Defective formation of the inner limiting membrane in laminin beta2- and gamma3-null mice produces retinal dysplasia - PubMed (original) (raw)
Defective formation of the inner limiting membrane in laminin beta2- and gamma3-null mice produces retinal dysplasia
Germán Pinzón-Duarte et al. Invest Ophthalmol Vis Sci. 2010 Mar.
Abstract
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 beta2 and gamma3 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 beta2- and gamma3-containing laminins affects retinal development. Methods. The function of the beta2- and gamma3-containing laminins was tested by producing a compound deletion of both the beta2 and the gamma3 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 beta2 and gamma3 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 beta2 and gamma3 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.
Figures
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 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 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 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 (* 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 Supplementary Fig. S1,
http://www.iovs.org/cgi/content/full/51/3/1773/DC1
). 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.
References
- Aumailley M, Bruckner-Tuderman L, Carter WG, et al. A simplified laminin nomenclature. Matrix Biol 2005;24:326–332 -PubMed
- Yurchenco PD, Amenta PS, Patton BL. Basement membrane assembly, stability and activities observed through a developmental lens. Matrix Biol 2004;22:521–538 -PubMed
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