Molecular Vision 2005; 11:1183-1199 <http://www.molvis.org/molvis/v11/a136/> Received 16 March 2005 | Accepted 22 December 2005 | Published 28 December 2005

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Localization of PDZ domain containing proteins discs large-1 and scribble in the mouse eye

Minh M. Nguyen, Charlene Rivera, Anne E. Griep
 
 

Department of Anatomy, University of Wisconsin Medical School, Madison, WI

Correspondence to: Dr. Anne E. Griep, Department of Anatomy, University of Wisconsin Medical School, 1300 University Avenue, Madison, WI, 53706; Phone: (608) 262-8988; FAX: (608) 262-7306; email: aegriep@facstaff.wisc.edu
 
Dr. Nguyen is now at the Department of Urology, Northwestern University, Chicago, IL.

Abstract

Purpose: In invertebrates such as Drosophila melanogaster and Caenorhabditis elegans, the PDZ domain containing proteins, Discs large (Dlg) and Scribble (Scrib), are found localized to specific junctional complexes and have been shown to be required for establishing and maintaining epithelial cell adhesion, polarity, and proliferation during development. In addition, they are known to be critical for neural development. However, the mechanisms and pathways through which they act in mammalian systems, especially in vivo, are poorly understood. The purpose of this study was to characterize the distribution of Dlg-1 and Scrib in various structures of the mouse eye and the regions where these proteins overlap with known adhesion proteins.

Methods: Embryos or mouse eyes were embedded, sectioned, subjected to immunofluorescence with antibodies to Dlg-1, Scrib, and E-cadherin, N-cadherin, or ZO-1, and stained sections viewed under confocal microscopy.

Results: Dlg-1 and Scrib were found widely distributed throughout the eye. In the lens, overlap was observed with E- and N-cadherin and ZO-1 in regions where adherens junctions are found, as well as in the complexes that attach lens cells to the underlying capsule. Overlap of Dlg-1 and Scrib with E-cadherin and ZO-1 was observed in the portions of the cornea and in the retinal pigment epithelium. However, in the neural retina, there appeared to be little, if any, overlap of Dlg-1 or Scrib with adhesion proteins, consistent with a role in synapse biology in the neural retina rather than adhesion.

Conclusions: The observed localization of Dlg-1 and Scrib with cadherins suggests that these proteins may play roles in cell adhesion, polarity and proliferation, as they do in invertebrates, suggesting cross-species conservation of function for these PDZ proteins. However, the broader distribution of these PDZ proteins within the eye suggests they may play more diverse roles in cell adhesion and differentiation.

Introduction

Cell-cell adhesion, cell communication and cell polarity are vital to the growth and differentiation of epithelial tissues and to maintaining their long-term integrity. These properties of epithelial cells are, in part, mediated via cell-cell junctional complexes such as occludens and adherens junctions. Although the major protein components of these junctional complexes have been studied widely, the mechanisms through which they are established, maintained and linked to the regulation of cell proliferation in mammalian systems are largely unexplored. Evidence from studies in invertebrates indicates that members of the PDZ (PSD95/Dlg/ZO-1) protein family play a role in these processes [1,2]. In this study we characterized the distribution and subcellular localization of two PDZ proteins, Discs Large-1 (Dlg-1) and Scribble (Scrib), relative to other known adhesion proteins, in the mouse eye.

PDZ proteins contain a common protein-protein recognition domain of approximately 80-90 amino acids, referred to as the PDZ domain [3]. These proteins are thought to affect epithelial cell structure, polarity, and growth behavior by directing the trafficking of proteins to proper plasma membrane surfaces of the cell, by organizing and stabilizing supramolecular adhesion and signaling complexes, and by acting as scaffolding/adaptor molecules [1-4]. Work in Drosophila has shown that a number of these PDZ proteins are required for the maintenance of cell growth, polarity, and adhesion of embryonic epithelial sheets of imaginal discs [5]. In Drosophila, null mutations in Discs large(Dlg) and Scribble (Scrib), the genes encoding the PDZ proteins Dlg and Scrib, result in neoplastic growth and multilayering in normally simple epithelia such as the epidermis, ovarian follicles, and imaginal discs [4,6], thus defining these proteins as tumor suppressors. Dlg, Scrib, and Lethal Giant Larvae (Lgl) are found localized along the basolateral membrane just basal to the septate junction and have been shown to be important for defining the apical and basolateral domains of the membrane and positioning cell junctions [7]. The loss of function of any one of these proteins results in the failure to properly form septate junctions as well as other cell-cell junctions such as adherens junctions [2]. In Caenorhabditis the loss of Dlg and LET-413 (Scrib) also results in the loss of proper localization of components of junctional complexes and defects in cell-cell adhesion [8-10].

Only recently have investigators begun to explore the role that the mammalian homologs of Drosophila Dlg and Scrib, Dlg-1 and Scrib, play in establishing and maintaining cell polarity and adhesion, in regulating cell proliferation in mammalian systems. Sequence comparison studies have shown that Dlg and Scrib are highly conserved among species and some studies have suggested that they also are conserved functionally [11]. In cultured mammalian epithelial cells, Dlg-1 has been associated with adherens junctions where it co-localizes with E-cadherin [12]. Cell culture studies also have shown overlap in the localization of Scrib with the tight junction protein, ZO-1, and that the loss of Scrib function results in the mislocalization of ZO-1 [13]. Very recently, it has been shown that intact Dlg-1 is necessary for proper craniofacial development in the mouse [14], a specific frame shift mutation in Scrib causes the neural tube defect craniorachischisis [15], and conditional knockout of Lgl in the nervous system leads to brain dysplasia and loss of cell polarity [16]. However, despite these recent findings, much remains unknown about the roles of Dlg-1 and Scrib in mammalian embryogenesis and in the maintenance of epithelial tissue structure and function in postnatal animals.

To begin to explore the role that these proteins might play in the growth and differentiation in mammalian model systems, we have chosen to look at the distribution of Dlg-1 and Scrib in the mouse eye. Many of its component parts such as lens, cornea, iris, ciliary body, and pigmented retinal epithelium contain epithelial tissue. Within these structures, the arrangement of junctional complexes and the major proteins associated with these junctions have been described, providing a context for evaluating the possible association of PDZ proteins with these structures. In addition to their role in epithelia, Dlg-1 and Scrib have also been shown to play important roles in neurons as synapse associated proteins [17,18], and the neural retina contains several different types of neurons and supporting glia.

Of particular interest is the lens because it is comprised entirely of epithelial cells derived from a single embryonic origin, the head ectoderm. Prior work has established that tight junctions are found only in the central epithelium, while adherens and gap junctions can be found in both the epithelium and fiber cells [19,20]. A specialized adhesion structure, called the basal membrane complex has been described and is presumed to be involved in attaching the fiber cells at their basal tips to the lens capsule [21,22]. Within the lens, E-cadherin is found specifically in the epithelium [23] whereas N-cadherin is found in both epithelium and fiber cells [19,24]. N-cadherin, the major adhesion protein found in the adherens junctions in the lens [19], has been suggested to be required for the switch from a proliferating epithelial cell to a postmitotic fiber cell in chick lens epithelial cultures [25]. Recently, it was reported that in the mouse lens ZO-1 is widely distributed. ZO-1 was found to be concentrated across the entire epithelial-fiber interface, the zonula adherens in the apical domain of the fiber cells, in the posterior tips of the fibers at the interface with the adhesions to the lens capsule and with the gap junction proteins, Cx46 and Cx50 [26]. Determining whether Dlg-1 and/or Scrib co-localize with these cell adhesion proteins in specific regions of the eye may provide some clues towards understanding their role in vivo in mammalian systems.

Having established previously that Dlg-1 and Scrib are expressed in the mouse lens [27], we now characterize their distribution within the various structures of the mouse eye and their overlap with E-cadherin, N-cadherin, and ZO-1. The results show that Dlg-1 and Scrib overlap in part with E-cadherin, N-cadherin, and ZO-1 in the epithelial tissues of many ocular structures, suggesting that Dlg-1 and Scrib may be involved in regulating cell polarity and cell adhesion. However, the broader distribution of Dlg-1 and Scrib suggests that they may play more varied roles in cell adhesion, migration, and differentiation in mammalian epithelial tissues than has been ascribed to date for these proteins in Drosophila and Caenorhabditis.

Methods

Mice

The FVB mouse strain was used. Embryos were staged designating midday on the day of vaginal plug as day 0.5 in development. Animals were staged by designating the day of birth as neonate (neo) and subsequent days as P1, P2, etc. All experiments using mice conformed to the Public Health Service Policy on Humane Care 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 Medical School.

Immunofluorescence

Embryos and eyes from E13.5 P10 FVB mice were fixed in 10% buffered formalin or 4% paraformaldehyde overnight at 4 °C, transferred to phosphate-buffered saline (PBS), dehydrated in increasing concentrations of ethanol and oriented in paraffin. Samples were embedded for sectioning (at 5 μm) either parallel or perpendicular to the optical axis to show the lens fiber cells in longitudinal or transverse orientations, respectively.

For protein detection by immunofluorescence, tissue sections were deparaffinized in xylenes, rehydrated through graded ethanols, and treated with trypsin (Sigma, catalog number T-7168, St. Louis, MO) for 30 min at room temperature. Sections were blocked with 5% horse serum/PBS for 1 h at room temperature. Excess blocking solution was removed and sections were incubated with either 1:300 dilution of affinity purified anti-SAP97 (Dlg-1) [28] (anti-SAP97 antibody provided by J. Hell, University of Iowa, Iowa City, IA), 1:100 dilution of anti-hScrib (catalog number sc-11049; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), 1:100 dilution of anti-E-cadherin (catalog number 610181; Transduction Laboratories, Los Angeles, CA), anti-N-cadherin (catalog number 610920; Transduction Laboratories) or 20 μg/ml of anti-ZO-1 (catalog number 33-9100; Zymed Laboratories Inc., San Francisco, CA) antibody in blocking buffer for 1 h at room temperature. Sections were washed in PBS and then incubated as follows with secondary reagents in a darkened chamber. For detecting Dlg-1, sections were incubated with 1:1000 dilution of Alexa Fluor 568 goat anti-rabbit (Molecular Probes, Eugene, OR). For detecting Scrib, sections were incubated with 1:100 Alex Fluor 568 donkey anti-goat or FITC-conjugated donkey anti-goat (Molecular Probes; Jackson ImmunoReasearch, West Grove, PA). For detecting E-cadherin, N-cadherin and ZO-1, sections were incubated with 1:100 dilution of FITC-conjugated horse anti-mouse (Vector Laboratories, Burlingame, CA). Following incubation with secondary antibodies, the sections were washed in the dark with PBS and mounted in 50% glycerol/PBS/0.4% propylgallate. Sections were stored at 4 °C until viewing. Sections were viewed on a Nikon Diaphot 200 confocal microscope. Images were captured using BioRad 1024 software. No staining was observed when sections were incubated with no primary antibody or pre-immune serum (Dlg-1, Scrib, E-cadherin, and ZO-1) or preincubated with blocking peptide (N-cadherin).

Results

Distribution of Dlg-1 protein in the lens

To characterize the distribution of Dlg-1 in the lens during embryonic and postnatal development, immunofluorescence with an anti-SAP97 (Dlg) antibody was performed on longitudinally oriented eye sections from mice of various ages and the stained sections were viewed using confocal microscopy (Figure 1A,C,E,G; Figure 2A,F). Staining for Dlg-1 was found in the epithelium (e) in lenses from embryonic day (E) 13.5 (Figure 1A,C, arrowhead), E15.5 (Figure 1E, arrowhead), neonatal (Figure 1G, arrowhead), and postnatal day (P) 10 mice (Figure 2A,F, arrowhead). Strong staining for Dlg-1 frequently was found in the apical region of the fiber cells in lenses from P10 mice (Figure 2F, double arrowheads). Strong staining also was noted in the basal tips of the fiber cells at all ages (Figure 1A,C,E,G, arrows; Figure 2A,F, arrows) and in the hyaloid vessels in the embryonic stages (Figure 1A-F, asterisks). Also notable was an apparent reduction in or loss of staining in the posterior portion of the newly differentiating fiber cells in the P10 lens (Figure 2A,F, asterisks; Figure 3A, asterisks).

To determine if Dlg-1 overlapped with E-cadherin and/or N-cadherin, sections were double stained with antibodies against Dlg-1 and antibodies against E-cadherin or N-cadherin. Sections were viewed by confocal microscopy and the overlap in staining assessed using double imaging. Overlap in staining for Dlg-1 and E-cadherin (yellow) was observed in the basal and lateral cell membrane in the epithelium (Figure 2D, arrowheads) and in the transition zone (Figure 2E, "tz" and arrowhead). Overlap in the staining patterns for Dlg-1 and N-cadherin was observed along the basal and lateral cell surfaces in the epithelium (Figure 2I, arrowheads) and in the basal cell surfaces of the transition zone (Figure 2J, tz, arrowhead). Overlap in the staining pattern of Dlg-1 and N-cadherin also was seen in the apical region of the fiber cells (Figure 2H, double arrowheads), at the apical interface of the epithelium and fiber cells (Figure 2J, arrowheads), in the fiber cells along the lateral surfaces (Figure 2H, double arrowheads), and at the basal tips of the fiber cells where distinct points of overlap were prominent on the basolateral surfaces (Figure 2K, arrows). In the posterior, overlap of Dlg-1 and N-cadherin staining was observed (Figure 2L, arrow). N-cadherin was present on both short and long sides of the fiber cells; however, it appears more highly concentrated on the short sides [21]. To determine on which surface of the fiber cells Dlg-1 and N-cadherin overlapped, transverse sections were immunostained and viewed under confocal microscopy at high resolution. Figure 2M-P show the double staining for Dlg-1 and N-cadherin on transverse sections in the same four regions of the lens shown in longitudinal sections in Figure 2I-L. Dlg-1 and N-cadherin strongly overlapped (yellow) on the short sides of the fibers in the anterior (Figure 2M, arrowheads), transition zone (Figure 2N, arrowheads), posterior fibers (Figure 2O, arrowheads) and posterior (Figure 2P, arrowheads) and at the basal attachments to the capsule (Figure 2O,P, arrowheads). Also apparent was Dlg-1 staining on the long sides of fibers, which did not appear to overlap with N-cadherin staining (Figure 2O). Finally, Dlg-1 appeared to be both membrane-associated and cytoplasmic along the posterior capsule (Figure 2O,P), but only membrane-associated in a region just interior to the capsule. This region corresponds to the region marked by the asterisk by the asterisk in Figure 2A,D where the intensity of staining for Dlg-1 appeared to be greatly reduced. Further to the interior cytoplasmic Dlg-1 staining once again became apparent (not shown), explaining the increase in intensity of staining for Dlg-1 to the interior observed in low magnification images (Figure 2A,F; Figure 3A).

To determine if Dlg-1 overlapped with ZO-1, double immunofluorescence experiments were carried out first on longitudinal sections of the eyes from P10 mice. Overlap in staining for Dlg-1 and ZO-1 was observed on the apical interface between epithelial and fiber cells in the transition zone (Figure 3E, arrowhead). In the posterior fiber cells, there were distinct non-overlapping regions (Figure 3F,G). Blood vessels outside the posterior capsule stained strongly for ZO-1 (Figure 3F, arrowhead). It has been reported that in the outer cortex of the fiber cell compartment ZO-1 is more predominant on the short sides of the fiber cells, but in the midcortex ZO-1 was predominantly localized on the long sides of the fiber cells [26,29]. Since Dlg-1 was found on both short and long sides of the fiber cells, we assessed Dlg-1 overlap with ZO-1 at high resolution on transverse sections. In transverse sections, Dlg-1 and ZO-1 overlapped strongly at the interface between epithelial and fiber cells and on the short sides of the fibers in the outer cortex in the anterior, transition zone, posterior fibers, and posterior (Figure 3H-K, arrowheads). Abundant Dlg-1 was observed on the long sides of the fibers; however, overlap of Dlg-1 with ZO-1 on the long sides was not observed. Thus, Dlg-1 was widely distributed in the lens including, but not limited to, regions where Dlg-1 overlapped with cadherins and/or ZO-1.

Distribution of Dlg-1 in other ocular structures

The distribution of Dlg-1 in other ocular structures also was assessed by immunofluorescence. During embryogenesis, Dlg-1 was widely distributed in the developing eye (Figure 1A,E,G). In the eye of the P10 mouse, Dlg-1 staining was found in the inner epithelium of the ciliary body (Figure 4A,D, "cbie") and the iris (Figure 4G, "i"). Dlg-1 staining also was observed in the corneal epithelium (cep) but not in the stroma (cs) or endothelium (Figure 4B,E,H, "cen"). In the retina, Dlg-1 staining was observed in the outer plexiform layer (opl), the outer nuclear layer (onl), the external limiting membrane (elm), the photoreceptor segments (ps), and the retinal pigment epithelium (rpe) but not in the ganglion cell layer (gcl), inner plexiform layer (ipl), or inner nuclear layer (inl; Figure 4C,F,I). Thus, specific patterns of Dlg-1 staining were observed in multiple structures within the eye.

Double immunofluorescence was used to assess overlap of Dlg-1 with E-cadherin, N-cadherin and ZO-1 in these ocular structures. Dlg-1 and E-cadherin overlapped in the corneal epithelium (Figure 4B, arrow) and the rpe (Figure 4C, arrowheads). Staining for Dlg-1 and N-cadherin was observed in the retina, particularly in the opl, onl, and elm, although direct overlap was observed only in the elm (Figure 4F, arrowheads). Overlap in staining for Dlg-1 and ZO-1 was observed in the corneal epithelium (Figure 4G,H) and dorsal iris (Figure 4G). There appeared to be little or no overlap in staining of Dlg-1 and ZO-1 in the ciliary body epithelium or retina (Figure 4G-I). Thus, a very restricted pattern of overlap in localization of Dlg-1 with E-cadherin, N-cadherin, and ZO-1 was observed.

Distribution of Scrib protein in the lens

To characterize the distribution of Scrib during eye development, immunofluorescence with an anti-Scrib antibody was performed on eye sections from mice of various ages, and the stained sections were viewed using confocal microscopy. Scrib was found in both the epithelium and the fiber cell compartments of the lens throughout the developmental time window tested (Figure 1B,D,F,H; Figure 5A,F). Staining for Scrib was found on the basal surface of the epithelium (Figure 1B,D,F,H), the epithelial-fiber interface (Figure 1B,D, double arrowheads) and basal region of the fiber cells (Figure 1B,D,F,H; Figure 5A,F, arrows). Scrib also was observed in the lateral membranes of the fiber cells (Figure 5A,H,J,K). Staining for Scrib was observed in the hyaloid vessels in embryonic time points (Figure 1B,D,F, asterisks). Unlike Dlg-1, Scrib appeared to be tightly localized to the membranes (Figure 5I-K).

To determine if Scrib overlapped with E-cadherin or N-cadherin, longitudinal sections were double stained with anti-Scrib antibodies and anti-E-cadherin or N-cadherin antibodies and viewed by confocal microscopy. Overlap was assessed using double imaging. Scrib overlapped with E-cadherin in the basal surface of the epithelial cells (Figure 5D, arrowhead) and this overlap extended into the transition zone (Figure 5E, arrowhead). Overlap in staining of Scrib and N-cadherin was observed in the basal surface of the epithelium (Figure 5I,J, arrowhead) and the apical surface interface of the epithelium and fiber cell compartment in the transition zone (Figure 5J, double arrowheads). Strong overlap in staining of N-cadherin and Scrib was seen in the basolateral surfaces of the fiber cells in the posterior region of the transition zone (Figure 5J, yellow just above asterisk) and the lateral surfaces of the fiber cells just adjacent to the basal surface in the posterior fiber region (Figure 5K, arrows). Notably, Scrib was present along the basal region of the fiber cells where they meet the lens capsule, just posterior to the transition zone while N-cadherin appeared reduced or absent from this region (Figure 5H,J, asterisks). In the extreme posterior, Scrib and N-cadherin staining overlapped along the basal surface, with a distinct band of N-cadherin just inside of the basal surface and another overlapping region just interior to the band of N-cadherin (Figure 5L). To determine on which side of the fiber cells Scrib and N-cadherin staining overlapped, transverse sections were double stained and viewed by confocal microscopy under high resolution (Figure 5M-P). Overlap was noted at the on the short sides of the fibers in the anterior (Figure 5M, arrowheads), transition zone (Figure 5N, arrowheads), posterior fibers (Figure 5O, arrowheads) and posterior (Figure 5P, arrowheads). Overlap also was noted at the basal attachments to the capsule (Figure 5O,P, arrowheads). Scrib appeared to be restricted to the short sides of the fiber cells where it overlapped with N-cadherin, except in the immediate region of attachments to the capsule (Figure 5O,P). Also, staining for Scrib was observed in the blood vessels on the outside of the posterior lens capsule (for example, Figure 5O, arrow).

To assess overlap of Scrib with ZO-1, double immunofluorescence experiments were carried out initially on longitudinal sections of eyes from P10 mice. Overlap in staining for Scrib and ZO-1 was seen at the apical surface of the epithelium in the transition zone (Figure 6C,E, arrowheads), and punctate overlap in staining was observed in the apico-lateral borders of the cells in the central epithelium (Figure 6D, arrowheads). A punctate overlap of Scrib and ZO-1 also was observed in the basolateral region of the fiber cells in the posterior (Figure 5G, arrow) with regions of non-overlap just to the interior of the lens. Overlap in staining for Scrib and ZO-1 also was observed in blood vessels outside the lens capsule (Figure 6F,G, arrowheads). To more completely assess overlap in staining of Scrib and ZO-1. immunofluorescence was carried out on transverse sections of eyes from P10 mice and viewed at high resolution. Overlap in staining for Scrib and ZO-1 was observed in the outer cortex on the short sides of the fibers in the anterior, transition zone, posterior fiber, and posterior regions, and the overlap faded to the interior (Figure 6H-K, arrowheads). Scrib and ZO-1 also overlapped intensely at the interface between epithelial and fiber cells in the anterior and transition zone (Figure 6H,I, arrowheads) and at the interface between the fibers and lens capsule in the posterior regions (Figure 6J,K, arrowheads). Thus, Scrib staining was widespread throughout the lens and specific regions of overlap with E- and N-cadherin and ZO-1 were observed.

Distribution of Scrib in other ocular structures

The distribution of Scrib in other ocular structure was assessed by immunofluorescence. Like Dlg-1, Scrib was widely distributed in the developing eye (Figure 1B,D,F,H). In the P10 mouse eye, staining for Scrib was seen both the inner and outer epithelia of the ciliary body (Figure 7A). Strong staining for Scrib was in the anterior epithelial layer of the iris with weaker staining in the posterior layer (Figure 7D). In the cornea, Scrib staining was observed in the epithelium (Figure 7D) and endothelium (Figure 7B,H). In the retina, staining for Scrib was seen in the ganglion cell layer (gcl) where it was very prominent in the blood vessels (Figure 7C,I, arrows), the inner plexiform layer (ipl), the inner nuclear layer (inl), the outer plexiform layer (opl), the external limiting membrane (elm) and the retinal pigment epithelium (Figure 7C,F,I, "rpe"). Thus, specific patterns of Scrib staining were observed in multiple structures within the eye.

Double immunofluorescence was used to characterize overlap between Scrib and E-cadherin, N-cadherin, and ZO-1. Overlap between Scrib and E-cadherin was found in the ciliary body (Figure 7A, "cboe") and corneal epithelium (Figure 7B, "cep"). Very little, if any, overlap of Scrib with N-cadherin was observed in the retina (Figure 7F). Some overlap of Scrib with ZO-1 also was seen in the blood vessels in the iris (Figure 7G, arrowheads), in the corneal endothelium (Figure 7H, arrowheads), and retinal ganglion cell layer (Figure 7I). Thus, specific regions of overlap of Scrib with cadherins or ZO-1 were found; however, the extent of the overlap was less than the overlap in the lens.

Co-localization of Dlg-1 and Scrib

In Drosophila, Dlg and Scrib co-localize on the membrane just basal to the septate junction [7]. To determine if Dlg-1 and Scrib are co-localized in the lens, double immunofluorescence analysis of the pattern of Dlg-1 and Scrib was carried out (Figure 8). Double immunofluorescence showed that staining for Dlg-1 and Scrib overlapped extensively (Figure 8). The overlap was particularly prominent along the apical and basal surfaces of the epithelium (Figure 8C, arrowheads), on the fibers in the posterior fiber region (Figure 8D, arrowhead), at the basal region of the fiber cells at the posterior capsule (Figure 8A,D, arrows) and at the posterior suture (Figure 8E, arrow). Overlap in staining for Dlg-1 and Scrib was found in the corneal epithelium, the iris, the ciliary body, and the gcl, opl, elm, and rpe in the retina (Figure 8A). Thus, there was extensive overlap of Dlg-1 and Scrib throughout the eye, although there also were regions where one but not the other was observed.

Discussion

Cell-cell adhesion, cell shape, and cell polarity are defining features of epithelial cells and tissues. In Drosophila, the establishment and maintenance of epithelial cell shape, polarity, and adhesion is dependent on the localization of specific PDZ-domain containing proteins, such as Dlg and Scrib, just basal to the septate junction. In this study, we showed that during both embryonic and postnatal development, high concentrations of Dlg-1 and Scrib are localized in regions known to contain vertebrate apical junctional complexes in various parts of the eye where they frequently overlapped with E-cadherin, N-cadherin, or ZO-1. However, Dlg-1 and Scrib also co-localized with N-cadherin and ZO-1 in the basal and lateral regions of the lens. We also showed that there are regions where Dlg-1 and Scrib do not overlap with each other or with these adhesion proteins. These data are summarized in Table 1. Based on co-localization of Dlg-1 and Scrib with cadherins and ZO-1, proteins known to be important adhesion factors, our data support the possibility that Dlg-1 and Scrib play a role in cell adhesion and polarity throughout the eye through their capacities as molecular scaffolds, a role conserved cross-species. However, the broader distribution of Dlg-1 and Scrib suggests that they may play additional roles in ocular biology.

Dlg-1 and Scrib co-localize with adhesion proteins in the lens

Prior work in Drosophila has shown that Dlg and Scrib are localized just basal to the septate junctions. In mammalian cells in culture Dlg-1 has been shown to be associated with E-cadherin, implicating its localization at the zonula adherens junction, and Scrib has been localized to tight junctions. In this study we found high, overlapping concentrations of Dlg-1 and N-cadherin at the apical tips of the fiber cells, suggesting that Dlg-1 is associated with the fiber cell zonula adherens [20]. We found that Scrib and ZO-1 co-localized in a punctate pattern along the apico-lateral borders of cells in the central epithelium (Figure 6D) suggesting that Scrib is associated with ZO-1 at tight junctions [19]. However, the pattern of co-localization of Dlg-1 and Scrib with N-cadherin and ZO-1 is more complex than these initial observations in mammalian tissue culture cells would predict. Most notable was the overlap of Scrib and ZO-1 in the apical membranes of epithelial and fiber cells in the transition zone (Figure 6E) where tight junctions are not found. Dlg-1 and Scrib overlapped with N-cadherin and ZO-1 at the basal tips of fiber cells suggesting their presence in the basal membrane complex, a specialized lens adhesion structure [21]. Finally, Dlg-1 and Scrib co-localized extensively in regions where each overlapped with N-cadherin and ZO-1. This suggests that Dlg-1 and Scrib may function cooperatively and in concert with other adhesion proteins in several distinct adhesion complexes. Interestingly, we also observed regions where Dlg-1 and Scrib overlapped with each other but not with N-cadherin or ZO-1. Thus, it is possible that Dlg-1 and Scrib play a role together that is independent of cadherin and/or ZO-1 containing complexes.

It is striking that much of the overlapping regions of high concentration of N-cadherin and Dlg-1 or Scrib were found in regions associated with the differentiation process. Prominent overlap of Dlg-1 and Scrib with N-cadherin is observed in the anterior portion of the transition zone where epithelial cells withdraw from the cell cycle and begin their differentiation into fiber cells (Figure 2J, Figure 5J, Figure 8C). It has been postulated that the association between adherens junctions and the cytoskeletal network is strengthened in the transition zone and that this strengthening of the linkage is important for fiber cell differentiation [25]. The apical adherens junction in the fibers and the basal attachments of the fibers to the capsule have also been considered to be important for proper differentiation. In typical epithelia, E-cadherin links to the actin cytoskeleton via β and α catenin [30,31]. In lens fiber cells, N-cadherin overlaps with β- and α-catenin and f-actin along the short sides of the cells [21,29]. Our data showing that the overlap between N-cadherin, and Dlg-1, and Scrib is found on the short sides of the fibers (Figure 2M-P; Figure 5M-P) is consistent with the possibility that Dlg-1 or Scrib are perhaps involved in strengthening the linkages of N-cadherin to the cytoskeleton during fiber differentiation. That β-catenin contains a PDZ ligand at its C terminus [32] suggests a possible means through which Dlg-1 or Scrib might be involved in regulating the cadherin-actin linkage. Overlap of Dlg-1 and Scrib with ZO-1, itself a PDZ protein, also was noted in these same regions (Figure 3H-K; Figure 6H-K). It has been reported that ZO-1 interacts with cadherins, catenins, and actin in other cell types [33-35]. The presence of ZO-1 on the short sides of the lens fibers in the outer cortex [26] where N-cadherin and catenins also are known to concentrate, suggests that ZO-1 may contribute to the formation of the strong adhesions proposed to be necessary for proper lens formation. The overlap of Dlg-1, Scrib, ZO-1, and N-cadherin in the apical regions of the fiber cells, in the transition zone and along the posterior capsule suggests that the three PDZ proteins may contribute to the formation of strong adhesion junctions and to the cadherin-associated downstream signaling cascades that may play a role in differentiation. Also striking is the overlap of ZO-1 with Dlg-1 and Scrib at the epithelial-fiber interface. Again, it is possible that these three PDZ proteins together contribute to the adhesion of the epithelial to fiber cells.

Interestingly, Dlg-1 was found on the long sides of the fiber cells, but did not overlap significantly on this face with N-cadherin or ZO-1. It is possible that Dlg-1 overlaps on this surface with other adhesion factors not known to overlap with N-cadherin or ZO-1, such as ezrin. Ezrin is known to be present on both short and long sides of lens fibers and its overlap with adhesion proteins other than cadherins especially on the long sides has led to the hypothesis that additional adhesion complexes exist and are important in the lens [29]. In retinal pigment epithelial cells, it has been shown by immunostaining that ezrin and Dlg-1 co-localize [36]. Thus, it is possible that Dlg-1 may overlap in the lens, particularly on the long sides of the fibers, with ezrin and therefore be involved in a type of adhesion complex distinct from a cadherin-containing adhesion complex. This role would necessarily be unique to Dlg-1 as opposed to Scrib since we did not detect Scrib on the long sides of the fibers (Figure 5M-P).

Suggested that adherens junctions may be important in regulating lens cell proliferation and differentiation because interfering with N-cadherin function in chick lens epithelial cell cultures results in continued proliferation and inhibition of differentiation in response to differentiation inducers [25]. Dlg-1 and Scrib have been shown to be tumor suppressor proteins in Drosophila [1,6], and it is suggested that they may affect cell proliferation in vertebrates as well [37,38]. It is possible that in the transition zone, Dlg-1 or Scrib through their scaffolding capacities are involved in regulating for proper cell cycle withdrawal and differentiation. In support of this hypothesis, it has recently been demonstrated that transgenic expression in the lens epithelium of the E6 oncoprotein from human papillomavirus type 16, which is known to bind to a number of PDZ domain containing proteins including Dlg-1 and Scrib, results in failure of cell cycle withdrawal and differentiation of secondary fiber cells [27]. Presently, however, the mechanism through which cell adhesion molecules and Dlg-1 or Scrib would regulate the switch from proliferation to differentiation is not known.

Although we observed that Scrib localization within cells appears to be highly membrane specific, Dlg-1 was found in membranes, cytoplasm (Figure 2), and nuclei (Figure 6J). The human homolog of Dlg-1, hDlg has six alternatively spliced exons and the different splice forms are expressed in different tissues and have different subcellular localizations [39], and the antibody to Dlg-1 used in our study recognizes all these splice variants [28,39]. Also, it has been shown that the differential phosphorylation state of Dlg-1 is correlated with different subcellular localization [40]. The distribution of Dlg-1 to multiple subcellular compartments that we observed is consistent with both possibilities. This prediction is further supported by the presence of multiple bands on immunoblots of lens extracts (data not shown). Our evaluation of Dlg-1 localization on transverse sections shows that at least in the posterior, sometimes Dlg-1 is found in both membrane and cytoplasm and other times only associated with the membrane (Figure 2O-P). The functional significance of these different patterns of subcellular localization of Dlg-1 specifically in these regions is not understood presently.

Dlg-1 and Scrib co-localize with adhesion proteins in other ocular structures

Other ocular structures such as the cornea, ciliary body, iris, and retinal pigment epithelium are composed at least in part of epithelia. Within these epithelia, the localization of tight junctions and adherens junctions, as well as E- and N-cadherin and ZO-1 are well documented [41-45]. Scrib and ZO-1 overlapped in regions that are documented to have tight junctions, including the corneal endothelium (Figure 7H) [43] and the apical interface between the inner and outer ciliary epithelia [44]. Interestingly, although both ZO-1 and Scrib were found in the retinal pigment epithelium, which is known to have tight junctions [42], there was only minimal co-localization on the apical surface (Figure 7I), and Scrib was uniquely observed along the basolateral aspects of the cells as well. While the punctate overlap on the apical surface suggests some overlap in tight junctions, Scrib may be involved in cell adhesion complexes in addition to tight junctions in the RPE. Overlap of Dlg-1 and Scrib with E-cadherin was observed most notably in the corneal epithelium (Figure 4B; Figure 7B). The overlap between Dlg-1 and E-cadherin was extensive, perhaps reflecting a role in the adherens junctions that are known to be present [41]. However, the overlap between E-cadherin and Scrib is more confined to the extreme apical border. Conversely, there was very little overlap of Dlg-1 or Scrib with cadherins in the epithelia of the iris or ciliary body and retinal pigment epithelium. The extensive overlap of cadherin with Dlg-1 and Scrib in the corneal epithelium is similar to that observed in the lens. This may reflect the common embryonic origin of the corneal epithelium and lens (that is the surface ectoderm).

We found that Dlg-1 and Scrib are widely distributed in the neural retina (Figure 4C,F,I; Figure 7C,F,I) as is N-cadherin, although the PDZ proteins do not appear to co-localize with N-cadherin. Interestingly, the specific distribution patterns of Dlg-1 and Scrib across the various layers of the neural retina differed. Both were strong in the opl and the elm, However, Dlg-1 was prominent in the onl and opl, whereas Scrib was prominent in the gcl and inl. It is not surprising to find widespread expression of Dlg-1 and Scrib in the mouse retina. PDZ proteins are well known for their role in organizing supramolecular membrane complexes and associated intracellular signaling molecules at the postsynaptic membrane and maintaining synaptic plasticity [17,18]. Furthermore, certain PDZ proteins have been shown to be essential for phototransduction in the Drosophila retina, presumably through their capacity to organize these membrane complexes [17]. The lack of co-localization of N-cadherin with Dlg-1 or Scrib would be consistent with this role for the PDZ protein rather than a role in adhesion. The co-localization of Dlg-1 and Scrib in some regions of the retina suggest they act cooperatively while the unique aspect of each protein's localization suggests that each also has distinct functions. It appears that Dlg-1 and Scrib may, at least in part, be associated with specific subsets of neurons within the retina. Analysis of co-localization of Dlg-1 and Scrib with cell type specific markers will be required to determine what these specific associations might be.

Both Dlg-1 and Scrib were found commonly in the various blood vessels of the eye ranging from the hyaloid vessels present along the posterior surface of the lens in embryos (Figure 1), to the small vessels associated with the posterior lens capsule in postnatal mice (Figure 3F,G; Figure 5O; Figure 6F,G), to the blood vessels of other ocular structures such as the iris (Figure 7D,G) and retina (Figure 7C,F,I). Overlap between Scrib and ZO-1 (Figure 5J,O; Figure 6F,G; Figure 7G,I) was particularly evident, and to a much lesser degree some overlap between Dgl-1 and ZO-1 was seen. Given the presence of tight junctions in the vascular endothelium, it is likely that the overlap between ZO-1 and these other PDZ proteins is found in those tight junctions. Overlap between Dlg-1 and Scrib was also observed in the vasculature (Figure 8D,E) suggesting a coordinated role for these two proteins.

In sum, the investigation of Dlg-1, Scrib, and other proteins containing PDZ domains that appear to have roles in cell polarity, proliferation, and adhesion is an emerging area of investigation in mammalian biology. In this study, we have provided the first comprehensive analysis of Dlg-1 and Scrib distribution and overlap with proteins found in junctional complexes in structures of the mammalian eye. Our data suggest that Dlg and Scrib may not only have functions that are conserved across species, but also may participate in additional ways in the regulation of cell adhesion, growth, and differentiation in mammalian organ systems such as the eye. Future studies will provide a better understanding of the roles of these proteins in the mouse and address their functional similarities and distinctions in different organisms.

Acknowledgements

We thank Johannes Hell (University of Iowa, Iowa City, IA) for the anti-SAP97 antibody, and Kate Hyde for critical reading of the manuscript. This work was supported by NIH grant R01 EY09091 (AEG).

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