Large is required for normal astrocyte migration and retinal vasculature development
- Min Zhou†1, 2,
- Herui Wang†3, 4,
- Hui Ren1, 2,
- Rui Jiang1, 2,
- Chi Zhang3,
- Xiaohui Wu3Email author and
- Gezhi Xu1, 2Email authorView ORCID ID profile
© The Author(s) 2017
Received: 20 September 2016
Accepted: 29 March 2017
Published: 17 April 2017
Persistent fetal vasculature (PFV) is a congenital developmental anomaly of the eye that accounts for about 5% of childhood blindness. The molecular mechanism of PFV remains unclear. As a glycosyltransferase of α-dystroglycan, LARGE mutations have been found in congenital muscular dystrophy patients with brain abnormalities. Spontaneous Large mutant mice displayed similar symptoms of human muscle–eye–brain disorders. However, the detailed roles of Large in ocular vasculature development still need to be uncovered.
In this paper, we report that a novel Large mutation generated by the piggyBac transposon insertion leads to PFV and abnormal retinal vasculature in mice. Glycosylation of α-DG, an essential component of the extracellular matrix, was significantly impaired in these Large mutants, leading to broken inner limiting membrane (ILM). As a guide of the retinal vasculature development, the distribution of retinal astrocytes became irregular within the retina, and many astrocytes abnormally migrated into the vitreous along with the hyaloid vessels in Large mutants.
Large is essential for ILM formation and retinal astrocyte migration. The novel Large mutant mouse can serve as a new PFV model to further dissect LARGE functions in ocular vasculature development.
KeywordsPersistent fetal vasculature (PFV) Large Inner limiting membrane (ILM) Retinal astrocytes
During early fetal development, hyaloid vasculature arises from the optic nerve head, extends through the vitreous, and surrounds the developing lens . Later the fetal vasculature normally regresses and is replaced by retinal vasculature (around mid-gestation in humans and around birth in rodents), resulting in an optically clear path between the cornea and the retina . Failure of the hyaloid vascular regression could lead to persistent fetal vasculature (PFV), a congenital developmental disorder of the eye that accounts for approximately 5% of the childhood blindness. Until now, the mechanisms underlying PFV formation remain unclear.
The inner limiting membrane (ILM) is a basement membrane that defines the border between the retina and the vitreous cavity. The presence and integrity of ILM is essential for normal astrocyte migration and retinal vasculature development . Retinal astrocytes forms a cellular network that provides a template for endothelial cell migration during angiogenesis . Mutation of Lama1, which encodes a basement membrane protein LAMININ α1, disrupts retinal vasculature development and inner limiting membrane formation, leading to vitreoretinal blood vessel formation, persistence of fetal vasculature, and epiretinal membrane formation in mice [3, 4]. These results indicate the pivotal roles of LAMININ in ILM formation and retinal vasculature development.
The interaction between α-dystroglycan (α-DG, a laminin receptor) and laminin is indispensable for the assembly and maintenance of ILM . Correct glycosylation of α-DG is essential for the interaction. Similar with Lama1 mutants, mutations in α-DG and in an enzyme that participates in glycosylation of α-DG (POMGnT1) also displayed defective ILM formation, abnormal astrocyte distribution and blood vessel formation [6, 7].
Except for POMGnT1, LARGE is another reported glycosyltransferase of α-DG . LARGE mutations have been found in congenital muscular dystrophy patients with brain abnormalities . Myd mice that carry a spontaneous deletion in Large (Large myd ), showed skeletal, cardiac, and tongue muscle dystrophies, defective retinal transmission, and neuronal migration defects, mimicking the human muscle–eye–brain disorders [10, 11]. Another intragenic deletion allele of Large (Large myd ) showed ocular vascular defects, including vitreal fibroplasia and retinal vessel tortuosity and fluorescein leakage . However, it’s still unclear about the details how Large mutation causes defective ocular vasculature.
Human genetics showed that the severity of the affected patients depends on the LARGE gene mutation type. A patient of Walker–Warburg syndrome, a severe form of dystroglycanopathy was reported to carry a homozygous intragenic loss-of-function deletion in LARGE . A less severely affected patient carried a compound heterozygous missense mutation and a heterozygous 1 bp insertion in LARGE . The residual function of mutant LARGE protein may be the reason for the milder phenotype in the second patient. Despite of the previously reported Large mutant mouse models, new Large mutants with different mutation types can expand our understanding of its role in the disease development.
In this study, we report a novel Large mutant (Large PB ) mouse line that shows defective retinal vasculature and persistent hyaloid vessels. Hypo-glycosylation of α-DG was found in mutant retina, leading to broken ILM. Retinal astrocytes abnormally migrate through ILM into vitreous along with the persistent hyaloid vessels in the mutants. These features make this mutant a useful model for further dissecting the roles of LARGE in ocular vasculature development.
Persistent fetal vasculature (PFV) in Large mutant mice
Abnormal astrocyte migration in Large mutant retinas
Broken inner limiting membrane (ILM) in Large mutants
To determine if the Müller cell end-feet, which normally attach to ILM, were affected in Large PB/PB mice, retinal sections were stained with an antibody against glutamine synthetase (GS). On P22, Müller cell processes extended through the entire length of the retina and terminated with end-feet below the ILM in Large PB/PB mice, similar with those of age-matched wild-type mice (Fig. 5c, d).
Laminin is an essential extracellular matrix component that binds with glycosylated α-DG to guide astrocyte migration and maintain the ILM integrity . We checked the expression pattern of Laminin α1 in the ILM, and did not find obvious differences between 2-month old wild-type and Large PB/PB mice (Fig. 5i, j). Since glycosylation of α-DG in Large PB/PB mice was disrupted, we assumed that the interaction between α-DG and Laminin was impaired. To prove this, we transferred 2-month old wild-type and Large PB/PB retinal protein lysate from gel to the PVDF membrane, and incubated the membrane with Laminin-1 solution. We then detected Laminin protein that bound to the membrane by western blot after extensive washing. The laminin overlay assay showed much weaker signal around the band size of α-DG in Large PB/PB group (Fig. 6c), confirming that the binding between Laminin and α-DG was impaired in Large PB/PB mice. These results indicate that the extracellular matrix is not well assembled in Large PB/PB mice, and the broken ILM in Large PB/PB mice is probably due to hypoglycosylated α-DG.
Abnormal astrocyte migration to vitreous in Large mutants
Large was reported as a causative gene for human muscle–eye–brain diseases characterized by severe congenital muscular dystrophy, eye abnormalities and neuronal migration defects in central nervous system [10, 11, 20]. However, its role in retinal vasculature development remains to be explored. In this study, we reported a novel Large mutant that exhibited PFV and retinal vasculature defects, which is likely due to the disorganization of ILM and consequent astrocyte migration defects.
Large myd and Large vls are two previously reported mouse mutants that have exon 5–7 and exon 3–5 of Large deleted, respectively . Large myd likely produces a truncated protein with the N-terminal transmembrane domain (TM) and coiled coil domain (CC), while Large vls likely generates a shorter truncated protein with only TM domain. RT-PCR result indicates that our PB allele produces the longest truncated protein with not only TM and CC, but also part of the catalytic domain. Due to the genetic differences, it’s reasonable that the ocular phenotypes in these three mutants are not exactly the same. To our knowledge, this is the first time that blood flow was observed in the persistent hyaloid vessels in the vitreous of Large PB/PB mice, while only fibroplasia was observed in Large myd/myd and Large vls/vls mutants. Besides, both α-DG and β-DG are disrupted in ILM of Large vls/vls , while only α-DG is affected in the Large PB/PB mutants.
In the retina, interaction between α-DG and laminins are crucial for ILM formation . Mutations in Lama1, α-DG, and POMGnT1 (another enzyme involved in glycosylation of α-DG) caused abnormal laminin deposition, resulting in defective formation, abnormal astrocyte distribution, and defective blood vessel formation [3, 6]. These results support our hypothesis that the broken ILM in Large mutants is probably due to the impaired interaction between hypo-glycosylated α-DG and laminins.
To our knowledge, this is the first report that retinal astrocyte can migrate into the vitreous and ensheathe the persistent hyaloid vessels in Large mutant mice. The protective mechanism of astrocytes in the maintenance of vitreous blood vessels could be a new direction for study of the persistent hyaloid vessel. Macrophages play critical roles in programmed hyaloid vessel regression. The macrophage WNT7b serves as a short-range paracrine signal to initiate the programmed cell death in the adjacent vascular endothelial cells of the temporary hyaloid vessels of the developing eye . In Large PB/PB mice, the astrocytes that ensheathe the hyaloid artery may prevent contact between the macrophages and vascular endothelial cells, thereby protecting the vascular endothelial cells from apoptosis and blocking involution of the hyaloid artery.
Our results indicate that Large is essential for ILM formation and retinal astrocyte migration. The novel Large mutant mouse line can be used as a new PFV model to further dissect LARGE functions in ocular vasculature development.
All animal experiments were performed in accordance with protocols approved by the Animal Care and Use Committee of the Institute of Developmental Biology and Molecular Medicine (IDM), Fudan University. Large mutant line (W146qRP) was generated by inserting a piggyBac transposon (PB) in Large during the process of a large-scale insertional mutagenesis project on the C57BL/6J background. In the Large PB allele, the PB insertion was mapped in the sixth intron (Chr: 8. 75490122, Ensembl release 54). The gene trap element in PB transposon contains splicing acceptor–IRES–lacZ coding sequence-polyA signal and can disrupt the expression of inserted gene efficiently. The PB insertion direction and inserted genomic sequence are also labeled in Fig. 1a.
Genotyping PCR was performed with a PB specific primer LB2 (5′-CTGAGATGTCCTAAATGCACAGCG-3′) and two flanking genomic primers W146qRP-L1 (5′-TTCACTGCCTTTTCCTCCAGC-3′) and W146qRP-R1 (5′-CCCCACAACTTTCCTGTTCATTAC-3′). RT-PCR was performed with the following primers: Large-F 5′-ACCAAAACTCTGCCTGCCAAC-3′, Large-R 5′-CTGCTCCCATTTCATCTTCCG-3′, Gapdh-F 5′-TGTTCCTACCCCCAATGTGTCC-3′, Gapdh-R 5′-GGAGTTGCTGTTGAAGTCGCAG-3′.
Mice were phenotyped by indirect ophthalmoscopy according to previously described methods .
Immunofluorescence staining of both cryosections and whole mount retina was performed as previously reported . Primary antibodies used on cryosections included anti-alpha DG (1:200, Millipore, Cat. 05-593), anti-beta DG (1:100, Abcam, ab49515), anti-LAMA1 (1:200, Millipore, MAB1903), anti-GFAP (1:500, DAKO Z0334), and anti-CD68 (1:100, Abcam, ab31630). Primary antibodies used for whole mount staining included anti-GFAP (1:100), and anti-G. simplicifolia isolectin (1:200, Invitrogen).
Light microscopy images were collected with Leica MZFLIII or DMRXA2. Electron microscopy imaging was performed as previously described . A Visualsonics Vevo 770 was used for the ultrasonic analysis of retinal defects in mutant mice.
LAMININ overlay assay
Laminin overlay assay was performed as previously reported . Briefly, PVDF membranes were incubated with TBS buffer containing 3% BSA, 1 mM CaCl2, and 1 mM MgCl2 for 1 h to block nonspecific binding. The membranes were then incubated with 1.25 µg/ml laminin-1 in TBST containing 1 mM CaCl2 and 1 mM MgCl2 overnight at 4 °C. After extensive washing, bound laminin was detected by standard Western blot procedures.
persistent fetal vasculature
- PB :
inner limiting membrane
Griffonia simplicifolia isolectin
outer plexiform layer
coiled coil domain
MZ and HW designed and performed experiments, interpreted data, and wrote the manuscript. HR, RJ, and CZ participated in phenotypic analysis. XW and GX conceived of and designed the studies, supervised the work, and wrote the manuscript. All authors read and approved the final manuscript.
We thank Yanyan Nie, Xiaorong Huang, and Zhiyan Xia for their assistance of animal care and experiments.
The authors declare that they have no competing interests.
Availability of data and materials
Please contact author for data and material requests.
Ethics approval and consent to participate
All animal experiments were performed in accordance with protocols approved by the Animal Care and Use Committee of the Institute of Developmental Biology and Molecular Medicine, Fudan University.
This work was supported by the following grants: the National Natural Science Foundation of China (30971650 and 81300805); the Chinese Hi-tech Research and Development Project (863) (2014AA021104); and the Science and Technology Commission of Shanghai Municipality (09PJ1402400 and 15XD1500500).
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- Fruttiger M. Development of the retinal vasculature. Angiogenesis. 2007;10:77–88.View ArticlePubMedGoogle Scholar
- Halfter W, Dong S, Dong A, Eller AW, Nischt R. Origin and turnover of ECM proteins from the inner limiting membrane and vitreous body. Eye. 2008;22:1207–13.View ArticlePubMedGoogle Scholar
- Edwards MM, Mammadova-Bach E, Alpy F, Klein A, Hicks WL, Roux M, Simon-Assmann P, Smith RS, Orend G, Wu J, et al. Mutations in Lama1 disrupt retinal vascular development and inner limiting membrane formation. J Biol Chem. 2010;285:7697–711.View ArticlePubMedPubMed CentralGoogle Scholar
- Edwards MM, McLeod DS, Grebe R, Heng C, Lefebvre O, Lutty GA. Lama1 mutations lead to vitreoretinal blood vessel formation, persistence of fetal vasculature, and epiretinal membrane formation in mice. BMC Dev Biol. 2011;11:60.View ArticlePubMedPubMed CentralGoogle Scholar
- Durbeej M, Henry MD, Campbell KP. Dystroglycan in development and disease. Curr Opin Cell Biol. 1998;10:594–601.View ArticlePubMedGoogle Scholar
- Takahashi H, Kanesaki H, Igarashi T, Kameya S, Yamaki K, Mizota A, Kudo A, Miyagoe-Suzuki Y, Takeda S, Takahashi H. Reactive gliosis of astrocytes and Muller glial cells in retina of POMGnT1-deficient mice. Mol Cell Neurosci. 2011;47:119–30.View ArticlePubMedGoogle Scholar
- Lee Y, Kameya S, Cox GA, Hsu J, Hicks W, Maddatu TP, Smith RS, Naggert JK, Peachey NS, Nishina PM. Ocular abnormalities in Large(myd) and Large(vls) mice, spontaneous models for muscle, eye, and brain diseases. Mol Cell Neurosci. 2005;30:160–72.View ArticlePubMedGoogle Scholar
- Inamori K, Yoshida-Moriguchi T, Hara Y, Anderson ME, Yu L, Campbell KP. Dystroglycan function requires xylosyl- and glucuronyltransferase activities of LARGE. Science. 2012;335:93–6.View ArticlePubMedPubMed CentralGoogle Scholar
- Longman C, Brockington M, Torelli S, Jimenez-Mallebrera C, Kennedy C, Khalil N, Feng L, Saran RK, Voit T, Merlini L, et al. Mutations in the human LARGE gene cause MDC1D, a novel form of congenital muscular dystrophy with severe mental retardation and abnormal glycosylation of alpha-dystroglycan. Hum Mol Genet. 2003;12:2853–61.View ArticlePubMedGoogle Scholar
- Holzfeind PJ, Grewal PK, Reitsamer HA, Kechvar J, Lassmann H, Hoeger H, Hewitt JE, Bittner RE. Skeletal, cardiac and tongue muscle pathology, defective retinal transmission, and neuronal migration defects in the Large(myd) mouse defines a natural model for glycosylation-deficient muscle–eye–brain disorders. Hum Mol Genet. 2002;11:2673–87.View ArticlePubMedGoogle Scholar
- Michele DE, Barresi R, Kanagawa M, Saito F, Cohn RD, Satz JS, Dollar J, Nishino I, Kelley RI, Somer H, et al. Post-translational disruption of dystroglycan-ligand interactions in congenital muscular dystrophies. Nature. 2002;418:417–22.View ArticlePubMedGoogle Scholar
- van Reeuwijk J, Grewal PK, Salih MA, de Bernabe DBV, McLaughlan JM, Michielse CB, Herrmann R, Hewitt JE, Steinbrecher A, Seidahmed MZ, et al. Intragenic deletion in the LARGE gene causes Walker–Warburg syndrome. Hum Genet. 2007;121:685–90.View ArticlePubMedPubMed CentralGoogle Scholar
- Ding S, Wu X, Li G, Han M, Zhuang Y, Xu T. Efficient transposition of the piggyBac (PB) transposon in mammalian cells and mice. Cell. 2005;122:473–83.View ArticlePubMedGoogle Scholar
- Sun LV, Jin K, Liu Y, Yang W, Xie X, Ye L, Wang L, Zhu L, Ding S, Su Y, et al. PBmice: an integrated database system of piggyBac (PB) insertional mutations and their characterizations in mice. Nucleic Acids Res. 2008;36:D729–34.View ArticlePubMedGoogle Scholar
- Dorrell MI, Aguilar E, Friedlander M. Retinal vascular development is mediated by endothelial filopodia, a preexisting astrocytic template and specific R-cadherin adhesion. Invest Ophthalmol Vis Sci. 2002;43:3500–10.PubMedGoogle Scholar
- Gnanaguru G, Bachay G, Biswas S, Pinzon-Duarte G, Hunter DD, Brunken WJ. Laminins containing the beta2 and gamma3 chains regulate astrocyte migration and angiogenesis in the retina. Development. 2013;140:2050–60.View ArticlePubMedPubMed CentralGoogle Scholar
- Hose S, Zigler JS Jr, Sinha D. A novel rat model to study the functions of macrophages during normal development and pathophysiology of the eye. Immunol Lett. 2005;96:299–302.View ArticlePubMedGoogle Scholar
- Zhang C, Asnaghi L, Gongora C, Patek B, Hose S, Ma B, Fard MA, Brako L, Singh K, Goldberg MF, et al. A developmental defect in astrocytes inhibits programmed regression of the hyaloid vasculature in the mammalian eye. Eur J Cell Biol. 2011;90:440–8.View ArticlePubMedPubMed CentralGoogle Scholar
- Hurskainen M, Eklund L, Hagg PO, Fruttiger M, Sormunen R, Ilves M, Pihlajaniemi T. Abnormal maturation of the retinal vasculature in type XVIII collagen/endostatin deficient mice and changes in retinal glial cells due to lack of collagen types XV and XVIII. FASEB J. 2005;19:1564–6.PubMedGoogle Scholar
- Mercuri E, Messina S, Bruno C, Mora M, Pegoraro E, Comi GP, D’Amico A, Aiello C, Biancheri R, Berardinelli A, et al. Congenital muscular dystrophies with defective glycosylation of dystroglycan: a population study. Neurology. 2009;72:1802–9.View ArticlePubMedGoogle Scholar
- Lobov IB, Rao S, Carroll TJ, Vallance JE, Ito M, Ondr JK, Kurup S, Glass DA, Patel MS, Shu W, et al. WNT7b mediates macrophage-induced programmed cell death in patterning of the vasculature. Nature. 2005;437:417–21.View ArticlePubMedPubMed CentralGoogle Scholar