Developmental Regulation of Barrier- and Non-Barrier Blood Vessels in the CNS

Abstract

The blood-brain barrier (BBB) is essential for maintaining tissue homeostasis in the central nervous system (CNS), which is crucial for proper neuronal function.
In vertebrates, the BBB is localized to microvascular endothelial cells that acquire barrier properties during angiogenesis of the neuroectoderm.
Complex tight junctions, lack of fenestrae, and low pinocytotic activity make the BBB endothelium a tight barrier for water-soluble molecules, which can only enter the CNS via specific transporters.
Differentiation of endothelial properties during embryonic development is initiated by endothelial-specific Wnt/ $β$ -catenin pathway.
The review summarizes cellular (neural precursor and endothelial cells) and molecular (VEGF and Wnt/ $β$ -catenin) mechanisms mediating brain angiogenesis and barrier formation.
It also introduces crosstalk with cellular and acellular elements within the developing CNS, such as the extracellular matrix.
Recent insights into the downstream molecular mechanisms of Wnt/ $β$ -catenin, like target genes Foxf2, Foxl2, Foxq1, Lef1, Ppard, Zfp551, Zic3, Sox17, Apcdd1 and Fgfbp1, are discussed concerning their involvement in refining and maintaining barrier characteristics in the mature BBB endothelium.
Barrier heterogeneity and differential endothelial barrier properties within the CNS are examined, focusing on the circumventricular organs and neurogenic niches in the subventricular zone and hippocampus.
Open questions and future BBB research directions are highlighted, aiming to leverage the understanding of BBB development for strategies to modulate BBB function under pathological conditions.

Keywords

BBB heterogeneity, blood–brain barrier, circumventricular organs, differentiation, tight junctions, Wnt/ $β$ -catenin signaling.

Introduction

The central nervous system (CNS) requires a specific micro-environment that differs in cellular and molecular composition from peripheral organs $[1]$ .
This specific brain milieu is maintained by the blood–brain barrier (BBB) $[2]$ , localized to brain capillary endothelial cells (BCECs) in most vertebrates except elasmobranches and chondrostei $[3–5]$ .
BCECs form complex and continuous interendothelial tight junctions (TJs), sealing off the paracellular pathway and controlling the passage of molecules $[6, 7]$ .
Brain ECs possess adherens junctions (AJs) formed by vascular endothelial cadherin (Cdh5, VE-cadherin, CD144) connected via $β$ - or $γ$ -catenin and $\alpha$ -catenin to the actin cytoskeleton, providing a signaling hub for barrier regulation $[8]$ .
BCECs lack fenestrae and show lower vesicular traffic, making the BBB endothelium a tight barrier for polar, water-soluble molecules larger than 450 Da $[2]$ .
The BBB endothelium is unique due to its barrier function, expression of specific enzymes and transporters, distinguishing it from other endothelial cells (ECs).
The differentiation of brain ECs towards the BBB phenotype is linked to their morphological connection to pericytes (PCs), perivascular fibroblasts (FBs), astrocytes (ACs), microglia (MGs) and nerve endings (Ns), together denominated as the neurovascular unit (NVU) $[9, 10]$ .
The NVU structure needs to be established during ontogenic development through precise interaction of cellular and molecular components.
Increasing evidence points to region-specific variations of BBB signature genes $[11, 12]$ .

Development of the BBB

Common Angiogenic Factors in CNS Vascularization

Vascular development in the brain starts from a preformed perineural vascular plexus (PNVP) surrounding the developing neural tube $[13]$ .
Pro- and anti-angiogenic factors control the angiogenic growth of blood vessels.
Vascular plexus are formed early in embryonic development by transformation of angioblasts into primitive endothelial vascular networks.
Vascular endothelial cell growth factor (VEGF) and its endothelial tyrosine kinase receptors VEGF-R1 (Flt-1) and VEGF-R2 (Flk-1) are crucial for CNS angiogenesis $[14]$ .
VEGF is expressed and released by neural progenitors, inducing ingrowth of capillaries from the PVNP $[15]$ .
Extracellular VEGF gradient formation and deposition within the extracellular matrix (ECM) regulate vascular branching during brain angiogenesis, guided by endothelial tip cells $[16, 17]$ .
Differentiation of tip cells and stalk cells depends on the interplay of VEGF/VEGFR with the Dll/Notch pathway $[18]$ .
Regulation of the VEGF-Notch signaling axis is achieved by Sema3E–Plexin-D1 signaling, negatively regulating the activity of VEGF-induced delta-like 4 (Dll4)–Notch signaling $[19]$ .
Sprouting endothelial cells produce platelet-derived growth factor B (PDGF-B), engaging its receptor PDGFR- $β$ expressed by pericytes, promoting pericyte recruitment.
Mice lacking PDGF-B or PDGFR- $β$ show pericyte loss from brain microvessels and develop lethal microaneurysms $[20]$ , indicating PDGF-B's involvement in brain vascularization.
Precise ECM deposition of PDGF-B is essential for pericyte recruitment to the developing CNS vasculature $[21]$ and BBB integrity $[22, 23]$ , suggesting pericytes' role in BBB differentiation and maturation $[24]$ .
Additional molecular mediators of angiogenesis include Tie-1 and Tie-2, angiopoietin-1 and angiopoietin-2 (Ang-1/-2), members of the transforming growth factor (TGF)- $β$ superfamily, and the EphB-receptor/ephrinB-ligand family $[25]$ .
Ang-1 via its receptor Tie2/Tek is implicated in BBB integrity $[1, 13]$ .
Junction proteins, like VE-cadherin, contribute to vascular stability and endothelial polarity and regulate the endothelial response to pro- and anti-angiogenic stimuli $[8]$ .
Vascular stability in the developing embryo is regulated by VE-cadherin, cytoskeletal components, and small GTPases Rac and Rho.
Erythrocyte-derived sphingosine phosphate-1 (S1P) and its receptors 1-5 (S1pr1-5) are important for vascular stability and integrity during early mouse development $[26, 27]$ .
Endothelial-specific S1P1 deletion in postnatal mice leads to BBB opening for small tracers $[27]$ .

CNS-Specific Regulators of Angiogenesis and Barrier Genesis: Morphogens Regulating BBB Differentiation

Avascular developing vertebrate brain can induce endothelial barrier properties $[28]$ , indicating the CNS provides essential cues for barrier induction.
BBB characteristics induction takes place in the preformed PVNP, indicated by endothelial expression of BBB-specific Glut1 and Wnt reporter gene expression at E9 in PVNP vessels of the mouse $[29, 30]$ .
The factor/s may be diffusible, leading to endothelial barrier induction beyond the glia limitans, or specifically secreted by radial glia endfeet.
Due to restricted induction of endothelial barrier properties, a limited diffusibility of the growth factor/s was considered a prerequisite.
The Wnt class of growth factor or morphogens fulfilled this prerequisite, and analysis of the $β$ -catenin-driven Wnt pathway demonstrated the importance of the Wnt pathway for brain angiogenesis and induction of endothelial barrier function $[29–31]$ .
Wnt ligands bind to a cysteine-rich domain in frizzled receptors, requiring interaction with LDL receptor-related protein 5 and 6 (Lrp5 and Lrp6).
Intracellularly, Wnt-receptor complex binding elicits recruitment of axin to the membrane via dishevelled (dvl), resulting in disintegration of the destruction complex for cytosolic $β$ -catenin.
The destruction complex comprises axin, adenomatous polyposis coli (APC), glycogen synthase kinase 3 $β$ (GSK3 $β$ ) and casein kinase 1 $\alpha$ (CK1 $\alpha$ ), ensuring rapid phosphorylation of cytosolic $β$ -catenin in the absence of pathway stimulation $[32]$ .
Phosphorylated $β$ -catenin allows binding of the E3 ubiquitin ligase beta-transducin repeat-containing proteins ( $β$ -TrCP), enabling proteasomal degradation upon ubiquitination.
Wnt ligands Wnt7a and Wnt7b specifically drive endothelial signaling within the brain, which is unique in the Wnt signaling landscape.
The linker region of Wnt7a/b sticks to the glycosylphosphatidylinositol (GPI)-anchored glycoprotein RECK, connected by the G protein-coupled receptor 124 (Gpr124) to dishevelled (Dsh) $[32–37]$ .
In ECs, Fzd4 binds Wnt7a/b and interacts with the co-receptor Lrp5 or Lrp6, resulting in $β$ -catenin stabilization $[13, 38, 39]$ .
Cytoplasmic $β$ -catenin level increases and facilitates its nuclear translocation, where it binds a member of the T-cell factor/lymphoid enhancer factor (TCF/LEF) transcription factor (TF) family and releases the transcriptional repressor transducin-like enhancer of split (TLE) or groucho(Gro)-related gene (Grg) proteins from their binding to TCF/LEF, leading to Wnt target gene expression $[40]$ .
Norrie disease protein (norrin, Ndp) was identified to equally drive endothelial Wnt/ $β$ -catenin signalling, requiring the co-receptor tetraspanin 12 (Tspan12) $[41, 42]$ .
Ndp is encoded by the norrie gene, mutated in Norrie disease (ND) patients $[43, 44]$ .
Mutations in the Fzd4 gene also lead to severe neurological symptoms known as familial exudative vitreoretinopathy (FEVR) $[41, 45, 46]$ .
The transcriptional activation of $β$ -catenin is the common signalling theme in both ligand-receptor systems $[47]$ .
Endothelial target genes of $β$ -catenin contribute to BBB characteristics, such as transporters and TJ genes.
Claudin-5 (Cldn5), Glut1, Apcdd1, TNF receptor superfamily member 19 (Tnfrsf19), Sox17 and Lef1 were shown to be induced by $β$ -catenin signaling $[48]$ .
Plasmalemma vesicle-associated protein (Plvap, Meca32) becomes repressed by Wnt/ $β$ -catenin activation $[49]$ .
The transcription factors Foxf2, Foxl2, Foxq1, Lef1, Ppard, Zfp551 and Zic3 are associated with the maturation of the BBB $[50]$ .
The Wnt/ $β$ -catenin pathway drives fibroblast growth factor-binding protein 1 (Fgfbp1) that influences ECM deposition and pericyte recruitment $[51]$ .
Wnt factors are likely presented to the PNVP beyond the glia limitans during embryonic development $[29–31, 52]$ .
Endothelial tip-cell determination in the PNVP depends upon Wnt7-induced $β$ -catenin signalling in ECs $[36]$ .
Premature sphingosine-1 phosphate receptor (S1pr) signalling becomes prematurely upregulated in PNVP ECs of the developing zebrafish in the absence of $β$ -catenin signalling, leading to reduced junctional localization of VE-cadherin and ESAM $[53]$ .
Wnt signalling suppresses S1pr signalling allowing for dynamic junction formation during anastomosis.
S1pr signalling may regulate BBB maturation and VE-cadherin stabilization.
Maturation of the BBB is a gradual process consisting of a differentiation phase with the recruitment of PCs and ACs and a maturation phase $[30, 31, 52]$ .
A functional restrictive murine BBB can be detected at E15.5 even before the appearance of astrocytes $[52, 54]$ .
Deletion of the evenness interrupted (Evi) gene from GFAP-expressing ACs does not result in detectable developmental defects $[55]$ , suggesting a maintenance role for AC-derived Wnt growth factors.
Sonic hedgehog (Shh) and retinoic acid (RA) participate in inducing or maintaining endothelial barrier function under steady-state and chronic inflammatory conditions $[56–59]$ .

Barrier Characteristics During Development

The BBB was considered nonfunctional in embryos or perinatal animals based on incorrect experimental settings $[60]$ .
Research examining the embryonic BBB has rejuvenated understanding of BBB development.
BCECs acquire their restrictive barrier properties along a gradual developmental process $[22, 54, 61–63]$ .
In the mouse spinal cord, BCECs are already restrictive by day E14 $[63]$ .
In the cortex, BCECs in ventrolateral regions are fully restrictive at E14, while those in dorsomedial regions reach this level of maturation only at E15 $[22, 54]$ .
Cerebral vessels mature in a centrifugal, wave-like fashion that results in sequential acquisition of restrictive barrier properties.
Maturation patterns align with an exit from a VEGF-dependent phase, with periventricular vessels being the last to mature $[64]$ .
Perturbing with VEGF signalling causing insults to periventricular vessels results in major deficits in inhibitory neurons in the cortical plate $[64]$ .
Developmental maturation happens in a different timing at different CNS locations.
There is heterogeneity in the function of adult BCECs in different brain regions.
There are regions within the CNS with vasculature possessing nonbarrier, peripheral properties.

Developmental Regulation of Sealing Properties

BCECs lack fenestrations and exhibit low rates of transcytosis.
Tight junctions (TJs) between neighboring ECs are responsible for intercellular restrictive barrier properties $[67]$ .
Sealing properties of brain capillaries are acquired in a gradual maturation process during embryonic and postnatal development.
During embryonic development, there is greater presence of TJs and disappearance of fenestrations, which correlate with decreased permeability during development.
Loss of the Mfsd2a gene enhances transcytosis in embryonic and adult BCEC’s $[54]$ and that MFSD2A transport function was shown to be essential for transcytosis inhibition $[68]$ .
The Mfsd2a transcript level is increased gradually from E11.5 reaching its maximum at E15.5, which aligns with the general sealing timeline of the embryonic BBB.
Complexity of the TJ network and P-face association of TJ particles were significantly increased between E18 and P1 in rat cortical vessels $[69]$ .
Maximal resistance is reached by E21 $[61]$ .
A recent mouse retina-vasculature study using HRP-EM showed that developmentally, the TJ pathway is already restrictive before the vesicular pathway is blocked $[74]$ .
Permeable TJs (in mice between E12-E15) become first restrictive to large molecules (around E15-E16) and only later to small molecules (between E17 to postnatal).
Claudin-5, claudin-11, occludin and Marveld2 were upregulated until approximately E15.5 $[50]$ and were dependent on Wnt signalling.
Cldn5 knock-out mouse embryos exhibited a leaky barrier only when challenged with small tracers such as sulfo-NHS-biotin (443 Da), Hoechst H33258 (562 Da) and gadolinium-dethylenetriamine-pentaacetic acid (742 Da) $[76]$ .
LSR knock-out mouse embryos (E14.5) exhibited a leaky barrier specifically in the spinal cord $[63]$ .

Regulation of Transporters

Polarized basal-apical membrane localization of transporters controls nutrient influx as well as waste and toxin efflux across the BBB.
Efflux is responsible for the clearance of CNS originating substances, toxins penetrating from the blood, and partitioning of endogenous substances such as hormones $[77]$ .
BBB endothelial cells express a highly specific set of transporters from the Slc, Slco, ATP, Abc and the major facilitator gene families $[22, 78–80]$ .
The expression of some transporters appears as early as E9 at the PVNP (e.g. Glut1) $[29, 60, 83]$ .
The expression of other transporters is upregulated only in the invading vascular sprouts from the PVNP into the neural tube (e.g. Mfsd2A) $[54]$ .
The complete repertoire of transporters is established after birth.
Wnt signalling influences the expression of BBB transporters and showed down-regulation of Glut1 upon perturbation of Wnt signalling in vivo during development, whereas Wnt signalling is able to induce expression of transporters in vitro $[29–31]$ .
Wnt signalling in the retina vasculature controls Mfsd2A transcription $[84]$ .
The expression of many if not all BBB-specific transporters is regulated by the Wnt/ $β$ -catenin pathway.
The overall expression of many BBB-related genes might indirectly depend on Wnt/ $β$ -catenin.
Ion gradients are established very early in brain development such as chloride gradients in the foetal sheep $[86]$ .

Development of Neurovascular Patterning

Stereotype neurovascular patterning is known to be established during embryonic development.
The vascular system has adopted signalling pathways such as VEGF, neuropilin 1/2, netrin/Dcc and semaphorin/plexin $[88–93]$ .
ECs release developmental cues, such as brain-derived growth factor (BDNF), that influence neurogenesis and neural cell fate $[87, 94, 95]$ .
Different brain regions have different vascular patterns.
Establishment of stereotyped vascular densities might be influenced by neuronal activity.
Ectopic VEGF overexpression induces an excessive angiogenic response that in turn results in the increased generation of basal progenitors, characterized by the transcription factor TBR2 $[98]$ .
Best evidence for the copatterning of the vascular and the neural tissue describes the interaction of angiogenic vessels and NPCs $[99]$ .

Site-Specific Regulation of BBB Development: Exceptions to the Rule

Some structures within the CNS, such as the choroid plexus (CP) and the circumventricular organs (CVOs), do not exhibit BBB vessels $[100, 101]$ .
The CVOs are small specialized midline structures localized around the third ventricle and fourth ventricle of the brain, harbouring a capillary plexus that is characterized by a high density of fenestrated vessels $[100, 101]$ .
The presence of CVOs is highly conserved in the chordate phylum, while their appearance might be diverse $[102–104]$ .
Vessels in the CVO structures do not undergo Wnt/ $β$ -catenin signalling $[103, 104, 106]$ .
The formation of an endothelial barrier phenotype becomes actively inhibited within the CVOs.
In the area postrema of the mouse, the inhibition of Wnt/ $β$ -catenin signalling might be achieved by Wnt inhibitory factor-1 (Wif1) $[104]$ .
In the zebrafish pituitary, vascular BBB properties become inhibited by a high expression of VEGF and Cyp26b that lead to endothelial fenestration and inhibition of Cldn5, respectively $[106]$ .
Recent data support an involvement of Wnt5a via Ryk and RhoA kinase Rock in CP morphogenesis $[107]$ .
BBB properties are induced during development by Ndp in the retina, olfactory bulb and the cerebellum.
In the white matter, oligodendrocyte precursor cells (OPCs) are the cell type releasing Wnt7a/b and thereby regulate white matter vascularization as well as BBB formation $[109]$ .
Sporadic individual endothelial cells in the hippocampus capillary network maintain a direct connection with neural stem cells $[110]$ .
These neural stem cells are then enabled to respond to systemic cues that naturally do not cross the BBB, also potentially explaining cryptic brain toxicity of specific chemotherapeutics.
There are specialized morphology and barrier properties in the subventricular zone (SVZ).

Cellular and Molecular Mechanisms Maintaining BBB Integrity Under Physiological Conditions

Vascular cells keep on undergoing epigenetic changes $[112]$ .
The BBB phenotype in adult mice was found to be plastic $[41]$ .
Ablation of Wnt signalling could revert BBB cells to a peripheral phenotype in adults $[30]$ .
Continuous tissue signalling is needed to maintain BBB integrity under physiological conditions.
BBB gene expression programmes might be hard wired into CNS vascular cells indicated by a clear epi-genetic signature and chromatin accessibility differences between CNS and non-CNS vascular cells $[113]$ .
Dominant-active $β$ -catenin does not re-induce a BBB-like expression profile and chromatin accessibility in long-term BECS in vitro $[114]$ .
The BBB phenotype is less rigid but more dynamic $[115, 116]$ .
The expression of some genes is subjected to circadian changes and are also influenced by neuronal activity $[115, 116]$ .

Outlook

Decisive contributions for understanding BBB development identified the avascular neuroectoderm as the source of barrier-inducing signals $[28]$ , demonstrated to belong to the Wnt pathway $[29–31]$ .
The detailed characterization of the sequential events of Wnt pathway activation is still incomplete $[117]$ .
Site-specific peculiarities of BBB induction, such as by norrin in the retina, olfactory bulb and cerebellum, as well as missing induction in the CVOs and CP, have only recently shown up on the BBB development landscape.
Several hot topics of BBB development remain to be addressed in the future which include the following:
- investigation of potential different ‘flavours’ of Wnt/ $β$ -catenin signalling in the CNS,
- site-specific regulation and molecular regulators of BBB development,
- interaction of Wnt/ $β$ -catenin with other pathways,
- timing and regulation of junctional gene expression and function,
- detailed characterization of junctional specificity in the BBB endothelium,
- timing and regulation of molecular players of the transcytosis pathway and
- mechanisms of BCEC’s differentiation including transcriptional and epi-genetic control of the ‘BBB cell identity’ during development and in the maintenance phase.
Understanding BBB development likely is a mining ground to understand BBB cell biology in general and therefore might well promote the identification of strategies for drug delivery and BBB restoration under pathological conditions.

The blood-brain barrier (BBB) in the CNS is vital for tissue homeostasis and neuronal function.
BBB is located in microvascular endothelial cells, gaining barrier properties during angiogenesis of the neuroectoderm.
Complex tight junctions, lack of fenestrae, and low pinocytotic activity make the BBB endothelium a tight barrier for water-soluble molecules, accessible via specific transporters.
Endothelial differentiation during development starts with the endothelial-specific Wnt/ $β$ -catenin pathway.
This review covers cellular and molecular mechanisms of brain angiogenesis and barrier formation, including interactions within the developing CNS and extracellular matrix.
Downstream Wnt/ $β$ -catenin mechanisms like Foxf2, Foxl2, Foxq1, Lef1, Ppard, Zfp551, Zic3, Sox17, Apcdd1, and Fgfbp1 refine and maintain BBB characteristics.
BBB heterogeneity and differential properties in circumventricular organs and neurogenic niches are examined.
Future research aims to use BBB development understanding to modulate its function in pathological conditions.

More concise summary

Development of the BBB

Common Angiogenic Factors in CNS Vascularization

Brain vascular development begins with a perineural vascular plexus (PNVP).
VEGF and its receptors VEGF-R1 and VEGF-R2 are critical for CNS angiogenesis.
VEGF is released by neural progenitors and induces capillaries from the PVNP.
VEGF gradient regulates vascular branching, guided by endothelial tip cells.
Tip and stalk cell differentiation depends on VEGF/VEGFR with the Dll/Notch pathway interplay.
Sema3E–Plexin-D1 signaling regulates the VEGF-Notch signaling axis.
Sprouting endothelial cells produce PDGF-B, engaging PDGFR- $β$ on pericytes, which promotes recruitment.
PDGF-B absence leads to pericyte loss and microaneurysms, indicating its role in brain vascularization and BBB integrity.
Ang-1 via Tie2/Tek is implicated in BBB integrity.
VE-cadherin contributes to vascular stability and regulates endothelial response to angiogenic stimuli.
Erythrocyte-derived S1P and its receptors are important for vascular stability.

CNS-Specific Regulators of Angiogenesis and Barrier Genesis: Morphogens Regulating BBB Differentiation

The avascular brain induces endothelial barrier properties.
BBB characteristics are induced in the PVNP, shown by Glut1 and Wnt reporter gene expression.
Wnt pathway is important for brain angiogenesis and endothelial barrier function.
Wnt ligands bind to frizzled receptors, requiring Lrp5 and Lrp6 interaction.
Intracellularly, Wnt-receptor binding recruits axin, disintegrating the destruction complex for cytosolic $β$ -catenin.
Wnt ligands Wnt7a and Wnt7b drive endothelial signaling within the brain.
Fzd4 binds Wnt7a/b and interacts with Lrp5 or Lrp6, resulting in $β$ -catenin stabilization.
Norrie disease protein (norrin, Ndp) also drives Wnt/ $β$ -catenin signaling, requiring tetraspanin 12 (Tspan12).
$β$ -catenin target genes, such as Claudin-5, Glut1, Apcdd1, Tnfrsf19, Sox17, and Lef1, contribute to BBB characteristics.
Plasmalemma vesicle-associated protein (Plvap, Meca32) is repressed by Wnt/ $β$ -catenin activation.
Transcription factors like Foxf2, Foxl2, Foxq1, Lef1, Ppard, Zfp551, and Zic3 are associated with BBB maturation.
Wnt factors are presented to the PNVP beyond the glia limitans.
S1pr signaling may regulate BBB maturation and VE-cadherin stabilization.
BBB maturation involves differentiation with PCs and ACs recruitment, and a maturation phase.
Shh and RA participate in inducing or maintaining endothelial barrier function.

Barrier Characteristics During Development

BCECs acquire restrictive barrier properties gradually.
Spinal cord BCECs are restrictive by E14 in mice.
Cortical BCECs mature in a centrifugal, wave-like fashion.
Maturation patterns align with exit from a VEGF-dependent phase.
Developmental maturation varies across CNS locations.
Heterogeneity exists in adult BCEC function in different brain regions.
Some CNS regions have vasculature with nonbarrier properties.

Developmental Regulation of Sealing Properties

BCECs lack fenestrations and have low transcytosis rates.
TJs between ECs provide intercellular restrictive barrier properties.
Sealing properties develop gradually.
Mfsd2a gene loss enhances transcytosis, and its transcript level aligns with the BBB sealing timeline.
TJ network complexity increases during development.
Claudin-5, claudin-11, occludin, and Marveld2 are upregulated and depend on Wnt signaling.
Cldn5 knock-out embryos show a leaky barrier to small tracers.
LSR knock-out embryos (E14.5) exhibited a leaky barrier specifically in the spinal cord.

Regulation of Transporters

Transporter localization controls nutrient influx and waste efflux.
BBB endothelial cells express specific transporters from Slc, Slco, ATP, Abc, and major facilitator gene families.
Some transporters appear early (e.g., Glut1), while others are upregulated later (e.g., Mfsd2A).
The complete transporter repertoire is established postnatally.
Wnt signaling influences BBB transporter expression.
Ion gradients are established early in brain development.

Development of Neurovascular Patterning

Stereotype neurovascular patterning is established during development.
The vascular system uses VEGF, neuropilin 1/2, netrin/Dcc, and semaphorin/plexin signaling pathways.
ECs release cues like BDNF that influence neurogenesis.
Different brain regions have different vascular patterns.
Neuronal activity might influence vascular densities.
VEGF overexpression induces basal progenitors.
Angiogenic vessels interact with NPCs.

Site-Specific Regulation of BBB Development: Exceptions to the Rule

Structures like the choroid plexus (CP) and circumventricular organs (CVOs) lack BBB vessels.
CVOs have fenestrated vessels and are located around the third and fourth ventricles.
Vessels in CVOs do not undergo Wnt/ $β$ -catenin signaling.
BBB phenotype formation is inhibited in CVOs.
Wif1 inhibits Wnt/ $β$ -catenin signaling in the area postrema.
VEGF and Cyp26b inhibit BBB properties in the zebrafish pituitary.
Wnt5a via Ryk and RhoA kinase Rock is involved in CP morphogenesis.
Ndp induces BBB properties in the retina, olfactory bulb, and cerebellum.
OPCs release Wnt7a/b and regulate white matter vascularization and BBB formation.
Some endothelial cells in the hippocampus connect with neural stem cells.
Specialized morphology and barrier properties exist in the subventricular zone (SVZ).

Cellular and Molecular Mechanisms Maintaining BBB Integrity Under Physiological Conditions

Vascular cells undergo epigenetic changes.
Adult BBB phenotype is plastic.
Wnt signaling ablation can revert BBB cells to a peripheral phenotype.
Continuous tissue signaling maintains BBB integrity.
BBB gene expression might be hardwired in CNS vascular cells.
$β$ -catenin does not re-induce BBB-like expression in BECS in vitro.
BBB phenotype is dynamic, influenced by circadian changes and neuronal activity.

Outlook

The neuroectoderm is the source of barrier-inducing Wnt pathway signals.
Detailed characterization of Wnt pathway activation is incomplete.
Site-specific BBB induction by norrin and lack of induction in CVOs and CP are recent discoveries.
Future research will focus on different Wnt/ $β$ -catenin signaling flavors, site-specific regulation, Wnt interactions with other pathways, junctional gene expression, transcytosis pathways, and BCEC differentiation mechanisms.
Understanding BBB development can promote drug delivery strategies and BBB restoration under pathological conditions.