BACKGROUND
the central nervous system  has developed specialized "barriers" to isolate neurons from the blood stream.
these barriers are critical for neurological function, as they maintain a stable environment with the regulation of ionic balance and nutrient transport and blockage of potentially toxic molecules.
the cns has two types of barriers: endothelial and epithelial  <cit> .
the blood-retinal barrier  consists of an inner brb, formed by endothelial cells lining the retinal blood vessels and the outer brb formed by the retinal pigment epithelium , a layer of epithelial cells between the retina and non-neuronal choroid  <cit> .
the blood-brain barrier  and the blood-spinal cord barrier are endothelial barriers located within the cerebral vessels of the brain and the spinal cord, whereas the barrier between blood and the cerebrospinal fluid  is formed by the epithelial cells of the choroid plexus  <cit> .
both the endothelial and epithelial barriers have tight junctions, which seal the intercellular cleft of endothelial or epithelial cells and restrict paracellular diffusion of water-soluble molecules <cit> .
a number of tight junction proteins have been identified <cit>  which include cytoplasmic adapter proteins such as zona occludens- <dig> , that link trans-membrane proteins such as occludin and claudins to the cytoskeleton.
while occludin and claudins are tight-junction-specific, zo- <dig> is also a component of adherens junctions <cit> .
studies on these proteins have determined that tight-junctions, initially regarded as static and rigid, are dynamic structures capable of rapid modulation in response to physiological or pathological signals.
claudin 5a has been recently shown to be essential for the establishment of a neuro-epithelial barrier and zebrafish brain ventricular lumen expansion <cit> .
the dearth of knowledge on bbb/brb development and disruption is likely due to the fact that bbb/brb research in the past three decades has been based mainly on in vitro models of cultured cells and experiments of in situ brain/eye perfusion.
an in vivo animal model, which can be studied without disrupting the organs, will be critical to address the pathophysiology of bbb/brb development.
almost all vertebrates, including teleosts, have a bbb with similar functional characteristics  <cit> .
the teleost zebrafish  has proven to be a powerful model system to study mechanisms of organogenesis  <cit> , including development of the circulatory system  <cit> .
recently, jeong et al  <cit>  have examined the bbb in zebrafish.
using molecular markers and injection assays they have demonstrated that a functional endothelial-based bbb is established as early as  <dig> dpf   <cit> .
the characteristics of the brb, including the molecular and cellular components, development, maintenance and function have not been studied as extensively, but are believed to be very similar to the bbb.
increased vascular permeability and breakdown of the brb underlies the vision loss in diseases such as retinopathy of prematurity, diabetic retinopathy and age-related macular degeneration  <cit> .
although a number of studies have suggested a role for tight junction or adherens junction <cit>  proteins such as occludin <cit> , claudin- <dig> <cit> , zo- <dig> <cit>  and ve-cadherin <cit>  in the maintenance of the brb, our understanding of the molecular mechanisms contributing to the brb breakdown in pathological conditions is incomplete.
because of its tissue transparency and rapid development, we hypothesized that the zebrafish would be a good model system to examine the molecular mechanisms regulating the development and maintenance of the brb.
we have determined that the embryos of zebrafish develop a functional brb in the hyaloid vessels by  <dig> dpf.
we have generated a transgenic zebrafish line that can display the formation, disruption and reconstruction of the bbb and brb in vivo.
it will have great advantages in studying the blood-neural barrier through forward-genetic screens and reverse-genetic techniques.
RESULTS
the blood retinal barrier  and blood brain barrier  is established at  <dig> dpf in zebrafish
to determine the temporal sequence of establishment of the brb and bbb in zebrafish, we injected two fluorescent dyes into the circulatory system of tg  <cit>  embryos at  <dig> dpf,  <dig>  dpf and  <dig> dpf.
the vasculature of flk1:mcherry embryos is labeled with a red fluorescent protein, mcherry.
two tracers were utilized for this purpose, fluorescein dextran  <dig> , to detect large molecule diffusion and sodium fluorescein , a small molecule tracer used as a marker of vascular permeability in routine clinical practice.
we observed that the brb in hyaloid vessels was established in 3-day  embryos, which could retain fd <dig> in the hyaloid vasculature , in contrast to the vessels in  <dig> and  <dig>  dpf embryos, which could not .
interestingly, fd <dig> leakage from the trunk vessels of  <dig> dpf embryos occurred immediately after injection.
no fd <dig> was present in the trunk vessels at  <dig> minutes after injection, being diffused over the entire trunk with some dye accumulation in the myotomal boundaries , and minimal fd <dig> in the intersegmental vessels .
in addition, we examined the permeability of the brain vasculature, particularly the central arteries such as middle mesencephalic central artery , posterior mesencephalic central artery , and cerebellar central artery .
in the 2dpf and  <dig> dpf embryos, although we observed some dye leakage over time with accumulation mostly in the brain ventricles , the boundaries of the fd4-infused mmcta  and ccta  remained sharp and clear at  <dig> or even  <dig> minutes after injection, indicating that the endothelial barrier against fd <dig> is established in the central arteries at  <dig> dpf.
in contrast to fd <dig>  most of the leaked sodium-fluorescein did not accumulate in the brain ventricles , but diffused evenly throughout the entire brain .
in the  <dig>  dpf embryos, after  <dig> minutes of injection, the boundaries of the central arteries could not be differentiated from adjacent brain tissue .
in  <dig> dpf embryos, although the diffusing fluorescein caused an increased background throughout the brain, the boundaries of the central arteries remained sharp and clear at  <dig> minutes after injection , suggesting that the endothelial barrier against fluorescein has been established in the vessels.
similar observations were made in the retina, which suggests that the brb against small molecules is formed in the hyaloid vasculature at  <dig> dpf as well .
claudin- <dig> is a marker for the cns vasculature
claudin- <dig> is a tight-junction protein expressed in the bbb.
we used a monoclonal antibody against the c-terminal region of mouse claudin- <dig>  to examine the spatial expression of claudin- <dig> in the zebrafish cns vasculature.
the antibody used in these experiments was raised against the 20-residue peptide of the c-terminal region of mouse claudin- <dig>  which has a similar sequence to two zebrafish claudin- <dig> genes.
we observed that the claudin- <dig> signal co-localizes with egfp expressing endothelial cells of the hyaloid and brain vasculature , but not of the trunk vessels .
the 1st to 4th branchial arches  and the anterior pronephros  express claudin.
these results demonstrate that the mouse claudin- <dig> antibody is a good marker for the bbb and brb of zebrafish.
at  <dig> dpf, claudin- <dig> immunoreactivity is present in the hyaloid  and brain vasculature  but not in the trunk vessels .
panels b-d and b'-d' are enlarged views of the dashed squares in a and a'.
the claudin- <dig> antibody binds to the central artery  and the primordial hindbrain channel .
besides the cns vasculature, claudin- <dig> is also present in the 1st to 4th branchial arches  and the anterior pronephros  that is localized near the 4th branchial arch .
the polygonal rpe cells  and epithelial cells in the third  and the fourth ventricles  as well as the mid plane of the brain  have a strong claudin- <dig> signal .
expression of claudin- <dig> in the developing bbb
temporal expression of claudin- <dig> was evaluated in the developing bbb.
four pairs of central arteries that develop via angiogenesis, sprout into brain parenchyma as early as 2dpf  <cit> .
at  <dig> dpf , the mmcta  and ccta , two pairs of newly developed central arteries, express claudin- <dig> while the basal communicating artery  that had developed during the earlier vasculogenesis stage does not express claudin- <dig> .
starting at  <dig>  dpf, vessels that have developed during the vasculogenesis stage start to express claudin- <dig>  this includes the middle cerebral vein   in the top layer and the bca  in the upper middle layer of the head.
the intensity of staining in blood vessels that develop via angiogenesis after  <dig> dpf such as mmcta  and ccta , increases over time.
like the mesencephalic vein , all the vessels in the top layer show the claudin- <dig> signal, although some only have a weak staining, such as the anterior cerebral vein .
.
at  <dig> dpf, all the brain vessels express claudin- <dig>  the spatial and temporal expression of claudin- <dig> is consistent with our results with leakage of injected dyes as well as those described previously  <cit>  in that the zebrafish bbb is fully developed by  <dig> dpf.
this result also suggests that claudin- <dig> expression is a useful marker for the development of the zebrafish bbb.
expression of claudin- <dig> in the developing brb
a claudin- <dig> signal is present at  <dig> dpf in the hyaloid vasculature of zebrafish  consistent with the presence of a vascular barrier.
we further analyzed the temporal distribution of claudin- <dig> protein from  <dig> to  <dig> dpf, at which time the brb is being established .
we found claudin- <dig> to be present in the net of the hyaloid vasculature  and the hyaloid artery  as early as  <dig>  dpf, with a cone shaped localization , with a diameter wider than that of the hyaloid artery .
at  <dig> dpf, the intensity of the claudin- <dig> signal is increased in the hyaloid vasculature .
at this time claudin- <dig> signal appears in the inner plexiform layer .
claudin- <dig> in the hyaloid artery appears as a line within the cone-shape staining until  <dig>  dpf, after which it overlaps with endothelial cell staining in the hyaloid artery .
at  <dig> dpf, claudin- <dig> staining is found at the points of penetration of the hyaloid artery into the retina .
overall, the hyaloid vessels, hyaloid artery and the vessel connecting the hyaloid vessels and the inner optic circle , but not the ioc itself and other choroidal vessels, express claudin- <dig> .
as indicated by the expression of claudin- <dig> in the rpe cells, the outer brb is also formed at  <dig> dpf .
the expression pattern of claudin- <dig> in the eye correlated with the observation that the brb is formed at  <dig> dpf in zebrafish .
we also examined the spatial and temporal distribution of zo- <dig>  a molecular component of both tight junctions and adherens junctions.
we observed that zo- <dig> was expressed earlier than claudin- <dig> in the brb and bbb .
zo- <dig> is not specific to the cns vasculature, since the zo- <dig> antibody binds to a number of endothelial vessels outside of the cns, such as the intersegmental vessels .
claudin-5a and 5b are expressed in the hyaloid vasculature
based on the sequence of the peptide used in generating the mouse claudin- <dig> antibody, we examined the spatial and temporal expression of five zebrafish claudin genes from  <dig> to  <dig> dpf.
claudin-5a, claudin-5b and claudin-h have the highest homology at the c-termini .
claudin-5a was expressed in the cns , from 1- <dig> dpf, which is in accordance with online data .
claudin-5a mrna was also present in the hyaloid vasculature from  <dig>  to  <dig>  dpf .
at  <dig> dpf, it could still be detected in the hyaloid vessels albeit at low levels .
mrna of claudin-5b was expressed throughout the vasculature system at  <dig> dpf , then gradually lost at  <dig> dpf, except in the blood vessels of the brain and cardiovascular system .
this is in agreement with the gene expression data available through the zebrafish information network .
here we extend this study to demonstrate expression of claudin-5b in the hyaloid vessels from  <dig>  to  <dig> dpf , similar to claudin-5a.
the partially overlapped expression patterns, as revealed by in situ hybridization and immunohistochemistry , suggest that the anti-mammalian claudin- <dig> antibody can recognize both claudin-5a and claudin-5b isoforms that constitute the zebrafish brb and bbb.
in situ hybridization experiments with zebrafish claudin h gene as well as two negative  controls for cldn 5a and cldn5b showed no expression in the hyaloid vasculature around the lens .
we have also used a  <dig>  kb dna fragment, upstream of the coding sequence of the claudin-5b to drive expression of egfp and determined expression in the hyaloid vessels .
both these approaches suggest that claudin 5a and 5b are present in the hyaloid vasculature.
no expression of the other three zebrafish claudin genes  was detected in the blood vessels of the brain and the hyaloid vessels .
the brb can be visualized in tg zebrafish
in order to visualize the blood retinal barrier and blood-brain barrier in vivo, we generated transgenic lines of zebrafish that express a vitamin d-binding protein fused with the enhanced green fluorescent protein  under the control of the liver-type fatty acid binding protein  promoter.
our goal was to generate a transgenic line that expresses a fusion protein in the plasma that could be used as an endogenous tracer for bbb or brb breakdown.
the l-fabp promoter can drive its expression in hepatocytes from  <dig>  dpf to adulthood  <cit> .
dbp is a member of the albumin family and in wild type zebrafish, is translated in the hepatic cells and secreted into the blood circulation.
by using the l-fabp promoter and the dbp-egfp, we generated a transgenic line that expresses the fusion protein in the plasma as an endogenous tracer.
the bbb and brb in tg can be displayed from  <dig> dpf to two months .
the presence of egfp fluorescence in all eggs laid by the l-fabp:dbp-egfp transgenic fish , indicates that the dbp-egfp is stored in oocytes as maternal material.
at  <dig>  dpf, the dbp-egfp is expressed at low levels and the protein accumulates mostly in the brain ventricles  and area of the heart .
at  <dig> dpf, there is a diffuse localization of egfp in the eye indicating leakage of dbp-egfp out of the hv .
in the brain the egfp-infused central arteries are the only distinguishable blood vessels .
at  <dig> hpf, blood vessels begin to grow into the liver  and the branchial arches  become distinguishable, but the boundaries of the hv are not sharp until  <dig> dpf .
at  <dig> dpf, the egfp-infused hyaloid and brain vessels as well as the intersegmental vessels and the dorsal aorta  can be easily differentiated from the fluorescent background.
however the boundaries of the blood vessels in the trunk are not as sharp and distinct , as those of the hyaloid and the brain vessels .
after  <dig> dpf, the increased fluorescence background in the brain prevents visualization of the brain vasculature , but the hv remains distinguishable in the fish up to  <dig> dpf .
bradykinin can disrupt the brb of zebrafish
in an effort to determine if l-fabp:dbp-egfp transgenic fish would be a useful tool to screen for inducers and/or inhibitors of the brb, we examined the ability of bradykinin to induce leakage of egfp.
bradykinin  is an oligopeptide hormone that can cause brb breakdown in vivo  <cit> .
extracellular carbonic anhydrase increases retinal vascular permeability through the prekallikrein-kinin pathway <cit> .
double transgenic l-fabp:dbp-egfp;flk1:mcherry larvae were treated with  <dig> to  <dig> μm bk from  <dig> to  <dig> dpf.
leaky hyaloid vessels were identified in 31%, 13% and 7% of the  <dig> μm-,  <dig> μm- and  <dig> μm-treated larvae respectively, but in none of the control or  <dig> μm-treated samples .
in a separate experiment, flk1:egfp larvae were stained with the claudin- <dig> antibody after treatment with  <dig> μm bk.
claudin  <dig> expression was lost in the hyaloid vasculature of 36% of the larvae .
the apparently normal morphology of the retinal vasculature as visualized by the red fluorescence , suggests the absence of generalized vascular pathology with bradykinin which likely has specific effects on the barrier function of the retinal vessels.
in addition, direct injection of bradykinin  into the eyes of l-fabp:dbp-egfp;flk1:mcherry fish resulted in leakage of dbp-egfp out of the hyaloid vasculature in 90% of injected eyes  with no effect in the contralateral control injected eyes .
discussion
disruption of the bbb or brb is a crucial event in the development and progression of a number of diseases.
loss of barrier function leading to increased vascular permeability in the brain or retina can be a cause or consequence of the pathology.
a detailed understanding of the physiological processes involved in the development and maintenance of the blood-neural barrier is critical for the identification of therapeutic targets.
one of the major limitations in this regard has been the absence of an easily regulated in vivo system that allows alterations of these barriers.
the bbb and brb, initially regarded as static and rigid, have now been proven to be dynamic structures with both paracellular and transcellular pathways capable of rapid modulation in response to physiological or pathological signals  <cit> .
development of the vascular system in the brain can be classified into three phases: vasculogenesis, angiogenesis and bbb formation.
during brain angiogenesis, sprouting vessels from the perineural plexus grow into the proliferating neuroectoderm and form a capillary network.
at this time, the vessels appear to be permeable to small molecules  <cit>  but not to plasma proteins  <cit> .
although there have been a large number of studies examining the molecular mechanisms involved in vasculogenesis and angiogenesis, very limited information is available for the development of the bbb  <cit> .
the src-suppressed c kinase substrate is a factor induced by high oxygen tension.
its expression in brain astrocytes leads to decreased vascular endothelial growth factor  and increased angiopoietin- <dig> secretion, which have been suggested to be important for the cessation of brain angiogenesis and formation of the bbb  <cit> .
more recent reports demonstrate that wnt signaling is required for cns angiogenesis  <cit>  and may play a role in the initiation of the development of the bbb in mice  <cit> .
hypoxia has been shown to regulate the barrier function of neural blood vessels by reducing the expression of claudin  <dig> in endothelial cells  <cit> .
in addition, claudin- <dig> deficient mice show a size selective  loosening of the blood-brain barrier <cit> .
in this report we describe l-fabp:dbp-egfp transgenic zebrafish, in which the bbb and brb can be visualized.
this model will be a critical tool for future studies related to the study of blood-neural barrier development and differentiation.
the molecular weight of the dbp-egfp fusion protein, both calculated by the macvector software and estimated by western blot data , is  <dig> kda.
since the zebrafish claudin-5a and claudin-5b are sufficient to block paracellular transport of any molecule of  <dig> da or larger, but not sufficient to block the passage of smaller molecules like sodium fluorescein  it is likely that dbp-egfp would be comparable to high mw compounds.
human albumin  has been used as an endogenous tracer for blood-brain barrier studies and as an indicator of compromised bbb function in a number of pathophysiological conditions.
under physiological conditions albumin crosses endothelial cell wall via transcytosis.
however, paracellular transport of albumin in various sized microvessels has been observed under inflammatory conditions and following treatment with reagents that affect the integrity of tight-junctions.
the zebrafish dbp is a member of albumin family and is highly homologous to the human dbp and thus a good marker to evaluate the integrity of the bbb and brb.
we have recently demonstrated that knock-down of claudin-5a results in the leakage of dbp-egfp out of the hyaloid vessels .
these results taken together with the results in fig.
<dig>  suggest that dbp-egfp, as an endogenous tracer, may be useful to evaluate the breakdown of the brb against high molecular weight compounds, either due to developmental defects or pathological conditions.
although the structures of the bbb and brb in vertebrates have been fairly well characterized  <cit> , information about their development during embryogenesis and their maintenance in adults is limited.
similarities between them suggest that some of the developmental and regulatory mechanisms involved in these two barrier systems may overlap.
many cns disorders, such as brain tumors, stroke, trauma, multiple sclerosis and neurodegenerative diseases, are associated with a dysfunction of the bbb <cit> .
on the other hand, the presence of the bbb presents a major challenge for delivery of therapeutic compounds to the brain, as most drugs do not cross the bbb  <cit> .
in the present study we used fd <dig> as a tracer of large molecular weight and fluorescein as a small molecular weight tracer.
in zebrafish, fd <dig>  but not fluorecein  could be retained in the vasculature at  <dig> dpf when the central arteries started to express claudin- <dig>  this suggests that claudin- <dig> is likely to be critical for the barrier against large molecules and some additional junction protein may be needed for the barrier against small molecules.
our results with tracer injections, the claudin- <dig> antibody staining, as well as the observations on the l-fabp:dbp-egfp embryos, revealed a subtle difference between the formation of brb and bbb.
the brb against both large and small molecules developed gradually in the hyaloid vessels and was formed by  <dig> dpf, similar to the bbb against small molecules.
in contrast the bbb against large molecules is formed in the central arteries at  <dig> dpf , and in other blood vessels at  <dig>  dpf.
the developmental process of the zebrafish bbb is similar to results obtained from other previously studied vertebrates, in that the brain vessels of the angiogenesis stage are permeable to small molecules but not to plasma proteins  <cit> .
however, not all cerebral blood vessels are impermeable in adult zebrafish.
the blood vessels of the circumventriclar organ lack a bbb structure  <cit> , as these vessels have special physiological functions.
consistent with this, the accumulation of egfp in the brain of the l-fabp:dbp-egfp larvae results in a high fluorescent background and the brain vasculature becomes indistinguishable from  <dig> dpf onward.
thus the l-fabp:dbp-egfp will need to be modified to allow conditional expression of dbp-egfp for studies on the bbb of zebrafish post  <dig> dpf.
in contrast, the zebrafish brb is easier to evaluate as all the hyaloid vessels develop an inner brb structure at  <dig> dpf; the outer brb also matures at  <dig> dpf and further seals off the eyes.
this is an added advantage in studying the brb as the hyaloid/retinal vessels can be observed in the transgenic l-fabp:dbp-egfp zebrafish up to  <dig> dpf.
the zebrafish has been widely used to study eye development and disease  <cit> , but the brb has not been studied in this model.
alvarez et al  <cit>  have recently examined the morphology and development of the hyaloid and retinal vasculature in zebrafish and identified an important distinction between mammals and zebrafish.
in mammals, the regression of the hyaloid vasculature by apoptosis and the formation of the retinal vasculature by angiogenesis are synchronized processes, while in the zebrafish, the hyaloid vessels develop into retinal vessels without regression.
in addition, adult zebrafish have tight-junctions in retinal endothelial cells and pericytes along the retinal vessels <cit> .
bradykinin is an oligopeptide hormone derived from proteolysis of kininogen and is involved in smooth muscle contraction and relaxation, increased vascular permeability, activation of pain sensory fibers, hypotension and inflammation  <cit> .
bradykinin activates two specific membrane receptors, b <dig> and b <dig> and plays a direct role in diabetes-induced breakdown of the brb  <cit> .
the kininogen and two bk receptors have been identified in zebrafish and two species of the pufferfish  <cit>  and shown to be localized to the brain and eye  <cit> .
the structure of bk is evolutionarily conserved between zebrafish and mouse with only two out of ten amino acids being substituted  <cit> .
previous studies have determined that mammalian bk has 16% potency of zebrafish bk in vitro assays  <cit> .
although the zebrafish bk receptor b <dig> is thought to have a ligand-interaction profile distinct from mammalian bk receptors  <cit> , we observed that 31% of the zebrafish larvae treated with  <dig> μm mammalian bk for four days demonstrated leaky hyaloid vessels.
these findings lend credence to the hypothesis that the regulatory mechanisms of brb may be conserved between zebrafish and humans.
the use of zebrafish as a model organism to study the bbb and brb has a number of advantages.
the ability to carry out forward-genetic screens in zebrafish is one of the models most attractive features.
a forward-genetic approach following mutagenesis of l-fabp:dbp-egfp transgenic zebrafish, and screening for a leaky brb phenotype will identify and characterize zebrafish mutants that affect the establishment and maintenance of the brb.
this phenotype-driven genome-wide screen, which makes no assumptions about the genes involved in the biological processes of interest, can likely reveal novel genetic pathways involved in the development of brb.
CONCLUSIONS
we have demonstrated that zebrafish have a bbb and brb structure which is formed at  <dig> dpf.
a transgenic zebrafish line, as well as a monoclonal claudin- <dig> antibody, can display the formation, disruption and reconstruction of the bbb and brb.
the l-fabp:dbp-egfp transgenic zebrafish will have great advantages in identifying the genes involved in development and maintenance of the brb, through reverse-genetic techniques and forward-genetic screens.
disruption of brb by bradykinin demonstrates that the transgenic zebrafish could also be used for experimental testing of therapeutic agents that could potentially be effective in the treatment of retinal or brain vascular leakage.

 the central nervous system has developed specialized "barriers" to isolate neurons from the blood stream. these barriers are critical for neurological function, as they maintain a stable environment with the regulation of ionic balance and nutrient transport and blockage of potentially toxic molecules. the cns has two types of barriers: endothelial and epithelial. the blood-retinal barrier consists of an inner brb, formed by endothelial cells lining the retinal blood vessels and the outer brb formed by the retinal pigment epithelium, a layer of epithelial cells between the retina and non-neuronal choroid. the blood-brain barrier and the blood-spinal cord barrier are endothelial barriers located within the cerebral vessels of the brain and the spinal cord, whereas the barrier between blood and the cerebrospinal fluid is formed by the epithelial cells of the choroid plexus. both the endothelial and epithelial barriers have tight junctions, which seal the intercellular cleft of endothelial or epithelial cells and restrict paracellular diffusion of water-soluble molecules. a number of tight junction proteins have been identified which include cytoplasmic adapter proteins such as zona occludens-, that link trans-membrane proteins such as occludin and claudins to the cytoskeleton. while occludin and claudins are tight-junction-specific, zo- is also a component of adherens junctions. studies on these proteins have determined that tight-junctions, initially regarded as static and rigid, are dynamic structures capable of rapid modulation in response to physiological or pathological signals. claudin 5a has been recently shown to be essential for the establishment of a neuro-epithelial barrier and zebrafish brain ventricular lumen expansion. the dearth of knowledge on bbb/brb development and disruption is likely due to the fact that bbb/brb research in the past three decades has been based mainly on in vitro models of cultured cells and experiments of in situ brain/eye perfusion. an in vivo animal model, which can be studied without disrupting the organs, will be critical to address the pathophysiology of bbb/brb development. almost all vertebrates, including teleosts, have a bbb with similar functional characteristics. the teleost zebrafish has proven to be a powerful model system to study mechanisms of organogenesis, including development of the circulatory system. recently, jeong et al have examined the bbb in zebrafish. using molecular markers and injection assays they have demonstrated that a functional endothelial-based bbb is established as early as dpf. the characteristics of the brb, including the molecular and cellular components, development, maintenance and function have not been studied as extensively, but are believed to be very similar to the bbb. increased vascular permeability and breakdown of the brb underlies the vision loss in diseases such as retinopathy of prematurity, diabetic retinopathy and age-related macular degeneration. although a number of studies have suggested a role for tight junction or adherens junction proteins such as occludin, claudin-, zo- and ve-cadherin in the maintenance of the brb, our understanding of the molecular mechanisms contributing to the brb breakdown in pathological conditions is incomplete. because of its tissue transparency and rapid development, we hypothesized that the zebrafish would be a good model system to examine the molecular mechanisms regulating the development and maintenance of the brb. we have determined that the embryos of zebrafish develop a functional brb in the hyaloid vessels by dpf. we have generated a transgenic zebrafish line that can display the formation, disruption and reconstruction of the bbb and brb in vivo. it will have great advantages in studying the blood-neural barrier through forward-genetic screens and reverse-genetic techniques.  the blood retinal barrier and blood brain barrier is established at dpf in zebrafish to determine the temporal sequence of establishment of the brb and bbb in zebrafish, we injected two fluorescent dyes into the circulatory system of tg embryos at dpf, dpf and dpf. the vasculature of flk1:mcherry embryos is labeled with a red fluorescent protein, mcherry. two tracers were utilized for this purpose, fluorescein dextran, to detect large molecule diffusion and sodium fluorescein, a small molecule tracer used as a marker of vascular permeability in routine clinical practice. we observed that the brb in hyaloid vessels was established in 3-day embryos, which could retain fd in the hyaloid vasculature, in contrast to the vessels in and dpf embryos, which could not. interestingly, fd leakage from the trunk vessels of dpf embryos occurred immediately after injection. no fd was present in the trunk vessels at minutes after injection, being diffused over the entire trunk with some dye accumulation in the myotomal boundaries, and minimal fd in the intersegmental vessels. in addition, we examined the permeability of the brain vasculature, particularly the central arteries such as middle mesencephalic central artery, posterior mesencephalic central artery, and cerebellar central artery. in the 2dpf and dpf embryos, although we observed some dye leakage over time with accumulation mostly in the brain ventricles, the boundaries of the fd4-infused mmcta and ccta remained sharp and clear at or even minutes after injection, indicating that the endothelial barrier against fd is established in the central arteries at dpf. in contrast to fd most of the leaked sodium-fluorescein did not accumulate in the brain ventricles, but diffused evenly throughout the entire brain. in the dpf embryos, after minutes of injection, the boundaries of the central arteries could not be differentiated from adjacent brain tissue. in dpf embryos, although the diffusing fluorescein caused an increased background throughout the brain, the boundaries of the central arteries remained sharp and clear at minutes after injection, suggesting that the endothelial barrier against fluorescein has been established in the vessels. similar observations were made in the retina, which suggests that the brb against small molecules is formed in the hyaloid vasculature at dpf as well. claudin- is a marker for the cns vasculature claudin- is a tight-junction protein expressed in the bbb. we used a monoclonal antibody against the c-terminal region of mouse claudin- to examine the spatial expression of claudin- in the zebrafish cns vasculature. the antibody used in these experiments was raised against the 20-residue peptide of the c-terminal region of mouse claudin- which has a similar sequence to two zebrafish claudin- genes. we observed that the claudin- signal co-localizes with egfp expressing endothelial cells of the hyaloid and brain vasculature, but not of the trunk vessels. the 1st to 4th branchial arches and the anterior pronephros express claudin. these results demonstrate that the mouse claudin- antibody is a good marker for the bbb and brb of zebrafish. at dpf, claudin- immunoreactivity is present in the hyaloid and brain vasculature but not in the trunk vessels. panels b-d and b'-d' are enlarged views of the dashed squares in a and a'. the claudin- antibody binds to the central artery and the primordial hindbrain channel. besides the cns vasculature, claudin- is also present in the 1st to 4th branchial arches and the anterior pronephros that is localized near the 4th branchial arch. the polygonal rpe cells and epithelial cells in the third and the fourth ventricles as well as the mid plane of the brain have a strong claudin- signal. expression of claudin- in the developing bbb temporal expression of claudin- was evaluated in the developing bbb. four pairs of central arteries that develop via angiogenesis, sprout into brain parenchyma as early as 2dpf. at dpf, the mmcta and ccta, two pairs of newly developed central arteries, express claudin- while the basal communicating artery that had developed during the earlier vasculogenesis stage does not express claudin-. starting at dpf, vessels that have developed during the vasculogenesis stage start to express claudin- this includes the middle cerebral vein in the top layer and the bca in the upper middle layer of the head. the intensity of staining in blood vessels that develop via angiogenesis after dpf such as mmcta and ccta, increases over time. like the mesencephalic vein, all the vessels in the top layer show the claudin- signal, although some only have a weak staining, such as the anterior cerebral vein.. at dpf, all the brain vessels express claudin- the spatial and temporal expression of claudin- is consistent with our results with leakage of injected dyes as well as those described previously in that the zebrafish bbb is fully developed by dpf. this result also suggests that claudin- expression is a useful marker for the development of the zebrafish bbb. expression of claudin- in the developing brb a claudin- signal is present at dpf in the hyaloid vasculature of zebrafish consistent with the presence of a vascular barrier. we further analyzed the temporal distribution of claudin- protein from to dpf, at which time the brb is being established. we found claudin- to be present in the net of the hyaloid vasculature and the hyaloid artery as early as dpf, with a cone shaped localization, with a diameter wider than that of the hyaloid artery. at dpf, the intensity of the claudin- signal is increased in the hyaloid vasculature. at this time claudin- signal appears in the inner plexiform layer. claudin- in the hyaloid artery appears as a line within the cone-shape staining until dpf, after which it overlaps with endothelial cell staining in the hyaloid artery. at dpf, claudin- staining is found at the points of penetration of the hyaloid artery into the retina. overall, the hyaloid vessels, hyaloid artery and the vessel connecting the hyaloid vessels and the inner optic circle, but not the ioc itself and other choroidal vessels, express claudin-. as indicated by the expression of claudin- in the rpe cells, the outer brb is also formed at dpf. the expression pattern of claudin- in the eye correlated with the observation that the brb is formed at dpf in zebrafish. we also examined the spatial and temporal distribution of zo- a molecular component of both tight junctions and adherens junctions. we observed that zo- was expressed earlier than claudin- in the brb and bbb. zo- is not specific to the cns vasculature, since the zo- antibody binds to a number of endothelial vessels outside of the cns, such as the intersegmental vessels. claudin-5a and 5b are expressed in the hyaloid vasculature based on the sequence of the peptide used in generating the mouse claudin- antibody, we examined the spatial and temporal expression of five zebrafish claudin genes from to dpf. claudin-5a, claudin-5b and claudin-h have the highest homology at the c-termini. claudin-5a was expressed in the cns, from 1- dpf, which is in accordance with online data. claudin-5a mrna was also present in the hyaloid vasculature from to dpf. at dpf, it could still be detected in the hyaloid vessels albeit at low levels. mrna of claudin-5b was expressed throughout the vasculature system at dpf, then gradually lost at dpf, except in the blood vessels of the brain and cardiovascular system. this is in agreement with the gene expression data available through the zebrafish information network. here we extend this study to demonstrate expression of claudin-5b in the hyaloid vessels from to dpf, similar to claudin-5a. the partially overlapped expression patterns, as revealed by in situ hybridization and immunohistochemistry, suggest that the anti-mammalian claudin- antibody can recognize both claudin-5a and claudin-5b isoforms that constitute the zebrafish brb and bbb. in situ hybridization experiments with zebrafish claudin h gene as well as two negative controls for cldn 5a and cldn5b showed no expression in the hyaloid vasculature around the lens. we have also used a kb dna fragment, upstream of the coding sequence of the claudin-5b to drive expression of egfp and determined expression in the hyaloid vessels. both these approaches suggest that claudin 5a and 5b are present in the hyaloid vasculature. no expression of the other three zebrafish claudin genes was detected in the blood vessels of the brain and the hyaloid vessels. the brb can be visualized in tg zebrafish in order to visualize the blood retinal barrier and blood-brain barrier in vivo, we generated transgenic lines of zebrafish that express a vitamin d-binding protein fused with the enhanced green fluorescent protein under the control of the liver-type fatty acid binding protein promoter. our goal was to generate a transgenic line that expresses a fusion protein in the plasma that could be used as an endogenous tracer for bbb or brb breakdown. the l-fabp promoter can drive its expression in hepatocytes from dpf to adulthood. dbp is a member of the albumin family and in wild type zebrafish, is translated in the hepatic cells and secreted into the blood circulation. by using the l-fabp promoter and the dbp-egfp, we generated a transgenic line that expresses the fusion protein in the plasma as an endogenous tracer. the bbb and brb in tg can be displayed from dpf to two months. the presence of egfp fluorescence in all eggs laid by the l-fabp:dbp-egfp transgenic fish, indicates that the dbp-egfp is stored in oocytes as maternal material. at dpf, the dbp-egfp is expressed at low levels and the protein accumulates mostly in the brain ventricles and area of the heart. at dpf, there is a diffuse localization of egfp in the eye indicating leakage of dbp-egfp out of the hv. in the brain the egfp-infused central arteries are the only distinguishable blood vessels. at hpf, blood vessels begin to grow into the liver and the branchial arches become distinguishable, but the boundaries of the hv are not sharp until dpf. at dpf, the egfp-infused hyaloid and brain vessels as well as the intersegmental vessels and the dorsal aorta can be easily differentiated from the fluorescent background. however the boundaries of the blood vessels in the trunk are not as sharp and distinct, as those of the hyaloid and the brain vessels. after dpf, the increased fluorescence background in the brain prevents visualization of the brain vasculature, but the hv remains distinguishable in the fish up to dpf. bradykinin can disrupt the brb of zebrafish in an effort to determine if l-fabp:dbp-egfp transgenic fish would be a useful tool to screen for inducers and/or inhibitors of the brb, we examined the ability of bradykinin to induce leakage of egfp. bradykinin is an oligopeptide hormone that can cause brb breakdown in vivo. extracellular carbonic anhydrase increases retinal vascular permeability through the prekallikrein-kinin pathway. double transgenic l-fabp:dbp-egfp;flk1:mcherry larvae were treated with to μm bk from to dpf. leaky hyaloid vessels were identified in 31%, 13% and 7% of the μm-, μm- and μm-treated larvae respectively, but in none of the control or μm-treated samples. in a separate experiment, flk1:egfp larvae were stained with the claudin- antibody after treatment with μm bk. claudin expression was lost in the hyaloid vasculature of 36% of the larvae. the apparently normal morphology of the retinal vasculature as visualized by the red fluorescence, suggests the absence of generalized vascular pathology with bradykinin which likely has specific effects on the barrier function of the retinal vessels. in addition, direct injection of bradykinin into the eyes of l-fabp:dbp-egfp;flk1:mcherry fish resulted in leakage of dbp-egfp out of the hyaloid vasculature in 90% of injected eyes with no effect in the contralateral control injected eyes. discussion disruption of the bbb or brb is a crucial event in the development and progression of a number of diseases. loss of barrier function leading to increased vascular permeability in the brain or retina can be a cause or consequence of the pathology. a detailed understanding of the physiological processes involved in the development and maintenance of the blood-neural barrier is critical for the identification of therapeutic targets. one of the major limitations in this regard has been the absence of an easily regulated in vivo system that allows alterations of these barriers. the bbb and brb, initially regarded as static and rigid, have now been proven to be dynamic structures with both paracellular and transcellular pathways capable of rapid modulation in response to physiological or pathological signals. development of the vascular system in the brain can be classified into three phases: vasculogenesis, angiogenesis and bbb formation. during brain angiogenesis, sprouting vessels from the perineural plexus grow into the proliferating neuroectoderm and form a capillary network. at this time, the vessels appear to be permeable to small molecules but not to plasma proteins. although there have been a large number of studies examining the molecular mechanisms involved in vasculogenesis and angiogenesis, very limited information is available for the development of the bbb. the src-suppressed c kinase substrate is a factor induced by high oxygen tension. its expression in brain astrocytes leads to decreased vascular endothelial growth factor and increased angiopoietin- secretion, which have been suggested to be important for the cessation of brain angiogenesis and formation of the bbb. more recent reports demonstrate that wnt signaling is required for cns angiogenesis and may play a role in the initiation of the development of the bbb in mice. hypoxia has been shown to regulate the barrier function of neural blood vessels by reducing the expression of claudin in endothelial cells. in addition, claudin- deficient mice show a size selective loosening of the blood-brain barrier. in this report we describe l-fabp:dbp-egfp transgenic zebrafish, in which the bbb and brb can be visualized. this model will be a critical tool for future studies related to the study of blood-neural barrier development and differentiation. the molecular weight of the dbp-egfp fusion protein, both calculated by the macvector software and estimated by western blot data, is kda. since the zebrafish claudin-5a and claudin-5b are sufficient to block paracellular transport of any molecule of da or larger, but not sufficient to block the passage of smaller molecules like sodium fluorescein it is likely that dbp-egfp would be comparable to high mw compounds. human albumin has been used as an endogenous tracer for blood-brain barrier studies and as an indicator of compromised bbb function in a number of pathophysiological conditions. under physiological conditions albumin crosses endothelial cell wall via transcytosis. however, paracellular transport of albumin in various sized microvessels has been observed under inflammatory conditions and following treatment with reagents that affect the integrity of tight-junctions. the zebrafish dbp is a member of albumin family and is highly homologous to the human dbp and thus a good marker to evaluate the integrity of the bbb and brb. we have recently demonstrated that knock-down of claudin-5a results in the leakage of dbp-egfp out of the hyaloid vessels. these results taken together with the results in fig. suggest that dbp-egfp, as an endogenous tracer, may be useful to evaluate the breakdown of the brb against high molecular weight compounds, either due to developmental defects or pathological conditions. although the structures of the bbb and brb in vertebrates have been fairly well characterized, information about their development during embryogenesis and their maintenance in adults is limited. similarities between them suggest that some of the developmental and regulatory mechanisms involved in these two barrier systems may overlap. many cns disorders, such as brain tumors, stroke, trauma, multiple sclerosis and neurodegenerative diseases, are associated with a dysfunction of the bbb. on the other hand, the presence of the bbb presents a major challenge for delivery of therapeutic compounds to the brain, as most drugs do not cross the bbb. in the present study we used fd as a tracer of large molecular weight and fluorescein as a small molecular weight tracer. in zebrafish, fd but not fluorecein could be retained in the vasculature at dpf when the central arteries started to express claudin- this suggests that claudin- is likely to be critical for the barrier against large molecules and some additional junction protein may be needed for the barrier against small molecules. our results with tracer injections, the claudin- antibody staining, as well as the observations on the l-fabp:dbp-egfp embryos, revealed a subtle difference between the formation of brb and bbb. the brb against both large and small molecules developed gradually in the hyaloid vessels and was formed by dpf, similar to the bbb against small molecules. in contrast the bbb against large molecules is formed in the central arteries at dpf, and in other blood vessels at dpf. the developmental process of the zebrafish bbb is similar to results obtained from other previously studied vertebrates, in that the brain vessels of the angiogenesis stage are permeable to small molecules but not to plasma proteins. however, not all cerebral blood vessels are impermeable in adult zebrafish. the blood vessels of the circumventriclar organ lack a bbb structure, as these vessels have special physiological functions. consistent with this, the accumulation of egfp in the brain of the l-fabp:dbp-egfp larvae results in a high fluorescent background and the brain vasculature becomes indistinguishable from dpf onward. thus the l-fabp:dbp-egfp will need to be modified to allow conditional expression of dbp-egfp for studies on the bbb of zebrafish post dpf. in contrast, the zebrafish brb is easier to evaluate as all the hyaloid vessels develop an inner brb structure at dpf; the outer brb also matures at dpf and further seals off the eyes. this is an added advantage in studying the brb as the hyaloid/retinal vessels can be observed in the transgenic l-fabp:dbp-egfp zebrafish up to dpf. the zebrafish has been widely used to study eye development and disease, but the brb has not been studied in this model. alvarez et al have recently examined the morphology and development of the hyaloid and retinal vasculature in zebrafish and identified an important distinction between mammals and zebrafish. in mammals, the regression of the hyaloid vasculature by apoptosis and the formation of the retinal vasculature by angiogenesis are synchronized processes, while in the zebrafish, the hyaloid vessels develop into retinal vessels without regression. in addition, adult zebrafish have tight-junctions in retinal endothelial cells and pericytes along the retinal vessels. bradykinin is an oligopeptide hormone derived from proteolysis of kininogen and is involved in smooth muscle contraction and relaxation, increased vascular permeability, activation of pain sensory fibers, hypotension and inflammation. bradykinin activates two specific membrane receptors, b and b and plays a direct role in diabetes-induced breakdown of the brb. the kininogen and two bk receptors have been identified in zebrafish and two species of the pufferfish and shown to be localized to the brain and eye. the structure of bk is evolutionarily conserved between zebrafish and mouse with only two out of ten amino acids being substituted. previous studies have determined that mammalian bk has 16% potency of zebrafish bk in vitro assays. although the zebrafish bk receptor b is thought to have a ligand-interaction profile distinct from mammalian bk receptors, we observed that 31% of the zebrafish larvae treated with μm mammalian bk for four days demonstrated leaky hyaloid vessels. these findings lend credence to the hypothesis that the regulatory mechanisms of brb may be conserved between zebrafish and humans. the use of zebrafish as a model organism to study the bbb and brb has a number of advantages. the ability to carry out forward-genetic screens in zebrafish is one of the models most attractive features. a forward-genetic approach following mutagenesis of l-fabp:dbp-egfp transgenic zebrafish, and screening for a leaky brb phenotype will identify and characterize zebrafish mutants that affect the establishment and maintenance of the brb. this phenotype-driven genome-wide screen, which makes no assumptions about the genes involved in the biological processes of interest, can likely reveal novel genetic pathways involved in the development of brb.  we have demonstrated that zebrafish have a bbb and brb structure which is formed at dpf. a transgenic zebrafish line, as well as a monoclonal claudin- antibody, can display the formation, disruption and reconstruction of the bbb and brb. the l-fabp:dbp-egfp transgenic zebrafish will have great advantages in identifying the genes involved in development and maintenance of the brb, through reverse-genetic techniques and forward-genetic screens. disruption of brb by bradykinin demonstrates that the transgenic zebrafish could also be used for experimental testing of therapeutic agents that could potentially be effective in the treatment of retinal or brain vascular leakage. 
the tg zebrafish will have great advantages in studying development and maintenance of the blood-neural barrier, which is a new application for the widely used vertebrate model. we have established an in vivo model to study the development and maintenance of these barriers by generating a transgenic zebrafish line that expresses a vitamin d-binding protein fused with enhanced green fluorescent protein in blood plasma, as an endogenous tracer. we also examined the expression of claudin- a component of tight junctions during the first days of development. the temporal establishment of the bbb and brb was examined using this transgenic line and the results were compared with that obtained by injection of fluorescent dyes into the sinus venosus of embryos at various stages of development. development and maintenance of the blood-brain and blood-retinal barrier is critical for the homeostasis of brain and retinal tissue. the hyaloid vasculature in the zebrafish retina develops a barrier function at dpf, which endows the zebrafish with unique advantages for studying the brb. zebrafish embryos develop bbb and brb function simultaneously by dpf, which is regulated by tight junction proteins. we observed that the bbb of zebrafish starts to develop by dpf, with expression of claudin- in the central arteries preceding it at dpf.
 development and maintenance of the blood-brain and blood-retinal barrier is critical for the homeostasis of brain and retinal tissue. despite decades of research our knowledge of the formation and maintenance of the blood-brain and blood-retinal barrier is very limited. we have established an in vivo model to study the development and maintenance of these barriers by generating a transgenic zebrafish line that expresses a vitamin d-binding protein fused with enhanced green fluorescent protein in blood plasma, as an endogenous tracer.  the temporal establishment of the bbb and brb was examined using this transgenic line and the results were compared with that obtained by injection of fluorescent dyes into the sinus venosus of embryos at various stages of development. we also examined the expression of claudin- a component of tight junctions during the first days of development. we observed that the bbb of zebrafish starts to develop by dpf, with expression of claudin- in the central arteries preceding it at dpf. the hyaloid vasculature in the zebrafish retina develops a barrier function at dpf, which endows the zebrafish with unique advantages for studying the brb.  zebrafish embryos develop bbb and brb function simultaneously by dpf, which is regulated by tight junction proteins. the tg zebrafish will have great advantages in studying development and maintenance of the blood-neural barrier, which is a new application for the widely used vertebrate model. 