Gradual Suppression of Transcytosis Governs Functional Blood-Retinal Barrier Formation

•Retinal vessels at P1 have functional tight junctions but display bulk transcytosis•Immature vessel leakage is entirely due to transcytosis and not via tight junctions•Gradual suppression of transcytosis governs functional blood-retinal barrier formation•Retinal vasculature is a tractable system to study CNS barriers Blood-central nervous system (CNS) barriers partition neural tissues from the blood, providing a homeostatic environment for proper neural function. The endothelial cells that form blood-CNS barriers have specialized tight junctions and low rates of transcytosis to limit the flux of substances between blood and CNS. However, the relative contributions of these properties to CNS barrier permeability are unknown. Here, by studying functional blood-retinal barrier (BRB) formation in mice, we found that immature vessel leakage occurs entirely through transcytosis, as specialized tight junctions are functional as early as vessel entry into the CNS. A functional barrier forms only when transcytosis is gradually suppressed during development. Mutant mice with elevated or reduced levels of transcytosis have delayed or precocious sealing of the BRB, respectively. Therefore, the temporal regulation of transcytosis governs the development of a functional BRB, and suppression of transcytosis is a principal contributor for functional barrier formation. Blood-central nervous system (CNS) barriers partition neural tissues from the blood, providing a homeostatic environment for proper neural function. The endothelial cells that form blood-CNS barriers have specialized tight junctions and low rates of transcytosis to limit the flux of substances between blood and CNS. However, the relative contributions of these properties to CNS barrier permeability are unknown. Here, by studying functional blood-retinal barrier (BRB) formation in mice, we found that immature vessel leakage occurs entirely through transcytosis, as specialized tight junctions are functional as early as vessel entry into the CNS. A functional barrier forms only when transcytosis is gradually suppressed during development. Mutant mice with elevated or reduced levels of transcytosis have delayed or precocious sealing of the BRB, respectively. Therefore, the temporal regulation of transcytosis governs the development of a functional BRB, and suppression of transcytosis is a principal contributor for functional barrier formation. Central nervous system (CNS) endothelial cells lining blood vessels form the blood-CNS barriers, which include the blood-brain barrier, blood-spinal cord barrier, and inner blood-retinal barrier (BRB) (Engelhardt and Coisne, 2011Engelhardt B. Coisne C. Fluids and barriers of the CNS establish immune privilege by confining immune surveillance to a two-walled castle moat surrounding the CNS castle.Fluids Barriers CNS. 2011; 8: 4Crossref PubMed Scopus (156) Google Scholar). These cells have two properties that limit the passage of substances between the blood and the CNS parenchyma: (1) specialized tight junction complexes between CNS endothelial cells prevent paracellular flux and (2) low rates of vesicular trafficking between the luminal and abluminal membrane, known as transcytosis, limit transcellular passage (Andreone et al., 2015Andreone B.J. Lacoste B. Gu C. Neuronal and vascular interactions.Annu. Rev. Neurosci. 2015; 38: 25-46Crossref PubMed Scopus (150) Google Scholar, Chow and Gu, 2015Chow B.W. Gu C. The molecular constituents of the blood-brain barrier.Trends Neurosci. 2015; 38: 598-608Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar, Raviola, 1977Raviola G. 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Mfsd2a is critical for the formation and function of the blood-brain barrier.Nature. 2014; 509: 507-511Crossref PubMed Scopus (570) Google Scholar, Knowland et al., 2014Knowland D. Arac A. Sekiguchi K.J. Hsu M. Lutz S.E. Perrino J. Steinberg G.K. Barres B.A. Nimmerjahn A. Agalliu D. Stepwise recruitment of transcellular and paracellular pathways underlies blood-brain barrier breakdown in stroke.Neuron. 2014; 82: 603-617Abstract Full Text Full Text PDF PubMed Scopus (372) Google Scholar). Barrier properties are not intrinsic to CNS endothelial cells but rather are acquired from the neural environment during development (Blanchette and Daneman, 2015Blanchette M. Daneman R. Formation and maintenance of the BBB.Mech. Dev. 2015; 138: 8-16Crossref PubMed Scopus (137) Google Scholar, Hagan and Ben-Zvi, 2015Hagan N. Ben-Zvi A. The molecular, cellular, and morphological components of blood-brain barrier development during embryogenesis.Semin. Cell Dev. 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The retinal vasculature is well suited to address many fundamental questions about CNS barriers. Physiologically analogous to the blood-brain barrier, the BRB regulates the optimal milieu for phototransduction. Unlike the complex brain vascular network, the retinal vasculature has a relatively simple, stereotypic development and architecture, consisting of a three-tiered, two-dimensional plexus (Stone et al., 1995Stone J. Itin A. Alon T. Pe’er J. Gnessin H. Chan-Ling T. Keshet E. Development of retinal vasculature is mediated by hypoxia-induced vascular endothelial growth factor (VEGF) expression by neuroglia.J. Neurosci. 1995; 15: 4738-4747Crossref PubMed Google Scholar). Mouse retinal angiogenesis occurs shortly after birth. CNS endothelial cells invade the optic nerve head and expand radially along the vitreal surface from the center toward the periphery, forming the entire primary plexus by postnatal day 8 (P8) (Figures 1A and 1B ) (Fruttiger, 2007Fruttiger M. Development of the retinal vasculature.Angiogenesis. 2007; 10: 77-88Crossref PubMed Scopus (368) Google Scholar). Sprouts from the primary plexus then penetrate into the retina, forming the deeper plexus, followed by further sprouting to form the intermediate plexus (Stahl et al., 2010Stahl A. Connor K.M. Sapieha P. Chen J. Dennison R.J. Krah N.M. Seaward M.R. Willett K.L. Aderman C.M. Guerin K.I. et al.The mouse retina as an angiogenesis model.Invest. Ophthalmol. Vis. Sci. 2010; 51: 2813-2826Crossref PubMed Scopus (440) Google Scholar). Here, we use the BRB as a model system to elucidate the relative contributions of tight junctions and transcytosis in regulating CNS barrier permeability during development. To our surprise, we found that functional tight junctions are already present when vessels first entered the CNS so the gradual suppression of transcytosis in CNS endothelial cells governs the development of a functional barrier. We first mapped the spatio-temporal formation of the functional BRB. To evaluate BRB permeability, we transcardially injected mice at several postnatal ages with Sulfo-NHS-Biotin (550 Da) or fluorescently labeled, 10-kDa dextran tracer. A functional BRB, as in adults, completely confines both tracers within the vasculature (Figure S1A). However, at P1, when blood vessels first enter the retina through the optic nerve head and are ensheathed by pericytes (Figure S2A), leakage of both tracers into the retinal parenchyma from the budding vessels was evident (Figure 1C), indicating that these developing vessels do not intrinsically have a functional barrier. In P3 and P5 retinas, both tracers leaked into the parenchyma and were taken up by non-vascular cells near nascent vessels at the angiogenic front, located distal to the optic nerve head (Figures 1D–1G, arrowheads), while both tracers were confined in the more mature vessels located proximal to the optic nerve head (Figures 1D–1G, arrows). Indeed, Sulfo-NHS-Biotin, 3-kDa dextran tracers, and 10-kDa dextran tracers at P5 reveal that a functional barrier is formed gradually in a proximal-to-distal fashion (Figures 1E–1H). At P8 and P9, although the angiogenic front reaches the retinal periphery, the nascent, distal vessels still exhibited leakage (Figures 1I and 1J), whereas the mature, proximal vessels confined Sulfo-NHS-Biotin and 10-kDa dextran tracers (Figures S1B and S1C). At P10, both tracers were completely confined in all vessels of the primary plexus, including distal vessels (Figures 1I and 1J; Figures S1B and S1C). Thus, these data demonstrate that developing vessels are initially leaky, that a functional barrier is formed gradually from proximal to distal, and that the primary plexus acquires a functional BRB by P10. We next examined when the functional BRB is established in the deeper plexus, which sprouts from the primary plexus between P7 and P12. Sprouting vessels of the deeper plexus showed leakage of both Sulfo-NHS-Biotin and 10-kDa dextran tracers at P8 and P9 (Figures S1D and S1E). However, at P10, when the deeper plexus vessels continue to sprout, both tracers were confined in newly formed vessels (Figures S1D and S1E). Finally, we investigated when the BRB becomes functional in the intermediate plexus, which sprouts from the deeper plexus between P12 and P17. At P12, sprouting nascent vessels, surprisingly, already confined both tracers; no leakage was observed at any ages examined (Figures S1F and S1G). Together, these data demonstrate that the developing retinal vasculature does not intrinsically have a functional barrier but instead gradually acquires barrier properties. Once barrier properties are attained by P10, nascent vessels sprouting from vessels with a functional BRB inherit and maintain these barrier properties. Thus, the three-tiered retinal vasculature acquires a functional BRB remarkably by one distinctive age: P10 (Figure 1; Figure S1). We next addressed the subcellular mechanisms underlying functional BRB development. We asked whether CNS endothelial cells form functional tight junctions and acquire low rates of transcytosis synchronously or sequentially to establish the functional BRB and what the relative contribution of each property is to the regulation of BRB permeability. To evaluate the functionality of tight junctions and suppression of transcytosis at subcellular resolution, we injected horseradish peroxidase (HRP) intravenously at several ages and imaged the retina with transmission electron microscopy (EM). In the lung and heart vasculature, where barrier properties are absent, luminal HRP leaks into the basement membrane and parenchyma both by passing through junctions, observed as HRP-filled tight junctions, and by high rates of transcytosis, evident as numerous HRP-filled intracellular vesicles (Karnovsky, 1967Karnovsky M.J. The ultrastructural basis of capillary permeability studied with peroxidase as a tracer.J. Cell Biol. 1967; 35: 213-236Crossref PubMed Scopus (1082) Google Scholar, Schneeberger-Keeley and Karnovsky, 1968Schneeberger-Keeley E.E. Karnovsky M.J. The ultrastructural basis of alveolar-capillary membrane permeability to peroxidase used as a tracer.J. Cell Biol. 1968; 37: 781-793Crossref PubMed Scopus (153) Google Scholar). However, in adult retinal endothelial cells, where barrier properties are present (Raviola, 1977Raviola G. The structural basis of the blood-ocular barriers.Exp. Eye Res. 1977; 25: 27-63Crossref PubMed Scopus (250) Google Scholar, Reese and Karnovsky, 1967Reese T.S. Karnovsky M.J. Fine structural localization of a blood-brain barrier to exogenous peroxidase.J. Cell Biol. 1967; 34: 207-217Crossref PubMed Scopus (1921) Google Scholar), we observed that luminal HRP is halted sharply at electron-dense “kissing points” between endothelial cells where adjacent membranes are tightly apposed (Figure 2A). Adult retinal endothelial cells also displayed few HRP-filled vesicles, indicating that transcytosis is suppressed (Figure 2B). At P1, we observed tracer extravasation from budding vessels, indicating leaky vessels (Figure 1C). Surprisingly, under EM, these CNS endothelial cells already had specialized tight junctions that halted the HRP at the kissing points (Figures 2C and 2E), as observed in adult, mature CNS endothelial cells (Figure 2A). However, these CNS endothelial cells had many HRP-containing vesicles, including luminal and abluminal membrane-associated vesicles and cytoplasmic vesicles (Figures 2D and 2F), similar to lung and heart endothelial cells, where suppression of transcytosis is absent (Schneeberger-Keeley and Karnovsky, 1968Schneeberger-Keeley E.E. Karnovsky M.J. The ultrastructural basis of alveolar-capillary membrane permeability to peroxidase used as a tracer.J. Cell Biol. 1968; 37: 781-793Crossref PubMed Scopus (153) Google Scholar). These results indicate that as early as vessel ingression at P1, functional tight junctions are present in budding CNS endothelial cells, but transcytosis has not been suppressed, suggesting that immature BRB leakiness is entirely due to uninhibited transcytosis. To test the hypothesis that suppression of transcytosis determines functional BRB development, we used EM to examine vesicular trafficking at later postnatal ages. At P5 and P8, in distal, leaky vessels, many HRP-filled vesicles were observed (Figures 2G and 2H), indicating that transcytosis has not been suppressed. However, in the proximal vessels that are impermeable to tracer, very few HRP-filled vesicles were observed, suggesting that suppression of transcytosis had occurred (Figures S3A and S3C). By P10, when all the retinal vessels have a functional BRB, negligible HRP-filled vesicles were observed in both distal and proximal vessels (Figures 2G and 2H; Figures S2A and S2C). Consistent with the observation at P1, functional tight junctions were present in all vessels, both proximal and distal, at all ages tested (Figures 2I and 2J; Figures S3B and S3D). These data demonstrate that a leaky, immature barrier is due entirely to high rates of bulk transcytosis in endothelial cells. A functional barrier is established days later, only after transcytosis is suppressed, coinciding with functional BRB development by P10. Therefore, the spatio-temporal regulation of transcytosis governs the development of a functional BRB. Given the significance of transcytosis in functional BRB formation, we next examined whether Mfsd2a, a known transcytosis regulator in blood-brain barrier formation (Ben-Zvi et al., 2014Ben-Zvi A. Lacoste B. Kur E. Andreone B.J. Mayshar Y. Yan H. Gu C. Mfsd2a is critical for the formation and function of the blood-brain barrier.Nature. 2014; 509: 507-511Crossref PubMed Scopus (570) Google Scholar), also plays a role in BRB formation. EM analysis of retinas from HRP-injected adult mice revealed increased HRP-filled vesicle density in the CNS endothelial cells of Mfsd2a−/− mice compared to Mfsd2a+/+ littermate controls (Figures 3A and 3B ), whereas tight junctions remained functional (Figures 3C and 3D). Thus, Mfsd2a plays a similar role at the BRB in suppressing transcytosis. We next examined Mfsd2a expression as a marker for suppressed transcytosis during BRB development by immunostaining retinas from dextran tracer-injected pups. At P7, Mfsd2a protein is only present in proximal, impermeable vessels but absent in distal, leaky vessels (Figures 3E and 3F). In contrast, Claudin-5 is fully present at the distal vessels (Figures 3E and 3F). Importantly, Mfsd2a is absent from leaky, distal vessels during development (Figure 3F), and only by P10, when tracer is completely confined in the vessels, is Mfsd2a expression present at the distal vessels (Figures 3E and 3F). Thus, the spatio-temporal expression of Mfsd2a correlates with the gradual suppression of transcytosis and the development of a functional BRB. If the timing of the gradual suppression of transcytosis governs the development of a functional BRB, then altered regulation of transcytosis should affect functional BRB development. To test this idea, we examined the time course of functional BRB formation in two mutant mice with either increased or decreased transcytosis. First, we injected 10-kDa dextran tracers into Mfsd2a−/− mice and wild-type littermates at P5, P10, and adult ages. In P5 Mfsd2a+/+ wild-type mice, tracer leaked from distal vessels but was confined in proximal vessels (Figures 3G and 3H). However, in P5 Mfsd2a−/− mice, tracer leaked from both distal and proximal vessels (Figures 3G and 3H), similar to what we observed in P1 wild-type pups (Figure 1C). Even at P10 and adulthood, this leaky phenotype persisted, indicating that failure to suppress transcytosis results in incomplete formation of a functional BRB (Figure S4). We next tested whether precocious suppression of transcytosis during development accelerates functional BRB formation. Among various transcytotic pathways, peripheral endothelial cells frequently utilize the caveolae-pathway (Tuma and Hubbard, 2003Tuma P. Hubbard A.L. Transcytosis: crossing cellular barriers.Physiol. Rev. 2003; 83: 871-932Crossref PubMed Scopus (507) Google Scholar). Caveolin-1 (Cav-1) knockout mice lack caveolae vesicles throughout the endothelium (Drab et al., 2001Drab M. Verkade P. Elger M. Kasper M. Lohn M. Lauterbach B. Menne J. Lindschau C. Mende F. Luft F.C. et al.Loss of caveolae, vascular dysfunction, and pulmonary defects in caveolin-1 gene-disrupted mice.Science. 2001; 293: 2449-2452Crossref PubMed Scopus (1313) Google Scholar, Razani et al., 2001Razani B. Engelman J.A. Wang X.B. 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To examine the impact of precocious suppression of transcytosis on functional BRB formation, we intravenously injected bovine serum albumin (BSA), known to be transported via caveolae vesicles in lung endothelial cells (Schubert et al., 2001Schubert W. Frank P.G. Razani B. Park D.S. Chow C.W. Lisanti M.P. Caveolae-deficient endothelial cells show defects in the uptake and transport of albumin in vivo.J. Biol. Chem. 2001; 276: 48619-48622Crossref PubMed Scopus (272) Google Scholar), in Cav-1+/+ and Cav-1−/− mice at P8. In Cav-1+/+ wild-type littermate mice, we observed BSA leakage into the retinal parenchyma from distal vessels (Figures 4C and 4D). However, in Cav-1−/− mice, BSA was confined throughout the vasculature, indicating that a functional BRB had formed at P8 instead of P10 (Figures 4C and 4D). This time shift in BRB impermeability demonstrates that precocious suppression of transcytosis results in an earlier formation of a functional BRB. This study provides the first spatio-temporal characterization of functional BRB formation and establishes the BRB as a tractable model system for future study of CNS barriers. Using this system, we unexpectedly found that suppression of transcytosis is a principal contributor in the normal development of a functional barrier. We demonstrated that during BRB development, budding CNS endothelial cells already display functional tight junctions as early as vessel ingression but have not yet suppressed transcytosis. Thus, the leakiness of the developing retinal vasculature is entirely due to transcytosis. A functional BRB is established days later, only after transcytosis is gradually suppressed in BRB endothelial cells (Figure S5). In contrast to the prevailing notion in the field emphasizing the role of tight junctions in barrier function, our findings support an emerging view that transcytosis also plays a key role in regulating CNS barrier permeability and indicate that while specialized tight junctions are intrinsic to retinal endothelial cells, suppression of transcytosis is induced by CNS environmental cues during development. Our findings revealed that CNS endothelial cells have a developmental program that actively inhibits transcytosis to ensure barrier function. Finally, unregulated BRB dysfunction is a pathological feature of many ocular diseases (Klaassen et al., 2013Klaassen I. Van Noorden C.J.F. Schlingemann R.O. Molecular basis of the inner blood-retinal barrier and its breakdown in diabetic macular edema and other pathological conditions.Prog. Retin. Eye Res. 2013; 34: 19-48Crossref PubMed Scopus (441) Google Scholar). 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This suggests that the differentiation state of pericytes or inductive factors derived from pericytes during development are likely the key mechanisms that induce CNS endothelial cells to suppress transcytosis for functional BRB formation. Indeed, the loss of function of two transcription factors, foxc1 and foxf2, in CNS pericytes actually increases CNS pericyte density but still results in blood-brain barrier breakdown (Reyahi et al., 2015Reyahi A. Nik A.M. Ghiami M. Gritli-Linde A. Pontén F. Johansson B.R. Carlsson P. Foxf2 is required for brain pericyte differentiation and development and maintenance of the blood-brain barrier.Dev. Cell. 2015; 34: 19-32Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, Siegenthaler et al., 2013Siegenthaler J.A. Choe Y. Patterson K.P. Hsieh I. Li D. Jaminet S.-C. Daneman R. Kume T. Huang E.J. Pleasure S.J. Foxc1 is required by pericytes during fetal brain angiogenesis.Biol. 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