GPR124 regulates murine brain embryonic angiogenesis and BBB formation by an intracellular domain-independent mechanism.

The GPR124/RECK/WNT7 pathway is an essential regulator of CNS angiogenesis and blood-brain barrier (BBB) function. GPR124, a brain endothelial adhesion seven-pass transmembrane protein, associates with RECK, which binds and stabilizes newly synthesized WNT7 that is transferred to frizzled (FZD) to initiate canonical ß-catenin signaling. GPR124 remains enigmatic: although its extracellular domain (ECD) is essential, the poorly conserved intracellular domain (ICD) appears to be variably required in mammals versus zebrafish, potentially via adaptor protein bridging of GPR124 and FZD ICDs. GPR124 ICD deletion impairs zebrafish angiogenesis, but paradoxically retains WNT7 signaling upon mammalian transfection. We thus investigated GPR124 ICD function using the mouse deletion mutant Gpr124ΔC. Despite inefficiently expressed GPR124ΔC protein, Gpr124ΔC/ΔC mice could be born with normal cerebral cortex angiogenesis, in comparison with Gpr124-/- embryonic lethality, forebrain avascularity and hemorrhage. Gpr124ΔC/ΔC vascular phenotypes were restricted to sporadic ganglionic eminence angiogenic defects, attributable to impaired GPR124ΔC protein expression. Furthermore, Gpr124ΔC and the recombinant GPR124 ECD rescued WNT7 signaling in culture upon brain endothelial Gpr124 knockdown. Thus, in mice, GPR124-regulated CNS forebrain angiogenesis and BBB function are exerted by ICD-independent functionality, extending the signaling mechanisms used by adhesion seven-pass transmembrane receptors.

Embryonic Pericytes Promote Microglial Homeostasis and Their Effects on Neural Progenitors in the Developing Cerebral Cortex.

Multifaceted microglial functions in the developing brain, such as promoting the differentiation of neural progenitors and contributing to the positioning and survival of neurons, have been progressively revealed. Although previous studies have noted the relationship between vascular endothelial cells and microglia in the developing brain, little attention has been given to the importance of pericytes, the mural cells surrounding endothelial cells. In this study, we attempted to dissect the role of pericytes in microglial distribution and function in developing mouse brains. Our immunohistochemical analysis showed that approximately half of the microglia attached to capillaries in the cerebral walls. Notably, a magnified observation of the position of microglia, vascular endothelial cells and pericytes demonstrated that microglia were preferentially associated with pericytes that covered 79.8% of the total capillary surface area. Through in vivo pericyte depletion induced by the intraventricular administration of a neutralizing antibody against platelet-derived growth factor receptor (PDGFR)ß (clone APB5), we found that microglial density was markedly decreased compared with that in control antibody-treated brains because of their low proliferative capacity. Moreover, in vitro coculture of isolated CD11b+ microglia and NG2+PDGFRα- cells, which are mostly composed of pericytes, from parenchymal cells indicated that pericytes promote microglial proliferation via the production of soluble factors. Furthermore, pericyte depletion by APB5 treatment resulted in a failure of microglia to promote the differentiation of neural stem cells into intermediate progenitors. Taken together, our findings suggest that pericytes facilitate microglial homeostasis in the developing brains, thereby indirectly supporting microglial effects on neural progenitors.SIGNIFICANCE STATEMENT This study highlights the novel effect of pericytes on microglia in the developing mouse brain. Through multiple analyses using an in vivo pericyte depletion mouse model and an in vitro coculture study of isolated pericytes and microglia from parenchymal cells, we demonstrated that pericytes contribute to microglial proliferation and support microglia in efficiently promoting the differentiation of neural stem cells into intermediate progenitors. Our present data provide evidence that pericytes function not only in the maintenance of cerebral microcirculation and blood brain barrier (BBB) integrity but also in microglial homeostasis in the developing cerebral walls. These findings will expand our knowledge and help elucidate the mechanism of brain development both in healthy and disease conditions.

Fgfbp1 promotes blood-brain barrier development by regulating collagen IV deposition and maintaining Wnt/ß-catenin signaling.

Central nervous system (CNS) blood vessels contain a functional blood-brain barrier (BBB) that is necessary for neuronal survival and activity. Although Wnt/ß-catenin signaling is essential for BBB development, its downstream targets within the neurovasculature remain poorly understood. To identify targets of Wnt/ß-catenin signaling underlying BBB maturation, we performed a microarray analysis that identified Fgfbp1 as a novel Wnt/ß-catenin-regulated gene in mouse brain endothelial cells (mBECs). Fgfbp1 is expressed in the CNS endothelium and secreted into the vascular basement membrane during BBB formation. Endothelial genetic ablation of Fgfbp1 results in transient hypervascularization but delays BBB maturation in specific CNS regions, as evidenced by both upregulation of Plvap and increased tracer leakage across the neurovasculature due to reduced Wnt/ß-catenin activity. In addition, collagen IV deposition in the vascular basement membrane is reduced in mutant mice, leading to defective endothelial cell-pericyte interactions. Fgfbp1 is required cell-autonomously in mBECs to concentrate Wnt ligands near cell junctions and promote maturation of their barrier properties in vitro Thus, Fgfbp1 is a crucial extracellular matrix protein during BBB maturation that regulates cell-cell interactions and Wnt/ß-catenin activity.

Development of the brain vasculature and the blood-brain barrier in zebrafish.

To ensure tissue homeostasis the brain needs to be protected from blood-derived fluctuations or pathogens that could affect its function. Therefore, the brain capillaries develop tissue-specific properties to form a selective blood-brain barrier (BBB), allowing the passage of essential molecules to the brain and blocking the penetration of potentially harmful compounds or cells. Previous studies reported the presence of this barrier in zebrafish. The intrinsic features of the zebrafish embryos and larvae in combination with optical techniques, make them suitable for the study of barrier establishment and maturation. In this review, we discuss the most recent contributions to the development and formation of a functional zebrafish BBB. Moreover, we compare the molecular and cellular characteristic of the zebrafish and the mammalian BBB.

Differential expression of Öbek controls ploidy in the Drosophila blood-brain barrier.

During development, tissue growth is mediated by either cell proliferation or cell growth, coupled with polyploidy. Both strategies are employed by the cell types that make up the Drosophila blood-brain barrier. During larval growth, the perineurial glia proliferate, whereas the subperineurial glia expand enormously and become polyploid. Here, we show that the level of ploidy in the subperineurial glia is controlled by the N-terminal asparagine amidohydrolase homolog Öbek, and high Öbek levels are required to limit replication. In contrast, perineurial glia express moderate levels of Öbek, and increased Öbek expression blocks their proliferation. Interestingly, other dividing cells are not affected by alteration of Öbek expression. In glia, Öbek counteracts fibroblast growth factor and Hippo signaling to differentially affect cell growth and number. We propose a mechanism by which growth signals are integrated differentially in a glia-specific manner through different levels of Öbek protein to adjust cell proliferation versus endoreplication in the blood-brain barrier.

Dynamic genome wide expression profiling of Drosophila head development reveals a novel role of Hunchback in retinal glia cell development and blood-brain barrier integrity.

Drosophila melanogaster head development represents a valuable process to study the developmental control of various organs, such as the antennae, the dorsal ocelli and the compound eyes from a common precursor, the eye-antennal imaginal disc. While the gene regulatory network underlying compound eye development has been extensively studied, the key transcription factors regulating the formation of other head structures from the same imaginal disc are largely unknown. We obtained the developmental transcriptome of the eye-antennal discs covering late patterning processes at the late 2nd larval instar stage to the onset and progression of differentiation at the end of larval development. We revealed the expression profiles of all genes expressed during eye-antennal disc development and we determined temporally co-expressed genes by hierarchical clustering. Since co-expressed genes may be regulated by common transcriptional regulators, we combined our transcriptome dataset with publicly available ChIP-seq data to identify central transcription factors that co-regulate genes during head development. Besides the identification of already known and well-described transcription factors, we show that the transcription factor Hunchback (Hb) regulates a significant number of genes that are expressed during late differentiation stages. We confirm that hb is expressed in two polyploid subperineurial glia cells (carpet cells) and a thorough functional analysis shows that loss of Hb function results in a loss of carpet cells in the eye-antennal disc. Additionally, we provide for the first time functional data indicating that carpet cells are an integral part of the blood-brain barrier. Eventually, we combined our expression data with a de novo Hb motif search to reveal stage specific putative target genes of which we find a significant number indeed expressed in carpet cells.

Mfsd2a is critical for the formation and function of the blood-brain barrier.

The central nervous system (CNS) requires a tightly controlled environment free of toxins and pathogens to provide the proper chemical composition for neural function. This environment is maintained by the 'blood-brain barrier' (BBB), which is composed of blood vessels whose endothelial cells display specialized tight junctions and extremely low rates of transcellular vesicular transport (transcytosis). In concert with pericytes and astrocytes, this unique brain endothelial physiological barrier seals the CNS and controls substance influx and efflux. Although BBB breakdown has recently been associated with initiation and perpetuation of various neurological disorders, an intact BBB is a major obstacle for drug delivery to the CNS. A limited understanding of the molecular mechanisms that control BBB formation has hindered our ability to manipulate the BBB in disease and therapy. Here we identify mechanisms governing the establishment of a functional BBB. First, using a novel tracer-injection method for embryos, we demonstrate spatiotemporal developmental profiles of BBB functionality and find that the mouse BBB becomes functional at embryonic day 15.5 (E15.5). We then screen for BBB-specific genes expressed during BBB formation, and find that major facilitator super family domain containing 2a (Mfsd2a) is selectively expressed in BBB-containing blood vessels in the CNS. Genetic ablation of Mfsd2a results in a leaky BBB from embryonic stages through to adulthood, but the normal patterning of vascular networks is maintained. Electron microscopy examination reveals a dramatic increase in CNS-endothelial-cell vesicular transcytosis in Mfsd2a(-/-) mice, without obvious tight-junction defects. Finally we show that Mfsd2a endothelial expression is regulated by pericytes to facilitate BBB integrity. These findings identify Mfsd2a as a key regulator of BBB function that may act by suppressing transcytosis in CNS endothelial cells. Furthermore, our findings may aid in efforts to develop therapeutic approaches for CNS drug delivery.

Roles of blood-brain barrier integrins and extracellular matrix in stroke.

Ischemicstroke is a leading cause of death and disability in the United States, but recent advances in treatments [i.e., endovascular thrombectomy and tissue plasminogen activator (t-PA)] that target the stroke-causing blood clot, while improving overall stroke mortality rates, have had much less of an impact on overall stroke morbidity. This may in part be attributed to the lack of therapeutics targeting reperfusion-induced injury after the blood clot has been removed, which, if left unchecked, can expand injury from its core into the surrounding at risk tissue (penumbra). This occurs in two phases of increased permeability of the blood-brain barrier, a physical barrier that under physiologic conditions regulates brain influx and efflux of substances and consists of tight junction forming endothelial cells (and transporter proteins), astrocytes, pericytes, extracellular matrix, and their integrin cellular receptors. During, embryonic development, maturity, and following stroke reperfusion, cerebral vasculature undergoes significant changes including changes in expression of integrins and degradation of surrounding extracellular matrix. Integrins, heterodimers with α and ß subunits, and their extracellular matrix ligands, a collection of proteoglycans, glycoproteins, and collagens, have been modestly studied in the context of stroke compared with other diseases (e.g., cancer). In this review, we describe the effect that various integrins and extracellular matrix components have in embryonic brain development, and how this changes in both maturity and in the poststroke environment. Particular focus will be on how these changes in integrins and the extracellular matrix affect blood-brain barrier components and their potential as diagnostic and therapeutic targets for ischemic stroke.

Zika virus infection disrupts neurovascular development and results in postnatal microcephaly with brain damage.

Zika virus (ZIKV) infection of pregnant women can result in fetal brain abnormalities. It has been established that ZIKV disrupts neural progenitor cells (NPCs) and leads to embryonic microcephaly. However, the fate of other cell types in the developing brain and their contributions to ZIKV-associated brain abnormalities remain largely unknown. Using intracerebral inoculation of embryonic mouse brains, we found that ZIKV infection leads to postnatal growth restriction including microcephaly. In addition to cell cycle arrest and apoptosis of NPCs, ZIKV infection causes massive neuronal death and axonal rarefaction, which phenocopy fetal brain abnormalities in humans. Importantly, ZIKV infection leads to abnormal vascular density and diameter in the developing brain, resulting in a leaky blood-brain barrier (BBB). Massive neuronal death and BBB leakage indicate brain damage, which is further supported by extensive microglial activation and astrogliosis in virally infected brains. Global gene analyses reveal dysregulation of genes associated with immune responses in virus-infected brains. Thus, our data suggest that ZIKV triggers a strong immune response and disrupts neurovascular development, resulting in postnatal microcephaly with extensive brain damage.

Perineurial Glial Plasticity and the Role of TGF-ß in the Development of the Blood-Nerve Barrier.

Precisely orchestrated interactions between spinal motor axons and their ensheathing glia are vital for forming and maintaining functional spinal motor nerves. Following perturbations to peripheral myelinating glial cells, centrally derived oligodendrocyte progenitor cells (OPCs) ectopically exit the spinal cord and myelinate peripheral nerves in myelin with CNS characteristics. However, whether remaining peripheral ensheathing glia, such as perineurial glia, properly encase the motor nerve despite this change in glial cell and myelin composition, remains unknown. Using zebrafish mutants in which OPCs migrate out of the spinal cord and myelinate peripheral motor axons, we assayed perineurial glial development, maturation, and response to injury. Surprisingly, in the presence of OPCs, perineurial glia exited the CNS normally. However, aspects of their development, response to injury, and function were altered compared with wildtype larvae. In an effort to better understand the plasticity of perineurial glia in response to myelin perturbations, we identified transforming growth factor-ß1 as a partial mediator of perineurial glial development. Together, these results demonstrate the incredible plasticity of perineurial glia in the presence of myelin perturbations.SIGNIFICANCE STATEMENT Peripheral neuropathies can result from damage or dysregulation of the insulating myelin sheath surrounding spinal motor axons, causing pain, inefficient nerve conduction, and the ectopic migration of oligodendrocyte progenitor cells (OPCs), the resident myelinating glial cell of the CNS, into the periphery. How perineurial glia, the ensheathing cells that form the protective blood-nerve barrier, are impacted by this myelin composition change is unknown. Here, we report that certain aspects of perineurial glial development and injury responses are mostly unaffected in the presence of ectopic OPCs. However, perineurial glial function is disrupted along nerves containing centrally derived myelin, demonstrating that, although perineurial glial cells display plasticity despite myelin perturbations, the blood-nerve barrier is compromised in the presence of ectopic OPCs.

The origins of the circumventricular organs.

The circumventricular organs (CVOs) are specialised neuroepithelial structures found in the midline of the brain, grouped around the third and fourth ventricles. They mediate the communication between the brain and the periphery by performing sensory and secretory roles, facilitated by increased vascularisation and the absence of a blood-brain barrier. Surprisingly little is known about the origins of the CVOs (both developmental and evolutionary), but their functional and organisational similarities raise the question of the extent of their relationship. Here, I review our current knowledge of the embryonic development of the seven major CVOs (area postrema, median eminence, neurohypophysis, organum vasculosum of the lamina terminalis, pineal organ, subcommissural organ, subfornical organ) in embryos of different vertebrate species. Although there are conspicuous similarities between subsets of CVOs, no unifying feature characteristic of their development has been identified. Cross-species comparisons suggest that CVOs also display a high degree of evolutionary flexibility. Thus, the term 'CVO' is merely a functional definition, and features shared by multiple CVOs may be the result of homoplasy rather than ontogenetic or phylogenetic relationships.

Developmental changes of l-arginine transport at the blood-brain barrier in rats.

l-Arginine is required for regulating synapse formation/patterning and angiogenesis in the developing brain. We hypothesized that this requirement would be met by increased transporter-mediated supply across the blood-brain barrier (BBB). Thus, the purpose of this work was to test the idea that elevation of blood-to-brain l-arginine transport across the BBB in the postnatal period coincides with up-regulation of cationic acid transporter 1 (CAT1) expression in developing brain capillaries. We found that the apparent brain-to-plasma concentration ratio (Kp, app) of l-arginine after intravenous administration during the first and second postnatal weeks was 2-fold greater than that at the adult stage. Kp, app of l-serine was also increased at the first postnatal week. In contrast, Kp, app of d-mannitol, a passively BBB-permeable molecule, did not change, indicating that increased transport of l-arginine and l-serine is not due to BBB immaturity. Double immunohistochemical staining of CAT1 and a marker protein, glucose transporter 1, revealed that CAT1 was localized on both luminal and abluminal membranes of brain capillary endothelial cells during the developmental and adult stages. A dramatic increase in CAT1 expression in the brain was seen at postnatal day 7 (P7) and day 14 (P14) and the expression subsequently decreased as the brain matured. In accordance with this, intense immunostaining of CAT1 was observed in brain capillaries at P7 and P14. These findings strongly support our hypothesis and suggest that the supply of blood-born l-arginine to the brain via CAT1 at the BBB plays a key role in meeting the elevated demand for l-arginine in postnatal brain.

The molecular, cellular, and morphological components of blood-brain barrier development during embryogenesis.

The blood brain barrier (BBB) is a hallmark of blood vessels in the brain and functions to protect the brain from unwanted blood born materials, support the unique metabolic needs of the brain, and define a stable environment crucial for brain homeostasis. The temporal profile of BBB development was long debated until recent studies produced convincing evidence demonstrating that the BBB is established and functional during embryogenesis. Here we review research focused on the molecular, cellular and morphological characteristics of BBB development. Our review discusses the precise temporal profile of BBB formation, the development of endothelial cell ultrastructure and the molecular components that provide sealing and transporting properties, the molecular pathways involved in the induction of BBB specific endothelial cell differentiation, the signaling pathways driving developmental angiogenesis versus barrier-genesis, and finally the contribution of other cell types to BBB formation. We examine aspects of BBB development that are still unresolved while highlighting research tools that could provide new insight to answer these open questions.

Glucocorticoids modify effects of TGF-ß1 on multidrug resistance in the fetal blood-brain barrier.

Transforming growth factor-ß1 (TGF-ß1) increases P-glycoprotein (P-gp; encoded by Abcb1) activity in fetal brain endothelial cells (BECs). P-gp is important for fetal brain protection against xenobiotics including synthetic glucocorticoids (sGC). We hypothesized that antenatal sGC would modify P-gp responsiveness to TGF-ß1 in fetal BECs. Pregnant guinea pigs were treated with dexamethasone or vehicle (N = 5/group) on gestational day (GD) 48-49 and BECs derived on GD50. In BECs from control fetuses, TGF-ß1 increased Abcb1 mRNA and P-gp function, by approximately 5-fold and 55% respectively, as well as tight junction function. In contrast, TGF-ß1 had no effect on these parameters in BECs from sGC-exposed fetuses. Moreover, levels of TGF-ß1 responsive gene, Smad7, were increased 3-fold in BECs from control fetuses after TGF-ß1 but not in sGC-exposed fetuses. In conclusion, antenatal sGC alters responsiveness to TGF-ß1 in fetal BECs. This study has identified novel mechanisms by which TGF-ß1 and sGC modulate fetal brain protection against xenobiotics and other P-gp substrates.

Mosaic Expression of Thyroid Hormone Regulatory Genes Defines Cell Type-Specific Dependency in the Developing Chicken Cerebellum.

The cerebellum is a morphologically unique brain structure that requires thyroid hormones (THs) for the correct coordination of key cellular events driving its development. Unravelling the interplay between the multiple factors that can regulate intracellular TH levels is a key step to understanding their role in the regulation of these cellular processes. We therefore investigated the regional/cell-specific expression pattern of TH transporters and deiodinases in the cerebellum using the chicken embryo as a model. In situ hybridisation revealed expression of the TH transporters monocarboxylate transporter 8 (MCT8) and 10 (MCT10), L-type amino acid transporter 1 (LAT1) and organic anion transporting polypeptide 1C1 (OATP1C1) as well as the inactivating type 3 deiodinase (D3) in the fourth ventricle choroid plexus, suggesting a possible contribution of the resulting proteins to TH exchange and subsequent inactivation of excess hormone at the blood-cerebrospinal fluid barrier. Exclusive expression of LAT1 and the activating type 2 deiodinase (D2) mRNA was found at the level of the blood-brain barrier, suggesting a concerted function for LAT1 and D2 in the direct access of active T3 to the developing cerebellum via the capillary endothelial cells. The presence of MCT8 mRNA in Purkinje cells and cerebellar nuclei during the first 2 weeks of embryonic development points to a potential role of this transporter in the uptake of T3 in central neurons. At later stages, together with MCT10, detection of MCT8 signal in close association with the Purkinje cell dendritic tree suggests a role of both transporters in TH signalling during Purkinje cell synaptogenesis. MCT10 was also expressed in late-born cells in the rhombic lip lineage with a clear hybridisation signal in the outer external granular layer, indicating a potential role for MCT10 in the proliferation of granule cell precursors. By contrast, expression of D3 in the first-born rhombic lip-derived population may serve as a buffering mechanism against high T3 levels during early embryonic development, a hypothesis supported by the pattern of expression of a fluorescent TH reporter in this lineage. Overall, this study builds a picture of the TH dependency in multiple cerebellar cell types starting from early embryonic development.

Pericytes are required for blood-brain barrier integrity during embryogenesis.

Vascular endothelial cells in the central nervous system (CNS) form a barrier that restricts the movement of molecules and ions between the blood and the brain. This blood-brain barrier (BBB) is crucial to ensure proper neuronal function and protect the CNS from injury and disease. Transplantation studies have demonstrated that the BBB is not intrinsic to the endothelial cells, but is induced by interactions with the neural cells. Owing to the close spatial relationship between astrocytes and endothelial cells, it has been hypothesized that astrocytes induce this critical barrier postnatally, but the timing of BBB formation has been controversial. Here we demonstrate that the barrier is formed during embryogenesis as endothelial cells invade the CNS and pericytes are recruited to the nascent vessels, over a week before astrocyte generation. Analysing mice with null and hypomorphic alleles of Pdgfrb, which have defects in pericyte generation, we demonstrate that pericytes are necessary for the formation of the BBB, and that absolute pericyte coverage determines relative vascular permeability. We demonstrate that pericytes regulate functional aspects of the BBB, including the formation of tight junctions and vesicle trafficking in CNS endothelial cells. Pericytes do not induce BBB-specific gene expression in CNS endothelial cells, but inhibit the expression of molecules that increase vascular permeability and CNS immune cell infiltration. These data indicate that pericyte-endothelial cell interactions are critical to regulate the BBB during development, and disruption of these interactions may lead to BBB dysfunction and neuroinflammation during CNS injury and disease.

Prenatal exposure to common environmental factors affects brain lipids and increases risk of developing autism spectrum disorders.

The prevalence of autism spectrum disorders (ASDs) has been on the rise over recent years. The presence of diverse subsets of candidate genes in each individual with an ASD and the vast variability of phenotypical differences suggest that the interference of an exogenous environmental component may greatly contribute to the development of ASDs. The lipid mediator prostaglandin E2 (PGE2 ) is released from phospholipids of cell membranes, and is important in brain development and function; PGE2 is involved in differentiation, synaptic plasticity and calcium regulation. The previous review already described extrinsic factors, including deficient dietary supplementation, and exposure to oxidative stress, infections and inflammation that can disrupt signaling of the PGE2 pathway and contribute to ASDs. In this review, the structure and establishment of two key protective barriers for the brain during early development are described: the blood-brain barrier; and the placental barrier. Then, the first comprehensive summary of other environmental factors, such as exposure to chemicals in air pollution, pesticides and consumer products, which can also disturb PGE2 signaling and increase the risk for developing ASDs is provided. Also, how these exogenous agents are capable of crossing the protective barriers of the brain during critical developmental periods when barrier components are still being formed is described. This review underlines the importance of avoiding or limiting exposure to these factors during vulnerable periods in development.

The ontogeny of P-glycoprotein in the developing human blood-brain barrier: implication for opioid toxicity in neonates.

BACKGROUND: Neonates have been shown to have a heightened sensitivity to the central depressive effects of opioids compared to older infants and adults. The limited development of P-glycoprotein (P-gp) may limit the ability of the neonate to efflux morphine from the brain back to the systemic circulation. The objective of the study was to determine the ontogeny of P-gp in the human brain. METHODS: Postmortem cortex samples from gestational age (GA) 20-26 wk, GA 36-40 wk, postnatal age (PNA) 0-3 mo, PNA 3-6 mo, and adults were immunostained for P-gp. RESULTS: The intensity of P-gp staining in adults was significantly higher compared to at GA 20-26 wk (P < 0.05), GA 36-40 wk (P < 0.05), and PNA 0-3 mo (P < 0.05). P-gp intensity at GA 20-26 wk (P < 0.05), GA 36-40 wk (P < 0.05), and PNA 0-3 mo (P < 0.05) was significantly lower compared to at PNA 3-6 mo. CONCLUSION: P-gp expression in the brain is limited at birth, increases with postnatal maturation, and reaches adult levels at ~3-6 mo of age. Given the immaturity of blood-brain barrier (BBB) P-gp after birth, morphine may concentrate in the brain. This provides mechanistic support to life threatening opioid toxicity seen with maternal codeine use during breastfeeding.

Retinoic acid induces blood-brain barrier development.

The blood-brain barrier (BBB) is crucial in the maintenance of a controlled environment within the brain to safeguard optimal neuronal function. The endothelial cells (ECs) of the BBB possess specific properties that restrict the entry of cells and metabolites into the CNS. The specialized BBB endothelial phenotype is induced during neurovascular development by surrounding cells of the CNS. However, the molecular differentiation of the BBB endothelium remains poorly understood. Retinoic acid (RA) plays a crucial role in the brain during embryogenesis. Because radial glial cells supply the brain with RA during the developmental cascade and associate closely with the developing vasculature, we hypothesize that RA is important for the induction of BBB properties in brain ECs. Analysis of human postmortem fetal brain tissue shows that the enzyme mainly responsible for RA synthesis, retinaldehyde dehydrogenase, is expressed by radial glial cells. In addition, the most important receptor for RA-driven signaling in the CNS, RA-receptor ß (RARß), is markedly expressed by the developing brain vasculature. Our findings have been further corroborated by in vitro experiments showing RA- and RARß-dependent induction of different aspects of the brain EC barrier. Finally, pharmacologic inhibition of RAR activation during the differentiation of the murine BBB resulted in the leakage of a fluorescent tracer as well as serum proteins into the developing brain and reduced the expression levels of important BBB determinants. Together, our results point to an important role for RA in the induction of the BBB during human and mouse development.