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Retinal Cell Biology  |   March 2010
Defective Formation of the Inner Limiting Membrane in Laminin β2- and γ3-Null Mice Produces Retinal Dysplasia
Author Affiliations & Notes
  • Germán Pinzón-Duarte
    From the Departments of Cell Biology and
  • Gerard Daly
    From the Departments of Cell Biology and
  • Yong N. Li
    the Graduate Program in Molecular Cellular and Developmental Biology, Sackler School of Graduate Biomedical Sciences, Tufts University, Boston, Massachusetts; and
  • Manuel Koch
    the Institute for Oral and Musculoskeletal Biology, Center for Biochemistry and Center for Molecular Medicine Cologne, University of Cologne, Cologne, Germany.
  • William J. Brunken
    From the Departments of Cell Biology and
    Ophthalmology, State University of New York, Downstate Medical Center, Brooklyn, New York;
    the Graduate Program in Molecular Cellular and Developmental Biology, Sackler School of Graduate Biomedical Sciences, Tufts University, Boston, Massachusetts; and
  • Corresponding author: William J. Brunken, SUNY Downstate Medical Center, Department of Cell Biology, 450 Clarkson Avenue, Box 5, Brooklyn, NY 11203; william.brunken@downstate.edu
Investigative Ophthalmology & Visual Science March 2010, Vol.51, 1773-1782. doi:10.1167/iovs.09-4645
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      Germán Pinzón-Duarte, Gerard Daly, Yong N. Li, Manuel Koch, William J. Brunken; Defective Formation of the Inner Limiting Membrane in Laminin β2- and γ3-Null Mice Produces Retinal Dysplasia. Invest. Ophthalmol. Vis. Sci. 2010;51(3):1773-1782. doi: 10.1167/iovs.09-4645.

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      © 2015 Association for Research in Vision and Ophthalmology.

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Abstract

Purpose.: Retinal basement membranes (BMs) serve as attachment sites for retinal pigment epithelial cells on Bruch's membrane and Müller cells (MCs) on the inner limiting membrane (ILM), providing polarity cues to adherent cells. The β2 and γ3 chains of laminin are key components of retinal BMs throughout development, suggesting that they play key roles in retinal histogenesis. This study was conducted to analyze how the absence of both β2- and γ3-containing laminins affects retinal development.

Methods.: The function of the β2- and γ3-containing laminins was tested by producing a compound deletion of both the β2 and the γ3 laminin genes in the mouse and assaying the effect on postnatal retinal development by using anatomic and electrophysiological techniques.

Results.: Despite the widespread expression of β2 and γ3 laminin chains in wild-type (WT) retinal BMs, the development of only one, the ILM, was disrupted. The postnatal consequence of the ILM disruption was an alteration of MC attachment and a resultant disruption in MC apical–basal polarity, which culminated in retinal dysplasia. Of importance, although their density was altered, retinal cell fates were unaffected. The laminin mutants have a markedly decreased visual function, resulting in part from photoreceptor dysgenesis.

Conclusions.: These data suggest that β2 and γ3 laminin isoforms are critical for the formation and stability of the ILM. These data also suggest that attachment of the MC to the ILM provides important polarity cues to the MC and for postnatal retinal histogenesis.

Laminins are extracellular matrix (ECM) glycoproteins composed of α, β, and γ subunits, which assemble into a single cruciform molecule. Five α, three β, and three γ laminin chains have been identified, forming 16 different isoforms. 1 Laminins bind to a variety of cell surface receptors, including integrins, dystroglycan, sulfated glycolipids, and syndecans. Laminins are key nucleating elements of the highly organized ECM known as BMs. 2,3 BMs and their receptors are indispensable during development, providing key polarity cues and conferring structural stability for macromolecular complexes by coupling the cytoskeleton to the ECM. 4,5 Several human diseases, including those of the central nervous system (CNS), which includes the eye, are linked to disruptions in laminin expression or deposition. 610  
In the CNS, at least three laminin isoforms have been identified: α3β3γ2, α4β2γ3, and α5β2γ3. 11 Both the β2 and γ3 laminin chains are present in retinal BMs (i.e., Bruch's membrane and the ILM). The temporal and spatial localization of β2 and γ3 laminin chains during development make them good candidates to participate in retinal histogenesis as organizing factors responsible for compartmentalization. 11 Therefore, to assess the function of β2- and γ3-containing laminins in development, we produced β2−/−γ3−/− mice by gene ablation via homologous recombination, and we analyzed the retina. Retinal disruptions in the β2−/−γ3−/− mice phenocopy some of the abnormalities described previously in the β2−/−γ3+/+ animal, such as perturbations in synaptogenesis and outer segment formation. 12 However, there are significant new aspects of the retinal phenotype in the β2−/−γ3−/− retina that reveal roles for β2 and γ3 laminins in ILM synthesis and MC maturation. Specifically, the data presented suggest that β2- and γ3- containing laminins are critical for the proper assembly of the ILM and MC attachment to the ILM and that retinal lamination is dependent on adequate contact between the endfeet of MCs and an intact ILM. Disruptions in the integrity of the ILM lead to retinal dysplasia but not to alterations in terminal cell differentiation. 
Materials and Methods
Animals
The animal procedures were in accordance with the institutional animal care and use committee and the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Targeted mutations of the Lamb2 and the Lamc3 genes were produced to generate two independent lines of mice (β2−/−γ3+/+ and β2+/+γ3−/−, respectively). 13,14 These lines were crossed to generate homozygous, compound-null mutants (β2−/−γ3−/−). The animals were genotyped by PCR on genomic DNA derived from tail samples. 14 Mice with β2+/+γ3+/+, β2−/−γ3+/+, β2+/+γ3−/−, and β2−/−γ3−/− genotypes were examined. The β2−/−γ3−/− mice were smaller and died at a younger age, but did not display significant external morphologic defects; this failure to thrive is similar to that in the laminin β2-deficient mouse. 13 Before the tissue was collected, the animals were anesthetized with CO2 and decapitated. Dissected retinas were examined by conventional histology, immunohistochemistry, and electron microscopy. To prevent confounding interpretation of the data, we matched eccentricity. 
Conventional Light and Electron Microscopy
Postnatal day (P)7, P15, and P20 eyecups were immersed in ice-cold Karnovsky's fixative for 12 hours, postfixed in 2% glutaraldehyde and 1% osmium tetroxide for 1 hour, dehydrated, embedded in plastic, and sectioned. For an overview of the gross morphology, Nissl-stained 0.5-μm sections were used. Ultrathin sections were viewed and photographed on a transmission electron microscope (CM-10; Philips, Eindhoven, The Netherlands). 
Immunohistochemistry
P15 retinas were used to assay neuronal cell differentiation, BM integrity, laminin, and β-dystroglycan expression. Postnatal day (P)10, P15, and P20 retinas were used to characterize MC maturation. The retinas were immersed for 30 minutes in ice-cold 4% paraformaldehyde, washed, cryoprotected, embedded (O.C.T. Compound; Finetek USA, Torrance, CA), and frozen. Radial sections (10 μm) were collected and incubated with primary antibodies (Table 1). Immunoreactions were visualized with Alexa (488 or 568)-conjugated secondary antibodies (1:500; Molecular Probes, Eugene, OR) and photographed (Eclipse E800 microscope; Nikon, Tokyo, Japan; with the Volocity suite of programs; Improvision Ltd., Lexington, MA). 
Table 1.
 
Primary Antibodies
Table 1.
 
Primary Antibodies
Cell Type Antibody Host Source Concentration Reference
Used to identify retinal cells
    Rod photoreceptor Rhodopsin Clone RET-P1 monoclonal Sigma-Aldrich, St. Louis, MO 1:500 15
    Rod bipolar αPKC Clone MC5 monoclonal Chemicon, Temecula, CA 1:500 16
    Horizontal Calbindin-D-28K Mouse monoclonal Sigma-Aldrich 1:500 16
    Amacrine Calretinin Mouse monoclonal Chemicon 1:500 16
    Ganglion Brn3a Mouse monoclonal Santa Cruz, Santa Cruz, CA 1:500 17
    Müller Glutamine synthetase Goat polyclonal Santa Cruz 1:500 16
GFAP Clone G-A-5 monoclonal Chemicon 1:500 18
Used to detect basement membranes
Collagen IV Rabbit polyclonal Chemicon 1:100 19
Laminin β2 chain Rabbit polyclonal Our laboratory 1:5,000 11
Laminin γ3 chain Rabbit polyclonal Our laboratory 1:5,000 11
Nidogen Rabbit polyclonal Ulrike Mayer* 1:3,000 19
Pan-laminin Rabbit polyclonal Sigma-Aldrich 1:1,000 20
Perlecan Rat polyclonal Santa Cruz 1:100,000 21
Used to detect basement membrane receptors
β-dystroglycan Rabbit polyclonal Dominique Mornet† 1:250 22
Electroretinography
P18 wild-type (WT) and β2−/−γ3−/− mice were dark-adapted for several hours and, in dim red light, anesthetized with 20 mg/kg ketamine HCl (Ketaset; Fort Dodge Animal Health, Fort Dodge, IA), followed by 65 mg/kg pentobarbital sodium (Nembutal; Ovation Pharmaceuticals Inc, Deerfield, IL). In a light-tight Faraday cage, visual responses were tested with 50-ms flashes; 10 flashes presented at a frequency of 0.5 Hz (PS22 Photopic stimulator; Grass Instruments, Quincy, MA) were averaged at a given stimulus intensity. For each animal, at least one set of recordings was made over a 4.4-log range of intensities, starting with the least bright flash. The responses were amplified (DAM 50 differential amplifier; World Precision Instruments, Sarasota, FL), recorded, and averaged (MacLab 8sp; AD Instruments, Mountain View, CA). 
Cell-Surface Ligand Adhesion Assay
The rat Müller cell line (rMC-1, gift of Vijay P. Sarthy, Northwestern University, Evanston, IL), 23 is an SV40 large-T antigen–transformed cell line. After dissociation, single cell suspensions were plated onto different substrates (Table 2) in DMEM-F12 (Life Technologies Inc., Carlsbad, CA) medium in an atmosphere of 5% CO2–balanced air at 37°C. After 2 hours, the cells were fixed in 4% paraformaldehyde in PBS for 20 minutes. The number of cells attached to each substrate was determined after staining with 0.1% cresyl violet. 
Table 2.
 
Protein Substrates for Adhesion Assays
Table 2.
 
Protein Substrates for Adhesion Assays
Substrate Source Concentration
Recombinant full-length netrin-4 Our laboratory 10 μg/mL
Recombinant netrin-4 without C domain Our laboratory 10 μg/mL
Recombinant laminin β2 short arm Our laboratory 10 μg/mL
Engelbreth-Holm-Swarm sarcoma derived laminin Invitrogen, San Diego, CA 10 μg/mL
Poly-l-lysine Sigma-Aldrich, St. Louis, MO 100 μg/mL
Bovine serum albumin Sigma-Aldrich 40 μg/mL
Results
Laminin β2 and γ3 Chains in ILM Development
In P15 WT retinas, β2 laminin immunoreactivity was observed in Bruch's membrane, the retinal blood vessels, and very strongly in the ILM (Fig. 1A). γ3-Laminin immunoreactivity had a similar distribution, albeit with more limited apparent deposition in the ILM (Fig. 1C). As expected, no immunolabeling was found for either the β2 or the γ3 chain in the compound-null animal (Figs. 1B, 1D), demonstrating that these animals were protein nulls. Similar results were obtained with Western blot (data not shown). To evaluate how the ablation of β2 and γ3 genes affect BM integrity, we examined the expression of canonical BM components in WT and mutant retinas (Table 1). In WT retinas, nidogen staining was observed as a thick, continuous band along the vitreal surface (i.e., the ILM), under the RPE (Bruch's membrane), and in the vascular BMs (Fig. 2A). In the β2−/−γ3+/+ retinas, nidogen staining in the ILM was largely intact; however, some spatial discontinuities were noted (Fig. 2B). In the β2+/+γ3−/− retinas, the ILM was intact (Fig. 2C). In contrast, the β2−/−γ3−/− retina was severely disrupted (Fig. 2D). Nidogen deposition was intact in the central retina (i.e., adjacent to the optic nerve head) but was completely absent over most of the vitreal surface. BM components (Table 1) in the retinal periphery were seen only in association with blood vessels (Fig. 2D, arrows; compare with perlecan in Fig. 7), suggesting that the ILM was absent from large regions of the β2−/−γ3−/− retina. This effect was specific for ILM formation or stability, in that nidogen and perlecan deposition in other BMs, Bruch's membrane, vascular BMs, and extraocular muscle BMs (Fig. 2, arrowheads) was intact. 
Figure 1.
 
β2 and γ3 laminin expression was absent in mutant retinas. (A, C) P15 WT retina; (B, D) P15 laminin-deficient retina. (A) β2 immunoreactivity was seen in all retinal BMs: Bruch's membrane, vascular BM, and the ILM. (B) This reactivity was completely absent in the mutant retina. (C) Strong deposition of γ3 chain was found in Bruch's membrane and vascular BM, but it was weakly present in the ILM. (D) No γ3 expression was seen in the mutant retina. Scale bar, 50 μm.
Figure 1.
 
β2 and γ3 laminin expression was absent in mutant retinas. (A, C) P15 WT retina; (B, D) P15 laminin-deficient retina. (A) β2 immunoreactivity was seen in all retinal BMs: Bruch's membrane, vascular BM, and the ILM. (B) This reactivity was completely absent in the mutant retina. (C) Strong deposition of γ3 chain was found in Bruch's membrane and vascular BM, but it was weakly present in the ILM. (D) No γ3 expression was seen in the mutant retina. Scale bar, 50 μm.
Figure 2.
 
β2γ3 Laminin deletion produced differential disruption of the ILM. BMs were visualized with nidogen immunostaining in all genotypes (as indicated). (A) Nidogen staining was seen as a continuous band along both BM and the ILM; vascular BM was also labeled. The deposition of nidogen, in Bruch's membrane and the vascular BM, was intact in all genotypes. In contrast, the ILM organization was affected by laminin deletion. Nidogen deposition was largely normal in the γ3−/− retina (C). In the β2−/− retina, nidogen deposition was more punctate and there were limited regions of discontinuity (B). In the β2−/−γ3−/− animals, nidogen deposition was largely lacking, with limited plaquelike staining associated with vitreal blood vessels (D, arrows). The extraocular muscle BM was intact in all genotypes (arrowheads). Scale bar, 50 μm.
Figure 2.
 
β2γ3 Laminin deletion produced differential disruption of the ILM. BMs were visualized with nidogen immunostaining in all genotypes (as indicated). (A) Nidogen staining was seen as a continuous band along both BM and the ILM; vascular BM was also labeled. The deposition of nidogen, in Bruch's membrane and the vascular BM, was intact in all genotypes. In contrast, the ILM organization was affected by laminin deletion. Nidogen deposition was largely normal in the γ3−/− retina (C). In the β2−/− retina, nidogen deposition was more punctate and there were limited regions of discontinuity (B). In the β2−/−γ3−/− animals, nidogen deposition was largely lacking, with limited plaquelike staining associated with vitreal blood vessels (D, arrows). The extraocular muscle BM was intact in all genotypes (arrowheads). Scale bar, 50 μm.
Disruptions in the ILM in the β2−/−γ3−/− Retina
In the first postnatal week, although the general organization of the β2−/−γ3−/− and WT retinas was similar (Figs. 3A, 3B), many abnormalities were evident in the β2−/−γ3−/− retinas. In the outer retina, the OPL was thinned and the photoreceptor inner/outer segments were shorter. The inner plexiform layer (IPL) was considerably thinned (Fig. 3B), and the retinal ganglion cell (RGC) layer was disorganized, with many ectopic cells extruding into the vitreal cavity (Fig. 3B, arrows). During the second postnatal week, retinal lamination was progressively disrupted (Figs. 3C, 3D). Although large regions of the P15 mutant retinas appeared to have the normal laminar organization, the relative thickness of the different layers differed considerably across the retina. In addition, focal abnormalities with different degrees of severity were seen. The most prominent was the formation in the outer nuclear layer of photoreceptor rosettes that very often disrupted the subjacent layers (Fig. 3D). Also, ectopic photoreceptor cells were present in the subretinal space; their presence was spatially coupled to the disruptions of the OLM (see Fig. 8). In the INL, rosette-like clusters of cells composed of interneurons were found in every retina and were associated with ILM breakdown (not shown). 
Figure 3.
 
The retina became progressively dysplastic in the first 2 postnatal weeks. Radial sections through WT (A, C) and mutant retinas (B, D) at P7 (A, B) and P15 (C, D). At P7, the mutant retina showed some sign of developmental disruptions. The OPL was underdeveloped as were the outer and inner segments, the IPL thickness was reduced, and the RGC layer was disrupted, with numerous ectopic cells in the vitreous (arrows). At P15, retinal lamination was disrupted in discrete patches, and large rosettes were common in the mutant retina (D); in addition, ectopic clusters of cells were present in the INL (not shown). Scale bar, 50 μm.
Figure 3.
 
The retina became progressively dysplastic in the first 2 postnatal weeks. Radial sections through WT (A, C) and mutant retinas (B, D) at P7 (A, B) and P15 (C, D). At P7, the mutant retina showed some sign of developmental disruptions. The OPL was underdeveloped as were the outer and inner segments, the IPL thickness was reduced, and the RGC layer was disrupted, with numerous ectopic cells in the vitreous (arrows). At P15, retinal lamination was disrupted in discrete patches, and large rosettes were common in the mutant retina (D); in addition, ectopic clusters of cells were present in the INL (not shown). Scale bar, 50 μm.
Effects of Discontinuities in the ILM and Disruptions of MC Endfeet
Next, we asked whether disruptions in the ILM produces changes in MC organization. In wholemount preparations of WT retina, the ILM was a thin sheet laying on the retinal surface (Fig. 4A), and the endfoot of each MC appeared to encircle the neurons of the RGC layer, forming a regular array of processes across the surface of the ILM (Fig. 4C). 24 In contrast, in β2−/−γ3−/− mice, the ILM was mostly absent (Fig. 4B), and MC endfeet did not show any particular relation to the RGC layer neurons (Fig. 4D). In particular, large regions of the vitreal surface were devoid of MC processes and, in other regions, the MC endfeet ended in a fibrotic tangle. In the radial sections (Figs. 4E, 4F), MC endfeet in the WT retinas terminated in a linear array of enlarged processes on the nidogen-positive ILM (Fig. 4E). In the β2−/−γ3−/− retinas, the ILM was disrupted, and there were large regions of retina with no MC processes (Fig. 4F, arrowheads) or with processes terminating on the remaining BM found on blood vessels at the vitreal border (Fig. 4F, arrows). 
Figure 4.
 
MC endfeet were disrupted where the ILM was discontinuous in the β2−/−γ3−/− retina. The ILM was visualized by nidogen deposition (red), MCs by GS staining (GS; green), and cell nuclei with DAPI (blue). Wholemount preparations of the retina (A–D) and radial sections (E, F) were shown. (A) The ILM was a seen as a continuous sheet with overlaying blood vessels; (B) in the mutant retina, there was no apparent ILM, and vascular defects were present. (C) MC endfeet (green processes) formed a lattice-like network surrounding the cells (DAPI) of the ganglion cell layer. (D) This regular array of endfeet was disrupted in the laminin mutant. The relationship of MC endfeet and the ILM was best seen in radial sections (E, F); in WT, MCs were perfectly radial and their endfeet sat on the ILM surface, whereas in the mutant retina, large regions of the retina were devoid of MC endfeet (arrowheads) and MC processes deviated toward and adhered to the intact vascular BM (E, arrows). Scale bar: (A–D) 100 μm; (E, F) 50 μm.
Figure 4.
 
MC endfeet were disrupted where the ILM was discontinuous in the β2−/−γ3−/− retina. The ILM was visualized by nidogen deposition (red), MCs by GS staining (GS; green), and cell nuclei with DAPI (blue). Wholemount preparations of the retina (A–D) and radial sections (E, F) were shown. (A) The ILM was a seen as a continuous sheet with overlaying blood vessels; (B) in the mutant retina, there was no apparent ILM, and vascular defects were present. (C) MC endfeet (green processes) formed a lattice-like network surrounding the cells (DAPI) of the ganglion cell layer. (D) This regular array of endfeet was disrupted in the laminin mutant. The relationship of MC endfeet and the ILM was best seen in radial sections (E, F); in WT, MCs were perfectly radial and their endfeet sat on the ILM surface, whereas in the mutant retina, large regions of the retina were devoid of MC endfeet (arrowheads) and MC processes deviated toward and adhered to the intact vascular BM (E, arrows). Scale bar: (A–D) 100 μm; (E, F) 50 μm.
MC differentiation was disrupted in β2−/−γ3−/− retinas. In WT retinas, MCs expressed glutamine synthetase (GS) and were well organized, with cell bodies in the inner layer of the INL and radial processes extending apically and basally. Apically, MCs processes formed a band of tight junctions with each other and photoreceptors, generating the outer limiting membrane (OLM). GS levels continued to increase from P10 through P20 (Fig. 5), concomitant with the maturation of synaptic function in the OPL. In the β2−/−γ3−/− retinas, MCs were markedly disrupted at P10 and P15. GS expression was reduced, and the organization of the cell was disrupted (Fig. 5). Specifically, the apical processes of MCs were disorganized and often expanded into the subretinal space (Fig. 5D, arrowheads), suggesting a disruption in OLM continuity. The basal endfeet formed fibrotic plaques that grew larger with age, particularly adjacent to vascular BMs (Fig. 5F). Patches of gliotic MCs, defined by upregulation of GFAP and assayed immunohistochemically, were seen in regions of retinal discontinuity (not shown). 
Figure 5.
 
MC maturation in the β2−/−γ3−/− retina was disrupted throughout development. GS was used to label MCs during development. (A, C, E) Radial sections of WT retina; (B, D, F) sections of age-matched mutant retinas. GS expression in the mutant retina lagged behind that of the WT (B vs. A). Moreover, MC morphology was disrupted over the whole age range. Early in development (B), the apparent density of cells was lower and the MC endfeet did not form a regular array. Later (D), apical processes from the MCs invaded the subretinal space at points where the OLM was disrupted (arrows). As development proceeded (F), fibrotic tangles on the vitreal surface became large, whereas the OLM remained disrupted and discontinuous. Scale bar, 50 μm.
Figure 5.
 
MC maturation in the β2−/−γ3−/− retina was disrupted throughout development. GS was used to label MCs during development. (A, C, E) Radial sections of WT retina; (B, D, F) sections of age-matched mutant retinas. GS expression in the mutant retina lagged behind that of the WT (B vs. A). Moreover, MC morphology was disrupted over the whole age range. Early in development (B), the apparent density of cells was lower and the MC endfeet did not form a regular array. Later (D), apical processes from the MCs invaded the subretinal space at points where the OLM was disrupted (arrows). As development proceeded (F), fibrotic tangles on the vitreal surface became large, whereas the OLM remained disrupted and discontinuous. Scale bar, 50 μm.
Adherence of MCs to Laminin Substrates
We used a short-term in vitro binding assay to determine substrate specificity for rat MCs (rMC-1 cells). Binding was normalized to no-substrate conditions; rMC-1 cells showed an eightfold increase in adhesion to EHS laminin (which is a mixture of several native laminins, including those containing the β2 chain), as well as to poly-l-lysine (Fig. 6). MCs showed a significant binding preference (3.75-fold) for the laminin β2 short arm. This somewhat decreased affinity for β2 short arm over EHS laminins (3.75 vs. 8) is not surprising, given the lack of several cell-binding domains provided by the associated LG domains in the native α chains present in EHS laminins. Of note, the rMC-1 cells did not bind to full-length netrin-4, which has considerable amino acid identity with laminin β chains. 25 These data clearly demonstrate that MCs recognize and adhere to laminins in general and, in particular, the β2 chain. 
Figure 6.
 
MCs showed preferential attachment to laminin substrates. Short-term adhesion assays were performed on glass coverslips (no substrate) or glass coverslips coated with poly-l-lysine (PLL), EHS laminin (EHS), laminin β2 short arm (laminin β2 SA), bovine serum albumin (BSA), or full-length netrin-4 (netrin 4). (A) Representative assays with EHS, β2 SA, BSA, and netrin-4 are shown. MCs adhered to the laminin substrates (EHS, β2SA) but not to the closely related netrin-4 or BSA. (B) In a histogram analysis of all conditions, rMC-1 cells exhibited a preference for laminin substrates.
Figure 6.
 
MCs showed preferential attachment to laminin substrates. Short-term adhesion assays were performed on glass coverslips (no substrate) or glass coverslips coated with poly-l-lysine (PLL), EHS laminin (EHS), laminin β2 short arm (laminin β2 SA), bovine serum albumin (BSA), or full-length netrin-4 (netrin 4). (A) Representative assays with EHS, β2 SA, BSA, and netrin-4 are shown. MCs adhered to the laminin substrates (EHS, β2SA) but not to the closely related netrin-4 or BSA. (B) In a histogram analysis of all conditions, rMC-1 cells exhibited a preference for laminin substrates.
Dystroglycan is reported to be a laminin receptor located on MC endfeet; thus, we used double immunostaining to study β-dystroglycan distribution and its colocalization with perlecan in WT and the β2−/−γ3−/− retinas. In agreement with previous reports, 22 in WT retina, we found β-dystroglycan immunoreactivity in the basal RPE, in the OPL, and at the ILM and surrounding blood vessels. In the mutant retinas, β-dystroglycan, and perlecan immunoreactivities were lost in the ILM, whereas co-staining appeared normal at Bruch's membrane and the vascular BMs (Fig. 7F, arrows). 
Figure 7.
 
β-Dystroglycan expression was disrupted in the MC endfeet. (A–C) Radial sections of WT and (D–F) laminin-deficient retina; perlecan was used as a BM marker in this study. Normally, β-dystroglycan was expressed in juxtaposition with retinal BMs. (A–C) Particularly noteworthy was the distribution surrounding the vascular BM and in the MC endfeet at the ILM (A; yellow in C). In the laminin-deficient retina, β-dystroglycan disappeared from the retinal surface but continued to surround the vascular BM (D, E, arrows). ILM continuity was disrupted specifically as perlecan staining disappeared from the mutant ILM, but it was present in Bruch's membrane and the vascular BM (E). Scale bar, 50 μm.
Figure 7.
 
β-Dystroglycan expression was disrupted in the MC endfeet. (A–C) Radial sections of WT and (D–F) laminin-deficient retina; perlecan was used as a BM marker in this study. Normally, β-dystroglycan was expressed in juxtaposition with retinal BMs. (A–C) Particularly noteworthy was the distribution surrounding the vascular BM and in the MC endfeet at the ILM (A; yellow in C). In the laminin-deficient retina, β-dystroglycan disappeared from the retinal surface but continued to surround the vascular BM (D, E, arrows). ILM continuity was disrupted specifically as perlecan staining disappeared from the mutant ILM, but it was present in Bruch's membrane and the vascular BM (E). Scale bar, 50 μm.
Ultrastructural Defects in Laminin-Null Retinas
Several abnormalities in the ultrastructure of BMs and the attached cells were apparent in the β2−/−γ3−/− retina (Fig. 8). Bruch's membrane was thickened and showed some disorganization in the arrays of collagen and elastin fibrils (Figs. 8A, 8B). The RPE varied from healthy-appearing cells to hyperplastic cells, with loss of pigment, enlarged vacuoles, and disruptions in the basal processes (Figs. 8C, 8D). Photoreceptor outer segments were shorter and less organized; ectopic cells were found in the subretinal space (Figs. 8E, 8F). The adherens junctions forming the OLM were irregular; in many regions they were completely absent (Figs. 8G, 8H). In the OPL, disruptions of rod photoreceptor synapses were apparent in the β2−/−γ3−/− retina (Figs. 8I, 8J). A significant number of ribbon synapses were dysmorphic in the β2−/−γ3−/− retinas; 30.7% of all ribbons were floating free in the cytoplasm of the rod terminal in the mutant compared with 10% in the WT retinas (n = 189, and 130, respectively). In the WT, 88.2% of the ribbon synapses in the OPL showed normal functional relationship with two or three postsynaptic contacts (dyads or triads, respectively) versus only 67.7% in the mutant retinas. These data are nearly identical with those reported for the β2−/−γ3+/+ animal, in which a profound disruption of synaptic integrity was noted. 12  
Figure 8.
 
Electron micrographs revealed ultrastructural abnormalities across the retina in the β2−/−γ3−/− retina. Ultrastructural analysis of WT and mutant retinas, the mutant retina displayed several morphologic disruptions compared with the control. Retinas from WT animals are shown in the left column (A, C, E, G, I, K); mutant retinas are shown in the right column (B, D, F, H, J, L). In the middle panels are low-power images of sections from WT and mutant retina for orientation. (A, B) In the mutant, Bruch's membrane was thickened and the collagen and elastin fibril organization was disrupted. (C, D) Mutant RPE was markedly vacuolated, and there were disruptions in the basal processes (not fully illustrated). (E, F) The outer segments in the mutant were disorganized and misaligned, with shorter disc membranes. (G, H) In the WT, the OLM was formed from a series of zonulae adherens. In the mutant, these junctions were disrupted; ectopic photoreceptor nuclei (Image Not Available in F and H; see also the orientation figure) were found at these sites of discontinuities. (I, J) There was considerable disruption of synaptic organization in the OPL of the mutant retina numerous floating ribbons (f) were found in the mutant. (K, L) MC processes sprouted into the vitreous cavity where the ILM was disrupted.
Figure 8.
 
Electron micrographs revealed ultrastructural abnormalities across the retina in the β2−/−γ3−/− retina. Ultrastructural analysis of WT and mutant retinas, the mutant retina displayed several morphologic disruptions compared with the control. Retinas from WT animals are shown in the left column (A, C, E, G, I, K); mutant retinas are shown in the right column (B, D, F, H, J, L). In the middle panels are low-power images of sections from WT and mutant retina for orientation. (A, B) In the mutant, Bruch's membrane was thickened and the collagen and elastin fibril organization was disrupted. (C, D) Mutant RPE was markedly vacuolated, and there were disruptions in the basal processes (not fully illustrated). (E, F) The outer segments in the mutant were disorganized and misaligned, with shorter disc membranes. (G, H) In the WT, the OLM was formed from a series of zonulae adherens. In the mutant, these junctions were disrupted; ectopic photoreceptor nuclei (Image Not Available in F and H; see also the orientation figure) were found at these sites of discontinuities. (I, J) There was considerable disruption of synaptic organization in the OPL of the mutant retina numerous floating ribbons (f) were found in the mutant. (K, L) MC processes sprouted into the vitreous cavity where the ILM was disrupted.
Last, consistent with the light microscopic observations, the vitreal surface of the β2−/−γ3−/− retina was markedly dysmorphic. The normal trilaminate structure of the ILM (Fig. 8K) was absent, and the remaining portions were loosely adherent to the retina (Fig. 8L). We observed processes of presumed MCs sprouting into the vitreal cavity (Fig. 8L) similar to the events of proliferative vitreoretinopathy. Consistent with the results reported thus far in the article, these data suggest that MCs adhere to ILM laminins. 
Disruption of the Laminar Arrangement in the Laminin β2γ3 Chain-Deficient Retina
Neuronal differentiation was examined in sections of P20 WT and mutant retinas with a panel of immunohistochemical markers (Fig. 9; Table 1). In the β2−/−γ3−/− retinas, we selected both regions with relatively normal organization and regions with impaired lamination for comparison; shown in Figure 9 were regions of moderate disorganization. Several generalizations can be made. First, all classes of cells appeared to be present. Second, the fundamental layered organization of the retina was largely intact. Third, the polarity of the cells was largely intact. The photoreceptors were aligned well and the rod bipolar cells and amacrine cells were properly oriented. 
Figure 9.
 
Retinal cells differentiate, but their laminar arrangement was disrupted in the laminin-deficient retina. Radial sections of WT (A, C, E, G, I) and mutant (B, D, F, H, J) P20 retina were examined for a variety of cell-specific markers (as indicated). All major cell types were present in mutant retinas, but their lamination was disrupted in patches (D, H, J). This disruption was most striking in the photoreceptors, which gathered in spheroid clusters with their outer segment oriented toward the center (B, rosette formation, arrows). Rhodopsin also showed a marked delocalization, staining the photoreceptor bodies (see IOVS_51_3.supp1  ). Finally, there were variable irregularities in the strict arrangement and laminar patterning of horizontal (C, D), bipolar (E, F), amacrine (G, H), and ganglion (I, J) cells and their processes. Scale bar, 50 μm.
Figure 9.
 
Retinal cells differentiate, but their laminar arrangement was disrupted in the laminin-deficient retina. Radial sections of WT (A, C, E, G, I) and mutant (B, D, F, H, J) P20 retina were examined for a variety of cell-specific markers (as indicated). All major cell types were present in mutant retinas, but their lamination was disrupted in patches (D, H, J). This disruption was most striking in the photoreceptors, which gathered in spheroid clusters with their outer segment oriented toward the center (B, rosette formation, arrows). Rhodopsin also showed a marked delocalization, staining the photoreceptor bodies (see IOVS_51_3.supp1  ). Finally, there were variable irregularities in the strict arrangement and laminar patterning of horizontal (C, D), bipolar (E, F), amacrine (G, H), and ganglion (I, J) cells and their processes. Scale bar, 50 μm.
Nevertheless, within individual cell types, several disruptions in organization were found. As noted earlier, rosettes formed in the outer retina, consisting largely of photoreceptors. These ectopic cells were polarized around an ectopic subretinal space, which was continuous with the normal subretinal space (Fig. 9B, arrows). These ectopic photoreceptors made outer segments and transported opsin into them (Fig. 9B and IOVS_51_3.supp1  ). In addition, rhodopsin was not confined to the outer segments in either normally positioned photoreceptors or photoreceptors within rosettes. Rather, there was significant rhodopsin immunoreactivity in the cell bodies of photoreceptors, and thus it appeared throughout the ONL (Fig. 9B, Supplementary Fig. S1). 
The organization of the rod bipolar cell terminals in the IPL appeared to be disrupted (Figs. 9E, 9F). In WT retinas, these cells ended in a tight band of terminals at the distalmost portions of the IPL (Fig. 9E); in the β2−/−γ3−/− retina, this band was widened and disorganized (Fig. 9F). Amacrine cells and RGCs, visualized with calretinin immunohistochemistry, revealed disruptions in cell positioning and in the characteristic trilaminate staining of the IPL (compare Fig. 9G with Fig. 9H). We have published a study of the effect on the dopaminergic system in this retina. 14 The ganglion cell layer was severely affected. RGC bodies were not properly organized in the RGC layer, with many RGCs extruding into the vitreous (compare Fig. 9J with Fig. 9I). 
Effect of Laminin β2γ3 Chain Deficiency on ERG a- and b-Waves
The physiological consequences of laminin β2 and γ3 ablation were analyzed in an electrophysiological approach. Figure 10 shows representative ERG recordings from P18 WT and β2−/−γ3−/− mice in response to flash stimuli delivered after prolonged dark adaptation. It was immediately evident that the postsynaptic responses (b-wave) were as severely disrupted in the β2−/−γ3−/− animals (Figs. 10A, 10B) as they were in the β2−/−γ3+/+ animals. 12 However, unlike in the β2−/−γ3+/+ mutants, the photoreceptor-generated currents (a-wave) in the β2−/−γ3−/− animals were also disrupted. In the β2−/−γ3−/− animals, the mean amplitudes of a-waves were reduced to ∼38% of control values, whereas the b-waves were reduced to ∼45% of the control (Figs. 10A, 10B). In addition to a reduction in the a-wave amplitude, the implicit time is increased from 30.2 ms in WT retinas to 35.7 ms in β2−/−γ3−/− retinas; the implicit time of the b-wave wave was largely unaltered (80.5 vs. 81.3 ms). Finally, there was a dramatic reduction in a-wave sensitivity in the compound nulls (Fig. 10C; ∼1.75-log unit shift) and the responsivity of both a- and b-waves was drastically reduced (Figs. 10C, 10D). 
Figure 10.
 
ERGs demonstrated profound dysfunction in photoreceptor transduction and output in the mutant retina. (A) Representative ERGs from WT (top) and laminin-deficient mice (bottom) are shown; the a- and b-wave amplitudes were reduced at all light intensities. (B) Histograms of a- and b-waves of all animals studied. (C) Response–intensity curves for a- and b-waves in WT and mutant mice; regression lines are drawn through the data points. In addition to profound reduction in the a-wave amplitude, the a-wave threshold was elevated considerably. The disruptions of the b-wave were consistent with the ultrastructural disruptions of the synapse in this mouse and in the β2−/− mouse. 12
Figure 10.
 
ERGs demonstrated profound dysfunction in photoreceptor transduction and output in the mutant retina. (A) Representative ERGs from WT (top) and laminin-deficient mice (bottom) are shown; the a- and b-wave amplitudes were reduced at all light intensities. (B) Histograms of a- and b-waves of all animals studied. (C) Response–intensity curves for a- and b-waves in WT and mutant mice; regression lines are drawn through the data points. In addition to profound reduction in the a-wave amplitude, the a-wave threshold was elevated considerably. The disruptions of the b-wave were consistent with the ultrastructural disruptions of the synapse in this mouse and in the β2−/− mouse. 12
It is also clear that cone function is diminished in β2−/−γ3−/− retinas. At higher intensity flashes, oscillations in the b-wave are commonly attributed to cone-activation. 26 These oscillations are diminished in the β2−/−γ3−/− retinas but not ablated, suggesting that cone function may be preserved (Fig. 10A, top left, compare WT and nulls). The dramatic reduction in the b-wave amplitude is most likely the result of the disruption of synaptic integrity in these animals (see the EM results described earlier), which was also seen in the β2−/−γ3+/+ retina. 12 Disruptions in MC function may also contribute to this reduction in the compound-null animal. 
Discussion
A key step in retinal development involves the functional subdivision of the tissue, not only the differentiation of glial and neural cell types, but also the ability to form different cell shapes and to subdivide the tissue into functionally distinct compartments. The resultant multilaminated structure is dependent on the adequate establishment of apical–basal cell polarity. The retina, like the rest of the CNS, is derived from the neural tube with origins in the ectoderm. In ectodermally derived tissues, apical–basal polarity is conferred by the adhesion of epithelial cells to the BM. Loss of BM integrity or misexpression of BM components has profound developmental consequences and, in adulthood, disruptions in BM integrity can lead to a variety of diseases, including kidney dysfunction, muscular dystrophies, and metastatic cancer. Disruptions in genes encoding laminin, ECM receptors (integrin, dystroglycan), their signaling molecules (ILK, FAK), or cytomatrix molecules (dystrophin) cause cortical and retinal dysplasia in animal models 2735 and humans. 36  
Laminins are dynamically regulated during development, and the regulation of their expression is complex. However, several important generalizations about the role of laminins in development have emerged. First, is the primacy of laminin in BM formation and stability. The laminin trimer, α1β1γ1 is expressed early, and the deletion of Lamc1 (the γ1 gene) results in preimplantation lethality 2 during epiblast formation. The addition of exogenous laminin to the Lamc1-null embryoid bodies restores BM formation and development through gastrulation. 37,38 Laminin-mediated assembly of the BM proceeds as follows: (1) binding of laminin via the LG domains to cell surface sulfated glycolipids; and (2) polymerizing via their LN terminals and incorporation of other ECM molecules to the nascent laminin polymer including collagen and HSPGs. 39 Another generalization that can be made is that early laminin isoforms are replaced in many tissues by different isoforms in a tissue-specific manner. 40  
Herein, we describe a series of histogenic effects in the retina, which arise from a common problem: the loss of integrity of a single BM—the ILM. We demonstrate that both β2- and γ3-containing laminins are components of the ILM and are essential for the stabilized assembly of the ILM; other BMs—vascular and Bruch's—are intact. The need for β2- and γ3-containing laminins in the ILM apparently arises late in development, as the central ILM is intact and formed in these animals, whereas the more peripheral ILM is disrupted. This situation is reminiscent of the temporal regulation of laminin expression in epithelial systems where a shift in isoforms precedes and parallels histogenic maturation. 41,42  
Multiple sources of ILM laminins are possible and include the lens, the neural retina (Müller and neuroepithelial cells), and the ciliary body. 43 As development proceeds and the vitreous body expands, distant sources of laminin for ILM synthesis seem unlikely. Thus, it may be that retinal sources become increasingly more important for ILM synthesis with advancing age. 
The single deletion of either Lamb2 or Lamc3 alone minimally disrupts the ILM. How then does their combined deletion so profoundly disrupt the ILM formation with apparent specificity? As the β3 and γ2 chains appear to be exclusive partners, only β1 and γ1 laminin chains can compensate for the loss of β2 and γ3, respectively. It is now emerging that the β2 and γ3 chains have some unique binding properties. For example, the presence of a γ3 chain in a laminin isoform reduces that isoform's apparent integrin binding. Similarly, β2 and β1 also have differential binding affinities for some integrins. 44,45 Moreover, the dynamics of laminin polymerization, which occurs as a result of the interaction among the LN (N-terminals) of the molecules, is no doubt different among the chains. 46 Thus, the biological properties of laminin β1- and γ1-containing laminins are fundamentally different from those of β2- and γ3-containing ones. 
The alterations during retinal histogenesis, as described for the β2−/−γ3−/− retina, are referred to as dysplasia (i.e., disturbances in development that result in the disruption of the normal tissue architecture). 47,48 The mechanisms that regulate normal retinal lamination are coming under increasing study. 49,50 A clear concept is emerging that the mechanisms of lamination and cell fate adoption are genetically separable. Retinal cell differentiation and lamination appear to proceed in the same time window, but use separate molecular mechanisms. 51,52 Our results are consistent with these observations: The deletion of both the β2 and γ3 chains of laminin, although it disrupts dendritogenesis, 14 does not affect cell fate. 
Moreover, although the β2- and γ3-deficient retina showed some anomalies at P0, the most dramatic dysplasia occurred during the second postnatal week, when rod photoreceptors and MCs were being born and differentiating. MCs were highly polarized with distinct subcellular compartments. A series of tight junctions were found at the apical surface of the MC; these were disrupted in the β2−/−γ3−/− mouse. Similarly, there were receptor complexes present at the basal endfoot that were disrupted also in the β2−/−γ3−/− mouse. MCs span the entire thickness of the neural retina, establishing morphologic and functional interactions with all retinal neurons. They were also intimately related with both limiting membranes as well as with the synaptic layers. Several studies have suggested that MC disruption causes disorganization of the normal retinal layering. 5359 The laminin receptor dystroglycan is expressed in MCs and mediates MC adhesion and migration as well as macromolecular clustering of the transport molecules aquaporin and Kir4.1 channels. 60 In reaggregated cultures of retinal neurons, the correct polarity of retinal layers is associated with the release of laminin by co-cultured RPE cells. 61 It seems reasonable to presume that BM proteins, laminin in particular, provide critical polarity cues in organizing the MCs. 
In the β2−/−γ3−/− retina, the integrity of the ILM was disrupted, which was accompanied by the loss of expression of β-dystroglycan (the transmembrane component of the dystroglycan complex) at the MC endfeet. This effect suggests that an intact ILM is necessary for proper assembly and/or localization of β-dystroglycan at the MC endfeet. The resulting lack of interaction between ECM proteins in the BM and β-dystroglycan at the endfeet of MCs may result in an abnormal maturation of these cells, preventing them from establishing adequate contacts with other cells, thus leading to retinal disorganization. In this regard, retinas of mice with mutations in α-dystroglycan, the ligand-binding component of the dystroglycan complex, are disorganized, similar to that in the β2−/−γ3−/− mouse. 62 Deletions of integrins α3 and α6 produce retinal dysplasia as well. All these studies suggest that adhesion to the BM is critical for retinal histogenesis. Further support comes from the work of Chen and Nathans, 63 who showed that outer retinal dysplasia in the rd7 mouse is probably the result of specific disruptions of cone photoreceptors with MCs and the ECM. 63 The results reported here identify β2 and γ3 laminins as key organizers of the ILM and implicated them as critical molecules for MC adhesion to the ILM. 
In addition to the profound dysplasia, β2- and γ3-deficient retinas showed abnormalities in photoreceptor morphogenesis (i.e., shorter and disorganized outer segments and detached or floating synaptic ribbons). There was also a disorganization of opsin targeting, resulting in misexpression of opsin in the ONL. Such delocalization of opsin is a common theme during photoreceptor injury and degeneration such as that in retinal detachment. 64 It remains to be determined whether laminins contribute directly to outer segment stabilization or these structural effects on the photoreceptor are mediated through MCs. These morphologic photoreceptor abnormalities phenocopy the effects of the laminin β2 chain-deficient retina 12 ; however, there were functional differences in the two mice as measured by the ERG. Unlike the β2−/− single mutant, the amplitude of the a-wave was reduced, and the implicit time was increased in the β2−/−γ3−/− mouse, suggesting profound dysfunction in the signal transduction process. This disruption may reflect a more profound intrinsic disruption of photoreceptor morphogenesis, in that the outer segments are shortened in both mutants. 12 Another possibility is that the photon capture rate in β2−/−γ3−/− retinas is impaired by MC disorganization. MCs have been shown to act as effective light guides in normal retinas. 65 Given the profound geometric rearrangement in the β2−/−γ3−/− animal and the near complete disruption of the array of MC endfeet, this light-gathering ability would be greatly impaired in these animals. 
The β2−/−γ3−/− and β2−/−γ3+/+ mice 12 both have profound reductions in the b-wave, which is accompanied by morphologic failure in synaptogenesis. This synaptic disruption may explain another aspect of the β2−/−γ3−/− phenotype. MCs are responsible for recycling glutamate to glutamine, via GS. GS expression is inducible and regulated by glutamate release. 66 The decrease in GS seen in the compound null is consistent with a disruption in synaptic function and the reduction of the b-wave. 
Taken together, these results suggest that the β2 and γ3 chains of laminin: (1) are critical for the normal organization of BMs and tissue histogenesis in the CNS and (2) are essential for normal polarity and maturation of MCs. These studies may be relevant to our understanding of the pathobiology of MCs and may provide the basis for ECM-targeted therapies. 
Supplementary Materials
Footnotes
 Supported by National Eye Institute Grant EY12676-09 (WJB), unrestricted funds from SUNY Downstate Medical Center, and a Deutsche Forschungsgemeinschaft (DFG) Grant SFB612 (MK B14).
Footnotes
 Disclosure: G. Pinzón-Duarte, None; G. Daly, None; Y.N. Li, None; M. Koch, P; W.J. Brunken, P
The authors thank Paul Witkovsky for critical reading of an earlier version of the manuscript and Galina Bachay for technical help. 
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Figure 1.
 
β2 and γ3 laminin expression was absent in mutant retinas. (A, C) P15 WT retina; (B, D) P15 laminin-deficient retina. (A) β2 immunoreactivity was seen in all retinal BMs: Bruch's membrane, vascular BM, and the ILM. (B) This reactivity was completely absent in the mutant retina. (C) Strong deposition of γ3 chain was found in Bruch's membrane and vascular BM, but it was weakly present in the ILM. (D) No γ3 expression was seen in the mutant retina. Scale bar, 50 μm.
Figure 1.
 
β2 and γ3 laminin expression was absent in mutant retinas. (A, C) P15 WT retina; (B, D) P15 laminin-deficient retina. (A) β2 immunoreactivity was seen in all retinal BMs: Bruch's membrane, vascular BM, and the ILM. (B) This reactivity was completely absent in the mutant retina. (C) Strong deposition of γ3 chain was found in Bruch's membrane and vascular BM, but it was weakly present in the ILM. (D) No γ3 expression was seen in the mutant retina. Scale bar, 50 μm.
Figure 2.
 
β2γ3 Laminin deletion produced differential disruption of the ILM. BMs were visualized with nidogen immunostaining in all genotypes (as indicated). (A) Nidogen staining was seen as a continuous band along both BM and the ILM; vascular BM was also labeled. The deposition of nidogen, in Bruch's membrane and the vascular BM, was intact in all genotypes. In contrast, the ILM organization was affected by laminin deletion. Nidogen deposition was largely normal in the γ3−/− retina (C). In the β2−/− retina, nidogen deposition was more punctate and there were limited regions of discontinuity (B). In the β2−/−γ3−/− animals, nidogen deposition was largely lacking, with limited plaquelike staining associated with vitreal blood vessels (D, arrows). The extraocular muscle BM was intact in all genotypes (arrowheads). Scale bar, 50 μm.
Figure 2.
 
β2γ3 Laminin deletion produced differential disruption of the ILM. BMs were visualized with nidogen immunostaining in all genotypes (as indicated). (A) Nidogen staining was seen as a continuous band along both BM and the ILM; vascular BM was also labeled. The deposition of nidogen, in Bruch's membrane and the vascular BM, was intact in all genotypes. In contrast, the ILM organization was affected by laminin deletion. Nidogen deposition was largely normal in the γ3−/− retina (C). In the β2−/− retina, nidogen deposition was more punctate and there were limited regions of discontinuity (B). In the β2−/−γ3−/− animals, nidogen deposition was largely lacking, with limited plaquelike staining associated with vitreal blood vessels (D, arrows). The extraocular muscle BM was intact in all genotypes (arrowheads). Scale bar, 50 μm.
Figure 3.
 
The retina became progressively dysplastic in the first 2 postnatal weeks. Radial sections through WT (A, C) and mutant retinas (B, D) at P7 (A, B) and P15 (C, D). At P7, the mutant retina showed some sign of developmental disruptions. The OPL was underdeveloped as were the outer and inner segments, the IPL thickness was reduced, and the RGC layer was disrupted, with numerous ectopic cells in the vitreous (arrows). At P15, retinal lamination was disrupted in discrete patches, and large rosettes were common in the mutant retina (D); in addition, ectopic clusters of cells were present in the INL (not shown). Scale bar, 50 μm.
Figure 3.
 
The retina became progressively dysplastic in the first 2 postnatal weeks. Radial sections through WT (A, C) and mutant retinas (B, D) at P7 (A, B) and P15 (C, D). At P7, the mutant retina showed some sign of developmental disruptions. The OPL was underdeveloped as were the outer and inner segments, the IPL thickness was reduced, and the RGC layer was disrupted, with numerous ectopic cells in the vitreous (arrows). At P15, retinal lamination was disrupted in discrete patches, and large rosettes were common in the mutant retina (D); in addition, ectopic clusters of cells were present in the INL (not shown). Scale bar, 50 μm.
Figure 4.
 
MC endfeet were disrupted where the ILM was discontinuous in the β2−/−γ3−/− retina. The ILM was visualized by nidogen deposition (red), MCs by GS staining (GS; green), and cell nuclei with DAPI (blue). Wholemount preparations of the retina (A–D) and radial sections (E, F) were shown. (A) The ILM was a seen as a continuous sheet with overlaying blood vessels; (B) in the mutant retina, there was no apparent ILM, and vascular defects were present. (C) MC endfeet (green processes) formed a lattice-like network surrounding the cells (DAPI) of the ganglion cell layer. (D) This regular array of endfeet was disrupted in the laminin mutant. The relationship of MC endfeet and the ILM was best seen in radial sections (E, F); in WT, MCs were perfectly radial and their endfeet sat on the ILM surface, whereas in the mutant retina, large regions of the retina were devoid of MC endfeet (arrowheads) and MC processes deviated toward and adhered to the intact vascular BM (E, arrows). Scale bar: (A–D) 100 μm; (E, F) 50 μm.
Figure 4.
 
MC endfeet were disrupted where the ILM was discontinuous in the β2−/−γ3−/− retina. The ILM was visualized by nidogen deposition (red), MCs by GS staining (GS; green), and cell nuclei with DAPI (blue). Wholemount preparations of the retina (A–D) and radial sections (E, F) were shown. (A) The ILM was a seen as a continuous sheet with overlaying blood vessels; (B) in the mutant retina, there was no apparent ILM, and vascular defects were present. (C) MC endfeet (green processes) formed a lattice-like network surrounding the cells (DAPI) of the ganglion cell layer. (D) This regular array of endfeet was disrupted in the laminin mutant. The relationship of MC endfeet and the ILM was best seen in radial sections (E, F); in WT, MCs were perfectly radial and their endfeet sat on the ILM surface, whereas in the mutant retina, large regions of the retina were devoid of MC endfeet (arrowheads) and MC processes deviated toward and adhered to the intact vascular BM (E, arrows). Scale bar: (A–D) 100 μm; (E, F) 50 μm.
Figure 5.
 
MC maturation in the β2−/−γ3−/− retina was disrupted throughout development. GS was used to label MCs during development. (A, C, E) Radial sections of WT retina; (B, D, F) sections of age-matched mutant retinas. GS expression in the mutant retina lagged behind that of the WT (B vs. A). Moreover, MC morphology was disrupted over the whole age range. Early in development (B), the apparent density of cells was lower and the MC endfeet did not form a regular array. Later (D), apical processes from the MCs invaded the subretinal space at points where the OLM was disrupted (arrows). As development proceeded (F), fibrotic tangles on the vitreal surface became large, whereas the OLM remained disrupted and discontinuous. Scale bar, 50 μm.
Figure 5.
 
MC maturation in the β2−/−γ3−/− retina was disrupted throughout development. GS was used to label MCs during development. (A, C, E) Radial sections of WT retina; (B, D, F) sections of age-matched mutant retinas. GS expression in the mutant retina lagged behind that of the WT (B vs. A). Moreover, MC morphology was disrupted over the whole age range. Early in development (B), the apparent density of cells was lower and the MC endfeet did not form a regular array. Later (D), apical processes from the MCs invaded the subretinal space at points where the OLM was disrupted (arrows). As development proceeded (F), fibrotic tangles on the vitreal surface became large, whereas the OLM remained disrupted and discontinuous. Scale bar, 50 μm.
Figure 6.
 
MCs showed preferential attachment to laminin substrates. Short-term adhesion assays were performed on glass coverslips (no substrate) or glass coverslips coated with poly-l-lysine (PLL), EHS laminin (EHS), laminin β2 short arm (laminin β2 SA), bovine serum albumin (BSA), or full-length netrin-4 (netrin 4). (A) Representative assays with EHS, β2 SA, BSA, and netrin-4 are shown. MCs adhered to the laminin substrates (EHS, β2SA) but not to the closely related netrin-4 or BSA. (B) In a histogram analysis of all conditions, rMC-1 cells exhibited a preference for laminin substrates.
Figure 6.
 
MCs showed preferential attachment to laminin substrates. Short-term adhesion assays were performed on glass coverslips (no substrate) or glass coverslips coated with poly-l-lysine (PLL), EHS laminin (EHS), laminin β2 short arm (laminin β2 SA), bovine serum albumin (BSA), or full-length netrin-4 (netrin 4). (A) Representative assays with EHS, β2 SA, BSA, and netrin-4 are shown. MCs adhered to the laminin substrates (EHS, β2SA) but not to the closely related netrin-4 or BSA. (B) In a histogram analysis of all conditions, rMC-1 cells exhibited a preference for laminin substrates.
Figure 7.
 
β-Dystroglycan expression was disrupted in the MC endfeet. (A–C) Radial sections of WT and (D–F) laminin-deficient retina; perlecan was used as a BM marker in this study. Normally, β-dystroglycan was expressed in juxtaposition with retinal BMs. (A–C) Particularly noteworthy was the distribution surrounding the vascular BM and in the MC endfeet at the ILM (A; yellow in C). In the laminin-deficient retina, β-dystroglycan disappeared from the retinal surface but continued to surround the vascular BM (D, E, arrows). ILM continuity was disrupted specifically as perlecan staining disappeared from the mutant ILM, but it was present in Bruch's membrane and the vascular BM (E). Scale bar, 50 μm.
Figure 7.
 
β-Dystroglycan expression was disrupted in the MC endfeet. (A–C) Radial sections of WT and (D–F) laminin-deficient retina; perlecan was used as a BM marker in this study. Normally, β-dystroglycan was expressed in juxtaposition with retinal BMs. (A–C) Particularly noteworthy was the distribution surrounding the vascular BM and in the MC endfeet at the ILM (A; yellow in C). In the laminin-deficient retina, β-dystroglycan disappeared from the retinal surface but continued to surround the vascular BM (D, E, arrows). ILM continuity was disrupted specifically as perlecan staining disappeared from the mutant ILM, but it was present in Bruch's membrane and the vascular BM (E). Scale bar, 50 μm.
Figure 8.
 
Electron micrographs revealed ultrastructural abnormalities across the retina in the β2−/−γ3−/− retina. Ultrastructural analysis of WT and mutant retinas, the mutant retina displayed several morphologic disruptions compared with the control. Retinas from WT animals are shown in the left column (A, C, E, G, I, K); mutant retinas are shown in the right column (B, D, F, H, J, L). In the middle panels are low-power images of sections from WT and mutant retina for orientation. (A, B) In the mutant, Bruch's membrane was thickened and the collagen and elastin fibril organization was disrupted. (C, D) Mutant RPE was markedly vacuolated, and there were disruptions in the basal processes (not fully illustrated). (E, F) The outer segments in the mutant were disorganized and misaligned, with shorter disc membranes. (G, H) In the WT, the OLM was formed from a series of zonulae adherens. In the mutant, these junctions were disrupted; ectopic photoreceptor nuclei (Image Not Available in F and H; see also the orientation figure) were found at these sites of discontinuities. (I, J) There was considerable disruption of synaptic organization in the OPL of the mutant retina numerous floating ribbons (f) were found in the mutant. (K, L) MC processes sprouted into the vitreous cavity where the ILM was disrupted.
Figure 8.
 
Electron micrographs revealed ultrastructural abnormalities across the retina in the β2−/−γ3−/− retina. Ultrastructural analysis of WT and mutant retinas, the mutant retina displayed several morphologic disruptions compared with the control. Retinas from WT animals are shown in the left column (A, C, E, G, I, K); mutant retinas are shown in the right column (B, D, F, H, J, L). In the middle panels are low-power images of sections from WT and mutant retina for orientation. (A, B) In the mutant, Bruch's membrane was thickened and the collagen and elastin fibril organization was disrupted. (C, D) Mutant RPE was markedly vacuolated, and there were disruptions in the basal processes (not fully illustrated). (E, F) The outer segments in the mutant were disorganized and misaligned, with shorter disc membranes. (G, H) In the WT, the OLM was formed from a series of zonulae adherens. In the mutant, these junctions were disrupted; ectopic photoreceptor nuclei (Image Not Available in F and H; see also the orientation figure) were found at these sites of discontinuities. (I, J) There was considerable disruption of synaptic organization in the OPL of the mutant retina numerous floating ribbons (f) were found in the mutant. (K, L) MC processes sprouted into the vitreous cavity where the ILM was disrupted.
Figure 9.
 
Retinal cells differentiate, but their laminar arrangement was disrupted in the laminin-deficient retina. Radial sections of WT (A, C, E, G, I) and mutant (B, D, F, H, J) P20 retina were examined for a variety of cell-specific markers (as indicated). All major cell types were present in mutant retinas, but their lamination was disrupted in patches (D, H, J). This disruption was most striking in the photoreceptors, which gathered in spheroid clusters with their outer segment oriented toward the center (B, rosette formation, arrows). Rhodopsin also showed a marked delocalization, staining the photoreceptor bodies (see IOVS_51_3.supp1  ). Finally, there were variable irregularities in the strict arrangement and laminar patterning of horizontal (C, D), bipolar (E, F), amacrine (G, H), and ganglion (I, J) cells and their processes. Scale bar, 50 μm.
Figure 9.
 
Retinal cells differentiate, but their laminar arrangement was disrupted in the laminin-deficient retina. Radial sections of WT (A, C, E, G, I) and mutant (B, D, F, H, J) P20 retina were examined for a variety of cell-specific markers (as indicated). All major cell types were present in mutant retinas, but their lamination was disrupted in patches (D, H, J). This disruption was most striking in the photoreceptors, which gathered in spheroid clusters with their outer segment oriented toward the center (B, rosette formation, arrows). Rhodopsin also showed a marked delocalization, staining the photoreceptor bodies (see IOVS_51_3.supp1  ). Finally, there were variable irregularities in the strict arrangement and laminar patterning of horizontal (C, D), bipolar (E, F), amacrine (G, H), and ganglion (I, J) cells and their processes. Scale bar, 50 μm.
Figure 10.
 
ERGs demonstrated profound dysfunction in photoreceptor transduction and output in the mutant retina. (A) Representative ERGs from WT (top) and laminin-deficient mice (bottom) are shown; the a- and b-wave amplitudes were reduced at all light intensities. (B) Histograms of a- and b-waves of all animals studied. (C) Response–intensity curves for a- and b-waves in WT and mutant mice; regression lines are drawn through the data points. In addition to profound reduction in the a-wave amplitude, the a-wave threshold was elevated considerably. The disruptions of the b-wave were consistent with the ultrastructural disruptions of the synapse in this mouse and in the β2−/− mouse. 12
Figure 10.
 
ERGs demonstrated profound dysfunction in photoreceptor transduction and output in the mutant retina. (A) Representative ERGs from WT (top) and laminin-deficient mice (bottom) are shown; the a- and b-wave amplitudes were reduced at all light intensities. (B) Histograms of a- and b-waves of all animals studied. (C) Response–intensity curves for a- and b-waves in WT and mutant mice; regression lines are drawn through the data points. In addition to profound reduction in the a-wave amplitude, the a-wave threshold was elevated considerably. The disruptions of the b-wave were consistent with the ultrastructural disruptions of the synapse in this mouse and in the β2−/− mouse. 12
Table 1.
 
Primary Antibodies
Table 1.
 
Primary Antibodies
Cell Type Antibody Host Source Concentration Reference
Used to identify retinal cells
    Rod photoreceptor Rhodopsin Clone RET-P1 monoclonal Sigma-Aldrich, St. Louis, MO 1:500 15
    Rod bipolar αPKC Clone MC5 monoclonal Chemicon, Temecula, CA 1:500 16
    Horizontal Calbindin-D-28K Mouse monoclonal Sigma-Aldrich 1:500 16
    Amacrine Calretinin Mouse monoclonal Chemicon 1:500 16
    Ganglion Brn3a Mouse monoclonal Santa Cruz, Santa Cruz, CA 1:500 17
    Müller Glutamine synthetase Goat polyclonal Santa Cruz 1:500 16
GFAP Clone G-A-5 monoclonal Chemicon 1:500 18
Used to detect basement membranes
Collagen IV Rabbit polyclonal Chemicon 1:100 19
Laminin β2 chain Rabbit polyclonal Our laboratory 1:5,000 11
Laminin γ3 chain Rabbit polyclonal Our laboratory 1:5,000 11
Nidogen Rabbit polyclonal Ulrike Mayer* 1:3,000 19
Pan-laminin Rabbit polyclonal Sigma-Aldrich 1:1,000 20
Perlecan Rat polyclonal Santa Cruz 1:100,000 21
Used to detect basement membrane receptors
β-dystroglycan Rabbit polyclonal Dominique Mornet† 1:250 22
Table 2.
 
Protein Substrates for Adhesion Assays
Table 2.
 
Protein Substrates for Adhesion Assays
Substrate Source Concentration
Recombinant full-length netrin-4 Our laboratory 10 μg/mL
Recombinant netrin-4 without C domain Our laboratory 10 μg/mL
Recombinant laminin β2 short arm Our laboratory 10 μg/mL
Engelbreth-Holm-Swarm sarcoma derived laminin Invitrogen, San Diego, CA 10 μg/mL
Poly-l-lysine Sigma-Aldrich, St. Louis, MO 100 μg/mL
Bovine serum albumin Sigma-Aldrich 40 μg/mL
Supplementary Figure S1
Copyright 2010 The Association for Research in Vision and Ophthalmology, Inc.
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