Several multispecific efflux ABC transporters have been located at the blood–brain interfaces where they limit the entry of harmful toxins, but also of pharmacologic agents such as anticancer drugs or antiretroviral protease inhibitors 16 17 39 .

Abcc1 and Abcc4 transcripts were abundant in choroid plexuses throughout development, and were moderately enriched in the adult compared to earlier stages. In contrast, the four other Abcc genes were expressed at a similar or higher level during development compared to adult. In particular, Abcc9 transcripts were strikingly enriched in earlier stages, with a 144-fold higher level in E15 compared to adult (p < 0.001). Expression levels of Abcb1b and Abcg2 were also higher at E15 and perinatal stages (qRT-PCR: p < 0.05) than in adult, in which they were apparently very low (Figure 3A).

These results indicate that the main Abc transporter genes expressed in choroid plexus belong to the Abcc subfamily. This is in accordance with a previous report showing the expression of Abcc1, Abcc4, Abcc5, and to a lesser extent Abcc3 in this tissue in adult rat 41 . It is also in agreement with the expression of these genes in both embryonic and adult mouse choroid plexuses 4 . Abcc1 protein was largely enriched in mouse, rat and human choroid plexuses compared to other brain tissues, as shown by immunohistochemistry and Western Blot 16 25 . In vivo transport experiments using knockout mice pointed to the role of this carrier in limiting the CSF concentration of drugs such as etoposide in the CSF 16 . In mouse, Abcc4 protein was located in both the luminal membrane of brain capillaries and the basolateral membrane of the choroidal epithelium 17 . Microdialysis studies showed that this carrier strongly restricts the passage of drugs such as topotecan from the blood into the CSF 17 . The developmental patterns observed also corroborate previous data showing substantial expression levels of Abcc1 and Abcc4 in newborn rat choroid plexus 23 , and the developmental up-regulation observed between E15 and adult for both Abcc genes 24 . Despite the developmental regulation of Abcc1 level of expression, Abcc1 protein level was already as high in P2 rats as in adult and was also located at the basolateral membrane of the choroid plexus 25 . Overall, the Abcc developmental profiles suggest that in addition to the main Abcc1, Abcc4 and other Abcc transporters may have a crucial function as efflux pumps during early brain development.

The low level of Abcb1 transcripts in adult rat choroid plexus is in line with earlier findings that showed, using Western blot analysis of adult rat and human tissues, that Abcb1 protein was far less abundant in choroid plexus by comparison to microvessels 25 . Similarly, the low level of expression of Abcg2 that we report in adult choroid plexus is accordant with the differential immunoreactivity of microvessels (highly positive) compared to choroid plexuses (negative), observed for this transporter in adult rat brain 24 42 . The higher expression of both Abcb1 and Abcg2 from early embryonic to postnatal stages (Figure 3A) suggests that these transporters fulfill age-related functions at the choroid plexus. A similar enrichment of Abcg2 transcripts in E15 compared to adult was described in mouse choroid plexuses 4 , and Abcg2 immunoreactivity was detected in embryonic rat choroid plexus, but not in the adult tissue 24 42 . The localization of the transporter at the basolateral membrane of the choroidal epithelial cells indicates that it could act as an efflux pump, similarly to Abcc1 and Abcc4. The precise localization of Abcb1 and other Abcc transporters identified in embryonic choroid plexus remains to be determined in order to understand the polarity of transport achieved by these proteins at the blood-CSF barrier.

Members of the Slco subfamily, referred to as organic anion transporting polypeptides, mediate sodium-independent transport of relatively large amphipathic substrates such as taurocholate, thyroid hormones, leukotrienes, and various drugs among which non-steroidal anti-inflammatory drugs, the synthetic opioid peptide DPDPE, digoxin, or dexamethasone. They also transport conjugates of steroids, drugs, and other xenobiotics 43 .

Within this family, Slco1a5 and Slco1c1 were highly expressed in rat choroid plexus (Figure 3B). They were both enriched in the adult compared to prenatal stages (RNA-Seq or qRT-PCR: p < 0.01). The expression level of Slco1a5, low at E15, rapidly increased to approach adult level by E19. Slco1c1 transcript level increased more steadily between E15 and adult. Four additional genes of this family, Slco1a4, Slco3a1, Slco2a1, and Slco2b1 were identified in choroid plexus, and expressed at equal or higher level in developing animals compared to adults.

Slco1a5 is a highly expressed choroidal transporter 18 41 , which is located at the apical membrane of the choroid plexus epithelium 18 41 . It appears to play a major role in CSF-to-blood transport of organic anions 44 . The already substantial expression of Slco1a5 at E19 compared to E15 suggests that the functional activity of this transporter is critical during the perinatal period. Slco1a4 protein has been previously detected in adult choroid plexus 45 where it is located at the basolateral membrane of choroidal epithelial cells 46 . Considering the partially overlapping substrate specificities of Slco1a5 and Slco1a4 47 , the polarized and opposite distribution of these transporters at both membrane domains of choroid plexus epithelial cells could be important for vectorial transport of ligands out of the CSF. Alternatively, the apical Slco1a5 protein may work in concert with basolateral Abcc transporters to achieve efficient CSF-to-blood efflux of organic anions.

Of note is the unexpected developmental regulation of Slco1c1 at the choroid plexus. This carrier is a high affinity transporter of thyroxine, a thyroid hormone important for multiple neurodevelopmental processes. Thyroid hormone deficiency in the brain during the fetal and neonatal period can cause mental retardation 48 49 . The low expression of choroidal Slco1c1 in the embryonic and perinatal stages suggests that other carriers are active at the blood-CSF barrier to provide for cerebral thyroxine requirements during early brain development 50 . Slco3a1, whose expression is slightly enriched in E19 and P2 compared to adult, and Slc16a10, expressed in fetal and newborn choroid plexuses, are potential candidates. Two different splice variants of the SLCO3A1 protein have been located at the apical and basolateral membranes of the human choroidal epithelium, and they both mediate thyroxine uptake in transfected cells and injected oocytes 51 . Slc16a10 is also an influx transporter for T3 and T4. It was recently identified in mouse embryo choroid plexus at a substantially (67-fold) higher level than in the adult 4 . Another likely candidate is transthyretin, whose synthesis in the brain is restricted to choroid plexus. The transthyretin gene is highly expressed throughout development in rat choroid plexus ( 52 and data not shown), and this thyroxin carrier was shown to be important and necessary for various aspects of normal brain development 53 .

Members of the Slc22 subfamily are classified as organic anion transporters, organic cation transporters, and organic cation/carnitine transporters. Organic anion transporters are anion exchangers, and accept various toxins and drugs as substrates, i.e. antineoplastics, antihypertensives, non-steroidal anti-inflammatory drugs, and ß-lactam antibiotics, such as benzylpenicillin 54 . Organic cation transporters function as membrane potential-dependent uniporters that mediate the facilitated diffusion of endogenous amines such as dopamine, or therapeutic drugs such as morphine and antihistamines. Organic cation/carnitine transporters carry carnitine with variable affinities. Two members of this subfamily, octn1 and octn2, also accept different cationic xenobiotics including drugs as substrates 55 .

Slc22a5, Slc22a8, and Slc22a17 were highly expressed in rat choroid plexus from E15 onwards. Seven additional organic cation and anion transporter genes, Slc22a2, Slc22a4, Slc22a6, Slc22a7, Slc22a12, Slc22a15, and Slc22a25 were found expressed in the choroidal tissue (Figure 3C). Transcripts were enriched in early embryos for four of these genes, and enriched in adult for four others. It should be noted that at E19, the expression levels of all ten Slc22 genes have almost reached adult levels. To the exception of Slc22a12, all Log₂ FC_ad absolute values at E19 were equal or lower than 2. Log₂ FC_ad values calculated at E15 were much more variable, ranging between −7.16 for Slc22a8, reflecting a 143-fold enrichment in adult, and 3.79 for Slc22a6, corresponding to a 13.8-fold enrichment in E15.

Our findings are in accordance with a previous study showing high constitutive mRNA levels for Slc22a5 and Slc22a8 in adult rat choroid plexus in comparison to other Slc22 transporters 41 . The Slc22a8 protein has been located at the apical membrane of the choroidal epithelium in rat 19 56 . Slc22a6 displayed the same cellular distribution when expressed as a fluorescent protein in adult rat choroid plexus tissue explants 19 56 . Inhibition experiments in choroidal cell monolayers pointed to apical Slc22 carriers, likely Slc22a8, as mediating the active efflux of antiviral nucleoside analogs across the blood-CSF barrier 57 . Slc22a6 and Slc22a8 transporters share a large number of exogenous substrates 58 . Our present data on their respective inverse profile of expression during development suggest that they sequentially contribute to organic anion clearance from the CSF. The switch from 22a6 to 22a8 in the embryo may reflect a need to adapt to changes in endogenous substrates at that period.

The expression of Slc22a5 in choroid plexus was high and steady in the developing brain. This organic cation transporter will mediate toxic efflux from the CSF if the site of cellular uptake is apical. Its membrane localization is presently unknown. Because Slc22a5 transports carnitine with the highest affinity among Slc22 proteins, it could participate in the control of carnitine homeostasis in the developing brain. Although carnitine is a necessary cofactor of mitochondrial β-oxidation that generates energy from fatty acids, this function is not essential for the developing brain, because peripheral ketone bodies, rather than fatty acids themselves, fuel the brain in suckling animals 59 . Carnitine may function as an antioxidant 60 or a promoting and modulatory agent for synaptic neurotransmission, notably cholinergic transmission 61 . The high and steady expression of Slc22a17 throughout development is noteworthy. This carrier is a cell surface receptor for the siderophore-binding protein 24p3, also known as lipocalin-2. In the brain the highest Slc22a17 transcript levels are found in choroid plexus 62 . Murine Slc22a17 protein, when expressed in HeLa cells, binds both the iron-free and Fe-containing 24p3. It enables their internalization by endocytosis and leads to either apoptosis or increase in intracellular iron-content. Through its binding to 24p3, Slc22a17 may influence infectious/inflammatory processes at the blood-CSF barrier. As a siderophore-binding protein, 24p3 has potential bacteriostatic properties. It also forms a complex with the matrix metalloproteinase MMP-9 63 . Both 24p3 and MMP9 are highly up-regulated in choroid plexus under inflammatory stimuli 64 65 . The relationship between Slc22a17, 24p3 and MMP9 at the choroid plexus, as well as the relevance of the high Slc22a17 expression during development needs further investigations.

The Slc15 subfamily comprises proton-coupled oligopeptide transporters 66 . Among these, Slc15a2 transports peptidomimetic drugs such as the antiviral agent aciclovir or the β-lactam antibiotic cefadroxil. Slc15a2 protein was shown by immunocytochemistry to distribute at the apical and subapical domain of rat choroid plexus epithelial cells in culture 67 . Accordingly, studies using knock-out mice indicated that this transporter removes peptide substrates from CSF into the choroidal tissue 66 . The present analysis showed high levels of expression for Slc15a2 that remained relatively unchanged during development (Figure 3D). These data support a protective role for this transporter in the developing brain toward neuropeptides in excess or peptidomimetic drugs. Slc15a3 and Slc15a4, which transport free histidine and endogenous di- or tripeptides, followed a developmental expression pattern similar to Slc15a2 (data not shown). Their membrane localization and functions are currently unknown.

Slc28 and Slc29 transporters play critical roles in cellular uptake and release of nucleosides that are precursors for nucleotide biosynthesis. Even though brain penetration of nucleoside reverse transcriptase inhibitors used for AIDS treatment is not mediated by choroidal nucleoside transporters 57 , these carriers can transport other anti-viral drugs and the anti-cancer drug gemcitabin 40 68 . Two concentrative Na⁺-coupled nucleoside transporters Slc28a2 and Slc28a3, and four equilibrative nucleoside transporters, Slc29a1 to Slc29a4 were identified in choroid plexus of E15 rats (Figure 3D). Most of these nucleoside transporters were expressed at levels close to adult levels from E15 onwards. Only Slc28a3 and Slc29a4 had lower transcript levels at E15 (p < 0.001). Functional transport and inhibition studies indicate a polarized localization of concentrative nucleoside transporters on the apical membrane, and equilibrative transporters on both the apical and basolateral membrane domains 69 . This subcellular distribution would imply that the concentrative transporters, possibly in conjunction with basolateral equilibrative transporters, fulfill neuroprotective functions by effluxing substrates from CSF rather than contributing to nucleoside entry into brain. This protective mechanism should be efficient already in the developing brain.

Among the more than 88 Cyps genes identified so far in the rat species (http://drnelson.uthsc.edu/rat.master.table.html), only four of them were detected in the lateral ventricle choroid plexus (Figure 4A). Cyp2d4 transcripts were enriched at the adult stage, while Cyp1b1 transcripts were enriched in embryos and perinatal stages. Cyp2j4 and Cyp2u1 expression did not change throughout development. Expression of microsomal epoxide hydrolase 1 (Ephx1) was high and steady in choroid plexus from E19 onwards, and only moderately lower (6-fold, p < 0.001) at E15. Ephx2 transcripts, coding for the soluble form of epoxide hydrolase, were more abundant in choroid plexus from developing animals compared to adults (Figure 4A). All Fmo genes (Fmo1 to −5) were expressed in choroid plexus from developing animals at levels close to adult levels, except for Fmo3, which was lower at P2 and E19 and undetectable at E15 (Figure 4B).

The present data suggest that Cyps play a marginal role in detoxification at the choroid plexus. This is in line with the low Cyp activities reported previously for choroidal tissue 70 , and with the lack of Cyp1a1 activity in choroid plexus, the protein being detected only after inductive treatment and only in choroidal vessels 71 72 . Of note, Cyp1b1, known as the main Cyp isoform expressed at the blood–brain barrier in both rat and human 73 74 , may have a specific detoxification function at the choroid plexus in early development, as its expression is highest in the pre- and postnatal period.

In contrast to Cyps, both Fmos and epoxide hydrolases appear to play significant detoxification roles at the blood-CSF barrier. The high level of expression of Ephx1 in choroid plexus is in line with the high mEphx1 activity measured in the rat tissue 70 and with the strong immunoreactivity of the mouse choroidal epithelium 75 . Ephx1 is well expressed throughout development, pointing to an efficient enzymatic protection towards carcinogenic epoxides and epoxide drug-intermediates from the embryonic period onwards. Ephx2 more specifically metabolizes lipid-derived epoxides, such as epoxyeicosatrienoic acids. Its age-dependent expression profile suggests a function in modulating lipid signaling during development.

Fmo1 and Fmo3, whose transcripts were both detected in choroid plexus, are considered as the most important members in the Fmo family with regard to detoxication of foreign compounds 76 . Fmo1 mRNA was previously detected in mouse choroid plexus by in situ hybridization 76 . The developmental profiles we observed for Fmo genes suggest that Fmo1-dependent biotransformation reactions support important choroidal detoxification pathways in the developing brain, which have never been explored. The significance of Fmo2 transcripts encoding a truncated non-functional protein 76 , and of Fmo4 and Fmo5 which were expressed at continuous but low level during development remains elusive in the context of detoxification.

Phase II of drug metabolism is catalyzed mainly by UDP-glucuronosyltransferases (Ugt), sulfotransferases (Sult), and glutathione-S-transferases (Gst). Similar to efflux transporters, the three enzyme families are multigenic families, composed of multiple isoforms with differential substrate specificities. This explains the broad substrate specificity of the overall detoxification process 21 .

Among detoxifying Ugts, the highly homologous genes of the Ugt1a subfamily could not be distinguished by the microarray probes. Transcripts of these genes were highly and steadily present from E19 to adult (Figure 4C). Their level of expression was lower at E15. This is in agreement with enzymatic data showing that planar molecules, which are typical substrates of Ugt1a enzymes, are efficiently conjugated by choroid plexus epithelial cells both in adult and newborn rat 22 70 . The Ugt1a1 isoform plays an important physiological role in the hepatic clearance of bilirubin in addition to xenobiotics. Conjugation of bilirubin at the blood-CSF barrier could represent an efficient mechanism to prevent the cerebral accumulation of bilirubin in hyperbilirubinemia 77 .

Sult1a1 displayed a similar profile of expression, its transcripts being enriched from E19 to adult, compared to E15. In line with this apparent high level of choroidal expression, Sult1a1-dependent enzymatic activity measured in various human brain regions from fetuses of 15–20 weeks of gestation was the highest in choroid plexus 27 . Sult1a1 is also responsible for the inactivation of thyroid hormones. In addition to xenobiotic conjugation, choroidal Sult1a1 could regulate the levels of these hormones critical for brain development.

Gsts are homo- or heterodimeric enzymes divided into three families, the cytosolic, microsomal and mitochondrial transferases. Within cytosolic Gsts, the alpha (Gsta), mu (Gstm), and pi (Gstp) classes are mainly involved in drug metabolism and detoxification pathways 30 . Some microsomal Gsts (Mgst) were also examined in this study. Although their best known functions are related to the biotransformation of arachidonic acid metabolites, these isoforms are also active in detoxification 30 78 . Five out of seven detoxifying isoforms, Gsta3, Gsta4, Gstm1, Gstm5, and Gstp1, were highly expressed at the choroid plexus (Figure 4D). All mu isoforms, as well as Gstp1 were expressed in choroid plexus of developing animals at similar or higher levels compared to adult. By contrast, transcripts of alpha Gsts were enriched in the adult (5- and 7-fold for Gsta3 and Gsta4 respectively) compared to E15. Mgst1, highly expressed in choroid plexus, showed a clear developmental up-regulation from E15 to adult, in contrast to Mgst3 whose mRNA level was highest at E15 (Figure 4E). The diversity of Gst genes that we found expressed in choroid plexus is in accordance with the previous immunodetection of all alpha, mu and pi Gst classes in the adult rat choroidal epithelium 79 80 81 . The overall high levels of Gst transcripts observed during the perinatal period is in line with earlier studies showing Gst-positive cells in rat choroid plexus at embryonic day 16 82 and with the presence of Gst pi in human fetal choroid plexus 83 . This transcriptional regulation parallels our previous enzymatic data showing that in both rat and human the choroidal conjugation of GSH with 1-chloro-2,4-dinitrobenzene, a substrate for multiple GSTs, was higher in fetus and newborn than in adult 22 23 .

The rate of glutahione synthesis is in part determined by the activity of glutamate-cysteine ligase, which is composed of a catalytic subunit (Gclc) and a modulatory subunit (Gclm). These two genes, as well as the glutathione synthase gene (Gss) were expressed in choroid plexus from E15 onwards (Figure 4F). Transcripts levels in perinatal stages were equal or slightly higher to those in adult. These profiles suggest that the large capacity of the choroidal tissue to synthesize glutathione described in adults 84 is already acquired in embryos.

Both Mgst1, previously localized in epithelial cells of rat choroid plexuses 85 , and Mgst3 have been described to have a peroxidase activity in addition to their glutathione-S-transferase activity 30 . In vitro experiments performed in MCF-7 breast carcinoma cells suggested that Mgst1 protects the cells against damages induced by cytostatic drugs or against SiO2 nanoparticle-induced oxidative stress and cytotoxicity 86 . Such antioxidant protection would be provided at the choroid plexus during development, by Mgst3 in earlier stages, then by Mgst1. These enzymes could reinforce the overall large antioxidant capacity of choroid plexus, which relies on various other enzymes present in this tissue (see below).

Cells forming the blood-CSF barrier are exposed to oxidative stress of various origins. First, they face blood-borne pro-oxidant compounds. They also have an overall high mitochondrial activity, which is a substantial local source of oxygen-derived species. In addition, these cells are exposed to the reactive and potentially deleterious intermediate metabolites that can form as a drawback of phase I oxidative and reductive metabolism and of selected phase II reactions. Choroidal cells possess an extensive enzymatic machinery to inactivate all these reactive molecules. It includes superoxide dismutases (Sod), which inactivate superoxide anions, catalase (Cat), which cleaves hydrogen peroxide, and glutathione peroxidases (Gpx), which metabolize a large spectrum of peroxide species. Some isoforms of the latter enzymes require glutathione in their catalytic cycle, and thus function in conjunction with glutathione reductase (Gsr), which regenerates the reduced form of the thiol-containing molecule (reviewed in 87 ).

Five out of eight Gpx genes identified in mammals (Gpx1, -3, -4, -7, and −8) and Gsr were detected in choroid plexuses (Figure 5A). All were expressed at similar or higher levels during the perinatal period compared to adult. At E15, Log₂Fc_ad absolute values were lower than 2, indicating only limited variation at this early embryonic stage. Gpx1 and Gpx4 were expressed at high levels at all stages. Gpx1 is mainly devoted to the metabolism of hydrogen peroxide when produced in excess, as occurring in infections, while Gpx4 specifically inactivates membrane-integrated hydroperoxy lipids. It thereby counteracts the activity of lipoxygenases, and can modulate inflammatory responses 87 . Very high Gpx activities have been measured in rat choroid plexuses isolated from adults 88 . It is expected from our data that these enzymes are already functional at earlier times and therefore contribute to protect the developing brain against oxidative and inflammatory insults. The soluble Cu/Zn superoxide dismutase genes Sod1 and Sod3, as well as the cat gene were also highly expressed in choroid plexus both during the perinatal period and in adult (Figure 5B). Variations in expression of Sod genes were more heterogeneous in choroid plexuses in E15 animals than in subsequent developmental stages. A strong immunoreactivity for catalase was previously detected in rat choroid plexus as early as at E14.5 89 .

Mammalian cells display several other means of protection against reactive species, including the heme oxygenase (Hmox) pathway. Hmox proteins catalyse the degradation of heme, resulting in the formation of biliverdin, which in turn can be converted via biliverdin reductases (Bvr) into bilirubin. Intracellular unconjugated bilirubin acts as antioxidant, and the biliverdin/bilirubin redox cycle is considered as an important constituent of the cellular antioxidant defense system acting mainly towards lipophilic reactive species 77 . Moderate hyperbilirubinemia was found neuroprotective against focal ischemia to which neonates are especially susceptible 90 . Both stress-inducible Hmox1 and constitutive Hmox2 genes, as well as the Bvra and Bvrb genes were expressed in choroid plexus (Figure 5C). For all four genes, transcripts were detected at similar or slightly higher levels during development than in adults (log₂ FC_ad values between −0.92 to 2.44). In line with our data, immunoreactivity of Hmox-2 usually considered as a neuronal isoform, has been reported in choroid plexuses of adult rats 91 . The steady expression of Hmox2 and the concurrent enriched levels of Hmox1 transcripts in embryonic stages represent a potent heme catabolic system able to generate biliverdin, not only in normal developmental conditions, but also in response to perinatal insults. In contrast to the liver, in which bvra and bvrb showed opposite age-dependent profiles of expression 92 , both genes were expressed at similar levels in choroid plexuses from E19 to the adult stage. Thus the bilirubin/biliverdin antioxidant redox cycle is likely to be efficient in choroidal cells throughout development.

Ligand and non-ligand activated transcription factors involved in the coordinated regulation of drug-metabolizing enzymes and transporters were incorporated in our study. They include the aryl hydrocarbon receptor (Ahr), the pregnane X receptor (Nr1i2), the constitutive androstane/activated receptor (Nr1i3), the peroxisome proliferator-activated receptor α (Ppara), the glucocorticoid receptor (Nr3c1), and the CCAAT/enhancer binding protein beta (Cebpb) reviewed in 29 , and 30 . Ahr dimerizes with the Ahr nuclear translocator (Arnt, also known as Hif1b). Nr1i2, Nr1i3 and Ppara heterodimerize with the retinoid X receptor α (Rxra). The tumor protein p53 (Tp53) was added to the study as it was shown to directly mediate the transcriptional induction of Abcb1 induced by genotoxic compounds 93 . The oxidative stress sensor nuclear factor erythroid 2-related factor 2 (Nfe2l2) and the hypoxia-inducible factor 1 alpha (Hif1a), all transcription factors involved in inductive processes mediated by redox imbalance and whose activation leads to the induction of detoxifying genes, are also reported. Four of these genes, Rxra, Nfe2l2, Hif1a, and Nr3c1 were expressed at high levels in choroid plexuses. Apart from Nr1i2, whose expression was clearly upregulated from E15 to adult stage (p < 0.001), the choroidal expression of all transcription factors was remarkably steady throughout development (Figure 6). Absolute log₂ FC_ad values determined at E19 were between 0 and 0.9.

Induction of choroidal drug metabolizing enzymes in choroid plexuses has been demonstrated only in adult rats, following treatment with polycyclic aromatic hydrocarbons, indicating that the AhR/Arnt-dependent pathway is functional at the blood-CSF barrier 72 94 . If functionality proves true for all factors throughout development, the panel of transcription factors that are expressed in choroid plexus suggests that following exposure to a large range of chemical xenobiotics the activity of drug metabolizing enzymes and transporters in the blood-CSF barrier can be induced in adult as well as in developing animals. Of note, the lower choroidal Nr1i2 expression at E15 could be compensated by Nr1i3 transcripts, as these two transcription factors regulate a number of common genes well expressed at the choroid plexus, such as Ugt1a, Abcc and cytosolic Gsts 95 . Similarly, the steady expression of Nfe2l2, Hif1a, and Arnt suggests that the neuroprotective functions of the blood-CSF barrier can be modulated in response to hypoxia and oxidative stress, whether occurring at early developmental age or in adult.