ScribLeCre and eye of Scrib-/-, but not in lens DNA from control mice (Figure 1D). A faint band corresponding to the floxed allele was observed in the ScribLeCre lens DNA. Protein lysates were prepared from the lenses of control and ScribLeCre mice as well as lysates from Scrib+/+ and Scrib-/- brain. Western blot analysis was performed using an anti-Scrib antibody and membranes were reblotted for Gapdh as a loading control. Scrib levels in ScribLeCre lens lysates were reduced compared to the control, although not completely absent (Figure 1E), consistent with the PCR results (Figure 1D). Scrib was not detected in the Scrib-/- brain protein lysate, indicating the specificity of the antibody.



11

Scrib was detected in the corneal lysates from control mice but not in the corneal lysates from ScribLeCre mice (Figure 1E). Together, these results indicate that Cre-mediated deletion of Scrib in the lens and cornea was efficient, although possibly not complete in the case of the lens.

Loss of Scrib Results in Lens and Cornea Defects Conditional loss of Scrib resulted in numerous ocular defects. By postnatal day 10 (P10) ScribLeCre eyes (Figure 2A) and the dissected ScribLeCre lenses (Figure 2B) were notably smaller than the controls; lenses were also misshapen and had central opacity, indicating the presence of a cataract (Figure 2B, arrow). Hematoxylin and eosin staining of lens sections showed that the ScribLeCre eyes had vacuolated lenses (Figure 2D, arrow/arrowhead) and a hyperplastic iris, which occluded the pupil (Figure 2D, asterisk). High magnification images of the lens epithelium revealed further defects. The control lens epithelium was a monolayer of cuboidal cells, whereas the ScribLeCre lens epithelium was unrecognizable as a lens epithelium and consisted of cells that appeared squamous rather than cuboidal (Figure 2E, F). The defects in the epithelial and/or fiber cells could account for the observed cataracts and small eye size. High magnification images of the corneal epithelium revealed defects. The control corneal epithelium was stratified with prominent cuboidal cells in the basal layer (Figure 2G). However, the ScribLeCre corneal epithelium had irregularly arranged, squamous cells (Figure 2H). These defects in corneal and lens epithelial shape in the ScribLeCre mice suggested a possible loss of epithelial identity.

Lens Epithelial Cells Lose Epithelial Characteristics and Acquire Mesenchymal Traits 

12

To assess the possibility that the lens epithelial cells were undergoing a cell fate change reflective of their abnormal morphology, we characterized the molecular nature of these cells. Lens sections from P10 mice were subjected to immunofluorescence assays for the lens epithelial proteins E-cadherin and, Pax6, a transcription factor required for expression of lens epithelial specific crystallin genes (Cvekl et al., 2004). Robust, nuclear staining for Pax6 (green) was observed in the epithelial cells of the control lenses where it overlapped with the nuclear counterstain, propidium iodide (PI, red) (Figure 3A, yellow).

However in the ScribLeCre

epithelial cells, some nuclei appeared red rather than yellow (Figure 3C, arrows) and in unmerged images, these same nuclei appeared to lack Pax6 staining (Figure 3D, arrows). Other nuclei were orange (Figure 3C) and showed reduced levels of Pax6 staining (Figure 3D). Western blot analysis of extracts from P2 control and ScribLeCre lenses and quantification of the results confirmed that Pax6 levels were reduced in ScribLeCre lenses by 55% (Figure 4). Immunoflourescence staining for E-cadherin (green) and -catenin (red) showed that these proteins colocalized along all membranes of control epithelial cells (Figure 3E, yellow), and unmerged image showed consistent E-cadherin staining along the membranes (Figure 3F). In contrast, E-cadherin and -catenin staining on ScribLeCre lenses showed regions in the epithelium where staining was absent or reduced (Figure 3G, arrows) which was also evident in the unmerged E-cadherin image (Figure 3H, arrows). Western blot analysis of extracts from P2 lenses and quantification of the results confirmed that E-cadherin levels were reduced by 35% (p<0.04) in the ScribLeCre lenses at that time point (Figure 4). The reduced expression of epithelial markers in the lens epithelium of the ScribLeCre mice prompted us to investigate if these cells were undergoing EMT. SMA is a myofibroblast protein that commonly appears during EMT in the lens (de Iongh et al., 2005). Therefore,



13

cryosections from control and ScribLeCre mice were immunostained with anti-SMA antibodies (red) and counterstained with To-Pro3 (blue) to identify nuclei (Figure 3I-L). While SMA was present only in the iris in control lenses (Figure 3I, J), in ScribLeCre eyes staining for SMA was observed in the lens as well as in the hyperplastic iris (Figure 3K, L). Thus, some cells in the lens epithelium of the ScribLeCre mice appear to have lost epithelial identity and acquired mesenchymal identity. EMT progression can be linked to activation of signaling through members of the TGFE superfamily. Normally, the lens epithelium does not respond to TGFE signaling; however, EMT in the lens is driven by TGFE (de Iongh et al., 2005; Wormstone et al., 2009). To determine if signaling through the TGFE superfamily was now active in lens epithelial compartment, sections from control and ScribLeCre mice were immunostained for Smad4, a component of the Smad complex that accumulates in the nucleus during signaling through TGFE superfamily members (Itoh et al., 2000). In control lenses, Smad4 was cytoplasmic in epithelial cells (Figure 3M, N). However, Smad4 was concentrated in the nuclei of epithelial cells in the ScribLeCre lenses (Figure 3O, P). Thus, cells in the ScribLeCre lens showed evidence for active signaling via the TGFE superfamily.

Loss of E-Cadherin in ScribLeCre Lenses Begins Early in Lens Development The loss of E-cadherin expression is a common feature of EMT (Kalluri and Weinberg, 2009; Thiery et al., 2009). As shown in Figures 3 and 4, Scrib deficiency resulted in reduced levels of E-cadherin within the epithelial cells of the ScribLecre lenses (Figures 3E-H and 4A). Because conditional deletion of Cdh1 in the lens placode resulted in an E-cadherin deficient lens 

14

vesicle and ultimately resulted in EMT (Pontoriero et al., 2009), we asked if loss of Scrib at this same stage in lens development resulted in changes in E-cadherin localization and/or levels at the time of lens vesicle formation. Double immunofluorescence experiments were carried out for Ecadherin and -catenin on lens sections from embryonic day 11.5 (E11.5) control and ScribLeCre embryos. At E11.5, control lens vesicles showed E-cadherin (green) colocalized with -catenin (red) along lateral membranes with a concentration of E-cadherin at the apical, anterior surfaces of the lens vesicle (yellow, Figure 5A, B, arrows). In contrast, colocalization was reduced in this area of ScribLeCre lens vesicles (Figure 5C). An examination of E-cadherin staining alone suggested that apparent loss of colocalization was due to reduced E-cadherin in the apical, anterior surface of the Scrib-deficient lens vesicle (Figure 5D).

To follow the

temporal progression of E-cadherin disruption, sections from E13.5-E17.5 embryos were immunostained. By E13.5 and E17.5, colocalization in the controls was restricted to the lateral and basal membranes of the epithelium (Figure 5E, I).

Punctate E-cadherin staining was

observed at the apical surfaces of cells (Figure 5F, J). In contrast, in regions of the E13.5 and E17.5 ScribLeCre epithelium, staining was red rather than yellow, suggesting that these regions lacked E-cadherin (Figure 5G, K, arrows). Unmerged images showed that in regions along the basal surface E-cadherin staining was absent and along the lateral membranes E-cadherin staining was reduced (Figure 5H, arrows). This trend continued through ScribLeCre E17.5 lenses (Figure 5L, arrows). Alterations in E-cadherin staining of E13.5 and E17.5 ScribLeCre lenses correlated well with morphological defects in the epithelium. In comparison to controls, cells in the epithelium of the E13.5 ScribLeCre mice were disorganized (Figure 5G); by E17.5, the epithelium was obviously flattened and characterized by irregularly shaped cells (Figure 5K).



15

Thus, beginning at very early stages of lens formation, the Scrib-deficient lens epithelium is characterized by the gradual loss of E-cadherin from the epithelium, a hallmark of EMT.

The Apical Marker, ZO-1, is Gradually Lost From Epithelial Cells of the ScribLeCre Lenses Another hallmark of EMT is the loss of the apically-restricted tight junction protein, ZO1 (Zeisberg and Neilson, 2009). In Cdh1 deficient lenses, both the localization and levels of ZO1 were disrupted (Pontoriero et al., 2009). Therefore, we asked if ZO-1 was similarly altered in the epithelium of ScribLeCre lenses. Paraffin sections from E11.5, E13.5, E15.5, and E17.5 lenses were subjected to immunofluorescence analysis using an anti-ZO-1 antibody. At E11.5 the pattern of ZO-1 staining in ScribLeCre lenses did not differ from that in the controls (data not shown). However, by E13.5 we observed differences in the pattern of ZO-1 staining. At this stage, in the epithelium of control lenses ZO-1 localized specifically to the apical membrane of the epithelial cells, which was evident where the fiber cells had not fully elongated (Figure 6A, arrows). At E15.5 and E17.5, intense ZO-1 staining was observed at the apical-apical interface of epithelial and fiber cells (Figure 6D, F arrows). In contrast, by E13.5, in the ScribLeCre lenses, ZO-1 staining was greatly reduced at the apical surface of the epithelial cells, which was evident in regions where the fiber cells had not fully elongated (Figure 6B, arrow). By E15.5, there were regions of ScribLeCre lens epithelium lacking ZO-1 (Figure 6D, arrows). By E17.5 there were regions lacking ZO-1 (Figure 6F, arrows) and, overall, the regular punctuate staining at the apical-apical interface of the epithelial and fiber cells was largely absent (Figure 6F). Thus, in the absence of Scrib, ZO-1 is gradually lost from the epithelium, a second hallmark of EMT. 

16

Downregulation of Pax6 Correlates Temporally with Upregulation of SMA Since we observed defects in E-cadherin and ZO-1 in the embryonic ScribLeCre lenses beginning at early stages in lens differentiation, we asked at what age loss of Pax6 first would be observed. Sections from day E13.5, E15.5 and E17.5 control and ScribLeCre lenses were immunostained for Pax6. At E13.5 and E15.5, no difference was observed in the intensity of staining between controls and ScribLeCre lenses (data not shown). However, by E17.5, reduced intensity of staining for Pax6 was observed in some nuclei in the epithelium from ScribLeCre lenses as compared to controls (Figure 7A-D). We then asked if Pax6 downregulation correlated temporally with upregulation of DSMA and indicators of TGFE signaling. Sections from E13.5, E15.5, and E17.5 control and ScribLeCre lenses were immunostained with antibodies against SMA and Smad4.

At E13.5 or E15.5, no staining for SMA was observed in the lens

epithelium of control or ScribLeCre mice and Smad4 staining was cytoplasmic (data not shown). However, at E17.5, staining for SMA (Figure 7G, H) was observed in ScribLeCre lenses, but not in controls (Figure 7E, F and I, J). Additionally, and staining for Smad4 was concentrated in the nuclei in the epithelium of ScribLeCre lenses (Figure 7K, L) TGFE signaling through Smads requires activation of Smad2/3 by phosphorylation, which forms a nuclear complex with activated Smad4 (Itoh et al., 2000). Therefore, to further define the signaling pathway that was now activated in the ScribLeCre lens epithelial cells, we asked if nuclear Smad3 could also be detected in ScribLeCre lenses. To do so, immunohistochemistry using anti-Smad3 antibodies was performed on eye sections from E17.5 and P10 control and ScribLeCre animals. No Smad3 staining was observed in control lenses (Figure 8A). However, Smad3 positive nuclei were observed in ScribLeCre lenses (Figure 8B, arrows). Total and Smad3 positive nuclei were



17

counted on 6-8 sections from 3 different eyes. In the ScribLeCre lens epithelia, 21.44% (p=0.02) of nuclei were positive for Smad3. Similarly, at P10, Smad3 staining was detected in the nuclei of lens epithelial cells of ScribLeCre eyes but not in the lens epithelial cells of controls (Figure 8C, D, arrows). TGFE activity induces the expression of zinc finger transcription factors, Snail, Slug, and Twist, which are transcriptional repressors of Cdh1 (Moustakas and Heldin, 2007). Because there was obvious loss of E-cadherin from lens epithelia of ScribLeCre mice from E17.5 through P10 (Figures 3 and 5), we asked if loss of E-cadherin at these time points coincided with the appearance of Snail in the lens epithelium. To do so, immunohistochemistry using anti-Snail antibody was performed on eye sections from E17.5 and P10 mice. In E17.5 controls, some background staining was observed only in the cytoplasm of epithelial cells (Figure 8E). However, in ScribLeCre lenses, cytoplasmic and nuclear staining was observed (Figure 8F, arrows). Total and Snail positive nuclei were counted on 6-8 sections from 3 different eyes. In ScribLeCre lens epithelia 20.87% (p=0.01) of nuclei were positive for Snail. At P10, nuclear staining for Snail was observed in ScribLeCre lenses but not in control lenses (Figure 8G, H, arrows). Thus, the appearance of SMA and TGF signaling molecules correlate temporally with the loss of lens epithelial protein, Pax6, and the continued loss of E-cadherin.

Molecular Changes in the Corneal Epithelium of ScribLeCre Mice As shown in Figure 2E and F, the corneal epithelium in the ScribLeCre mice appeared disorganized and basal layer cells appeared flattened rather than cuboidal. We have previously determined that Scrib is expressed in the corneal epithelium (Nguyen et al., 2005) and that Scrib



18

protein was not detected in the corneas of P10 ScribLeCre mice (Figure 1E). Given the effect of loss of Scrib in the lens, we asked if loss of Scrib in the corneal epithelium also resulted in EMT in that tissue. At P10, the stratified corneal epithelium is characterized by E-cadherin (Figure 9A,B; green), which normally localizes at cell membranes of the basal and superficial layers of the corneal epithelium (Takahashi et al., 1992).

In the ScribLeCre corneal epithelium, E-

cadherin staining was reduced and absent from many cells in the superficial layers (Figure 9C,D). The corneal epithelium is also marked by the uniform expression of cytokeratin K12 in the suprabasal and superficial layers of the epithelium (Kao et al., 1996). Staining for K12 was observed throughout all membranes in the epithelium of control corneas at P10 (Figure 9E, F; red). However, K12 staining in the ScribLeCre corneal epithelium was not uniform, with regions showing reduced staining (Figure 9G, H), suggesting that the integrity of this epithelium was compromised. To determine if cells in the corneal epithelium were in transition to a mesenchymal state, sections were immunostained with anti-SMA antibodies. Whereas the corneal epithelium from control mice was negative for SMA, SMA staining was present in the ScribLeCre corneal epithelium and endothelium (Figure 9K, L). Therefore, loss of Scrib is in a second epithelium, the corneal epithelium, leads to molecular changes consistent with EMT.

DISCUSSION Epithelial cells are distinguished from other cell types in part by the presence of strong cell-cell adhesion and apical-basal polarity. The loss of the epithelial proteins, E-cadherin and ZO-1, major components of adherens and tight junctions, respectively, from epithelial cells and appearance of mesenchymal proteins such as DSMA and activated TGFE signaling intermediates are characteristics found in many examples of EMT (Kalluri and Weinberg, 2009; Thiery et al., 2009). Studies from invertebrate systems indicate that scrib is one factor playing an important 

19

role in the genesis and maintenance of epithelial adherens junctions and apical polarity complexes. In this study, using the mouse lens as a model epithelial tissue, we found that loss of Scrib at the lens placode stage resulted in loss of E-cadherin and ZO-1, components of adherens junctions and apical polarity complexes, indicating that Scrib plays a conserved role cross species. Furthermore, the loss of these factors, which are also lost in EMT, was accompanied by other molecular changes characteristic of EMT (summarized in Figure S1). Molecular changes consistent with EMT also occurred in a second ocular derivative of the surface ectoderm, the corneal epithelium. Thus, these data suggest that Scrib may generally be required for establishing or maintaining the specialized characteristics that define epithelial cells, which are necessary for maintaining the integrity of an epithelium in vivo and for preventing EMT.

Scrib is an Upstream Regulator of Epithelial Identity in the Lens In the lens, conditional deletion of Cdhl1 at the lens vesicle stage leads to the loss of cell adhesion and shape, the gradual loss of ZO-1, and, ultimately, EMT (Pontoriero et al., 2009), indicating the importance of E-cadherin in maintaining epithelial identity. In ScribLeCre lenses, the first phenotypic changes we observed were altered cell morphology and a reduced accumulation of E-cadherin at the apical borders of the cells in lens vesicle. These initial changes were followed by gradual loss of ZO-1. Given the similarities in the lens phenotype between the Cdh1 conditional mutant and ScribLeCre mice, we suggest that Scrib functions upstream of Ecadherin as a regulator of epithelial identity as it is required to maintain normal cell-cell adhesion and apical polarity, two defining characteristics of epithelial cells.



20

Interestingly, the developmental time point at which we first observed reduced accumulation of E-cadherin, E11.5, is only one day after the lens vesicle normally separates from the overlying ectoderm. For detachment of the lens vesicle from the overlying ectoderm to occur, E-cadherin-containing cell-cell adherens junctions must be broken and subsequently reformed between new neighboring cells (Wiley et al., 2010). It is possible, therefore, that reestablishing adherens junctions to form the lens vesicle is impaired in the absence of Scrib. In keeping with this concept, deletion of Scrib after the lens vesicle has formed does not result in EMT (unpublished data), suggesting Scrib may be required for unique events involving E-cadherin function at the time of lens vesicle formation. The loss of ZO-1 from the apical domain of the epithelial cells beginning at E13.5 suggests that disrupting adherens junctions leads to the destabilization of the tight junction complexes. Similarly, reduced levels of another apical polarity protein, Par3, which is part of the Par3/Par6/aPKC complex (Humbert et al., 2008; Wodarz and Nathke, 2007), was observed on the central epithelial cells of ScribLeCre lens and in the transition zone Par3 was also was observed on the lateral membranes (data not shown), indicating that restriction of Par3 to the apical domain was lost. This is reminiscent of findings in Drosophila where loss of lgl, a component of the scrib polarity complex, leads to expansion of the apical domain into the basolateral domain (Kaplan et al., 2009). Our data suggest that the dynamic stability of these complexes in vivo in the mouse lens epithelium requires Scrib and that these are required to maintain epithelial identity. However, Scrib may also be responsible at the time of lens vesicle formation and shortly thereafter for other cellular and molecular events that are crucial for forming and maintaining a lens epithelium. For example, we have noticed that ScribLeCre lenses show defects cell viability and proliferation (Yamben and Griep, manuscript in preparation). These changes could, along with the fiber cell defects, contribute to the overall



21

phenotype of the ScribLeCre lens. Additional investigations are needed to further elucidate Scrib’s involvement with early lens formation and how this impacts on maintaining epithelial identity as well as the overall integrity of the lens. Following initial disruption in E-cadherin localization and loss of ZO-1, we observed evidence for active TGFE signaling and the appearance of, DSMA. Thus, further decreases in Ecadherin expression observed in late embryonic and postnatal ScribLeCre lenses may be the result of Snail activity, as Snail is a known negative regulator of Cdh1 transcription (Moustakas and Heldin, 2007). Loss of the lens epithelial transcription factor, Pax6, also was observed. This likely contributes to the loss of lens epithelial identity in the Scrib-deficient lenses, since lenses of mice haplo-insufficient for Pax6 exhibit EMT (Lovicu et al., 2004) and Pax6 is a regulator of expression of the lens specific crystallin genes (Cvekl et al., 2004). It has been shown that downregulation of Pax6 occurs in the TGFE overexpressing transgenic mouse lens (Lovicu et al., 2004) and activated Smad3 can negatively regulate Pax6 expression (Grocott et al., 2007). The timepoint (E17.5) when Pax6 loss was first noticed in the ScribLeCre lenses suggests TGFE signaling may be responsible. Thus, we suggest that in the Scrib deficient lenses, EMT initiates with the destabilization of the junctional complexes and apical-basal polarity and then is driven through the acquisition of active TGFEsignaling. Scrib, EMT and Disease The loss of epithelial identity and EMT is associated with two types of cataract in human and mouse models, ASC (Hales et al., 1995; Lovicu et al., 2004; Saika et al., 2009; Srinivasan et al., 1998) and PCO, (Wormstone et al., 2006; Wormstone et al., 2002; Wormstone et al., 2009), which commonly occurs after cataract surgery. Phenotypically, the lenses of ScribLeCre mice



22

mimic many of the defects observed in these models of cataract. Additionally, defects in fiber cell adhesion have been shown to be a contributing factor to cortical cataract in experimental models (Zhou et al., 2007). ScribLeCre lenses also exhibit defects in cell adhesion in the fiber cell compartment suggesting that Scrib function may be required to maintain normal fiber cell characteristics and prevent cortical cataractogenesis. It is not known if Scrib expression and/or function is compromised in any of these examples of mouse or human cataract. Further studies will be required to address this question. The loss of Scrib led to EMT not only in the lens but also in the corneal epithelium, as evidenced by the reduced corneal epithelial proteins, E-cadherin and K12, and the expression of DSMA. This suggests that Scrib may be required to maintain epithelial identity, thus preventing EMT and disease in many different organs. In the eye, EMT of corneal epithelial cells in the limbal region has been suggested to be involved in the pathogenesis of pterygium (Kato et al., 2007). EMT of retinal pigment epithelial cells is observed in proliferative vitreoretinopathy, a consequence of retinal detachment (Hiscott et al., 1999; Nagasaki et al., 1998; Thiery et al., 2009). EMT is associated with tissue fibrosis in a variety of other organs such as kidney, liver, and lung (Kalluri and Weinberg, 2009). EMT and the targeting of cell polarity complexes by EMT inducers have been associated with increased invasiness and metastasis in cancer (Banks et al., 2012; Elsum et al., 2012; Moreno-Bueno et al., 2008; Olmeda et al., 2008). For example, the human papillomavirus oncogenes have been shown to downregulate epithelial proteins while upregulating mesenchymal proteins in human keratinocytes (Hellner et al., 2009). Of particular relevance to our study, expression of the E6 oncoprotein, dependent on an intact PDZ binding domain through which E6 binds to and promotes the degradation of Scrib (Thomas et al., 2005), has been shown to result in phenotypic changes in cultured keratinocytes that are indicative of 

23

EMT (Watson et al., 2003). In cervical cancer specimens, greatly reduced levels of Scrib are observed in high-grade squamous intraepithelial lesions (SIL) and invasive cancers as compared to low grade SIL lesions (Nakagawa et al., 2004), showing an inverse correlation between Scrib levels and the degree of invasiveness of the tumor. Also, reduced levels of Scrib are associated with loss of epithelial polarity and tissue architecture in colon adenocarcinoma (Gardiol et al., 2006), metastasis in endometrial cancer (Ouyang et al., 2010), and breast cancer (Zhan et al., 2008). Thus, it is possible that the loss of Scrib function results in EMT that drives the metastatic progression of many types of cancers. CONCLUSIONS Together, these data suggest that, in vivo, mammalian Scrib is required to maintain epithelial identity and its loss allows epithelial cells to be susceptible to EMT. Scrib may maintain epithelial identity primarily through its effect on junctional complexes. An examination of molecular changes during the early stages of lens formation in ScribLeCre lenses may provide further insight into roles of Scrib in EMT and human disease.

ACKNOWLEDGEMENTS The authors thank Ruth Ashery-Padan for generously providing the LeCre mice. The authors also thank Lance Roderick of the Keck Imaging Facility, Toshi Kinoshita of the Pathology Department Histology Core, the McArdle Laboratories Histology Core and Denis Lee and Susan Moran of the McArdle Laboratories. The authors thank Angela Verdoni and Aki Ikeda for guidance with the cornea experiments. The authors thank Paul Lambert for helpful discussions and his critical reading of the manuscript. I.F.Y. was supported by the NIH training grant



24

GM07215. This work was supported by NIH grants EY09091, CA98428, CA14520, and EY016665.

REFERENCES Acloque, H., Adams, M.S., Fishwick, K., Bronner-Fraser, M., Nieto, M.A., 2009. Epithelialmesenchymal transitions: the importance of changing cell state in development and disease. J Clin Invest 119, 1438-1449. Ashery-Padan, R., Marquardt, T., Zhou, X., Gruss, P., 2000. Pax6 activity in the lens primordium is required for lens formation and for correct placement of a single retina in the eye. Genes Dev 14, 2701-2711. Banks, L., Pim, D., Thomas, M., 2012. Human tumour viruses and the deregulation of cell polarity in cancer. Nat Rev Cancer 12, 877-886. Baum, B., Georgiou, M., 2011. Dynamics of adherens junctions in epithelial establishment, maintenance, and remodeling. J Cell Biol 192, 907-917. Bilder, D., 2004. Epithelial polarity and proliferation control: links from the Drosophila neoplastic tumor suppressors. Genes Dev 18, 1909-1925. Bilder, D., Li, M., Perrimon, N., 2000. Cooperative regulation of cell polarity and growth by Drosophila tumor suppressors. Science 289, 113-116. Bilder, D., Perrimon, N., 2000. Localization of apical epithelial determinants by the basolateral PDZ protein Scribble. Nature 403, 676-680. Cvekl, A., Yang, Y., Chauhan, B.K., Cveklova, K., 2004. Regulation of gene expression by Pax6 in ocular cells: a case of tissue-preferred expression of crystallins in lens. Int J Dev Biol 48, 829844. de Iongh, R.U., Wederell, E., Lovicu, F.J., McAvoy, J.W., 2005. Transforming growth factorbeta-induced epithelial-mesenchymal transition in the lens: a model for cataract formation. Cells Tissues Organs 179, 43-55. Dwivedi, D.J., Pino, G., Banh, A., Nathu, Z., Howchin, D., Margetts, P., Sivak, J.G., West-Mays, J.A., 2006. Matrix metalloproteinase inhibitors suppress transforming growth factor-betainduced subcapsular cataract formation. Am J Pathol 168, 69-79. Elsum, I., Yates, L., Humbert, P.O., Richardson, H.E., 2012. The Scribble-Dlg-Lgl polarity module in development and cancer: from flies to man. Essays Biochem 53, 141-168. 

25

Gardiol, D., Zacchi, A., Petrera, F., Stanta, G., Banks, L., 2006. Human discs large and scrib are localized at the same regions in colon mucosa and changes in their expression patterns are correlated with loss of tissue architecture during malignant progression. Int J Cancer 119, 12851290. Giepmans, B.N., van Ijzendoorn, S.C., 2009. Epithelial cell-cell junctions and plasma membrane domains. Biochim Biophys Acta 1788, 820-831. Grocott, T., Frost, V., Maillard, M., Johansen, T., Wheeler, G.N., Dawes, L.J., Wormstone, I.M., Chantry, A., 2007. The MH1 domain of Smad3 interacts with Pax6 and represses autoregulation of the Pax6 P1 promoter. Nucleic Acids Res 35, 890-901. Hales, A.M., Chamberlain, C.G., McAvoy, J.W., 1995. Cataract induction in lenses cultured with transforming growth factor-beta. Invest Ophthalmol Vis Sci 36, 1709-1713. Harris, B.Z., Lim, W.A., 2001. Mechanism and role of PDZ domains in signaling complex assembly. J Cell Sci 114, 3219-3231. Hellner, K., Mar, J., Fang, F., Quackenbush, J., Munger, K., 2009. HPV16 E7 oncogene expression in normal human epithelial cells causes molecular changes indicative of an epithelial to mesenchymal transition. Virology 391, 57-63. Hiscott, P., Sheridan, C., Magee, R.M., Grierson, I., 1999. Matrix and the retinal pigment epithelium in proliferative retinal disease. Prog Retin Eye Res 18, 167-190. Humbert, P., Russell, S., Richardson, H., 2003. Dlg, Scribble and Lgl in cell polarity, cell proliferation and cancer. Bioessays 25, 542-553. Humbert, P.O., Grzeschik, N.A., Brumby, A.M., Galea, R., Elsum, I., Richardson, H.E., 2008. Control of tumourigenesis by the Scribble/Dlg/Lgl polarity module. Oncogene 27, 6888-6907. Itoh, S., Itoh, F., Goumans, M.J., Ten Dijke, P., 2000. Signaling of transforming growth factorbeta family members through Smad proteins. Eur J Biochem 267, 6954-6967. Johnson, K., Wodarz, A., 2003. A genetic hierarchy controlling cell polarity. Nat Cell Biol 5, 1214. Kalluri, R., Weinberg, R.A., 2009. The basics of epithelial-mesenchymal transition. J Clin Invest 119, 1420-1428. Kao, W.W., Liu, C.Y., Converse, R.L., Shiraishi, A., Kao, C.W., Ishizaki, M., Doetschman, T., Duffy, J., 1996. Keratin 12-deficient mice have fragile corneal epithelia. Invest Ophthalmol Vis Sci 37, 2572-2584.



26

Kaplan, N.A., Liu, X., Tolwinski, N.S., 2009. Epithelial polarity: interactions between junctions and apical-basal machinery. Genetics 183, 897-904. Kato, N., Shimmura, S., Kawakita, T., Miyashita, H., Ogawa, Y., Yoshida, S., Higa, K., Okano, H., Tsubota, K., 2007. Beta-catenin activation and epithelial-mesenchymal transition in the pathogenesis of pterygium. Invest Ophthalmol Vis Sci 48, 1511-1517. Klymkowsky, M.W., Savagner, P., 2009. Epithelial-mesenchymal transition: a cancer researcher's conceptual friend and foe. Am J Pathol 174, 1588-1593. Lewandoski, M., Meyers, E.N., Martin, G.R., 1997. Analysis of Fgf8 gene function in vertebrate development. Cold Spring Harb Symp Quant Biol 62, 159-168. Lovicu, F.J., Ang, S., Chorazyczewska, M., McAvoy, J.W., 2004. Deregulation of lens epithelial cell proliferation and differentiation during the development of TGFbeta-induced anterior subcapsular cataract. Dev Neurosci 26, 446-455. Michaelis, U.R., Chavakis, E., Kruse, C., Jungblut, B., Kaluza, D., Wandzioch, K., Manavski, Y., Heide, H., Santoni, M.J., Potente, M., Eble, J.A., Borg, J.P., Brandes, R.P., 2013. The polarity protein scrib is essential for directed endothelial cell migration. Circ Res 112, 924-934. Montcouquiol, M., Rachel, R.A., Lanford, P.J., Copeland, N.G., Jenkins, N.A., Kelley, M.W., 2003. Identification of Vangl2 and Scrb1 as planar polarity genes in mammals. Nature 423, 173177. Moreno-Bueno, G., Portillo, F., Cano, A., 2008. Transcriptional regulation of cell polarity in EMT and cancer. Oncogene 27, 6958-6969. Moustakas, A., Heldin, C.H., 2007. Signaling networks guiding epithelial-mesenchymal transitions during embryogenesis and cancer progression. Cancer Sci 98, 1512-1520. Murdoch, J.N., Henderson, D.J., Doudney, K., Gaston-Massuet, C., Phillips, H.M., Paternotte, C., Arkell, R., Stanier, P., Copp, A.J., 2003. Disruption of scribble (Scrb1) causes severe neural tube defects in the circletail mouse. Hum Mol Genet 12, 87-98. Nagasaki, H., Shinagawa, K., Mochizuki, M., 1998. Risk factors for proliferative vitreoretinopathy. Prog Retin Eye Res 17, 77-98. Nakagawa, S., Yano, T., Nakagawa, K., Takizawa, S., Suzuki, Y., Yasugi, T., Huibregtse, J.M., Taketani, Y., 2004. Analysis of the expression and localisation of a LAP protein, human scribble, in the normal and neoplastic epithelium of uterine cervix. Br J Cancer 90, 194-199. Nakaya, Y., Sheng, G., 2008. Epithelial to mesenchymal transition during gastrulation: an embryological view. Dev Growth Differ 50, 755-766.



27

Nguyen, M.M., Rivera, C., Griep, A.E., 2005. Localization of PDZ domain containing proteins Discs Large-1 and Scribble in the mouse eye. Mol Vis 11, 1183-1199. Olmeda, D., Montes, A., Moreno-Bueno, G., Flores, J.M., Portillo, F., Cano, A., 2008. Snai1 and Snai2 collaborate on tumor growth and metastasis properties of mouse skin carcinoma cell lines. Oncogene 27, 4690-4701. Ouyang, Z., Zhan, W., Dan, L., 2010. hScrib, a human homolog of Drosophila neoplastic tumor suppressor, is involved in the progress of endometrial cancer. Oncol Res 18, 593-599. Pearson, H.B., Perez-Mancera, P.A., Dow, L.E., Ryan, A., Tennstedt, P., Bogani, D., Elsum, I., Greenfield, A., Tuveson, D.A., Simon, R., Humbert, P.O., 2011. SCRIB expression is deregulated in human prostate cancer, and its deficiency in mice promotes prostate neoplasia. J Clin Invest 121, 4257-4267. Piatigorsky, J., 1981. Lens differentiation in vertebrates. A review of cellular and molecular features. Differentiation 19, 134-153. Pontoriero, G.F., Smith, A.N., Miller, L.A., Radice, G.L., West-Mays, J.A., Lang, R.A., 2009. Co-operative roles for E-cadherin and N-cadherin during lens vesicle separation and lens epithelial cell survival. Dev Biol 326, 403-417. Qin, Y., Capaldo, C., Gumbiner, B.M., Macara, I.G., 2005. The mammalian Scribble polarity protein regulates epithelial cell adhesion and migration through E-cadherin. J Cell Biol 171, 1061-1071. Rivera, C., Yamben, I.F., Shatadal, S., Waldof, M., Robinson, M.L., Griep, A.E., 2009. Cellautonomous requirements for Dlg-1 for lens epithelial cell structure and fiber cell morphogenesis. Dev Dyn 238, 2292-2308. Saika, S., Yamanaka, O., Flanders, K.C., Okada, Y., Miyamoto, T., Sumioka, T., Shirai, K., Kitano, A., Miyazaki, K., Tanaka, S., Ikeda, K., 2008. Epithelial-mesenchymal transition as a therapeutic target for prevention of ocular tissue fibrosis. Endocr Metab Immune Disord Drug Targets 8, 69-76. Saika, S., Yamanaka, O., Okada, Y., Tanaka, S., Miyamoto, T., Sumioka, T., Kitano, A., Shirai, K., Ikeda, K., 2009. TGF beta in fibroproliferative diseases in the eye. Front Biosci (Schol Ed) 1, 376-390. Srinivasan, Y., Lovicu, F.J., Overbeek, P.A., 1998. Lens-specific expression of transforming growth factor beta1 in transgenic mice causes anterior subcapsular cataracts. J Clin Invest 101, 625-634. Sugiyama, Y., Akimoto, K., Robinson, M.L., Ohno, S., Quinlan, R.A., 2009. A cell polarity protein aPKClambda is required for eye lens formation and growth. Dev Biol 336, 246-256.



28

Symonds, J.G., Lovicu, F.J., Chamberlain, C.G., 2006. Posterior capsule opacification-like changes in rat lens explants cultured with TGFbeta and FGF: effects of cell coverage and regional differences. Exp Eye Res 82, 693-699. Takahashi, M., Fujimoto, T., Honda, Y., Ogawa, K., 1992. Distributional change of fodrin in the wound healing process of the corneal epithelium. Invest Ophthalmol Vis Sci 33, 280-285. Thiery, J.P., Acloque, H., Huang, R.Y., Nieto, M.A., 2009. Epithelial-mesenchymal transitions in development and disease. Cell 139, 871-890. Thomas, M., Massimi, P., Navarro, C., Borg, J.P., Banks, L., 2005. The hScrib/Dlg apico-basal control complex is differentially targeted by HPV-16 and HPV-18 E6 proteins. Oncogene 24, 6222-6230. Tsuji, T., Ibaragi, S., Hu, G.F., 2009. Epithelial-mesenchymal transition and cell cooperativity in metastasis. Cancer Res 69, 7135-7139. Watson, R.A., Thomas, M., Banks, L., Roberts, S., 2003. Activity of the human papillomavirus E6 PDZ-binding motif correlates with an enhanced morphological transformation of immortalized human keratinocytes. J Cell Sci 116, 4925-4934. Wiley, L.A., Dattilo, L.K., Kang, K.B., Giovannini, M., Beebe, D.C., 2010. The tumor suppressor merlin is required for cell cycle exit, terminal differentiation, and cell polarity in the developing murine lens. Invest Ophthalmol Vis Sci 51, 3611-3618. Wodarz, A., Nathke, I., 2007. Cell polarity in development and cancer. Nat Cell Biol 9, 10161024. Woods, D.F., Hough, C., Peel, D., Callaini, G., Bryant, P.J., 1996. Dlg protein is required for junction structure, cell polarity, and proliferation control in Drosophila epithelia. J Cell Biol 134, 1469-1482. Wormstone, I.M., Anderson, I.K., Eldred, J.A., Dawes, L.J., Duncan, G., 2006. Short-term exposure to transforming growth factor beta induces long-term fibrotic responses. Exp Eye Res 83, 1238-1245. Wormstone, I.M., Tamiya, S., Anderson, I., Duncan, G., 2002. TGF-beta2-induced matrix modification and cell transdifferentiation in the human lens capsular bag. Invest Ophthalmol Vis Sci 43, 2301-2308. Wormstone, I.M., Wang, L., Liu, C.S., 2009. Posterior capsule opacification. Exp Eye Res 88, 257-269. Yates, L.L., Schnatwinkel, C., Hazelwood, L., Chessum, L., Paudyal, A., Hilton, H., Romero, M.R., Wilde, J., Bogani, D., Sanderson, J., Formstone, C., Murdoch, J.N., Niswander, L.A.,



29

Greenfield, A., Dean, C.H., 2013. Scribble is required for normal epithelial cell-cell contacts and lumen morphogenesis in the mammalian lung. Dev Biol 373, 267-280. Zeisberg, M., Neilson, E.G., 2009. Biomarkers for epithelial-mesenchymal transitions. The Journal of clinical investigation 119, 1429-1437. Zhan, L., Rosenberg, A., Bergami, K.C., Yu, M., Xuan, Z., Jaffe, A.B., Allred, C., Muthuswamy, S.K., 2008. Deregulation of scribble promotes mammary tumorigenesis and reveals a role for cell polarity in carcinoma. Cell 135, 865-878. Zhao, M., Szafranski, P., Hall, C.A., Goode, S., 2008. Basolateral junctions utilize warts signaling to control epithelial-mesenchymal transition and proliferation crucial for migration and invasion of Drosophila ovarian epithelial cells. Genetics 178, 1947-1971. Zhou, J., Leonard, M., Van Bockstaele, E., Menko, A.S., 2007. Mechanism of Src kinase induction of cortical cataract following exposure to stress: destabilization of cell-cell junctions. Mol Vis 13, 1298-1310.

FIGURE LEGENDS Figure 1. Generation of Scrib conditional knockout mice. A: Shown are exons 1-10 of the wildtype (WT) Scrib allele. The targeting allele (TA) shown illustrates loxP sites that flank exons 2-8 and frt sites that flank a positive selection neomycin (NEO) marker. Mice containing the targeting allele were mated to mice expressing Flpase, which removed the neo marker and generated the floxed (F) allele.

Arrows indicate locations of PCR primers a (ScribF), b

(ScribwtR), and c (ScribckoR) used to identify if exons 2-8 had been deleted and d (Scrib1-X2R) used in combination with a to routinely genotype the floxed and wt alleles. The mutant allele was generated through mating with Lens-Cre. B: Immunofluoresence demonstrating localization of Scrib (red) in the E10.5 lens vesicle (lv). Arrow indicates concentrated apical localization in the lens vesicle. oc, optic cup. C: GFP fluorescence indicating cre activity specifically in the E10.5 ScribLecre vesicle. D: Lens specific cre-mediated deletion of exons 2-8. PCR primer a, 

30

b, and c were used to amplify DNA fragments from lens, eye and tail samples.

The wt

band=437bp, the null band=193bp and the floxed band=325bp. ScribLeCre lenses had the 193bp null band. E: Whole cell lysates from lenses of P2 and corneas of P10 control and ScribLeCre mice were subjected to immunoblot analysis for Scrib and reprobed for Gapdh as a loading control. Scrib was reduced in lenses from ScribLeCre mice as compared controls and absent in the cornea lysate from ScribLeCre mice. Protein lysates from Scrib wt and null brain were included to demonstrate the specificity of the antibody.

Figure 2: Morphological defects in the eyes of postnatal day 10 (P10) ScribLeCre mice. AB: Eyes (A) and lenses (B) were isolated from control and ScribLeCre mice and viewed under a dissecting microscope.

A: P10 ScribLeCre eyes were noticeably smaller (right) compared to

controls (left). B: ScribLeCre lenses were smaller than controls and also had an opaque center indicating a cataract (arrow). C-J: Longitudinally oriented paraffin embedded sections of eyes from P10 control (C, E, G) and ScribLeCre (D, F, H) mice were stained with hematoxylin and eosin. C-D: Sections of controls (C) and ScribLeCre eyes (D) showing that mutant lenses were vacuolated (arrow) and nuclei were scattered throughout the lens (arrowhead). Also, the iris was hyperplastic (asterisk). E-F: Higher magnification of the lens epithelium in control (E) and ScribLeCre (F) lenses highlighting the flattened and elongated cells in the epithelium of the mutant as compared to controls (arrowheads). G-H: Higher magnification image of control (G) and ScribLeCre (H) corneas showing that the epithelium of mutant corneas lacked an organized stratified epithelium with cuboidal shaped cells in the basal layer (arrowheads). Insets show higher magnification images of the regions indicated by black boxes. ac, anterior chamber; c,



31

cornea; e, epithelium; en, endothelium; f, fiber cells; i, iris; l, lens; s, stroma.

Bar = 50μm for

C, D and 25μm for E-H.

Figure 3:

Epithelial proteins are downregulated and mesenchymal proteins are

upregulated in the P10 ScribLeCre lens epithelium. Longitudinally oriented, paraffin embedded (A-H, M-P) or cryogenic (I-L) sections from P10 control and ScribLeCre eyes were immunostained with antibodies against epithelial proteins (Pax6; A-D and E-Cadherin; E-H), the mesenchymal protein, SMA (I-L), and Smad4 (M-P). Sections were counterstained with either propidium iodide (red, A, C), To-Pro3 (blue, I, K, M, O) or anti-D-catenin antibodies (E, G). AD: Pax6 (green) was found consistently in the nuclei of epithelial cells from control lenses (AB) whereas nuclear Pax6 staining was absent from (arrows; C, D) or reduced in nuclei of ScribLeCre epithelial cells. E-F: E-cadherin (green) colocalized (yellow) with -catenin (red) along all membranes of control epithelium. G-H: In the flattened ScribLeCre epithelium there were areas where E-cadherin and D-catenin were absent from basal and apical membranes (arrows). I-L: SMA was observed only in the iris (i) of control lenses (I, L), but was found in both the iris and the epithelium (arrow) of ScribLeCre lenses (K-L). The red box inset (L) shows a higher magnification image of the region indicated by the white box. M-P: Smad4 (red) was cytoplasmic in control epithelial cells (N) but was concentrated in the nuclei of ScribLeCre epithelial cells (O, P, arrows). e, epithelium; f, fiber cells; i, iris. Bar = 50μm.

Figure 4. E-cadherin and Pax6 levels are reduced in ScribLeCre lenses. Protein lysates from P2 control and ScribLeCre mice were subjected to western blot analysis using anti-E-cadherin 

32

(A) and anti-Pax6 (B) antibodies. Blots were reprobed for Gapdh as a loading control. For Ecadherin, three independent pools of protein were analyzed on three different blots. For Pax6 two independent pools were analyzed on two different blots. Bands were quantified by phosphorimager analysis and for E-cadherin statistical analysis was conducted using the twosided Wilcoxon Rank Sum test. The levels of E-Cadherin were reduced 35% (p=0.04) in ScribLeCre lenses as compared to controls. The levels of Pax6 were reduced 55% in ScribLeCre lenses as compared to controls.

Figure 5: E-cadherin is progressively lost from ScribLeCre lenses. Longitudinally oriented, paraffin embedded sections from E11.5 (A-D), E13.5 (E-H) and E17.5 (I-L) control (A, B, E, F, I, J) and ScribLeCre (C, D, G, H, K, L) embryos (A-H) and eyes (I-L) were immunostained with anti-E-cadherin (green) and anti--catenin (red) antibodies.

A-B: Colocalization (yellow) was

prominent at the apical surface of cells in the anterior lens vesicle (A, arrow). E-cadherin was punctate in this region (B). C-D: E-cadherin staining was reduced in the anterior region of the ScribLeCre lens vesicles (arrows). E-F: Colocalization was observed along the basal and lateral membranes of controls at E13.5 (E). E-cadherin was punctate along the apical surface (F, arrow). G-H: In E13.5 ScribLeCre lenses, E-cadherin was specifically reduced along the basal and lateral membranes surface (arrows). I-J: In E17.5 control lenses, colocalization was found along all surfaces of the epithelium (I). E-cadherin was punctate at the apical surface (J). K-L: In E17.5 ScribLeCre lenses, E-cadherin was absent or reduced along some basal, lateral membranes (arrows). Red boxes show higher magnification images of the regions indicated by white boxes. ce, corneal epithelium; le, lens epithelium; lf, fiber cells; lv, lens vesicle; s, corneal stroma. Bar= 50 μm. 

33

Figure 6: The tight junction protein, ZO-1, is gradually lost from the apical surface of ScribLeCre lens epithelial cells. Longitudinally oriented, paraffin embedded sections of E13.5 embryos (A, B), E15.5 heads (C, D), and E17.5 eyes (E, F) from control (A, C, E) and ScribLeCre (B, D, F) mice were immunostained with anti-ZO-1 antibodies (green) and the nuclei counterstained with propidium iodide (red). A-B: At E13.5, ZO-1 staining was observed at the apical surfaces of epithelial cells in the control lenses (arrows) but was reduced or absent (B, arrow) on the apical surface of the ScribLeCre lenses. C-D: At E15.5, ZO-1 staining was observed at the epithelial-fiber interface in control lenses (C, arrows) but was reduced or absent (D, arrows) on the apical surface of the epithelial cells of the ScribLeCre lenses. E-F: At E17.5, punctate ZO-1 staining was observed on the apical membrane of the epithelial cells at the epithelial-fiber interface (E, arrows) but was further reduced or absent (F, arrows) on the apical membrane of the epithelial cells of the ScribLeCre lenses. The staining pattern in the lens fiber cells was also disrupted at E17.5. e, epithelium; f, fiber cells. Bar= 50 μm.

Figure 7: Downregulation of Pax6 and upregulation of SMA and nuclear Smad4 are observed in the lens epithelium of E17.5 ScribLeCre embryos. Longitudinally oriented, paraffin embedded (A-D, I-L) or cryogenic (E-H) sections from eyes of E17.5 control (A, B, E, F, I, J) and ScribLeCre (C, D, G, H, K, L) embryos were immunostained for Pax6 (A-D, green), DSMA (E-H, red) or Smad4 (I-L, red) and the nuclei counterstained with propidium idodide (A, C, red) or To-Pro3 (E, G, I, K, blue). A-D: Pax6 staining was observed uniformly in the nuclei of lens epithelium from control mice (A, B) whereas the intensity of Pax6 staining was variable in



34

the nuclei of the epithelium from ScribLeCre mice with some nuclei exhibiting markedly reduced staining (C, D) and some of these nuclei appeared flattened rather than rounded (C, D, arrowhead). E-H: Staining for SMA was observed in the iris but not the lens of control eyes (E, F) whereas staining was observed in both the lens epithelium (arrows) and iris of the ScribLeCre eyes (G, H). Note that the iris does not extend across the entire ScribLeCre eye at E17.5 as it does in P10 ScribLeCre eyes (see Figure 2D). The hyperplastic iris was only observed in eyes of postnatal ScribLeCre mice. Red boxes show higher magnification images of regions indicated by white boxes. I-L: Smad4 staining (red) was cytoplasmic in control epithelial cells (I, J) whereas it was concentrated in the nuclei of some of the ScribLeCre epithelial cells (arrows) and the nuclei appeared flattened rather than rounded (K, L). e, epithelium f, fibers i, iris. Bar = 50μm.

Figure 8: Nuclear Smad3 and Snail are detected in lens epithelium of ScribLeCre mice. Longitudinally oriented, paraffin embedded eye sections from control (A, C, E, G) and ScribLeCre (B, D, F, H) E17.5 embryos (A, B, E, F) or P10 mice (C, D, G, H) were immunostained for Smad3 (A-D) or Snail (E-H) and counterstained with hematoxylin. A-D: Nuclear immunoreactivity for Smad3 was not observed in the lens epithelium of E17.5 (A) or P10 (C) control eyes whereas nuclear Smad3 staining was observed in some nuclei of the lens epithelium of E17.5 (B, arrows) and P10 (D, arrows) ScribLeCre mice.

E-H:

Nuclear

immunoreactivity for Snail was not observed in nuclei of E17.5 (E) or P10 (G) control lens epithelium whereas nuclear staining was observed in ScribLeCre lenses at both E17.5 (F, arrows) and P10 (H, arrows). ce, corneal epithelium, le, lens epithelium; lf, lens fiber cells; s, corneal stroma. Bar = 25μm.



35

Figure 9: The corneal epithelium in the ScribLeCre mice acquires EMT characteristics. Longitudinally oriented, cryogenic sections from P10 control and ScribLeCre mice were immunostained for E-cadherin (A-D, green), cytokeratin 12 (K12, E-H, red) and DSMA (I-L, red). Nuclei were counterstained with propidium iodide (red, A, C) or To-Pro3 (blue, E, G, I-L). A-D: E-cadherin (green) is strongly expressed along all cell surfaces of the corneal epithelial cells of controls (A-B), but is weakly expressed or absent in the corneal epithelial cells from the ScribLeCre mice (D arrows). E-H: K12 (red) is expressed throughout the corneal epithelium of control mice but is reduced and discontinuous in corneal epithelium of ScribLeCre mice. (G, H, arrows).

I-L: SMA (red) was expressed only in ScribLeCre corneal epithelium and

endothelium (K-L, arrows).

Red boxes show higher magnification images of epithelium

indicated by the white boxes. e, epithelium; s, stroma; en, endothelium Bars=50μm.



36

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9