BACKGROUND
the retinal vasculature, which nourishes the inner retina, consists of three plexi: the superficial in the nerve fiber layer, the intermediate in the inner plexiform layer, and the deep in the outer plexiform layer.
the photoreceptors in the outer retina are maintained by the choroidal vessels.
retinal vessels develop in man during fetal development  <cit>  but in mice during the first  <dig> post-natal weeks  <cit> .
prior to the formation of retinal vessels, the lens and developing inner retina are nourished by the hyaloid vasculature.
this three component vasculature  lies in the vitreous, a gel-like structure separating the retina and the lens.
the hyaloid vessels regress as retinal vessels form and are gone in humans by birth and by three weeks in the mouse.
the portion of the hyaloid vasculature closest to the retina is the vasa hyaloidea propria  which is also the last to regress.
although occurring prior to birth in the human and post-natally in the mouse, the retinal vasculature develops in a similar pattern in these species.
the primary vascular plexus forms first and, once complete, branching vessels dive into the inner retina to form the deep plexus and then the intermediate plexus .
the stimuli guiding the formation of retinal vessels, has yet to be confirmed.
while in the mouse retina, endothelial cells migrate and proliferate across the retina guided by an astrocyte template  <cit> , other guidance systems determine the pattern in dog  <cit>  and man  <cit> .
furthermore, many studies have pointed towards vegf as a key regulator in vascular development  <cit> , but it has recently been demonstrated that retinal vessels form normally without astrocyte-derived vegf  <cit> .
together, these data suggest that other proteins and, potentially, cells other than astrocytes guide endothelial cell migration in forming the superficial retinal vasculature.
the proximity of the internal limiting membrane  to retinal astrocytes and the superficial vasculature suggests that this structure may be important for development of retinal blood vessels.
indeed, several mouse mutants with disruptions to the ilm have an abnormal retinal vasculature  <cit> .
laminin α <dig> is a primary component of laminin  <dig>  which is believed to be important for basement membrane formation  <cit> .
in addition, this laminin chain links the ilm to receptors, such as integrins and dystroglycan  <cit> , which are found on müller cells in the retina  <cit> .
retinal vascular defects have recently been described in two mouse lines with mutations in lama <dig>  the gene which encodes laminin α <dig>  <cit> .
while the vascular defects are similar, the two mutants differ in their generation and effect on laminin α <dig>  one being a recessive point mutation  and the other being a conditional knockout that leads to the complete loss of lama <dig>  but bypasses the embryonic lethality normally associated with laminin α <dig> deletion.
in the lama1nmf <dig> mice, a chemically-induced mutation causes the replacement of a tyrosine with a cysteine at amino acid  <dig>  in the n-terminal domain, which includes sites for receptors binding as well as polymerization  <cit> .
here forward, lama1nmf <dig> and lama1Δ refer to mice homozygous for the respective mutations.
the present study investigates the development of retinal vessels in the previously described lama <dig> mutants.
focus was placed on the lama1nmf <dig> because a random point mutation has greater potential to be associated with human disease than the complete deletion of lama <dig>  which is embryonic lethal in mice  <cit>  and likely to cause lethality in humans as well.
the observation that a point mutation in this large gene causes retinal disease in mice suggests that it may also do so in humans.
transmission electron microscopy  was used for ultrastructural analysis of the ilm, glial cells, and blood vessels in these mice.
finally, epiretinal membranes are described in both of these mutants that have characteristics similar to human persistent fetal vasculature  and proliferative vitreoretinopathy .
RESULTS
retinal vessels form in the lama1nmf <dig> retina at p1
blood vessel and astrocyte development was first investigated in cross sections by labeling with anti-pan laminin, anti-platelet-derived growth factor receptor α  and gs isolectin at postnatal day   <dig> when the primary retinal vasculature has just begun to form.
in the wild type  retina, astrocytes and blood vessels extended from the optic nerve head under the laminin-positive ilm .
although some astrocytes were also observed along the base of the hyaloid artery, the vast majority of these glial cells were observed under the ilm.
no astrocytes were observed in the vitreous away from the hyaloid artery.
in the lama1nmf <dig> sections, blood vessels and pdgfrα-positive astrocytes were observed on the retinal side of the ilm in the peripapillary region, the area surrounding the optic nerve head .
adjacent to this region, however, astrocytes were observed traversing the ilm into the vitreous .
in addition, some sections demonstrated astrocytes migrating into the vitreous along the hyaloid vessels .
sequential confocal z stacks were used to evaluate these sections and revealed openings in the ilm through which astrocytes entered the vitreous .
peripheral to these "break" points, astrocytes were observed in a linear arrangement on the vitreal aspect of the ilm and only an occasional astrocyte was observed in the retina .
unlike those in the retina, vitreal astrocytes expressed laminin.
flatmount wt retinas were labeled with fluorescein isothiocyanate conjugated griffonia simplicifolia isolectin b <dig>  to visualize blood vessels and anti-glial fibrillary acidic protein  to visualize astrocytes.
this labeling revealed a vascular apron surrounding the optic nerve head with endothelial cell filopodia extending out over an astrocyte template .
when present, hyaloid vessels were easily distinguished from those in the retina by the plane of focus and their gs isolectin intensity.
a similar vascular apron was observed in lama1nmf <dig> mice .
in addition, a number of individual gs isolectin positive cells were observed in both the wt and lama1nmf <dig> retinas.
some of these cells are microglia, which gs isolectin also labels, as they also were positive for iba- <dig> .
jb- <dig> sections taken from wt eyes that were stained with periodic acid-schiff  and hematoxylin revealed that all astrocytes were under the fully intact ilm .
jb- <dig> sections also demonstrated astrocytes leaving the retina in the peripapillary region of lama1nmf <dig> mice .
peripheral to this point, astrocytes were not observed in the retina .
ultrastructure analysis with tem confirmed that astrocytes migrate into the vitreous through openings/breaks in the ilm.
müller cell processes were also seen extending into the vitreous at these break points .
although they express gfap, the astrocytes in the vitreous have few, if any, well-defined intermediate filaments at this stage, demonstrating that they are still maturing .
the migration of astrocytes into the vitreous near the peripapillary region and on the hyaloid artery was observed consistently in all eyes examined with the different techniques.
lama1nmf <dig> retinal vessels migrate into the vitreous by p3
examination of retinal flatmounts at p <dig> revealed the extension of blood vessels in the wt retina to the midperiphery behind a fully-formed astrocytic template .
the hyaloid vessels, which detached from the retina during dissection, were still present but were not ensheathed with retinal astrocytes .
immunohistochemical labeling of cross sections demonstrated that retinal vessels and astrocytes were located on the retinal side of the ilm .
one or two astrocytes were observed along the hyaloid artery right at the optic nerve head .
despite the close proximity of the vhp vessels to the ilm, they did not cross this barrier or make contact with retinal astrocytes in the wt eyes .
by contrast, the vessels in the lama1nmf <dig> retinas had not progressed from that seen at p <dig> in retinal flatmounts .
there were also less astrocytes in the mutant retina  compared to the wt .
a number of gs isolectin-positive cells were, however, observed throughout both the wt  and lama1nmf <dig> retinas .
these cells were more prominent in the avascular regions of both the wt and the lama1nmf <dig> retinas.
imaging of retinas with the vitreous intact demonstrated that most of the lama1nmf <dig> astrocytes had migrated into the vitreous where they invested the vhp .
astrocytes were also observed forming thin glial bridges connecting blood vessels within the vitreous .
capillary networks were seen branching from the lama1nmf <dig> vhp .
as was observed at p <dig>  cross sectional analysis revealed astrocytes migrating from the retina into the vitreous both at the optic nerve head and through breaks in the ilm .
these astrocytes extended in a linear fashion along the vitreal aspect of the ilm.
at this stage, astrocytes were clearly enveloping vhp vessels and forming a laminin-positive membrane-like structure.
the retinal vessels observed in the peripapillary region at p <dig> had also migrated into the vitreous.
jb- <dig> cross sections demonstrated that these vessels had begun to anastomose with vessels of the vhp .
only intravitreal vessels are evident in lama1nmf <dig> mice at p7
at p <dig>  the primary vascular plexus was complete in the wt retina with the exception of the extreme periphery in some retinas .
this vasculature included arteries and veins as well as the typical chicken wire-like capillary plexus.
the astrocyte template was complete in wt mice at this stage with astrocytes ensheathing retinal vessels .
the hyaloid vasculature and vhp had begun regressing and detached easily from the retina during dissection.
when present, these vessels had gs isolectin-positive cells, likely hyalocytes, on top of them .
by contrast, the lama1nmf <dig> retinal vasculature resembled that of the p <dig> retina, being limited to an apron in the peripapillary region.
retinal vessels were absent beyond this point and individual gs isolectin-positive cells were prominent .
there was a large reduction in the number of astrocytes within the mutant retina  compared to the control .
while some peripheral retinal astrocytes formed a normal honeycomb-like pattern, most extended long thin processes across the retina.
when the vitreous was left intact, a much different image was revealed .
a large number of astrocytes were observed in the vitreous where they ensheathed vessels and formed a dense membrane-like structure.
the intravitreal capillary networks, which had begun forming at p <dig>  were more extensive.
in sections, anti-pan laminin and gs isolectin labeling demonstrated the peripapillary retinal vessels traversing the ilm into the vitreous where they continued to extend towards ora serrata .
a membrane-like laminin positive structure was forming in association with these vitreal vessels .
peripheral to break points in the ilm, no intraretinal vessels were observed .
it is important to note that the laminin-positive ilm was also observed on either side of blood vessels traversing into the vitreous .
labeling of adjacent sections with anti-pdgfrα in addition to anti-pan laminin and gs isolectin revealed that astrocytes crossed the ilm and were part of the vitreal membrane .
these astrocytes were positive for laminin.
these astrocytes extended along the vitreal side of the ilm and towards ora serrata in a fashion similar to that observed within the inner retina of the wt mice .
intravitreal vessels invade the lama1nmf <dig> retina by p10
in the p <dig> wt retina, the superficial chicken wire capillary network was remodeled into a spoke-wheel-like system with an organized branching pattern .
astrocytes had a distinct stellate morphology with thin processes that contacted and ensheathed retinal blood vessels .
superficial vessels could be seen diving to form the deep capillary plexus .
gs isolectin labeling of the lama1nmf <dig> flatmount retinas demonstrated an extensive vascular network with an unusual branching pattern in the vitreous .
when the vitreous was left intact and retinas double-labeled with gs isolectin and anti-gfap, the appearance was similar to that at p <dig> although the astrocyte "membrane" appeared to be denser.
at this stage, branches from intravitreal vessels extended into the retina to form an intraretinal capillary plexus .
this abnormal branching was best appreciated with confocal z stack images through the retina .
labeling of whole eye cross sections with anti-pdgfrα, anti-pan laminin, and gs isolectin more clearly demonstrated vessels within the vitreous and their branches that had invaded the inner and outer retina and formed a capillary network in the outer plexiform layer .
in addition to astrocytes migrating near the vitreoretinal surface, some had migrated further into the vitreous .
many of these astrocytes possessed long, thin processes and created glial bridges between intravitreal blood vessels.
jb- <dig> sections of the wt retina revealed that astrocytes resided under the ilm and that the superficial vessels were diving into the deeper retinal layers .
in the lama1nmf <dig> p <dig> jb- <dig> sections, long thin processes from the glial bridges mentioned above as well as their frequent contact with blood vessels could be appreciated .
in some cases, they created tractional attachments with the vessels .
astrocytes extended across the vitreal surface of the ilm to the far periphery.
some astrocytes appeared to have made contact with cells in the retina.
intravitreal vessels that had invaded the retina, extended into both the nerve fiber layer and into the deeper retina .
ultrastructural analysis demonstrated breaks in the ilm similar to those seen at p <dig> .
astrocytes and blood vessels were closely associated in the vitreous .
additionally, intermediate filaments were present in intravitreal astrocytes at this age .
adult lama1nmf223
fundus examination of four-week old lama1nmf <dig> mice demonstrated a white membrane-like structure surrounding hyaloid vessels in the vitreal plane of focus, .
the position of this membrane in relation to the vitreal blood vessels suggested it was the astrocytic membrane observed in flatmounts and sectioned retinas.
fluorescein angiography confirmed the presence of large patent vessels within the vitreous .
these vessels could be seen branching into the retina to form the inner retinal capillary system.
flatmount retinas labeled with anti-gfap and gs isolectin confirmed the continuance of the astrocyte membrane in the vitreous of lama1nmf <dig> mutants .
labeling with anti-pdgfrα, anti-pan laminin, and gs isolectin clearly showed a chain of pdgfrα-positive astrocytes across the vitreal surface of the ilm as well as the glial bridges between intravitreal blood vessels .
the vitreal vessels dove into the retina where they branched to form the inner and outer retinal capillary networks .
abnormal retinal vascular development in the lama1Δ mice is similar to that in lama1nmf223
transmission electron microscopy revealed a double layered ilm which spanned the entire retina in the wt mouse at p <dig> .
by contrast, the p <dig> lama1Δ mice revealed a thin, fragmented ilm that contained frequent breaks through which astrocytes, müller cells, and ganglion cells seemed to be protruding .
astrocytes were observed on the vitreal side of the ilm .
the vascular development in lama1Δ retinas was examined at key time points determined by investigation of the lama1nmf <dig> mutants: p <dig>  p <dig>  and p <dig>  at p <dig>  a small apron of vessels was evident at the optic nerve head, similar to that seen in the lama1nmf <dig> mutants .
when the vitreous was left intact, astrocytes were observed on the hyaloid vessels at p <dig>  at p <dig>  the gfap and gs isolectin labeling produced images similar to those from the lama1nmf <dig> mice showing astrocytes associating with the vhp  and no vessels within the retina .
labeling of the p <dig> retina revealed a dense astrocyte network present in the vitreous of the lama1Δ mice that was more dense than in the lama1nmf <dig> eye  and intravitreal vessels extending into the retina .
in some retinas, gfap-positive processes were also observed traversing the retina along with the blood vessels.
the deep vasculature was developing from diving vitreal vessels at p <dig> .
in summary, the same changes occurred in the lama1Δ mice as in the lama1nmf <dig> mice.
the progression, however, was slightly faster and the severity of the pathology was greater at least in terms of astrocyte coverage of the vhp.
discussion
the previous study reported that few, if any, retinal vessels developed in the lama <dig> mutants and that these were replaced by persistent intravitreal vessels which penetrate the retina to form the intraretinal capillary networks  <cit> .
using confocal microscopy, additional immunohistochemical markers, cross sectional analysis, and tem, the present study redefines the development of retinal vessels and astrocytes in the lama <dig> mutants.
in addition, the previously described "vitreal fibroplasia" is identified as a vitreal membrane containing laminin-positive astrocytes, similar to human persistent fetal vasculature.
the ilm in the lama1nmf <dig> retina is normal in appearance throughout much of the retina but has small frequent breaks.
by contrast, the lama1Δ mice have a very thin ilm with a diffuse, nonlinear appearance throughout much of the retina.
in addition, breaks in the lama1Δ retina generally extended over larger areas of the retina.
in many areas, the ilm was lacking completely.
these findings are not surprising because an ilm lacking laminin α <dig> would likely not be bound well to the retina as this protein contains the binding sites for receptors on müller cell endfeet  <cit> .
the preservation of the ilm in some areas suggests that other alpha chains present in the ilm, such as α <dig>  <cit> , assist in binding this structure to the retina.
despite the differing ilm structure, the two lama <dig> mutants have very similar abnormalities with regards to retinal vascular development.
both mutants have a vascular apron around the optic nerve head at p <dig>  suggesting that neither the y265c mutation nor the complete loss of this protein affected endothelial cell differentiation and emigration from the optic nerve.
beginning around p <dig>  however, it is evident that the retinal blood vessels cease to develop in the lama1nmf <dig> retina and have entered the vitreous where they remain until p7-p <dig>  these vessels, which originate within the peripapillary retina grow along the retinal surface and eventually anastomose with those of the vhp.
together, these vessels form a dense vascular network in the vitreous that persists into adulthood and is ensheathed by astrocytes.
these vitreal vessels sprout and invade the retina around p <dig> to form the retinal vascular capillary networks.
despite their abnormal origin, the lama <dig> mutant retinal capillary networks at p <dig>  are correctly placed within the retina.
therefore, the cues guiding these diving vessels are still present.
it appears, however, as though the intermediate plexus forms early in the lama1nmf <dig> retina as vessels are evident in the inner plexiform layer at p <dig> compared to p <dig> in the wt retina.
the formation of the inner capillary networks further indicates that neither the y265c point mutation in lama <dig> nor the complete deletion of this protein alters the ability of endothelial cells to form blood vessels.
rather, astrocyte migration across the vitreal surface of the ilm precedes and likely causes the misguided migration of the blood vessels into the vitreous.
in all retinas investigated at various ages, astrocytes were observed in the vitreous before retinal vessels traversed the ilm.
in addition, only the vhp vessels were observed in the vitreous posterior to astrocytes on the vitreal aspect of the ilm.
it is possible that if a normal template of astrocytes was present in the retina, the blood vessel development would also be normal.
therefore, identifying and neutralizing the factor which stimulate astrocyte invasion into the vitreous may also alleviate the vascular defects in these mice.
most astrocytes from the lama <dig> mutant retinas migrate towards and eventually envelop the vhp vessels.
in addition, a subpopulation of these glial cells migrates from the optic nerve head directly into the vitreous along the hyaloid artery and its branches.
these observations suggest that an attractant within the persistent and proliferating intravitreal vessels stimulates astrocyte migration.
the previous report demonstrated that the tyrosine to cysteine mutation in the lama1nmf <dig> mice significantly decreases the binding ability of laminin α <dig>  <cit> .
it is logical to hypothesize, therefore, that this mutation disrupts the strength by which laminin α <dig> and, consequently, the entire ilm, bind to receptors on müller cell endfeet.
as a result, the ilm may become less of a barrier for cells in the retina.
astrocytes may, therefore, respond to chemoattractants in the vitreous more easily than those in the wt retina.
astrocyte migration into the vitreous in response to these chemoattractants may create breaks in the ilm.
this idea is supported by the fact that in most cases, both laminin immunohistochemistry and tem show the ilm pushed inwardly toward the vitreous where the cells exit the retina rather than there being a clear opening in the ilm through which cells migrate .
indeed, a number of proteins within the normal vitreous are known to stimulate astrocyte migration, including hyaluronic acid  <cit> , endothelin- <dig>  <cit> , tgf-beta, and pdgf-aa  <cit>
.
among these, pdgf-aa is the best candidate as it is known to stimulate astrocyte migration and is believed to be generated by ganglion cells in the retina  <cit> .
furthermore, pdgf-aa overexpression by the lens stimulates astrocyte migration into the vitreous, resulting in a pattern similar to that observed in the lama <dig> mutants  <cit> .
once within the vitreous, most astrocytes migrate along the vitreal surface of the ilm towards the ora serrata.
this migration pattern is similar to that seen within wt retinas with the exception that it occurs on the vitreal side of the ilm.
this observation indicates that the migratory cues normally present inside the retina are on the vitreal surface of the ilm in lama <dig> mutants.
it could also be hypothesized that proteins constituting the ilm act as guidance cues and astrocytes in the mutant retina respond to these but remain on the vitreal side once they have traversed the ilm.
yet another theory is that müller cells, the other retinal glial cell, guide astrocyte migration.
although müller cells may not be fully differentiated, these glial cells are in place prior to astrocyte migration into and across the retina.
furthermore, müller cell processes in both lama <dig> mutants extend into the vitreous where chemoattractants they produce could spill out.
in addition, some müller cell endfeet are properly positioned, potentially contributing to frequent contact between vitreal astrocytes and the retina.
further investigation is required to identify the stimuli that guide astrocytes across the retina and, perhaps more importantly, what cell type provide these stimuli.
the association of astrocytes with hyaloid vessels occurs in other rodent mutants with persistent fetal vasculature, including collagen 15a1/18a <dig> double knockouts  <cit> , lif transgenics  <cit> , and the nuc <dig> rat  <cit> .
the question arises then, do astrocytes associate with vitreal vessels as a consequence of their failure to regress or is the astrocyte association preventing their regression by stabilizing the vasculature?
in the case of the lama <dig> mutants, the astrocytes migrate into the vitreous and associate with hyaloid and vhp by p <dig>  before vhp regression has normally begun.
this suggests that the astrocyte ensheathment of vitreal blood vessels subsequently inhibits the vessel regression and may even stimulate their proliferation.
astrocyte ensheathment of the fetal vasculature in vitreous also occurs in the human disease pfv  <cit> .
pfv can be a blinding disorder because the retinal vasculature is incomplete and the persistent fvv and membrane pulls on the retina causing detachment  <cit> .
preretinal glial membranes are often found in patients with pfv and likely contribute to retinal detachment  <cit> .
it is not uncommon for patients with pfv to develop cataracts and glaucoma as well  <cit> .
few animal models exist for this condition and treatment involves surgery.
the lama <dig> mutants described herein have two hallmark features of this disease: persistence of the fetal vasculature and preretinal glial membranes, making them potential models for studying this disease.
the membranes formed by astrocytes in lama <dig> mutants also resemble epiretinal membranes seen in patients with proliferative vitreoretinopathy , a complication of retinal detachment in which retinal cells and macrophages enter the vitreous.
the ultrastructure of the glial-containing membrane in lama <dig> mutants is similar to that described for epiretinal membranes  <cit> .
in some areas, astrocytes extend thin, delicate processes across the vitreoretinal surface and into the vitreous.
these created structures similar to those which foos called glial bridges  <cit> .
in others areas, numerous astrocytes overlap one another to form a dense mesh-like structure.
while the glial membranes in lama <dig> mutants contain mainly tightly associated astrocytes, frequent irregularly large openings are also observed .
the astrocytes associated with this membrane make laminin, suggesting they may also produce other extracellular matrix proteins, a key feature in pvr.
current animals models for pvr are invasive, either injection of fibroblasts into vitreous or surgical disruption of the ilm to stimulate membrane formation  <cit> .
the lama <dig> mutants described herein provide novel, genetic models for studying pvr and pfv.
the production and expression of laminin by vitreal astrocytes in lama1nmf <dig> mutants but not those in the retina indicates that they could contribute to the generation, expansion, and stabilization of the preretinal membranes.
astrocyte expression of laminin α <dig> and γ <dig> has been observed in association with glial activation during cns disease  <cit> .
furthermore, activated astrocytes express and secrete many cytokines which could alter the development of retinal vessels and promote membrane formation.
a better understanding of how these vitreal membranes develop could help stop their formation or aid in treating them without surgery.
CONCLUSIONS
the present study describes a novel vascular phenotype in which vessels form initially the retina but then traverse the ilm and anastomose with hyaloid vessels.
interestingly, these vessels are able to re-enter the retina later in development and produce properly placed retinal capillary networks .
the data presented in this report clearly demonstrates the importance of a fully functional ilm to retinal vascular development.
the present study strongly suggests that mutations in lama <dig> or other ilm components in humans could cause retinal diseases such as pfv, pvr, and retinal detachment.
indeed, some lama1Δ mutants experience retinal detachments.
finally, the observation that astrocyte ensheathment of hyaloid vessels prior to their normal regression indicates that these glial cells may contribute to the persistence of these vessels.
these models could increase understanding and aid in finding treatments for pfv and pvr.

 the retinal vasculature, which nourishes the inner retina, consists of three plexi: the superficial in the nerve fiber layer, the intermediate in the inner plexiform layer, and the deep in the outer plexiform layer. the photoreceptors in the outer retina are maintained by the choroidal vessels. retinal vessels develop in man during fetal development but in mice during the first post-natal weeks. prior to the formation of retinal vessels, the lens and developing inner retina are nourished by the hyaloid vasculature. this three component vasculature lies in the vitreous, a gel-like structure separating the retina and the lens. the hyaloid vessels regress as retinal vessels form and are gone in humans by birth and by three weeks in the mouse. the portion of the hyaloid vasculature closest to the retina is the vasa hyaloidea propria which is also the last to regress. although occurring prior to birth in the human and post-natally in the mouse, the retinal vasculature develops in a similar pattern in these species. the primary vascular plexus forms first and, once complete, branching vessels dive into the inner retina to form the deep plexus and then the intermediate plexus. the stimuli guiding the formation of retinal vessels, has yet to be confirmed. while in the mouse retina, endothelial cells migrate and proliferate across the retina guided by an astrocyte template, other guidance systems determine the pattern in dog and man. furthermore, many studies have pointed towards vegf as a key regulator in vascular development, but it has recently been demonstrated that retinal vessels form normally without astrocyte-derived vegf. together, these data suggest that other proteins and, potentially, cells other than astrocytes guide endothelial cell migration in forming the superficial retinal vasculature. the proximity of the internal limiting membrane to retinal astrocytes and the superficial vasculature suggests that this structure may be important for development of retinal blood vessels. indeed, several mouse mutants with disruptions to the ilm have an abnormal retinal vasculature. laminin α is a primary component of laminin which is believed to be important for basement membrane formation. in addition, this laminin chain links the ilm to receptors, such as integrins and dystroglycan, which are found on müller cells in the retina. retinal vascular defects have recently been described in two mouse lines with mutations in lama the gene which encodes laminin α. while the vascular defects are similar, the two mutants differ in their generation and effect on laminin α one being a recessive point mutation and the other being a conditional knockout that leads to the complete loss of lama but bypasses the embryonic lethality normally associated with laminin α deletion. in the lama1nmf mice, a chemically-induced mutation causes the replacement of a tyrosine with a cysteine at amino acid in the n-terminal domain, which includes sites for receptors binding as well as polymerization. here forward, lama1nmf and lama1Δ refer to mice homozygous for the respective mutations. the present study investigates the development of retinal vessels in the previously described lama mutants. focus was placed on the lama1nmf because a random point mutation has greater potential to be associated with human disease than the complete deletion of lama which is embryonic lethal in mice and likely to cause lethality in humans as well. the observation that a point mutation in this large gene causes retinal disease in mice suggests that it may also do so in humans. transmission electron microscopy was used for ultrastructural analysis of the ilm, glial cells, and blood vessels in these mice. finally, epiretinal membranes are described in both of these mutants that have characteristics similar to human persistent fetal vasculature and proliferative vitreoretinopathy.  retinal vessels form in the lama1nmf retina at p1 blood vessel and astrocyte development was first investigated in cross sections by labeling with anti-pan laminin, anti-platelet-derived growth factor receptor α and gs isolectin at postnatal day when the primary retinal vasculature has just begun to form. in the wild type retina, astrocytes and blood vessels extended from the optic nerve head under the laminin-positive ilm. although some astrocytes were also observed along the base of the hyaloid artery, the vast majority of these glial cells were observed under the ilm. no astrocytes were observed in the vitreous away from the hyaloid artery. in the lama1nmf sections, blood vessels and pdgfrα-positive astrocytes were observed on the retinal side of the ilm in the peripapillary region, the area surrounding the optic nerve head. adjacent to this region, however, astrocytes were observed traversing the ilm into the vitreous. in addition, some sections demonstrated astrocytes migrating into the vitreous along the hyaloid vessels. sequential confocal z stacks were used to evaluate these sections and revealed openings in the ilm through which astrocytes entered the vitreous. peripheral to these "break" points, astrocytes were observed in a linear arrangement on the vitreal aspect of the ilm and only an occasional astrocyte was observed in the retina. unlike those in the retina, vitreal astrocytes expressed laminin. flatmount wt retinas were labeled with fluorescein isothiocyanate conjugated griffonia simplicifolia isolectin b to visualize blood vessels and anti-glial fibrillary acidic protein to visualize astrocytes. this labeling revealed a vascular apron surrounding the optic nerve head with endothelial cell filopodia extending out over an astrocyte template. when present, hyaloid vessels were easily distinguished from those in the retina by the plane of focus and their gs isolectin intensity. a similar vascular apron was observed in lama1nmf mice. in addition, a number of individual gs isolectin positive cells were observed in both the wt and lama1nmf retinas. some of these cells are microglia, which gs isolectin also labels, as they also were positive for iba-. jb- sections taken from wt eyes that were stained with periodic acid-schiff and hematoxylin revealed that all astrocytes were under the fully intact ilm. jb- sections also demonstrated astrocytes leaving the retina in the peripapillary region of lama1nmf mice. peripheral to this point, astrocytes were not observed in the retina. ultrastructure analysis with tem confirmed that astrocytes migrate into the vitreous through openings/breaks in the ilm. müller cell processes were also seen extending into the vitreous at these break points. although they express gfap, the astrocytes in the vitreous have few, if any, well-defined intermediate filaments at this stage, demonstrating that they are still maturing. the migration of astrocytes into the vitreous near the peripapillary region and on the hyaloid artery was observed consistently in all eyes examined with the different techniques. lama1nmf retinal vessels migrate into the vitreous by p3 examination of retinal flatmounts at p revealed the extension of blood vessels in the wt retina to the midperiphery behind a fully-formed astrocytic template. the hyaloid vessels, which detached from the retina during dissection, were still present but were not ensheathed with retinal astrocytes. immunohistochemical labeling of cross sections demonstrated that retinal vessels and astrocytes were located on the retinal side of the ilm. one or two astrocytes were observed along the hyaloid artery right at the optic nerve head. despite the close proximity of the vhp vessels to the ilm, they did not cross this barrier or make contact with retinal astrocytes in the wt eyes. by contrast, the vessels in the lama1nmf retinas had not progressed from that seen at p in retinal flatmounts. there were also less astrocytes in the mutant retina compared to the wt. a number of gs isolectin-positive cells were, however, observed throughout both the wt and lama1nmf retinas. these cells were more prominent in the avascular regions of both the wt and the lama1nmf retinas. imaging of retinas with the vitreous intact demonstrated that most of the lama1nmf astrocytes had migrated into the vitreous where they invested the vhp. astrocytes were also observed forming thin glial bridges connecting blood vessels within the vitreous. capillary networks were seen branching from the lama1nmf vhp. as was observed at p cross sectional analysis revealed astrocytes migrating from the retina into the vitreous both at the optic nerve head and through breaks in the ilm. these astrocytes extended in a linear fashion along the vitreal aspect of the ilm. at this stage, astrocytes were clearly enveloping vhp vessels and forming a laminin-positive membrane-like structure. the retinal vessels observed in the peripapillary region at p had also migrated into the vitreous. jb- cross sections demonstrated that these vessels had begun to anastomose with vessels of the vhp. only intravitreal vessels are evident in lama1nmf mice at p7 at p the primary vascular plexus was complete in the wt retina with the exception of the extreme periphery in some retinas. this vasculature included arteries and veins as well as the typical chicken wire-like capillary plexus. the astrocyte template was complete in wt mice at this stage with astrocytes ensheathing retinal vessels. the hyaloid vasculature and vhp had begun regressing and detached easily from the retina during dissection. when present, these vessels had gs isolectin-positive cells, likely hyalocytes, on top of them. by contrast, the lama1nmf retinal vasculature resembled that of the p retina, being limited to an apron in the peripapillary region. retinal vessels were absent beyond this point and individual gs isolectin-positive cells were prominent. there was a large reduction in the number of astrocytes within the mutant retina compared to the control. while some peripheral retinal astrocytes formed a normal honeycomb-like pattern, most extended long thin processes across the retina. when the vitreous was left intact, a much different image was revealed. a large number of astrocytes were observed in the vitreous where they ensheathed vessels and formed a dense membrane-like structure. the intravitreal capillary networks, which had begun forming at p were more extensive. in sections, anti-pan laminin and gs isolectin labeling demonstrated the peripapillary retinal vessels traversing the ilm into the vitreous where they continued to extend towards ora serrata. a membrane-like laminin positive structure was forming in association with these vitreal vessels. peripheral to break points in the ilm, no intraretinal vessels were observed. it is important to note that the laminin-positive ilm was also observed on either side of blood vessels traversing into the vitreous. labeling of adjacent sections with anti-pdgfrα in addition to anti-pan laminin and gs isolectin revealed that astrocytes crossed the ilm and were part of the vitreal membrane. these astrocytes were positive for laminin. these astrocytes extended along the vitreal side of the ilm and towards ora serrata in a fashion similar to that observed within the inner retina of the wt mice. intravitreal vessels invade the lama1nmf retina by p10 in the p wt retina, the superficial chicken wire capillary network was remodeled into a spoke-wheel-like system with an organized branching pattern. astrocytes had a distinct stellate morphology with thin processes that contacted and ensheathed retinal blood vessels. superficial vessels could be seen diving to form the deep capillary plexus. gs isolectin labeling of the lama1nmf flatmount retinas demonstrated an extensive vascular network with an unusual branching pattern in the vitreous. when the vitreous was left intact and retinas double-labeled with gs isolectin and anti-gfap, the appearance was similar to that at p although the astrocyte "membrane" appeared to be denser. at this stage, branches from intravitreal vessels extended into the retina to form an intraretinal capillary plexus. this abnormal branching was best appreciated with confocal z stack images through the retina. labeling of whole eye cross sections with anti-pdgfrα, anti-pan laminin, and gs isolectin more clearly demonstrated vessels within the vitreous and their branches that had invaded the inner and outer retina and formed a capillary network in the outer plexiform layer. in addition to astrocytes migrating near the vitreoretinal surface, some had migrated further into the vitreous. many of these astrocytes possessed long, thin processes and created glial bridges between intravitreal blood vessels. jb- sections of the wt retina revealed that astrocytes resided under the ilm and that the superficial vessels were diving into the deeper retinal layers. in the lama1nmf p jb- sections, long thin processes from the glial bridges mentioned above as well as their frequent contact with blood vessels could be appreciated. in some cases, they created tractional attachments with the vessels. astrocytes extended across the vitreal surface of the ilm to the far periphery. some astrocytes appeared to have made contact with cells in the retina. intravitreal vessels that had invaded the retina, extended into both the nerve fiber layer and into the deeper retina. ultrastructural analysis demonstrated breaks in the ilm similar to those seen at p. astrocytes and blood vessels were closely associated in the vitreous. additionally, intermediate filaments were present in intravitreal astrocytes at this age. adult lama1nmf223 fundus examination of four-week old lama1nmf mice demonstrated a white membrane-like structure surrounding hyaloid vessels in the vitreal plane of focus,. the position of this membrane in relation to the vitreal blood vessels suggested it was the astrocytic membrane observed in flatmounts and sectioned retinas. fluorescein angiography confirmed the presence of large patent vessels within the vitreous. these vessels could be seen branching into the retina to form the inner retinal capillary system. flatmount retinas labeled with anti-gfap and gs isolectin confirmed the continuance of the astrocyte membrane in the vitreous of lama1nmf mutants. labeling with anti-pdgfrα, anti-pan laminin, and gs isolectin clearly showed a chain of pdgfrα-positive astrocytes across the vitreal surface of the ilm as well as the glial bridges between intravitreal blood vessels. the vitreal vessels dove into the retina where they branched to form the inner and outer retinal capillary networks. abnormal retinal vascular development in the lama1Δ mice is similar to that in lama1nmf223 transmission electron microscopy revealed a double layered ilm which spanned the entire retina in the wt mouse at p. by contrast, the p lama1Δ mice revealed a thin, fragmented ilm that contained frequent breaks through which astrocytes, müller cells, and ganglion cells seemed to be protruding. astrocytes were observed on the vitreal side of the ilm. the vascular development in lama1Δ retinas was examined at key time points determined by investigation of the lama1nmf mutants: p p and p at p a small apron of vessels was evident at the optic nerve head, similar to that seen in the lama1nmf mutants. when the vitreous was left intact, astrocytes were observed on the hyaloid vessels at p at p the gfap and gs isolectin labeling produced images similar to those from the lama1nmf mice showing astrocytes associating with the vhp and no vessels within the retina. labeling of the p retina revealed a dense astrocyte network present in the vitreous of the lama1Δ mice that was more dense than in the lama1nmf eye and intravitreal vessels extending into the retina. in some retinas, gfap-positive processes were also observed traversing the retina along with the blood vessels. the deep vasculature was developing from diving vitreal vessels at p. in summary, the same changes occurred in the lama1Δ mice as in the lama1nmf mice. the progression, however, was slightly faster and the severity of the pathology was greater at least in terms of astrocyte coverage of the vhp. discussion the previous study reported that few, if any, retinal vessels developed in the lama mutants and that these were replaced by persistent intravitreal vessels which penetrate the retina to form the intraretinal capillary networks. using confocal microscopy, additional immunohistochemical markers, cross sectional analysis, and tem, the present study redefines the development of retinal vessels and astrocytes in the lama mutants. in addition, the previously described "vitreal fibroplasia" is identified as a vitreal membrane containing laminin-positive astrocytes, similar to human persistent fetal vasculature. the ilm in the lama1nmf retina is normal in appearance throughout much of the retina but has small frequent breaks. by contrast, the lama1Δ mice have a very thin ilm with a diffuse, nonlinear appearance throughout much of the retina. in addition, breaks in the lama1Δ retina generally extended over larger areas of the retina. in many areas, the ilm was lacking completely. these findings are not surprising because an ilm lacking laminin α would likely not be bound well to the retina as this protein contains the binding sites for receptors on müller cell endfeet. the preservation of the ilm in some areas suggests that other alpha chains present in the ilm, such as α, assist in binding this structure to the retina. despite the differing ilm structure, the two lama mutants have very similar abnormalities with regards to retinal vascular development. both mutants have a vascular apron around the optic nerve head at p suggesting that neither the y265c mutation nor the complete loss of this protein affected endothelial cell differentiation and emigration from the optic nerve. beginning around p however, it is evident that the retinal blood vessels cease to develop in the lama1nmf retina and have entered the vitreous where they remain until p7-p these vessels, which originate within the peripapillary retina grow along the retinal surface and eventually anastomose with those of the vhp. together, these vessels form a dense vascular network in the vitreous that persists into adulthood and is ensheathed by astrocytes. these vitreal vessels sprout and invade the retina around p to form the retinal vascular capillary networks. despite their abnormal origin, the lama mutant retinal capillary networks at p are correctly placed within the retina. therefore, the cues guiding these diving vessels are still present. it appears, however, as though the intermediate plexus forms early in the lama1nmf retina as vessels are evident in the inner plexiform layer at p compared to p in the wt retina. the formation of the inner capillary networks further indicates that neither the y265c point mutation in lama nor the complete deletion of this protein alters the ability of endothelial cells to form blood vessels. rather, astrocyte migration across the vitreal surface of the ilm precedes and likely causes the misguided migration of the blood vessels into the vitreous. in all retinas investigated at various ages, astrocytes were observed in the vitreous before retinal vessels traversed the ilm. in addition, only the vhp vessels were observed in the vitreous posterior to astrocytes on the vitreal aspect of the ilm. it is possible that if a normal template of astrocytes was present in the retina, the blood vessel development would also be normal. therefore, identifying and neutralizing the factor which stimulate astrocyte invasion into the vitreous may also alleviate the vascular defects in these mice. most astrocytes from the lama mutant retinas migrate towards and eventually envelop the vhp vessels. in addition, a subpopulation of these glial cells migrates from the optic nerve head directly into the vitreous along the hyaloid artery and its branches. these observations suggest that an attractant within the persistent and proliferating intravitreal vessels stimulates astrocyte migration. the previous report demonstrated that the tyrosine to cysteine mutation in the lama1nmf mice significantly decreases the binding ability of laminin α. it is logical to hypothesize, therefore, that this mutation disrupts the strength by which laminin α and, consequently, the entire ilm, bind to receptors on müller cell endfeet. as a result, the ilm may become less of a barrier for cells in the retina. astrocytes may, therefore, respond to chemoattractants in the vitreous more easily than those in the wt retina. astrocyte migration into the vitreous in response to these chemoattractants may create breaks in the ilm. this idea is supported by the fact that in most cases, both laminin immunohistochemistry and tem show the ilm pushed inwardly toward the vitreous where the cells exit the retina rather than there being a clear opening in the ilm through which cells migrate. indeed, a number of proteins within the normal vitreous are known to stimulate astrocyte migration, including hyaluronic acid, endothelin-, tgf-beta, and pdgf-aa. among these, pdgf-aa is the best candidate as it is known to stimulate astrocyte migration and is believed to be generated by ganglion cells in the retina. furthermore, pdgf-aa overexpression by the lens stimulates astrocyte migration into the vitreous, resulting in a pattern similar to that observed in the lama mutants. once within the vitreous, most astrocytes migrate along the vitreal surface of the ilm towards the ora serrata. this migration pattern is similar to that seen within wt retinas with the exception that it occurs on the vitreal side of the ilm. this observation indicates that the migratory cues normally present inside the retina are on the vitreal surface of the ilm in lama mutants. it could also be hypothesized that proteins constituting the ilm act as guidance cues and astrocytes in the mutant retina respond to these but remain on the vitreal side once they have traversed the ilm. yet another theory is that müller cells, the other retinal glial cell, guide astrocyte migration. although müller cells may not be fully differentiated, these glial cells are in place prior to astrocyte migration into and across the retina. furthermore, müller cell processes in both lama mutants extend into the vitreous where chemoattractants they produce could spill out. in addition, some müller cell endfeet are properly positioned, potentially contributing to frequent contact between vitreal astrocytes and the retina. further investigation is required to identify the stimuli that guide astrocytes across the retina and, perhaps more importantly, what cell type provide these stimuli. the association of astrocytes with hyaloid vessels occurs in other rodent mutants with persistent fetal vasculature, including collagen 15a1/18a double knockouts, lif transgenics, and the nuc rat. the question arises then, do astrocytes associate with vitreal vessels as a consequence of their failure to regress or is the astrocyte association preventing their regression by stabilizing the vasculature? in the case of the lama mutants, the astrocytes migrate into the vitreous and associate with hyaloid and vhp by p before vhp regression has normally begun. this suggests that the astrocyte ensheathment of vitreal blood vessels subsequently inhibits the vessel regression and may even stimulate their proliferation. astrocyte ensheathment of the fetal vasculature in vitreous also occurs in the human disease pfv. pfv can be a blinding disorder because the retinal vasculature is incomplete and the persistent fvv and membrane pulls on the retina causing detachment. preretinal glial membranes are often found in patients with pfv and likely contribute to retinal detachment. it is not uncommon for patients with pfv to develop cataracts and glaucoma as well. few animal models exist for this condition and treatment involves surgery. the lama mutants described herein have two hallmark features of this disease: persistence of the fetal vasculature and preretinal glial membranes, making them potential models for studying this disease. the membranes formed by astrocytes in lama mutants also resemble epiretinal membranes seen in patients with proliferative vitreoretinopathy, a complication of retinal detachment in which retinal cells and macrophages enter the vitreous. the ultrastructure of the glial-containing membrane in lama mutants is similar to that described for epiretinal membranes. in some areas, astrocytes extend thin, delicate processes across the vitreoretinal surface and into the vitreous. these created structures similar to those which foos called glial bridges. in others areas, numerous astrocytes overlap one another to form a dense mesh-like structure. while the glial membranes in lama mutants contain mainly tightly associated astrocytes, frequent irregularly large openings are also observed. the astrocytes associated with this membrane make laminin, suggesting they may also produce other extracellular matrix proteins, a key feature in pvr. current animals models for pvr are invasive, either injection of fibroblasts into vitreous or surgical disruption of the ilm to stimulate membrane formation. the lama mutants described herein provide novel, genetic models for studying pvr and pfv. the production and expression of laminin by vitreal astrocytes in lama1nmf mutants but not those in the retina indicates that they could contribute to the generation, expansion, and stabilization of the preretinal membranes. astrocyte expression of laminin α and γ has been observed in association with glial activation during cns disease. furthermore, activated astrocytes express and secrete many cytokines which could alter the development of retinal vessels and promote membrane formation. a better understanding of how these vitreal membranes develop could help stop their formation or aid in treating them without surgery.  the present study describes a novel vascular phenotype in which vessels form initially the retina but then traverse the ilm and anastomose with hyaloid vessels. interestingly, these vessels are able to re-enter the retina later in development and produce properly placed retinal capillary networks. the data presented in this report clearly demonstrates the importance of a fully functional ilm to retinal vascular development. the present study strongly suggests that mutations in lama or other ilm components in humans could cause retinal diseases such as pfv, pvr, and retinal detachment. indeed, some lama1Δ mutants experience retinal detachments. finally, the observation that astrocyte ensheathment of hyaloid vessels prior to their normal regression indicates that these glial cells may contribute to the persistence of these vessels. these models could increase understanding and aid in finding treatments for pfv and pvr. 
astrocytes continue proliferating within the vitreous to form a dense mesh that resembles epiretinal membranes associated with persistent fetal vasculature and proliferative vitreoretinopathy. the lama mutants described in this report are potential models for studying the human conditions persistent fetal vasculature and proliferative vitreoretinopathy. two such models are the recently described mouse lines with mutations in lama an important component of the retinal internal limiting membrane. although astrocytes and blood vessels initially migrate into lama mutant retinas, both traverse the peripapillary ilm into the vitreous by p 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. valuable insights into the complex process of retinal vascular development can be gained using models with abnormal retinal vasculature. lama and a fully intact ilm are required for normal retinal vascular development. these mutants have a persistence of the fetal vasculature of vitreous but lack a primary retinal vascular plexus. the present study provides a detailed analysis of astrocyte and vascular development in these lama mutants. mutations in lama allow developing retinal vessels to enter the vitreous where they anastomose with vessels of the hyaloid system which persist and expand.
 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 lama an important component of the retinal internal limiting membrane. these mutants have a persistence of the fetal vasculature of vitreous but lack a primary retinal vascular plexus. the present study provides a detailed analysis of astrocyte and vascular development in these lama mutants.  although astrocytes and blood vessels initially migrate into lama mutant retinas, both traverse the peripapillary ilm into the vitreous by p 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.  lama and a fully intact ilm are required for normal retinal vascular development. mutations in lama 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 lama mutants described in this report are potential models for studying the human conditions persistent fetal vasculature and proliferative vitreoretinopathy. 