Lama1 mutations lead to vitreoretinal blood vessel formation, persistence of fetal vasculature, and epiretinal membrane formation in mice

Abstract

Background: Valuable insights into the complex process of retinal vascular development can be gained using models with abnormal retinal vasculature. Two such models are the recently described mouse lines with mutations in Lama1, an important component of the retinal internal limiting membrane (ILM). These mutants have a persistence of the fetal vasculature of vitreous (FVV) but lack a primary retinal vascular plexus. The present study provides a detailed analysis of astrocyte and vascular development in these Lama1 mutants.

Results: Although astrocytes and blood vessels initially migrate into Lama1 mutant retinas, both traverse the peripapillary ILM into the vitreous by P3. Once in the vitreous, blood vessels anastomose with vessels of the vasa hyaloidea propria, part of the FVV, and eventually re-enter the retina where they dive to form the inner and outer retinal capillary networks. Astrocytes continue proliferating within the vitreous to form a dense mesh that resembles epiretinal membranes associated with persistent fetal vasculature and proliferative vitreoretinopathy.

Conclusions: Lama1 and a fully intact ILM are required for normal retinal vascular development. Mutations in Lama1 allow developing retinal vessels to enter the vitreous where they anastomose with vessels of the hyaloid system which persist and expand. Together, these vessels branch into the retina to form fairly normal inner retinal vascular capillary plexi. The Lama1 mutants described in this report are potential models for studying the human conditions persistent fetal vasculature and proliferative vitreoretinopathy.

Figure 1

A schematic of retinal vascular development. The key stages in vascular development of the normal mouse retina (top) and the Lama1^nmf223 mutant retina (bottom) are represented in this diagram. Solid red lines depict blood vessels while blue lines indicate vessels which originate in the Lama1 mutant retinas but traverse the ILM into the vitreous beyond the peripapillary region. Broken red lines depict the regressing hyaloid vasculature. At P1, vessels have begun forming in both the control and Lama1 mutant retinas. The fetal vessels of vitreous (FVV), which includes the tunica TVL and the VHP, are regressing in the control by P7 and the primary retinal plexus is complete, while in the Lama1 mutants, the FVV have undergone some regression but those closest to the retina have proliferated. Retinal vessels (blue) have traversed the ILM to anastomose with the VHP vessels in P7 Lama1 mutants. The retinal vasculature is complete at P21 in the control retina and no hyaloid vessels remain in the vitreous. The FVV in the Lama1 retina at P21 persist and have proliferated and formed a retinal vasculature. (TVL-Tunica vasculosa lentis; VHP-vasa hyaloidea propria).

Figure 2

Cross sections of whole eyes from P1 mice labeled with anti-PDGFRα (light blue), GS isolectin (green), anti-pan laminin (red), and DAPI (blue). Shown are both merged images (A, E, I) and individual labels. In the WT retina (A-D), astrocytes (arrowheads) and blood vessels (arrows) extended radially from the optic nerve head (asterisks) but, with the exception of a few astrocytes on the hyaloid artery, were present under the laminin-positive ILM (C). In the Lama1^nmf223 mice (E-H), astrocytes began migrating across the retina under the laminin-positive ILM (upward-facing arrowhead) but exited the retina once outside the peripapillary region (downward facing arrowhead). Blood vessels (arrow) were also present in the peripapillary retina. Individual optical slices taken from high magnification confocal Z stack images of the same area (I-L) revealed openings or breaks in the ILM through which astrocytes migrated. Astrocytes were seen in the retina (upward-facing arrowheads) and on the vitreal surface of the retina (downward-facing arrowheads) near a break in the ILM. The double arrow indicates the VHP, which in this area is devoid of astrocyte ensheathment. Scale bars indicate (A-H: 50 μm; I-L: 10 μm).

Figure 3

P1 and P3 flatmount retinas labeled with anti-GFAP (red) and GS isolectin (green). At P1, removal of the vitreous revealed an apron of vessels around the optic nerve head behind an astrocyte template in both the WT (A) and the Lama1^nmf223 retinas (B). A similar image was observed in the Lama1^nmf223 retina at P3 when the vitreous was removed (C). The WT vasculature (arrow) extended into the mid retina at P3 (D). Higher magnification more clearly demonstrated the endothelial filopodia following the GFAP-positive astrocyte template in the WT retina (E). When imaged with the vitreous intact, it is clear that astrocytes ensheath the retinal vessels but not the hyaloid (arrow) in the WT (F). By contrast, imaging of the Lama1^nmf223 retina with the vitreous still intact, demonstrated the migration of astrocytes into the vitreous (G). The hyaloid vessels are proliferating at this stage rather than regressing. Removal of the vitreous demonstrated that very few astrocytes (arrowhead) remained in the retina beyond the peripapillary region (H). Astrocytes could be seen ensheathing the vitreal blood vessels (arrows) in the Lama1^nmf223 retinas (I). A capillary network had started to form in the vitreous of the Lama1^nmf223 mutants (G). Scale bars indicate A- D: 100 μm; D-I: 50 μm).

Figure 4

Plastic sections of P1 and P3 WT and Lama1^nmf223 eyes. JB-4 sections of P1 WT peripapillary (A) and midperiphery (B) retina show astrocytes (arrowheads) in retina and VHP (arrows) in vitreous. Sections from P1 Lama1^nmf223 retinas stained with hematoxylin and PAS (C, D) showed the VHP in vitreous (arrows) as well as astrocytes in the retina (upward arrowhead) and on the vitreal surface of the ILM (downward arrowhead). Astrocytes were only observed in the retina within the peripapillary region (C). Beyond this point, these glial cells (downward arrowheads) were found on the vitreal aspect of the ILM (C). Astrocytes were not observed in the retina peripheral to traversing the ILM (D). TEM analysis of P1 Lama1^nmf223 retina (E-G) showed the ILM (open arrowheads) and astrocytes on both the vitreal (solid downward arrowheads) and retinal sides of the ILM (solid upward arrowhead). The ILM is incomplete in this area having only a single lamina. Müller cell endfeet (asterisk) can also be seen protruding through the ILM in an area where this structure is otherwise complete (G). JB-4 analysis of P3 whole eyes demonstrated the migration of retinal blood vessels (arrows) from the retina into the vitreous (H). In a neighboring section, the retinal vessels and VHP (paired arrows) can be seen anastomosing in the vitreous (I). Open arrowheads indicate the ILM. Scale bars indicate (A-D, E-F: 20 μm; C, D: 1 μm, G: 2 μm).

Figure 5

Cross sections of whole eyes from P3 mice labeled with anti-PDGFRα (light blue), GS isolectin (green), anti-pan laminin (red), and DAPI (blue). Shown are both merged images (A, E, I, M) and individual labeling. In the WT retina (A-D), astrocytes (upward arrowheads) and blood vessels extended out from the optic nerve head but were contained under the laminin-positive ILM. The VHP and hyaloid vessels within vitreous (paired arrows) were still present. Higher magnification images of the WT retina confirmed that, while there were a few astrocytes associated with the VHP (arrow) at the optic nerve head (D), most astrocytes (arrowheads) were found within the retina (E-H). In the P3 Lama1^nmf223 retina (I-L), the density of intraretinal astrocytes (upward arrowheads) was similar to that seen at P1 but there appeared to be more astrocytes within the vitreous (downward arrowheads) ensheathing the VHP and hyaloid vessels (paired arrows). Higher magnification images (M-P) demonstrated astrocytes (arrowheads) associating with VHP (arrow). As seen at P1, astrocytes within the vitreous were positive for anti-pan laminin (O). Scale bars indicate (A-D, I-L: 50 μm; E-H, M-P: 20 μm).

Figure 6

P7 flatmount retinas labeled with anti-GFAP (red) and GS isolectin (green). A complete primary retinal vascular network and astrocyte template were observed in the WT mouse at P7 (A, D = peripapillary region, B, E = midperiphery C, F = far periphery). The GS isolectin positive cells in A are likely hyalocytes on the VHP (arrows). Remodeling of the retinal blood vessels had also already occurred in the control retina (A). The astrocyte template was more evident with the green channel turned off (D-F). By contrast, retinal vessels were observed only at the optic nerve head (bottom left) in the Lama1^nmf223 retina (G) and not in mid (H) or far periphery (I). Isolated GS isolectin positive cells could be seen across the retina. Astrocytes in the mutant retina (G-I) were reduced in number and did not have the honeycomb-like pattern observed in the WT (D-F). When the vitreous was left intact, a dense astrocyte mesh was observed along with a vitreal capillary network in all regions (J = peripapillary, K = mid retina, and L = peripheral retina). Scale bars indicate 100 μm.

Figure 7

Cross sections of whole eyes from Lama1^nmf223 P7 mice were labeled with GS isolectin (green), anti-pan laminin (red), and DAPI (blue). A panoramic composite of a representative Lama1^nmf223 retina demonstrated the migration of blood vessels out of the retina towards the VHP (paired arrows) and across the vitreal surface of the ILM (A). Higher magnification of this area (B, C) demonstrated the presence of a laminin-positive membrane-like structure surrounding the exiting blood vessels (arrows) and exiting through the ILM (opposing open arrowheads). Labeling of an adjacent section (D) with anti-PDGFRα (light blue) along with GS isolectin (green), anti-pan laminin (red), and DAPI (blue) showed astrocytes within retina (upward solid arrowhead) and on the vitreal side (downward arrowhead) of the ILM (open arrowhead). Higher magnification (E) showed an astrocyte (solid arrowhead) migrating from the retina to vitreous through the ILM (open arrowhead). Scale bars indicate (A: 50 μm; B, C, E: 10 μm; D: 20 μm).

Figure 8

P10 flatmount retinas labeled with anti-GFAP (red) and GS isolectin (green). A complete and remodeled primary or superficial vasculature was observed in the WT mouse (A) and the deep vascular plexus was forming at this stage (B). A panoramic image of the Lama1^nmf223 retina with only GS isolectin labeling (C) demonstrated the abnormal persistence of the VHP, growth of vessels into vitreous from the peripapillary retina and the formation of a complex capillary network within the retina. Double labeling with anti-GFAP and GS isolectin (D) showed continued association of astrocytes with the hyaloid vessels. There appeared to be two layers of astrocytes within the vitreous, one which ensheathed the persistent VHP and one below the vessels on the surface of the retina. Also evident is branching from the intravitreal vessels (arrow) into the retina (D) to form the intraretinal plexus (E). Asterisks mark the optic nerve head. Scale bars indicate (A-C: 100 μm; D, E: 50 μm).

Figure 9

The diving of vitreal vessels into the retina is evident with both flatmount and cross section analysis. Sequential frames from a confocal Z stack image of a P10 flatmount Lama1^nmf223 retina labeled with GFAP and GS isolectin demonstrated the diving of vessels (arrows and arrowhead) into the retina (A-D). Cross sections from P10 Lama1^nmf223 eyes were labeled with anti-PDGFRα (light blue), GS isolectin (green), anti-pan laminin (red), and DAPI (blue) to support this observation. Vessels from the vitreous were branching into the retina at this stage, primarily in the inner plexiform layer, as the deep vascular plexus was forming (paired arrows) (E-H). Laminin-positive astrocytes (solid arrowhead) were also observed on the vitreal side of the ILM (open arrowhead) near the VHP (arrows) (E-H). Scale bars indicate (A-D: 20 μm; E-H: 50 μm).

Figure 10

A cross section from a P10 Lama1^nmf223 mouse labeled with GS isolectin (green), anti-pan laminin (red), anti-PDGFRα (light blue) and DAPI (blue). A PDGFRα-positive astrocyte bridge (solid arrowheads) (A, C), is attached to GS isolectin (green) positive intravitreal blood vessels (paired and single arrows) (A, D). The ILM (open arrowhead) and the basement membrane of the intravitreal vessels were laminin-positive (B). Scale bars indicate 20 μm.

Figure 11

Plastic sections of the P10 WT and Lama1^nmf223 eyes. JB-4 cross sections revealed the diving of superficial retinal vessels (paired arrows) in the WT retina (A, B). In the Lama1^nmf223 retinas, astrocytes (arrowheads) and blood vessels (paired arrows) were in the vitreous and entering the retina (C, D). TEM confirms the presence of astrocytes (arrowhead) and blood vessels (arrow) in the vitreous (E) with intermediate filaments evident in astrocytes at high magnification (F). Scale bars indicate (A-D: 20 μm; E: 3 μm; F: 250 nm).

Figure 12

The retinal phenotype in the adult Lama1^nmf223 mouse. Fundus image (A) of the adult Lama1^nmf223 mouse reveals a membrane-like structure in the vitreous (arrowhead) surrounding persistent VHP near the optic nerve head (asterisk). Fluorescein angiography (B) confirms the presence of patent vessels (arrow) within the vitreous along with the inner retinal capillaries. Anti-GFAP (red) and GS isolectin (green) labeled flatmount retina (C) shows the extension of an astrocyte membrane (arrowheads) across the retina along the persistent VHP (arrow). Asterisks indicate the optic nerve head. Cross sections (D, E) labeled with anti-PDGFRα (light blue), anti-pan laminin (red) and GS isolectin (green), and DAPI (blue) further demonstrate the astrocytes in the vitreous (solid arrowhead) above the ILM (open arrowhead) and the presence of superficial and deep capillaries in the retina (paired arrows). Diving of vitreal vessels (arrow) into the retina was also observed (E). Scale bars indicate (C: 100 μm; D: 40 μm; E: 20 μm).

Figure 13

Summary of vascular and astrocyte development in the Lama1^Δ retina TEM analysis of P1 WT retina demonstrates a double layered ILM (A). In contrast, the ILM in a Lama1^Δ mutant mouse contains frequent openings or breaks in this structure through which Müller cell processes extended into the vitreous (B). Open arrowheads point to the ILM. Examination of the Lama1^Δ mouse reveals a similar vascular development pattern similar to that seen in the Lama1^nmf223 mouse. (C) Anti-GFAP (red) and GS isolectin (green) labeling show the presence of a vascular apron (arrow) around the optic nerve head (asterisk) as well as large blood vessels of the hyaloid vasculature in the P1 Lama1^Δ mouse. At P7, astrocytes have entered the vitreous where they associate with hyaloid vessels (D). A dense astrocyte membrane and capillary network are observed in the vitreous across the entire retina at P10 (E). Diving vitreal vessels (arrows) can be also observed (F). A deep retinal plexus is forming from these diving vessels (G). Asterisks indicate the optic nerve head. Scale bars indicate (A: 500 nm; B: 2 μm; C, F: 50 μm; G, E, G: 100 μm).