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Open Access

Peer-reviewed

Research Article

Requirement for Dlgh-1 in Planar Cell Polarity and Skeletogenesis during Vertebrate Development

  • Charlene Rivera ,

    Contributed equally to this work with: Charlene Rivera, Sara J. S. Simonson

    Current address: Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, North Carolina, United States of America

    Affiliation Department of Cell and Regenerative Biology, University of Wisconsin-Madison, Madison, Wisconsin, United States of America

    ⨯
  • Sara J. S. Simonson ,

    Contributed equally to this work with: Charlene Rivera, Sara J. S. Simonson

    Current address: Center for Commercialization, University of Washington, Seattle, Washington, United States of America

    Affiliation Department of Oncology, University of Wisconsin-Madison, Madison, Wisconsin, United States of America

    ⨯
  • Idella F. Yamben,

    Current address: Kelly Scientific Resources, Madison, Wisconsin, United States of America

    Affiliation Department of Cell and Regenerative Biology, University of Wisconsin-Madison, Madison, Wisconsin, United States of America

    ⨯
  • Shalini Shatadal,

    Affiliation Department of Cell and Regenerative Biology, University of Wisconsin-Madison, Madison, Wisconsin, United States of America

    ⨯
  • Minh M. Nguyen,

    Current address: Lillehei Heart Institute, University of Minnesota School of Medicine, Minneapolis, Minnesota, United States of America

    Affiliation Department of Cell and Regenerative Biology, University of Wisconsin-Madison, Madison, Wisconsin, United States of America

    ⨯
  • Maryline Beurg,

    Affiliation Institut National de la Santé et de la Recherche Médicale, Hôpital Pellegrin, Bordeaux, France

    ⨯
  • Paul F. Lambert,

    Affiliation Department of Oncology, University of Wisconsin-Madison, Madison, Wisconsin, United States of America

    ⨯
  • Anne E. Griep

    aegriep@wisc.edu

    Affiliation Department of Cell and Regenerative Biology, University of Wisconsin-Madison, Madison, Wisconsin, United States of America

    ⨯

Abstract

The development of specialized organs is tightly linked to the regulation of cell growth, orientation, migration and adhesion during embryogenesis. In addition, the directed movements of cells and their orientation within the plane of a tissue, termed planar cell polarity (PCP), appear to be crucial for the proper formation of the body plan. In Drosophila embryogenesis, Discs large (dlg) plays a critical role in apical-basal cell polarity, cell adhesion and cell proliferation. Craniofacial defects in mice carrying an insertional mutation in Dlgh-1 suggest that Dlgh-1 is required for vertebrate development. To determine what roles Dlgh-1 plays in vertebrate development, we generated mice carrying a null mutation in Dlgh-1. We found that deletion of Dlgh-1 caused open eyelids, open neural tube, and misorientation of cochlear hair cell stereociliary bundles, indicative of defects in planar cell polarity (PCP). Deletion of Dlgh-1 also caused skeletal defects throughout the embryo. These findings identify novel roles for Dlgh-1 in vertebrates that differ from its well-characterized roles in invertebrates and suggest that the Dlgh-1 null mouse may be a useful animal model to study certain human congenital birth defects.

Citation: Rivera C, Simonson SJS, Yamben IF, Shatadal S, Nguyen MM, Beurg M, et al. (2013) Requirement for Dlgh-1 in Planar Cell Polarity and Skeletogenesis during Vertebrate Development. PLoS ONE 8(1): e54410. https://doi.org/10.1371/journal.pone.0054410

Editor: Xin Zhang, Indiana University School of Medicine, United States of America

Received: June 20, 2012; Accepted: December 13, 2012; Published: January 22, 2013

Copyright: © 2013 Rivera et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: This work was supported by NIH grants EY09091 (AEG), CA908428 (PFL/AEG). The University of Wisconsin Carbone Cancer Center (P30 CA14520) provided shared service support for the project. The funders had no role in the study design, data collection and analysis, decision to publish or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

The development of specialized organs in vertebrates is tightly linked to the regulation of cell growth, apical-basal cell polarity and cell-cell adhesion during embryogenesis. In addition, the directed movements of cells and their orientation in the same direction within the plane of a tissue, termed planar cell polarity (PCP), appear to be crucial for the proper formation of the body plan. Of great interest has been to decipher the mechanisms involved in regulating these critical aspects of development. From studies in invertebrates it is known that certain PSD95/Dlg/ZO-1 (PDZ) domain containing proteins such as Discs-large (Dlg) play prominent roles in regulating apical-basal polarity [1], [2] while different PDZ proteins are part of a genetic network that regulates PCP [3]. Recent work in vertebrates supports the contention that the function of many polarity factors has been conserved cross-species. However, studies also suggest that certain factors have different or additional roles in vertebrates as compared to invertebrates [4]. In this study, we address the role of Dlgh-1, the mouse homolog of Drosophila dlg, in mouse development.

In Drosophila melanogaster, mutations in dlg and scrib, the gene encoding a second PDZ protein, Scribble, result in neoplastic overgrowth, multilayering and loss of cell shape in various epithelial tissues including the imaginal discs [1], the embryonic epidermis, and the follicular epithelia [2], suggesting a role for these factors in cell proliferation, adhesion and apico-basal polarity. In Drosophila and Caenorhabditis elegans, Dlg and Scrib are thought to be required for properly localizing and maintaining adherens junctions and apical determinants [1], [2], [5], [6], [7], [8], [9]. In Drosophila, Dlg localizes to the septate junctions where it forms a complex with Scrib and Lethal giant larvae (Lgl) [1], [2]. This Dlg/Scrib/Lgl complex antagonizes other PDZ complexes, thereby restricting adherens junctions and apical determinants, such as Crumbs, to the appropriate compartment [10]. Finally, the Partner of Inscuteable/Discs-large complex is required for planar polarity during the asymmetric division of the sensory organ precursor (pl) cell [11].

Dlg is a member of the membrane-associated guanylate kinase (MAGUK) family [12], and, as a scaffolding protein, is capable of assembling supramolecular complexes at specific sites within and proximal to the cytoplasmic membrane. Dlg may accomplish this by interacting with other molecules through its multiple protein-protein interaction domains, which include three PDZ domains, a src-homology 3 (SH3) domain, a band 4.1 domain, and a guanylate kinase homolog (GUK) domain [12]. Homologs of Drosophila Dlg are found in humans (hDlg), rat (SAP97), and mouse (Dlgh-1, also referred to as Dlg-1), and the high degree of conservation among its multiple domains suggests conserved function cross-species [13], [14], [15]. In mammals, Dlgh-1 is ubiquitously expressed and is found at sites of cell-cell contacts in epithelial and neuronal cells [16], [17], [18], [19], [20], [21]. It interacts with a wide variety of proteins, which include the tumor suppressors adenomatous poli (APC) [22] and protein tyrosine phosphatase and tensin homologue (PTEN) [23], Shaker-type K+ channel α-subunits [24], the G-protein coupled receptor homologue tumor endothelial marker 5 (TEM5) [25], and the MAGUK Lin-2/CASK [26]. In addition to interacting with endogenous cellular proteins, Dlgh-1 is also targeted by multiple human viral oncoproteins including the human papillomavirus (HPV) E6 [27], the adenovirus E4-ORF1 [28], and the human T cell leukemia virus type 1 (HTLV-1), Tax 1, [29]. These interactions have been implicated in the transforming and tumorigenic properties of the viral oncoproteins [27], [29], [30], [31], [32], [33].

The role of Dlgh-1 in mouse development has begun to be explored. Caruana and Bernstein [34] reported that mice carrying a gene trap insertion in Dlgh-1 (Dlgh-1g/gtt mice), which results in a Dlgh-1 protein lacking the SH3, band 4.1, and GUK domains, exhibited a cleft palate and a shorter mandible than Dlgh-1 wild type mice. Additional studies on this Dlgh-1gt/gt mouse showed that these mice also exhibited a decrease in the number of nephrons in the developing kidney [35] and cell cycle misregulation in the epithelium of the ocular lens [36]. Since the L27 and three PDZ domains of Dlgh-1 were left intact in the gene trap allele [34], [37], it is possible that the fusion protein generated from this gene trap allele may retain some or acquire new activities of Dlgh-1 and, therefore, the phenotypes observed may not fully reflect the null phenotype. Consistent with this possibility, lacZ staining of Dlgh-1gt/gt lenses and control Dlgh-1+/+ lenses showed similar staining patterns (Rivera and Griep, unpublished observations). Furthermore, two recent reports show that deletion of Dlgh-1 in mice caused abnormalities in the ureteric smooth muscle [38] and absence of vagina and seminal vesicles [39]. Additionally, conditional deletion of Dlgh-1 in the lens led to more severe phenotype than was observed in the lenses of the Dlgh-1gt/gt mice including epithelial and fiber cell specific defects in cell adhesion and polarity [40].

In the present study, we generated mice carrying a germline null mutation in Dlgh-1 by deleting one of the exons encoding the first PDZ domain and characterized the wide-ranging effects of this mutation on mouse embryogenesis. Loss of Dlgh-1 not only recapitulated the craniofacial and kidney defects observed in the Dlgh-1gt/gt mouse but also resulted in reduced ossification in the skull, maxilla, and middle ear, and shortening of the long bones. Furthermore, deletion of Dlgh-1 resulted in eyelid closure and neural tube closure defects, and the misorientation of the stereociliary bundles of the cochlear hair cells, all of which suggest defects in PCP. Thus, our analysis identifies novel roles for Dlgh-1 in mouse development, places Dlgh-1 in the group of factors that regulate PCP in the mouse, and provides new insight into the distinct in vivo requirements for this gene in vertebrates, as compared to invertebrates.

Results

Generation of Dlgh-1 Null Mice

To generate Dlgh-1 null mice, mice carrying a mutant allele of Dlgh-1, in which exon 8 and a neomycin resistance-cassette are flanked by loxP sites [40] (Fig. 1A), were mated to EIIA-Cre transgenic mice [41]. The Dlgh-1 null allele was moved onto the FVB/NJ and C57BL/6J genetic backgrounds by backcrossing to either FVB/NJ or C57BL/6J mice for at least three generations before use. Mice heterozygous for the null allele were intercrossed to generate Dlgh-1−/− mice and Southern blot analysis was used to confirm the genotype of the progeny (Fig. 1B).

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Figure 1. Generation of Dlgh-1 null mice.

(A) Schematic representation of the targeting of the Dlgh-1 allele. WT, wild-type allele; C, construct, plasmid containing loxP sites flanking exon 8 of Dlgh-1 and neo cassette, and the TK cassette; F, the floxed allele allele, containing lox P sites flanking Dlgh-1′s exon 8 and the neo cassette; R, recombined allele, desired recombination event lacking exon 8 and neo. Abbreviations: 7, exon 7; 8, exon 8; 9, exon 9; P, PstI; triangles, lox P sequences; neo, neomycin; TK, thymidine kinase. 3′ probe for Southern blot analysis is denoted by black bars. Fragments expected from PstI digestion and 3′ probe hybridization are depicted by double-arrowed lines. (B) Southern blot analysis of PstI digested genomic DNA isolated from mice that were WT (+/+), heterozygote (+/−), or null (−/−) for the Dlgh-1 allele. Sizes of hybridizing bands are shown in kb. (C) Western blot analysis of Dlgh-1 protein levels. Protein lysates of brain, heart, lung, kidney, skin, eye, liver, and mouse embryo fibroblasts (MEF) from Dlgh-1 wild type (+/+), heterozygote (+/−), or null (−/−) mice were resolved by SDS-PAGE and immunoblotted with an anti-Dlgh-1 antibody, as described in Materials and Methods. Membranes were reprobed with an anti-β-actin antibody as a loading control.

https://doi.org/10.1371/journal.pone.0054410.g001

To determine if the mutated Dlgh-1 allele was indeed a null allele, Dlg-1 protein levels in brain, heart, lung, kidney, skin, liver, and eyes of E18.5 Dlgh-1+/+, Dlgh-1+/−, and Dlgh-1−/− mice were assessed by immunoblot analysis using an N-terminal specific anti-Dlgh-1 antibody. The Dlgh-1 protein levels were reduced by approximately 50% in the Dlgh-1+/− mice compared to the Dlgh-1+/+ mice and Dlgh-1 protein was not detected in any of the tissues analyzed from the Dlgh-1−/− mice or in mouse embryo fibroblasts (MEFs) derived from day E14.5 Dlgh-1 null embryos (Fig. 1C). These results confirmed that the Dlgh-1 mutant allele is a null allele.

Effect of Loss of Dlgh-1 on Mouse Embryogenesis

It has been reported that mice homozygous for the Dlgh-1gt allele die at or near the time of birth [12]. To determine if nullizygosity for Dlgh-1 also would be lethal, we intercrossed Dlgh-1+/− mice and examined litters for live Dlgh-1−/− pups. Of the 47 pups genotyped, 10 were Dlgh-1+/+ (21%), 22 (47%) were Dlgh-1+/−, and 15 (32%) were Dlgh-1−/−. Of the 15 Dlgh-1−/− pups, all of which exhibited severe craniofacial abnormalities, 13 were found dead on the day of birth and the remaining two died shortly after being found. These data indicate that loss of Dlgh-1 confers perinatal lethality with 100% penetrance and support the hypothesis that Dlgh-1 plays an important role in mouse development.

To understand the impact of the loss of Dlgh-1 on mouse development, Dlgh-1+/+, Dlgh-1+/−, and Dlgh-1−/− mice were collected at embryonic stages E11.5, E14.5, E16.5, and E18.5. Dlgh-1−/− embryos were viable at all embryonic stages examined. However, at the gross level Dlgh-1−/− embryos exhibited an abnormal head shape that included a shortened snout (Figs. 2A, H–I). Dlgh-1−/− mice were smaller than their Dlgh-1+/+ counterparts. This reduction in size was apparent overtly (Fig. 2A) and in histological section by E14.5 (Figs. 2B, C). At the gross anatomic level, Dlgh-1−/− mice also exhibited a secondary cleft-palate (Figs. 2D, E) that was consistent with the lack of a nasopharynx in these mutant animals observed in histological sections (Figs. 2F–I). All of these phenotypes of the Dlgh-1−/− mice were observed with 100% penetrance on both the FVB/NJ and C57BL/6J genetic backgrounds and were similar to those observed in the Dlgh-1gt/gt mice [34]. Additionally, the kidneys of the Dlgh-1−/− mice were smaller in size and exhibited a reduced tubule to mesenchyme ratio (data not shown), consistent with a defect in branching morphogenesis, which was reported for the previously described Dlgh-1 mutant mice [35], [38]. Finally, defects in the ocular lens were also noted in the Dlgh-1−/− mice (data not shown) as reported previously for the Dlgh-1gt/gt mice [36] and mice in which Dlgh-1 was specifically deleted in the lens [40]. Together, these data show that Dlgh-1 is required for mouse survival, likely in part through its contribution to palate formation, and for multiple aspects of mouse development.

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Figure 2. Phenotype of Dlgh-1−/− mice.

(A) Newborn Dlgh-1−/− mice are smaller then their Dlgh-1+/+ counterparts and have a shortened snout and mandible (arrows). (B–C) Hematoxylin and eosin stained paraffin sections of FVB/NJ E14.5 Dlgh-1+/+ (B) and Dlgh-1−/− (C) mice. At this developmental stage, the reduced size of the Dlgh-1−/− is noticeable. (D–E). Dissected heads of Dlgh-1+/+ and Dlgh-1−/− C57BL/6J E18.5 embryos showing the cleft palate (asterisk*) in Dlgh-1−/− mice. (F–G) Frontal sections of Dlgh-1+/+ (F) and Dlgh-1−/− (G) E18.5 embryos on the C57BL/6J background showing the absence of the nasopharyngeal passage (NP, arrow in F). (H–I) Sagittal H&E stained sections of E18.5 Dlgh-1+/+ (H) and Dlgh-1−/− (I) embryos on the FVB/NJ background showing the dome-shaped head of Dlgh-1−/− mice, the absence of the nasopharyngeal passage (NP), and the shortened snout and mandible (arrows). NC, nasal cavity; NP, nasopharyngeal passage; P, pharynx; T, tongue. Scale bar = 250 µm.

https://doi.org/10.1371/journal.pone.0054410.g002

Effect of Loss of Dlgh-1 on Eyelid and Neural Tube Closure

The cleft palate and reduced size of the Dlgh-1−/− mice (Fig. 2) were observed on both the FVB/NJ or C57BL/6J backgrounds. However, some defects were noted exclusively on the C57BL/6J background. First, eyelid closure defects were apparent in 100% of C57BL/6J Dlgh-1−/− mice at E18.5 (Fig. 3). The severity of this phenotype varied. In some cases the epidermis had extended and fused; however, the dermis had not filled in (Fig. 3E). In other cases, the epidermis had failed to extend, leading to a complete failure of eyelid closure (Fig. 3F). To determine if the effect of Dlgh-1 deficiency on eyelid closure may be a direct effect, head sections from Dlgh-1+/+ and Dlgh-1−/− mice were subjected to double immunofluorescence experiments using anti-Dlgh-1 and anti-Vangl2 antibodies (Fig. 4). Staining for Dlgh-1 was observed in the Dlgh-1+/+ eyelid epithelium (Fig. 4A, B) but was absent in the Dlgh-1−/− eyelid (Fig. 4D, E), suggesting that the open eyelid defect may be a direct result of loss of Dlgh-1 function.

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Figure 3. Dlgh-1−/− mice display defects in eyelid closure.

E18.5 Dlgh-+/+− and Dlgh-1−/− mice on the C57BL/6 background were collected and analyzed for the presence of closed eyelids. Dlgh-1+/+ mice (A) form closed eyelids while Dlgh-1−/− (B, C) littermates show defects in eyelid closure. (D–F) Hematoxylin and eosin staining of histological sections collected from E18.5 mice show completely closed eyelids in Dlgh-1+/+ mice (D) whereas eyelids in Dlgh-1−/− mice were only partially closed (E) or completely open (F). Insets show a higher magnification of the eyelids. Arrows show the position of the tips of the dermis in Dlgh-1+/+ (D) and Dlgh-1−/− (E) embryos, which failed to extend fully in the Dlgh-1−/− eye. Scale bar = 250 µm and 150 µm for insets. c, cornea; e, eyelids; l, lens.

https://doi.org/10.1371/journal.pone.0054410.g003

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Figure 4. Expression of Dlgh-1 and Vangl2 proteins in the eyelid of Dlgh-1+/+ and Dlgh-1−/− mice.

Sections of sagittally oriented, paraffin embedded heads from newborn Dlgh-1+/+ (A–C) and Dlgh-1−/− (D–F) mice on the C57BL/6J background were subjected to double immunofluorescence analysis with anti-Dlgh-1 (green) and anti-Vangl2 (red) antibodies. Shown are representative merged and unmerged images of the staining in the eyelid of Dlgh-1+/+ (A–C) and Dlgh-1−/− (D–F) eyelids. Dlgh-1 (A, B) and Vangl2 (A, C) localized to the membranes of the epithelium (arrow in A) including the site of eyelid fusion, in Dlgh-1+/+ mice. Staining for Dlgh-1 was absent in eyelid of the Dlgh-1−/− mice (E) and Vangl2 accumulated in the cytoplasm rather than in the membrane (D, F). e, epithelium; ep, epidermis; f, eyelid fusion. Scale bar = 50 µm.

https://doi.org/10.1371/journal.pone.0054410.g004

Interestingly, the failure of eyelid closure is also noted in the circletail (Crc/Crc) mouse, in which a defective Scrib protein lacking the C-terminal two PDZ domains is expressed [42], and in the looptail (Vangl2Lp/Lp) mouse, in which a defective Vangl2 protein carrying an amino acid substitution mutation (S464N) is expressed [43], [44]. The Vangl2 protein is normally expressed in the eyelid epithelium [43], [44], [45] and is primarily localized to the plasma membrane [45]. However, the mutant Vangl2 protein in Vangl2Lp/Lp mice fails to localize to the membrane; rather it is cytoplasmic [45]. Immunofluorescent staining of eyelids from Dlgh-1+/+ mice showed that Vangl2 was tightly localized to the membranes in the eyelid epithelium (arrow, Fig. 4A, C) where there was overlap with Dlgh-1 staining. In contrast, Vangl2 was cytoplasmic in the eyelid of the Dlgh-1−/− mice (Fig. 4D, F). Thus, the correct subcellular localization of Vangl2 in the eyelid epithelium depends on Dlgh-1.

Vangl2Lp/Lp and Crc/Crc mice also exhibit craniorachischisis [42], [44], [46], a severe neural tube closure defect in which the neural tube is open from the midbrain-hindbrain boundary to the tail. This same neural tube defect was observed in C57BL/6J Dlgh-1−/− mice at both E16.5 (Figs. 5C, D) and E18.5 (not shown). Unlike the open eyelid phenotype, however, craniorachischisis occurred with low penetrance; only 6.7% (2 out of 30) of the C57BL/6J Dlgh-1 null mice displayed this defect. Dlgh-1−/− mice that exhibited craniorachischisis also exhibited gastroschisis, an externalization of the gut (Fig. 5A, C). Closure of the eyelid and neural tube involves a process of polarized cell movement known as convergent extension [3], [47]. Thus, the defects in eyelid and neural tube closure exhibited in the Dlgh-1−/− mice suggest a new role for Dlgh-1 in the mouse in the regulation of convergent extension.

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Figure 5. A fraction of Dlgh-1−/− mice display defects in neural tube closure.

E16.5 Dlgh-1+/+ and Dlgh-1−/− embryos on the C57BL/6J background were collected, examined for overt defects, and photographed under a dissecting microscope. Profile (A) and dorsal (B) views of an E16.5 Dlgh-1+/+ embryo. Profile (C) and dorsal (D) views of an E16.5 Dlgh-1−/− embryo. In addition to craniorachischisis (C, D arrows), the Dlgh-1−/− embryo exhibits gastroschisis (C, arrowhead) and a rightward kink to the body axis (D).

https://doi.org/10.1371/journal.pone.0054410.g005

Effect of the Loss of Dlgh-1 on Cochlear Hair Cell Stereociliary Bundle Orientation

Recent studies in vertebrates have shown that convergent extension is regulated, at least in part, by a group of proteins that are involved in planar cell polarity (PCP) in Drosophila [3]. PCP refers to the polarization of cells within the plane of a cell sheet. In Drosophila, PCP regulates wing hair, body bristle, and eye ommitidial cluster orientation. In mammals, the uniform orientation of stereociliary bundles of the hair cells within the cochlea represents a distinctive example of PCP. In Vangl2Lp/Lp mice, the stereociliary bundles in the inner (IHC) and outer (OHC) hair cell layers are misoriented [4]. Similarly, in Crc/Crc mice the stereociliay hair bundles in the second and third row of OHCs are misoriented [4], Together, these data identify a role for Vangl2 and Scrib in the regulation of the PCP pathway in vertebrates.

Because dlg and scrib are known to interact in Drosophila [2] and because the open eyelid, neural tube, and gut phenotypes of the Dlgh-1−/− embryos (Fig. 4) resemble that of Crc/Crc mutant mice [42], we asked if stereociliary bundle orientation was affected in the Dlgh-1 null mice. Cochlear sensory epithelia (organs of Corti) from E18.5 Dlgh-1+/+ and Dlgh-1−/− embryos were stained with phalloidin to label hair cell sterocilia, a structure rich in filamentous actin, and the specimens analyzed by confocal microscopy. Normally, the stereociliary bundles are arranged uniformly around the primary cilia and oriented toward the outer edge of the organ. The IHC bundles are arranged in a curved shape while the OHC bundles are arranged in a v-shape [48]. The stereociliary bundles in the IHC row of the Dlgh-1−/− mice were uniformly polarized, as in the Dlgh-1+/+ mice (Fig. 6A, B). The stereociliary bundles in all three rows of OHCs of the Dlgh-1+/+ mice were uniformly oriented (Fig. 6A). However, many stereociliary bundles in the third row of OHCs in the Dlgh-1−/− cochleae were misoriented (Fig. 6B, arrows). Occasionally, stereociliary bundles in the second OHC layer were also misoriented (Fig. 6B). The angle of orientation of individual stereociliary bundles in the OHC3 layer relative to the neural-abneural axis in Dlgh-1+/+ and Dlgh-1−/− cochlea were measured and the degrees of deviation from this axis calculated (Fig. 6C). The average degrees of deviation for bundles in the Dlgh-1−/− mice (33.7+/−1.87 degrees) was significantly higher (p = 1.5×10−9) than in the Dlgh-1+/+ mice (17.3+/−0.87 degrees). To determine if the effect of Dlgh-1 deficiency on the cochlea might be a direct effect, head sections from day E15.5 Dlgh-1+/+ embryos were immunostained with an anti-Dlg-1 antibody (Fig. 6D). Immunoreactivity was observed in the epithelial cells of the duct, which give rise to the sensory cells, and cartilage within the cochlea, indicating the presence of Dlgh-1 protein in these structures. To further determine if the effect of Dlgh-1 deficiency on hair cell orientation might be a direct effect, cochlear explants from P2 control mice were subjected to immunofluorescence for Dlgh-1. Immunoreactivity was observed in the basal-lateral membrane domain of the outer hair cells (Fig. 6F, arrows). The expression of Dlgh-1 in tissues such as the eyelid and hair cells, which show defects in the Dlgh-1−/− mouse, together with the open eyelid, mislocalization of PCP protein Vangl2 in the open eyelid, craniorachischisis, and misorientation of cochlear stereociliary in the Dlgh-1−/− mice (Figs. 3, 4, 5, 6) suggest a role for Dlgh-1 in the regulation of PCP in the mouse.

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Figure 6. Dlgh-1−/− mice display defects in outer cell hair polarity.

(A, B) Representative images of the OHC and IHC layers in the cochlea of Dlgh-1+/+ and Dlgh-1−/− mice. The organ of Corti was isolated from the cochleae of E18.5 Dlgh-1+/+ (A) and Dlgh-1−/− (B) embryos, stained with phalloidin to visualize the actin bundles of the inner and outer hair cells, and viewed by confocal microscopy. Arrows indicate cells with misoriented stereociliary bundles in OHC3 row of the Dlgh-1−/− mice. (C) Measurement of misorientation of sterociliary bundles in OHC3 layer of Dgh-1+/+ and Dlgh-1−/− mice. The angle of sterociliary bundles in OHC3 of Dlgh-1+/+ and Dlgh-1−/− mice was measured in reference to a line parallel to the neural-abneural axis and perpendicular to the plane of the pillar cells. Plotted is the deviation in degrees from the neural-abneural axis for each OHC3 bundle measured in (4 cochleae from 3 mice of each genotype, 36 cells for Dlgh-1+/+ and 33 cells for Dlgh-1−/− mice). No deviation from the neural-abneural axis was assigned a value of 0°. The average angle deviation from 0° for OHC3 bundles in Dlgh-1−/− cochlea (33.7°+/−1.869, red bar) was significantly higher (p = 1.5×10−9) than that of the Dlgh-1+/+ (17.3°+/−0.869, red bar). (D, E) Immunohistochemical detection of Dlgh-1 protein in the cochlea of E15.5 mouse embryos. Transverse sections of paraffin embedded heads from control Dlgh-1+/+ mice were subjected to immunohistochemistry using (D) an anti-Dlgh-1 specific antibody and alkaline phosphatase detection or (E) no primary antibody. The pink color in the epithelium of the cochlear duct and cartilage in (D) is indicative of the presence of Dlgh-1 protein in these structures. (F) Immunofluorescent detection of Dlgh-1 protein in the cochlear hair cells of P2 mice control. Fixed cochlear explants from Dlgh-1+/+ mice were subjected to immunofluorescence using an anti-Dlgh-1 (green) antibody. Arrows show concentration of Dlgh-1 on the basal-lateral membranes of the OHC cells. IHC, inner hair cells, OHC1, 2, 3, outer hair cell rows 1, 2, and 3. c, cartilage in cochlea, d, cochlear duct. Scale bar = 50 µm.

https://doi.org/10.1371/journal.pone.0054410.g006

Effect of the Loss of Dlgh-1 in Skeletogenesis

The Dlgh-1gt/gt mice exhibited a cleft palate and a hypotrophic mandible [34], suggesting that Dlgh-1 may be required for the formation of at least certain craniofacial bones. To test this hypothesis, the skeletal frames of E18.5 Dlgh-1+/+, Dlgh-1+/−, and Dlgh-1−/− mice on both the FVB/NJ and C57BL/6J background were stained with alizarin red and alcian blue to visualize bone and cartilage, respectively. Dlgh-1−/− mice on both the FVB/NJ (Fig. 7) and C57BL/6J (not shown) genetic backgrounds had shorter mandibles (MA) and maxillas (MX), which was consistent with the short snout phenotype. However, uniquely on the FVB/NJ background, Dlgh-1−/− mice also showed additional skeletal defects. Dlgh-1−/− embryos exhibited reduced ossification of multiple craniofacial structures including the nasal bone (N), the retrotympanic process (RTP), and the tympanic bone (T) (Figs. 7A, C). Interestingly, the developing calvarium was also affected as the ossification of the frontal bone (F) and the parietal bone (P) were reduced or absent in the Dlgh-1−/− mice (Fig. 7C). Furthermore, the parietal bone was absent in one Dlgh-1+/− specimen on the FVB/NJ background, suggesting a dosage-dependent effect of Dlgh-1 on calvarium development (Fig. 7B).

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Figure 7. Dlgh-1−/− mice display defects in skeletogenesis.

Eviscerated carcasses of E18.5 Dlgh-1+/+, Dlgh-1+/−, and Dlgh-1−/− mice on the FVB/NJ background were prepared and stained for cartilage and bone with alcian blue and alizarin red, respectively. Craniofacial skeleton in (A) Dlgh-1+/+, (B) Dlgh-1+/−, and (C) Dlgh-1−/− mice. Arrows show loss of bone formation in Dlgh-1+/− and Dlgh-1−/− mice. F, frontal bone; IP, intraparietal bone; P, parietal bone; N, nasal; Ro, rostral bone; RTP, retrotemporal process; T, temporal bone. (D–G) Axial structures of Dlgh-1+/+ (D) and Dlgh-1−/− mice (E). Forelimb (F) and hindlimb (G) structures. Arrows show the absence of bone formation in Dlgh-1−/− mice. S, sternebrae; H, humerus; R, radius; U, ulna; F, femur; Fi, fibula; T, tibia. Scale bar in A-E = 1000 µm, in F = 650 µm and in G = 500 µm.

https://doi.org/10.1371/journal.pone.0054410.g007

The skeletal defects in the Dlgh-1−/− mice on the FVB/NJ background extended to a reduction in the ossification of the ribs, as well as the second, third, and fourth sternebrae (S) (Fig. 7E). The long bones of the fore- and hindlimbs were also shortened by approximately 20% and 18%, respectively, in the Dlgh-1−/− mice when compared to their Dlgh-1+/+ counterparts (Figs. 7F, G). Calcification in the phalanges, while apparent in the limbs of the Dlgh-1+/+ mice, was absent in the limbs of the Dlgh-1−/− mice (Figs. 6F, G, arrow). Taken together, these results suggest that Dlgh-1 is required for proper skeletogenesis of craniofacial, axial and long bones. Furthermore, as the calvarium forms through intramembranous bone formation whereas the other craniofacial bones and the axial bones form through endochondral ossification [49], these data show that both forms of bone development are affected when Dlgh-1 is ablated in the mouse.

Discussion

The MAGUK protein Dlgh-1, which is highly conserved cross-species, has emerged as an important factor in the regulation of cell-cell adhesion, apical-basal polarity, and cell proliferation in Drosophila [1], [2]. The regulation of these cellular aspects is critical for the establishment of specialized organs during embryogenesis. In the current study, we assess the role of Dlgh-1 in mouse embryogenesis (see Table 1 for a summary of phenotypes). We provide evidence that loss of Dlgh-1 affects PCP during mouse embryogenesis. We further provide evidence that loss of Dlgh-1 causes defects in skeletal structures arising through both endochondral and intramembranous ossification. These findings demonstrate newly discovered roles for Dlgh-1 in vertebrate development and suggest that the Dlgh-1−/− mouse may serve as an animal model to study major congenital birth defects in humans such as defects in neural tube closure and skeletal malformations.

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Table 1. Summary of Phenotypes in Dlgh-1 Null Mice.

https://doi.org/10.1371/journal.pone.0054410.t001

Role of Dlgh-1 in Mouse Organogenesis

The results presented in this study suggest that Dlgh-1 plays a vital role during mouse organogenesis. Defects in the mandible, the kidneys, and the lens have been previously described in the Dlgh-1gt/gt mice [34], [35], [36]. The formation of these structures and organs was likewise affected in our Dlgh-1 null mice (Fig. 2 and data not shown). More recently, novel defects in the ureteric smooth muscle and the urogenital tract have been observed in Dlgh-1−/− mice [38], [39]. In the present study, we provide evidence for Dlgh-1 playing a role in the skeletogenesis of trunk and limb structures, in neural tube and eyelid closure, and in the organization of the stereociliary bundles in the cochlea. These latter phenotypes were not reported previously in mice carrying the gene trap insertion in Dlgh-1 [34] and we have not observed open eyelids or open neural tubes in C57BL/6 Dlgh-1gt/gt mice in our laboratory (M.M. Nguyen, C. Rivera, and A.E. Griep, unpublished observations). As the N-terminal portion of Dlgh-1 including the L27 and three PDZ domains are intact in the Dlgh-1gt fusion protein [34], [37], it is likely that the function of one or more of these protein-protein interaction domains is required for Dlgh-1′s role in the novel phenotypes we have observed.

Role of Dlgh-1 in Planar Cell Polarity

Planar cell polarity is the mechanism through which the orientation of cells is coordinated within the plane of a cell sheet. In vertebrates, PCP has been suggested to be required for several developmental processes that involve convergent extension, such as lengthening of the body axis, eyelid closure, neural tube closure, branching morphogenesis in the kidney and lung, and the sterociliary cell hair bundle orientation in cochlea [3]. Dlgh-1−/− mice, with variable penetrance, exhibited a reduced body length to width ratio, open eyelids (Fig. 3), craniorachischisis (Fig. 5), and misorientation of stereocilia in the third row of cochlear OHCs (Fig. 6). Moreover, Dlgh-1 is expressed in the eyelid epithelium (Fig. 4A, B) and cochlear hair cells (Fig. 6F) and Dlgh-1 deficiency led to mislocalization of the core PCP protein, Vangl2, in the mutant eyelid (Fig. 4D, F). Thus, this study provides the first evidence in support of a role for Dlgh-1 in the regulation of PCP in the mouse in vivo.

Although much remains to be learned about the molecular and genetic mechanisms regulating PCP, from studies in the Drosophila eye and wing, factors including Frizzled (Fz), Dishevelled (Dsh), Strabismus/Van Gogh (stbm/Vang), and Starry night/Flamingo (Stan/Fmi) are thought to mediate PCP through non-canonical Wnt signaling [3]. This pathway appears to be highly conserved in vertebrates. Wnt signaling has been shown to be required for mediating the organization of cochlear OHCs into their distinctive unidirectional orientation during development [48], [50]. Fz3−/−;Fz6−/−, Dvl1−/−;Dvl2−/−, and Lp/Lp mice show reduced body length to width ratio, craniorachischisis, failure of eyelid closure, and misorientation of stereociliary bundles at the apices of sensory hair cells [4], [42], [51], [52], [53] Additionally, this small group of factors in vertebrates includes Celsr1 [54], a protocadherin and homolog of Stan/Fmi, and PTK 7, a protein tyrosine kinase [55].

Interestingly, Fz3, Fz6, and Vangl2 contain C-terminal PDZ binding motifs whereas Dvl1 and Dvl2 are PDZ domain proteins, suggesting that PDZ domain proteins and their ligands interact to play a major role in PCP. Dvl2 genetically interacts with Vangl2 and Vangl2 is required for the proper membrane localization of Dvl2 in the cochlear hair cells [52]. Vangl2 interacts with Fz3 and is required for targeting of Fz3 to the membrane of the cochlear hair cells [56]. It has recently been shown that these core PCP proteins also interact with other PDZ proteins in vertebrate systems. Fz4 and Fz7 have been shown to interact with the MAGUK protein, MAGI-3, through the PDZ binding motif of Fz and the first PDZ domain of MAGI-3 in vitro and in epithelial cells [57] and Dlgh-1 has been shown to interact through its PDZ domain with the C-terminal PDZ binding motif in Fz1, Fz4, and Fz7 at least in yeast two hybrid experiments [58], [59]. Mice defective for the PDZ protein Scrib (Crc/crc mice) exhibit the same phenotypes as mouse Fz, Dvl, and Vangl2 mutants. Scrib been shown to genetically interact with Vangl2 to modulate PCP in the cochlea [4] and Scrib binds to Vangl2 [56]. In this study, we have shown that the phenotype of Dlgh-1 null mice bears many similarities to the phenotypes of PCP mutant mice. Furthermore, we have shown that Dlgh-1 is expressed in the eyelid epithelium (Fig. 4A, B) and the outer hair cells of the cochlea (Fig. 6F) and is required for correct subcellular localization of Vangl2 in the eyelid (Fig. 4D, F). Therefore, we suggest that Dlgh-1 also is a member of this group of factors involved in the regulation of PCP in the mouse, which is has very recently been shown to include another mouse Dlg, Dlg-3 [60]. The role for Dlgh-1 in PCP in properly localizing Vangl2 to the membrane may be a critical function for PCP as Vangl2 has been shown to be required for proper localization of Fz3 [56] and Dvl2 [61], [62].

We observed differences in the phenotypes of Dlgh-1−/− embryos depending on the genetic background. On the C57BL/6J background, eyelid closure defects were present in 100% and craniorachischisis was present in 6.7% of Dlgh-1−/− embryos (Figs. 3, 5). Neither of these phenotypes was observed in Dlgh-1−/− embryos on the FVB/NJ background. The low penetrance of craniorachischisis in Dlgh-1−/− embryos on the C57BL/6J background is similar to observations of low penetrance of craniorachischisis in Dvl1−/−;Dvl2−/− mice on the C57BL/6J background. By contrast the penetrance of craniorachischisis is high on the 129 background [52]. The observation that the penetrance of some phenotypes associated with PCP is dependent on mouse genetic background suggests either that there are additional genetic modulators of PCP that have yet to be identified or that polymorphisms in one or more of the already identified genes on these different genetic backgrounds influences PCP.

Role of Dlgh-1 in Skeletogenesis

The cleft palate and hypotrophic mandible in the Dlgh-1gt/gt mice indicated that this PDZ domain protein might be important in the development of craniofacial skeletal structures [34]. The Dlgh-1−/− mice not only recapitulated the craniofacial defects observed in the Dlgh-1gt/gt mice, but also displayed reduced ossification of the maxilla, middle ear, frontal, and parietal bones and overall reduced size of the skeleton (Fig. 7).

One mechanism through which loss of Dlgh-1 may affect craniofacial development is through disruption of WNT/PCP signaling [63]. Recently, it has been shown that certain core Wnt/PCP proteins are expressed in cranial neural crest (CNC) cells [64], [65], which give rise to most of the skeletal structures of the head and neck, and these core PCP proteins are required for CNC migration [66]. As noted above, Dlgh-1 has been shown to bind to several Fz receptors, including those commonly associated with Wnt/PCP signaling [58], [59]. Likewise, Wnt5a, which has been suggested to be a regulator of Wnt/PCP signaling in the mouse [50], is expressed in outgrowing regions of the facial primordia and Wnt5a mutant mice display abnormally shaped head and shortened snout and mandible [67]. Finally, Wnt5a deficiency in mice results in cleft palate formation due to inhibition of the directional migration of cells that is required for palate formation [68]. Thus, the similarity of the craniofacial defects in Wnt5a−/−, Vangl2Lp/Lp, and Dgh-1−/− mice suggests that Dlgh-1 may be a regulator of craniofacial skeletogenesis through a mechanism that involves modulating Wnt/PCP.

The Dlgh-1−/− mice exhibited defects not only in skeletal structures of the head, but also of the trunk and limbs and the skeletons were reduced in size overall. In particular, the forelimbs and hindlimbs of Dlgh-1−/− mice were approximately 20% shorter than those of Dlgh-1+/+ mice and the humerus was clearly wider in the mutants as compared to controls (Fig. 6). One possibility is that the reduced size is due to an inhibition of proliferation, as Dlgh-1 has been associated with regulating proliferation [22]. However, Wnt/PCP also has been shown to play a role in limb development [69], [70]. Wnt5a mutant mice exhibit truncated limbs [67] and the directional outgrowth of the limb has been shown to require Wnt5a/JNK signaling in the distal mesenchyme [71], [72]. Similarly, Vangl2Lp/Lp mice display shorter and wider limbs [73]. Recent studies have demonstrated a genetic and mechanistic link between Wnt5a and Vangl2 through the frizzled-like receptor, Ror2 [73], which binds Wnt5a [74], clearly establishing a connection between Wnt5a and a core PCP protein. Thus, the similarities in the limb phenotypes of these mouse mutants and the Dlgh-1−/− mice combined with the known mechanistic link between Dlgh-1 and Vangl2 suggest that the mechanism through which Dlgh-1 exerts its effects may be through modulating Wnt/PCP.

Although, the most likely explanation for the defects in skeletogenesis in the Dlgh-1 mutant mice is that Dlgh-1 is required the Wnt/PCP-dependent development of these skeletal structures, analysis of mouse mutants has demonstrated that numerous other signaling pathways play a role in skeletogenesis. These include FGFR [75], [76], TGFβII [75], [77], BMP [78], and Wnt/β-catenin [79], [80], [81], [82]. Further studies will be required to determine if the mechanism through which Dlgh-1 affects bone formation is through an effect on Wnt/PCP and how Wnt/PCP signaling coordinates with these other signaling pathways to direct the proper formation of the skeleton.

In summary, we provide evidence of new roles for Dlgh-1 in the regulation of PCP and both endochondral and intramembranous bone formation in the mouse.

Materials and Methods

Ethics Statement

All procedures using mice conformed to the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health and the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. The protocol covering these studies (protocol #M00712) was approved by the Institutional Animal Care and Use Committee of the University of Wisconsin School of Medicine and Public Health (Animal Welfare Assurance #A3368-01).

Generation of the Dlgh-1 Null Mouse

The generation of the mice carrying the gene targeted Dlgh-1 conditional allele (Fig. 1A) has been described in detail previously (Rivera et al., 2009). To generate mice carrying a germline null mutation in Dlgh-1, female Dlgh-1fn/+ mice were mated to male EIIA-Cre transgenic mice [41] (Fig. 1). Removal of exon 8, which encodes a portion of the first PDZ domain of the Dlgh-1 protein, results in a frameshift mutation that generates a termination codon 3 codons downstream of the deletion. Male F1 progeny were mated to stock C57BL/6J or FVB/NJ females. Tail DNAs from the F2 progeny were screened by PCR for both the WT allele and the null allele using the Dlgh-1 5′ primer CATCATGGTTGAAGTGCTCTGGGC paired to the Dlgh-1 3′ primer GGAAGGAAACTCACGGATGGTCC (Fig. 1).

Gross and Microscopic Analyses

For examination of morphological defects at the gross level, newborn mice were examined for overt defects and photographed under a dissecting microscope. To examine palate formation, E18.5 mice were collected from pregnant dams and an incision made through the temporal-mandibular joint to expose the palate surface. Embryos were staged by designating the morning of the vaginal plug as day E0.5.

For histological analysis, E14.5, E16.5, and E18.5 embryos and neonates from Dlgh-1+/+, Dlgh-1+/−, and Dlgh-1−/− animals were fixed in 4% paraformaldehyde at 4°C for 2–3 days, dehydrated through graded alcohols and xylenes, and embedded in paraffin for transverse, coronal, and sagittal sectioning. E18.5 day embryos were decalcified prior to dehydration. Serial 5 µm paraffin sections were stained with hematoxylin and eosin and viewed by light microscopy. For detection of Dlgh-1 protein by immunohistochemistry, transverse oriented sections from E15.5 embryos were rehydrated and stained with an anti-SAP97 antibody (1∶500 dilution, obtained from J. Hell, University of Iowa), followed by Vectastain ABC-AP and Vector Red (Vector Labs) according to manufacturer’s instructions. For detection of Dlgh-1 and Vangl2 proteins by immunofluorescence, sagittal sections from paraffin embedded newborn Dlgh-1+/+ and Dlgh-1−/− mice were rehydrated, boiled in a rice cooker for 30 minutes in a solution of 10 mM Tris/1 mM EDTA/0.05% Tween-20 pH 9.0, blocked in 5% donkey serum diluted in 1X PBS, incubated with an anti-Dlgh-1 antibody (1∶1000 dilution, catalog number NBP1-48054, Novus Biologicals) and an anti-Vangl2 antibody (1∶100 dilution, catalog number sc-46560, Santa Cruz Biotechnology) overnight at 4°C. The next day, sections were washed and incubated with fluorescein conjugated horse anti-mouse and Alexa Fluor 568 conjugated goat anti-rabbit secondary antibodies for 1 hour at room temperature. Stained sections were viewed on a Zeiss Axioimager M2 microscope and images captured using Axiovision 4.8.2 software.

Cochlear Hair Cell Stereociliary Bundle Staining

The intact organ of Corti from E18.5 Dlgh-1+/+, Dlgh-1+/−, and Dlgh-1−/− embryos was isolated in Hank’s Balanced Salt Solution (HBSS, Gibco, Invitrogen) and fixed in 4% paraformaldehyde for 3 hours at 4°C. After initial fixation, the stria vascularis and tectorial membrane were removed and then the explants were fixed in 4% paraformaldehyde for overnight at 4°C. The fixed explants were permeabilized with 0.5% triton-X100 in HBSS for 30 minutes at room temperature, then stained with Alexa Fluor 568-phalloidin (1 µg/ml, Molecular Probes) for 1 hour, and finally mounted as a surface preparation on a glass slide. Stained sections were viewed on a Nikon Diaphot 200 confocal microscope and images were captured using BioRad 1024 software. The orientation of individual stereociliary bundles was determined relative to a line parallel to the neural-abneural axis and perpendicular to the row of pillar cells, as described by Montcouquiol et al. [4]. The deviation from the neural-abneural axis was measured in degrees with a protractor. A cell with the vertex of the sterociliary bundle parallel to the neural-abneural axis was assigned 00 deviation. A total of 4 cochleae from 3 different mice of each genotype were analyzed. The data were subjected to statistical analysis using the two-sided Wilcoxon Rank Sums test and p<.05 was considered statistically significant. For detection of Dlgh-1 in the hair cells by immunofluorescence, fixed explants were immersed in 150 mM Tris-HCl, pH 9.0 at 65°C for 15 minutes followed by acetone treatment at −20°C for 20 minutes. After washing, explants were blocked in 10% serum diluted in 0.5% Triton X-100 in PBS for 2 hours at room temperature. Explants then were incubated with anti-Dlgh-1 antibody (1∶500 dilution, Novus Biologicals) overnight at 4°C followed by incubation with fluorescein conjugated horse anti-mouse secondary antibody for 2 hours at room temperature. Immunostained explants were viewed and photographed as described above.

Immunoblot Analysis

Whole tissues and MEFs were lysed in RIPA Buffer [1% NP-40, 0.1% sodium dodecyl sulfate (SDS), 0.5% deoxycholatate sodium (DOC), 150 mM NaCl, 50 mM Tris, pH 8.0] and centrifuged at 13,200 rpm for 15 minutes at 4°C. Protein concentrations in cell extracts were determined by the bicinchoninic acid (BCA) assay. Fifty µg of RIPA-soluble lysate from E18.5 Dlgh-1+/+, Dlgh-1+/−, and Dlgh-1−/− animals were run on a 7.5% acrylamide gel and transferred to a PVDF membrane. The blots were blocked in 5% nonfat dry milk 1X PBS containing 0.1% Tween-20 and then incubated with mouse anti-Dlgh-1 (catalog number 610874, BD Transduction Laboratories), which was raised against amino acids 5–213 of human Dlgh-1 (amino acids upstream of the first PDZ domain), for 3 hours at room temperature in block solution. After washing and incubation with HRP-coupled goat-mouse (Pierce), detection was performed with ECL plus (Amersham). Blots were reprobed with mouse anti-β-actin (Sigma) as loading control.

Skeletal Staining

Eviscerated carcasses from E18.5 Dlgh-1+/+, Dlgh-1+/−, and Dlgh-1−/− animals were fixed in 95% ethanol for 5 days at room temperature. After fixation, samples were cleared in 1% KOH for 2 days at RT. Samples were stained in 0.5% alcian blue 8GS (in 70% ethanol, 5% glacial acetic acid) and 0.2% alizarin red S solution (95% ethanol, 5% glacial acetic acid) for 5 days at room temperature. Samples were destained in 1% KOH for 2 days at room temperature and in graded glycerol/1% KOH at room temperature.

Acknowledgments

We thank the McArdle Laboratory Histology Core for tissue processing and the Keck Facility for confocal microscopy. We thank Heidi Rosenbaum and Malinda Waldof for technical assistance, Ruth Sullivan for helpful discussions on the histopathology of the Dlgh-1−/− mice, and Robert Fettiplace for helpful discussions and insight.

Author Contributions

Conceived and designed the experiments: CR SJSS IFY SS MMN MB PFL AEG. Performed the experiments: CR SJSS IFY SS MMN MB. Analyzed the data: CR SJSS SS MB PFL AEG. Wrote the paper: CR SJSS IFY SS MMN MB PFL AEG.

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