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Retinal ganglion cell (RGC) death causes irreversible vision loss in patients with glaucoma and other forms of optic neuropathy because the mammalian retina and optic nerve lack endogenous regenerative capacity. RGC transplantation and optic nerve regeneration represent a potential translational approach to vision restoration in glaucoma secondary to RGC loss. Functional RGC replacement requires that 1) donor RGCs integrate into the recipient retina and receive synaptic input from afferent bipolar and amacrine cells and that 2) donor RGCs extend their axons and establish synaptic connections to appropriate neurons in the brain. Here, in an effort to address retinal integration, we demonstrate that the internal limiting membrane (ILM) acts as a physical barrier to the integration of transplanted human stem cell-derived RGCs (hRGCs) into the recipient retina following intravitreal transplantationin vivo. To circumvent the ILM barrier, we intravitreally injected the nonspecific protease pronase-E in immunosuppressed adult C57BL/6J mice prior to transplantation of hRGCs. Separately, we also transplanted hRGCs into adultLama1nmf223mice, which harbor a point mutation in theirLama-α1gene that causes developmental ILM dysgenesis. We assessed donor hRGC survival and engraftment using 3D reconstructions of confocal z-stacks in retinal flatmounts. Migration of surviving donor RGC somas into the recipient RGC layer significantly increased after proteolytic or developmental ILM disruption. Moreover, lamination of dendritic arbors into the recipient inner plexiform layer was observed exclusively following ILM disruption. To assess the clinical translatability of this finding, we transplanted hRGCs onto postmortem organotypic human retinal explant cultures and observed significantly increased engraftment following proteolytic ILM digestion. These findings enhance our understanding of the barriers faced by transplanted hRGCs in the adult murine and human retina and provide an avenue for clinically translatable regenerative medicine approaches to vision restoration in optic neuropathy.

Purpose This study evaluates whether topical ketotifen fumarate (KTF) can prevent geographic atrophy (GA)-like phenotypes in a rat model. Methods Pharmacokinetics (PKs) of KTF after topical administration twice daily for 5 days was analyzed in rat retina, retinal pigment epithelium (RPE)/choroid/sclera, and in plasma by an liquid chromatography tandem mass spectrometry (LC-MS/MS) method. Rats were then given hydrogel implants +/- 48/80 in the superior subconjunctival space and topically treated with 1% and 0.25% of KTF or phosphate buffer saline (PBS) twice daily. Rats were euthanized at 1, 2, 4, and 8 weeks postinjection. Choroidal mast cells (MCs) were stained with nonspecific esterase and the RPE monolayer was labeled with RPE65 and ZO-1 in whole mount choroids. Retinal and choroidal areas were determined in cryosections stained with picrosirius red. Dark-adapted electroretinogram (ERG) was also performed to evaluate retinal function. Results PK results showed the highest level of KTF (average 5.6 nM/mg) in the RPE/choroid/sclera in rats given topical 1% KTF. Topical 1% KTF significantly reduced choroidal MC degranulation at 1 week and 2 weeks (both P < 0.001) and RPE loss at 4 weeks (P < 0.001) as well as retinal and choroidal thinning (both P < 0.001) and reduction in ERG amplitude at 8 weeks (P < 0.05) compared to PBS. Similar results were obtained with 0.25% KTF. Conclusions Both 1% and 0.25% KTF eye drops effectively reduced MC degranulation, RPE loss, and retinal and choroidal thinning while preventing the decline of ERG amplitude in a GA-like rat model. These data suggest that topical KTF might be a new therapeutic drug for treating GA. Translational Relevance The results of this study demonstrate that topical KTF successfully reduced GA-like phenotypes in a rat model and may provide a novel therapy for GA.

Purpose The present study investigated retinal glia and choroidal vessels in flatmounts and sections from individuals with clinically diagnosed Stargardt disease (STGD). Methods Eyes from three donors clinically diagnosed with STGD were obtained through the Foundation Fighting Blindness (FFB). Genetic testing was performed to determine the disease-causing mutations. Eyes were enucleated and fixed in 4% paraformaldehyde and 0.5% glutaraldehyde. After imaging, retinas were dissected and immunostained for glial fibrillary acidic protein, vimentin, and peanut agglutin. Following RPE removal, the choroid was immunostained with Ulex europaeus agglutinin lectin. For each choroid, the area of affected vasculature, percent vascular area, and choriocapillaris luminal diameters were measured. The retina from one donor was hemisected and cryopreserved or embedded in JB-4 for cross-section analysis. Results Genetic testing confirmed the STGD diagnosis in donor 1, whereas a mutation in peripherin 2 was identified in donor 3. Genetic testing was not successful on donor 2. Therefore, only donor 1 can definitively be classified as having STGD. All donors had areas of RPE atrophy within the macular region, which correlated with underlying choriocapillaris loss. In addition, Muller cells formed pre- and subretinal membranes. Subretinal gliotic membranes correlated almost identically with RPE and choriocapillaris loss. Conclusions Despite bearing different genetic mutations, all donors demonstrated choriocapillaris loss and Muller cell membranes correlating with RPE loss. Muller cell remodeling was most extensive in the donor with the peripherin mutation, whereas choriocapillaris loss was greatest in the confirmed STGD donor. This study emphasizes the importance of genetic testing when diagnosing macular disease.

Although chronic inflammation is believed to contribute to the pathology of age-related macular degeneration (AMD), knowledge regarding the events that elicit the change from para-inflammation to chronic inflammation in the pathogenesis of AMD is lacking. We propose here that lipocalin-2 (LCN2), a mammalian innate immunity protein that is trafficked to the lysosomes, may contribute to this process. It accumulates significantly with age in retinal pigment epithelial (RPE) cells of Cryba1 conditional knockout (cKO) mice, but not in control mice. We have recently shown that these mice, which lack βA3/A1-crystallin specifically in RPE, have defective lysosomal clearance. The age-related increase in LCN2 in the cKO mice is accompanied by increases in chemokine (C-C motif) ligand 2 (CCL2), reactive gliosis, and immune cell infiltration. LCN2 may contribute to induction of a chronic inflammatory response in this mouse model with AMD-like pathology.

The mechanisms controlling vascular development, both normal and pathological, are not yet fully understood. Many diseases, including cancer and diabetic retinopathy, involve abnormal blood vessel formation. Therefore, increasing knowledge of these mechanisms may help develop novel therapeutic targets. The identification of novel proteins or cells involved in this process would be particularly useful. The retina is an ideal model for studying vascular development because it is easy to access, particularly in rodents where this process occurs post-natally. Recent studies have suggested potential roles for laminin chains in vascular development of the retina. This review will provide an overview of these studies, demonstrating the importance of further research into the involvement of laminins in retinal blood vessel formation.

Purpose Abnormal retinal angiogenesis leads to visual impairment and blindness. Understanding how retinal vessels develop normally has dramatically improved treatments for people with retinal vasculopathies, but additional information about development is required. Abnormal neuron patterning in the outer retina has been shown to result in abnormal vessel development and blindness, for example, in people and mouse models with Crumbs homologue 1 (CRB1) mutations. In this study, we report and characterize a mouse model of inner retinal lamination disruption and bleeding, the Down syndrome cell adhesion molecule (Dscam) mutant, and test how neuron-neurite placement within the inner retina guides development of intraretinal vessels. Methods Bax mutant mice (increased neuron cell number), Dscam mutant mice (increased neuron cell number, disorganized lamination), Fat3 mutant mice (disorganized neuron lamination), and Dscam gain-of-function mice (Dscam(GOF)) (decreased neuron cell number) were used to manipulate neuron placement and number. Immunohistochemistry was used to assay organization of blood vessels, glia, and neurons. In situ hybridization was used to map the expression of angiogenic factors. Results Significant changes in the organization of vessels within mutant retinas were found. Displaced neurons and microglia were associated with the attraction of vessels. Using Fat3 mutant and Dscam(GOF) retinas, we provide experimental evidence that vessel branching is induced at the neuron-neurite interface, but that other factors are required for full plexus layer formation. We further demonstrate that the displacement of neurons results in the mislocalization of angiogenic factors. Conclusions Inner retina neuron lamination is required for development of intraretinal vessels.

Nephronophthisis (NPHP) is an autosomal recessive kidney disease that is often associated with vision and/or brain defects. To date, 11 genes are known to cause NPHP. The gene products, while structurally unrelated, all localize to cilia or centrosomes. Although mouse models of NPHP are available for 9 of the 11 genes, none has been described for nephronophthisis 4 (Nphp4). Here we report a novel, chemically induced mutant, nmf192, that bears a nonsense mutation in exon 4 of Nphp4. Homozygous mutant Nphp4(nmf192/nmf192) mice do not exhibit renal defects, phenotypes observed in human patients bearing mutations in NPHP4, but they do develop severe photoreceptor degeneration and extinguished rod and cone ERG responses by 9 weeks of age. Photoreceptor outer segments (OS) fail to develop properly, and some OS markers mislocalize to the inner segments and outer nuclear layer in the Nphp4(nmf192/nmf192) mutant retina. Despite NPHP4 localization to the transition zone in the connecting cilia (CC), the CC appear to be normal in structure and ciliary transport function is partially retained. Likewise, synaptic ribbons develop normally but then rapidly degenerate by P14. Finally, Nphp4(nmf192/nmf192) male mutants are sterile and show reduced sperm motility and epididymal sperm counts. Although Nphp4(nmf192/nmf192) mice fail to recapitulate the kidney phenotype of NPHP, they will provide a valuable tool to further elucidate how NPHP4 functions in the retina and male reproductive organs.

The Neuromutagenesis Facility at the Jackson Laboratory generated a mouse model of retinal vasculopathy, nmf223, which is characterized clinically by vitreal fibroplasia and vessel tortuosity. nmf223 homozygotes also have reduced electroretinogram responses, which are coupled histologically with a thinning of the inner nuclear layer. The nmf223 locus was mapped to chromosome 17, and a missense mutation was identified in Lama1 that leads to the substitution of cysteine for a tyrosine at amino acid 265 of laminin alpha1, a basement membrane protein. Despite normal localization of laminin alpha1 and other components of the inner limiting membrane, a reduced integrity of this structure was suggested by ectopic cells and blood vessels within the vitreous. Immunohistochemical characterization of nmf223 homozygous retinas demonstrated the abnormal migration of retinal astrocytes into the vitreous along with the persistence of hyaloid vasculature. The Y265C mutation significantly reduced laminin N-terminal domain (LN) interactions in a bacterial two-hybrid system. Therefore, this mutation could affect interactions between laminin alpha1 and other laminin chains. To expand upon these findings, a Lama1 null mutant, Lama1(tm1.1Olf), was generated that exhibits a similar but more severe retinal phenotype than that seen in nmf223 homozygotes. The increased severity of the Lama1 null mutant phenotype is probably due to the complete loss of the inner limiting membrane in these mice. This first report of viable Lama1 mouse mutants emphasizes the importance of this gene in retinal development. The data presented herein suggest that hypomorphic mutations in human LAMA1 could lead to retinal disease.

We have previously reported that in the Nuc1 rat, which has a spontaneous mutation in Cryba1 (the gene encoding βA3/A1-crystallin), astrocytes exhibit decreased Notch signaling, leading to reduced promoter activity for glial fibrillary acidic protein (GFAP). Interestingly, in both Nuc1 astrocytes and in wild type astrocytes following knockdown of Cryba1, vascular endothelial growth factor (VEGF) secretion is decreased. This has led us to explore signaling mediators that could be regulated by βA3/A1-crystallin to modulate both GFAP and VEGF. Several studies have shown that the signal transducer and activator of transcription 3 (STAT3) is involved in the co-regulation of GFAP and VEGF. We show that STAT3 and βA3/A1-crystallin may co-regulate each other in astrocytes. Such co-regulation would create a positive feedback circuit; i.e., in the cytosol of astrocytes, βA3/A1-crystallin is necessary for the phosphorylation of STAT3, which then dimerizes and translocates to the nucleus to form DNA-binding complexes, activating transcription of Cryba1. This stoichiometric co-regulation of STAT3 and Cryba1 could potentiate expression of GFAP and secretion of VEGF, both of which are essential for maintaining astrocyte and blood vessel homeostasis in the retina. Consistent with this idea, Cryba1 knockout mice exhibit an abnormal astrocyte pattern and defective remodeling of retinal vessels.

Purpose Muller cells create the external limiting membrane (ELM) by forming junctions with photoreceptor cells. This study evaluated the relationship between focal photoreceptors and RPE loss in geographic atrophy (GA) and Muller cell extension into the subretinal space. Methods Human donor eyes with no retinal disease or geographic atrophy (GA) were fixed and the eye cups imaged. The retinal posterior pole was stained for glial fibrillary acidic protein (GFAP; astrocytes and activated Muller cells) and vimentin (Muller cells) while the submacular choroids were labeled with Ulex Europaeus Agglutinin lectin (blood vessels). Choroids and retinas were imaged using a Zeiss 710 confocal microscope. Additional eyes were cryopreserved or processed for transmission electron microscopy (TEM) to better visualize the Muller cells. Results Vimentin staining of aged control retinas (n = 4) revealed a panretinal cobblestone-like ELM. While this pattern was also observed in the GA retinas (n = 7), each also had a distinct area in which vimentin+ and vimentin+/GFAP+ processes created a subretinal membrane. Subretinal glial membranes closely matched areas of RPE atrophy in the gross photos. Choroidal vascular loss was also evident in these atrophic areas. Smaller glial projections were noted, which correlated with drusen in gross photos. The presence of glia in the subretinal space was confirmed by TEM and cross cross-section immunohistochemistry. Conclusions In eyes with GA, subretinal Muller cell membranes present in areas of RPE atrophy may be a Muller cell attempt to replace the ELM. These membranes could interfere with treatments such as stem cell therapy.

Purpose The choriocapillaris (CC), the capillary network of the choroid, is positioned adjacent to Bruch's membrane (BM) and the RPE. The aim of this study was to clarify the mechanism(s) for transport of serum albumen from CC lumen to RPE. Methods Alexa647 conjugated to BSA (BSA-A647) or PBS was administrated via the femoral vein to young and aged wild-type (WT; C57BL/6J) mice and Caveolin-1 knockout mice (Cav1(-/-)). Mice were perfused with PBS and killed at 30 minutes, 1 hour, and 4 hours after injection. Eyecups were cryopreserved, and cryosections were analyzed on a Zeiss 710 confocal microscope. Bovine serum albumin conjugated to gold nanoparticles (BSA-GNP) was administrated through the left common carotid artery. Mice were perfused with PBS and killed at 30 minutes after injection. Eyecups were embedded after fixation, and 70-nm-thick sections were analyzed on a Hitachi H7600 transmission electron microscope. Results In eyes of WT young mice, BSA-A647 was transported to the RPE at 30 minutes and diffused to the photoreceptor layer by 1 hour. In contrast, most BSA-A647 was found in the CC in Cav1(-/-) eyes. The majority of BSA-GNP found in the CC of young WT mice was on the luminal side in caveolae at 30 minutes after injection. In aged WT mice, BSA-GNPs were found in defective tight junctions between endothelial cells and appeared trapped at the diaphragm of fenestrations. Conclusions Normally, CC carefully regulates transport system of BSA from lumen to BM by caveolae-mediated transcytosis; however, endothelium cells of aged control WT mice have leaky tight junctions and lacked regulated BSA transport.

VEGF(165) b is an anti-angiogenic form of VEGF(165) produced by alternative splicing. The localization of pro-angiogenic VEGF(165) and anti-angiogenic VEGF(165) b was investigated during development of the vasculatures in fetal human eyes from 7 to 21 weeks gestation (WG). The fetal vasculature of vitreous, which includes tunica vasculosa lentis (TVL), had moderate VEGF(165) immunoreactivity at 7WG and very little VEGF(165) b. Both forms were elevated at 12WG. VEGF(165) then decreased around 17WG when the TVL regresses but VEGF(165) b remained elevated. In choroid, VEGF(165) was present in forming choriocapillaris (CC) and retinal pigment epithelium (RPE) at 7WG while VEGF165b was present in CC and mesenchymal precursors within the choroidal stroma. By 21WG, both forms were elevated in RPE and choroidal blood vessels but VEGF(165) b was apical and VEGF(165) basal in RPE. Diffuse VEGF(165) immunoreactivity was prominent in 12WG innermost retina where blood vessels will form while VEGF(165) b was present in most CXCR4(+) progenitors in the inner neuroblastic layer and migrating angioblasts in the putative nerve fiber layer. By 21WG, VEGF(165) was present in nerve fibers and VEGF(165) b in the inner Muller cell process. The localization of VEGF(165) b was distinctly different from VEGF(165) both spatially and temporally and it was often associated with nucleus in progenitors.

Retinal vascular development is a complex process that is not yet fully understood. The majority of research in this area has focused on astrocytes and the template they form in the inner retina, which precedes endothelial cells in the mouse retina. In humans and dogs, however, astrocyte migration follows behind development of blood vessels, suggesting that other cell types may guide this process. One such cell type is the ganglion cell, which differentiates before blood vessel formation and lies adjacent to the primary retinal vascular plexus. The present study investigated the potential role played by ganglion cells in vascular development using Math5(-/-) mice. It has previously been reported that Math5 regulates the differentiation of ganglion cells and Math5(-/-) mice have a 95% reduction in these cells. The development of blood vessels and glia was investigated using Griffonia simplicifolia isolectin B4 labeling and GFAP immunohistochemistry, respectively. JB-4 analysis demonstrated that the hyaloid vessels arose from choriovitreal vessels adjacent to the optic nerve area. As previously reported, Math5(-/-) mice had a rudimentary optic nerve. The primary retinal vessels did not develop post-natally in the Math5(-/-) mice, however, branches of the hyaloid vasculature eventually dove into the retina and formed the inner retinal capillary networks. An astrocyte template only formed in some areas of the Math5(-/-) retina. In addition, GFAP(+) Müller cells were seen throughout the retina that had long processes wrapped around the hyaloid vessels. Transmission electron microscopy confirmed Müller cell abnormalities and revealed disruptions in the inner limiting membrane. The present data demonstrates that the loss of ganglion cells in the Math5(-/-) mice is associated with a lack of retinal vascular development.

We had developed a conditional Laminin α 1 knockout-mouse model (Lama1(cko)) bypassing embryonic lethality of Lama1 deficient mice to study the role of this crucial laminin chain during late developmental phases and organogenesis. Here, we report a strong defect in the organization of the adult cerebellum of Lama1(cko) mice. Our study of the postnatal cerebellum of Lama1(cko) animals revealed a disrupted basement membrane correlated to an unexpected excessive proliferation of granule cell precursors in the external granular layer (EGL). This was counteracted by a massive cell death occurring between the postnatal day 7 (P7) and day 20 (P20) resulting in a net balance of less cells and a smaller cerebellum. Our data show that the absence of Lama1 has an impact on the Bergmann glia scaffold that aberrantly develops. This phenotype is presumably responsible for the observed misplacing of granule cells that may explain the overall perturbation of the layering of the cerebellum and an aberrant folia formation.

Mutations in the Rhodopsin (Rho) gene can lead to autosomal dominant retinitis pigmentosa (RP) in humans. Transgenic mouse models with mutations in Rho have been developed to study the disease. However, it is difficult to know the source of the photoreceptor (PR) degeneration in these transgenic models because overexpression of wild type (WT) Rho alone can lead to PR degeneration. Here, we report two chemically mutagenized mouse models carrying point mutations in Rho (Tvrm1 with an Y102H mutation and Tvrm4 with an I307N mutation). Both mutants express normal levels of rhodopsin that localize to the PR outer segments and do not exhibit PR degeneration when raised in ambient mouse room lighting; however, severe PR degeneration is observed after short exposures to bright light. Both mutations also cause a delay in recovery following bleaching. This defect might be due to a slower rate of chromophore binding by the mutant opsins compared with the WT form, and an increased rate of transducin activation by the unbound mutant opsins, which leads to a constitutive activation of the phototransduction cascade as revealed by in vitro biochemical assays. The mutant-free opsins produced by the respective mutant Rho genes appear to be more toxic to PRs, as Tvrm1 and Tvrm4 mutants lacking the 11-cis chromophore degenerate faster than mice expressing WT opsin that also lack the chromophore. Because of their phenotypic similarity to humans with B1 Rho mutations, these mutants will be important tools in examining mechanisms underlying Rho-induced RP and for testing therapeutic strategies.

Variability in onset, progression, severity, and phenotypic expression is commonly observed in many retinal diseases (Tables 1 and 2). Although interfamily variability may be caused by environmental or allelic differences, intrafamily variability, when a common mutation is segregating, may also be due to genetic modifiers (1, 2, 3). In contrast to independently acting alleles that may lead to an additive effect on disease severity or age of onset, genetic modifiers are defined as background genes that epistatically interact with a given disease genotype to affect phenotypic outcome. In general, allelic variability at modifier loci does not in itself produce a phenotype. A single gene or possibly a combination of genes in the same or parallel pathways as the mutant gene may act to create a final effect on the expression of the disease phenotype. These modifiers may enhance the effect of the mutation to cause a more severe mutant phenotype or an earlier onset, or conversely, delay or reduce the mutant phenotype even to the extent of completely restoring the wild-type (WT) condition.

Purpose The mode of development of the human hyaloid vascular system (HVS) remains unclear. Early studies suggested that these blood vessels formed by vasculogenesis, while the current concept seems to favor angiogenesis as the mode of development. We examined embryonic and fetal human HVS using a variety of techniques to gain new insights into formation of this vasculature. Methods Embryonic and fetal human eyes from 5.5 to 12 weeks gestation (WG) were prepared for immunohistochemical analysis or for light and electron microscopy. Immunolabeling of sections with a panel of antibodies directed at growth factors, transcription factors, and hematopoietic stem cell markers was employed. Results Light microscopic examination revealed free blood islands (BI) in the embryonic vitreous cavity (5.5-7 WG). Giemsa stain revealed that BI were aggregates of mesenchymal cells and primitive nucleated erythroblasts. Free cells were also observed. Immunolabeling demonstrated that BI were composed of mesenchymal cells that expressed hemangioblast markers (CD31, CD34, C-kit, CXCR4, Runx1, and VEGFR2), erythroblasts that expressed embryonic hemoglobin (Hb-e), and cells that expressed both. Few cells were proliferating as determined by lack of Ki67 antigen. As development progressed (12 WG), blood vessels became more mature structurally with pericyte investment and basement membrane formation. Concomitantly, Hb-e and CXCR4 expression was down-regulated and von Willebrand factor expression was increased with the formation of Weibel-Palade bodies. Conclusions Our results support the view that the human HVS, like the choriocapillaris, develops by hemo-vasculogenesis, the process by which vasculogenesis, erythropoiesis, and hematopoiesis occur simultaneously from common precursors, hemangioblasts.

PURPOSE. To determine the molecular basis and the pathologic consequences of a chemically induced mutation in a mouse model of photoreceptor degeneration, nmf240. METHODS. Mice from a G3 N-ethyl-N-nitrosourea mutagenesis program were screened by indirect ophthalmoscopy for abnormal fundi. A chromosomal position for the recessive nmf240 mutation was determined by a genome-wide linkage analysis by use of simple sequence length polymorphic markers in an F2 intercross. The critical region was refined, and candidate genes were screened by direct sequencing. The nmf240 phenotype was characterized by histologic analysis of the retina, brain, and male reproductive organs and by electroretinogram (ERG)-based studies of the retina and retinal pigment epithelium (RPE). RESULTS. Clinically, homozygous nmf240 mutants exhibit a grainy retina that progresses to panretinal patches of depigmentation. The mutation was localized to a region on chromosome 16 containing Clcn2, a gene associated with retinal degeneration. Sequencing identified a missense C-T mutation at nucleotide 1063 in Clcn2 that converts a glutamine to a stop codon. Mice homozygous for the Clcn2 nmf240 mutation experience a severe loss of photoreceptor cells at 14 days of age that is preceded by an elongation of RPE apical microvilli. Homozygous mutants also experience leukoencephalopathy in multiple brain areas and male sterility. Despite a normal retinal histology in nmf240 heterozygotes, the ERG light peak, generated by the RPE, is reduced. CONCLUSIONS. The nmf240 phenotype closely resembles that reported for Clcn2 knockout mice. The observation that heterozygous nmf240 mice present with a reduced ERG light peak component suggests that CLCN2 is necessary for the generation of this response component. (Invest Ophthalmol Vis Sci. 2010;51:3264‐3272) DOI:10.1167/iovs.09-4887

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.