Spatiotemporal arising of molecular and functional Blood-Brain Barrier properties in the developing Gallus gallus optic tectum

Spatiotemporal arising of molecular and functional Blood-Brain Barrier properties in the developing Gallus gallus optic tectum | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Spatiotemporal arising of molecular and functional Blood-Brain Barrier properties in the developing Gallus gallus optic tectum Jesús Juárez-Balarezo, María Jesús Garrido-Muñoz, Benjamín Reuse-Benavente, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8911082/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 9 You are reading this latest preprint version Abstract Background The cerebrovascular endothelium possesses unique properties that strictly regulate the neural microenvironment. While mammalian Blood Brain Barrier (BBB) development is well-characterized, the avian embryo ( Gallus gallus ) offers a unique, highly accessible model for real-time developmental imaging and manipulation. However, the precise molecular and functional timeline of BBB maturation in the avian brain remains poorly explored. Here, we provide the first comprehensive spatiotemporal characterization of BBB development in the Gallus gallus optic tectum (OT). Methods We performed a multi-modal analysis between developmental stages HH32 and HH44, combining transcriptional analysis of BBB-associated genes, confocal and lightsheet microscopy, and quantitative functional assays for permeability and efflux activity. Results We identified a critical developmental window between stages HH36 and HH40 that marks the transition to a specialized barrier. This period is characterized by the significant upregulation of tight junction proteins ( CLDN5, OCLN ) and efflux transporters ( ABCB1 ), accompanied by the downregulation of fenestration markers ( PLVAP ). Also, tight junction assembly protein ZO-1 undergoes a progressive redistribution from a discontinuous pattern to continuous linear expression along the vessel walls during this period. Functionally, we demonstrate that paracellular restriction to 10 kDa dextran develops in a region-dependent manner, while quantitative assays reveal a sharp reduction in Evans Blue permeability starting at HH38. Furthermore, we detected the onset of P-glycoprotein efflux activity from HH36 onwards. Conclusions Altogether, our findings redefine the chicken embryo as a tractable model for barrier genesis research, pinpointing the HH36-40 developmental window as the discrete period where the molecular and functional BBB properties are acquired. This establishes a robust baseline for future mechanistic investigations into neurovascular development and pathology. Blood-Brain Barrier Chicken embryo Barrier-genesis Optic tectum Permeability ABC Transporters Tight junctions Figures Figure 1 Figure 2 Figure 3 Figure 4 Background The Blood Brain Barrier (BBB) is a set of properties that strictly regulates the movement of substances between the circulating blood and the brain parenchyma, ensuring central nervous system (CNS) homeostasis. Formed by specialized endothelial cells, the BBB is maintained by complex interactions within the neurovascular unit (NVU), which includes pericytes, astrocytes, microglia and neurons 1 , 2 . This unique endothelial phenotype is characterized by the presence of continuous tight junctions, the absence of fenestrations, suppressed rates of transcytosis, the expression of specific influx and efflux transporters, and the low expression of adhesion molecules that restrict the entry of immune cells into the brain 3 – 5 . In vertebrates, the development of the BBB proceeds through distinct phases: angiogenesis, differentiation and maturation 6 , 7 . Angiogenesis initiates when endothelial cells from the perineural vascular plexus invade the neuroectoderm 7 . The onset of anti-angiogenic signaling, combined with the recruitment of pericytes and subsequent astrocyte interaction, drives the transition from sprouting angiogenesis to barrier differentiation. Finally, the maturation phase is reached with the persistent expression and redistribution of tight junction proteins in BBB vessels and the downregulation of transcytosis, while the close interaction with the other NVU components sustains its integrity and function 4 , 8 . While general CNS angiogenesis is driven by conserved signaling programs, including Hypoxia-Inducible Factor (HIF), Vascular Endothelial Growth Factor (VEGF), and Notch signaling, the specific acquisition and maintenance of barrier properties is primarily orchestrated by the Wnt/beta-catenin pathway 9 – 12 . Canonical Wnt signaling strictly regulates the expression of essential transporters (e.g., Glucose transporter 1 or GLUT-1) and tight junction proteins (Claudin-3, Claudin-5), while simultaneously repressing the fenestration marker plasmalemma vesicle-associated protein (PLVAP) 9 – 11 . Complementing Wnt, the Hedgehog pathway, specifically Sonic Hedgehog (SHH), has also been described as a critical modulator of barrier integrity 13 , 14 . Secreted largely by astrocytes, SHH promotes BBB formation by inducing Claudin-5 and Occludin expression and maintaining endothelial immune quiescence 13 . However, while SHH is well-documented in embryonic morphogenesis and neural guidance, its precise contribution to the developmental timeline of the BBB remains barely explored. Although the acquisition of BBB properties has been mostly characterized in mammals, the existence of a barrier that protects the CNS is found even in invertebrates, with variations in some structural and functional components 5 , 15 , 16 . Among vertebrates the avian embryo, particularly Gallus gallus , offers a powerful, accessible model for vascular research due to its external development and amenability to real-time imaging. The aforementioned Wnt/beta-catenin and SHH pathways are both conserved in avians and mammals in the context of neural and vascular development 17 – 19 , suggesting a conserved role in BBB development and homeostasis. However, our current understanding of the avian BBB relies largely on electron microscopy and qualitative dye-injection studies 20 – 24 . While these early works established that barrier functions develop prior to hatching, they lacked the resolution to integrate signaling dynamics with molecular data and modern functional assays. Consequently, there is a critical gap in our knowledge regarding the molecular mechanisms that drive avian BBB development. In this study, we bridge this gap by defining a particular developmental period where the molecular and functional properties of the chicken BBB are developed. Focusing on the optic tectum (OT), a bilateral mesencephalic structure that shows a cortical architecture organized in 15 layers 25 with a well-characterized angiogenic pattern 6 , 26 , we combined transcriptional analysis, confocal and lightsheet microscopy, and quantitative permeability assays to study the chicken BBB development. Here, we show that the Gallus gallus BBB acquires its properties during a specific, narrow developmental window between HH36 and HH40. Complementing the widely established utility of the chicken chorioallantoic membrane (CAM) for general vascular studies, our work establishes a new framework for using the avian embryo to investigate neurovascular development and disease. Methods Embryos Fertilized eggs of Gallus gallus domesticus were obtained from Agricola Chorombo S.A. (Chile) and maintained under controlled temperature conditions (15 ± 1°C) at the facilities of the Faculty of Sciences, University of Chile. Prior to incubation, eggs were equilibrated at room temperature (± 22°C) for one hour. Incubation was conducted in experimental egg incubators (GQF 1520) at 37 ± 2°C with 70% relative humidity, in darkness, without rotation. Embryos were staged according to Hamburger and Hamilton 1951 27 and collected between stages HH32 and HH44 (E7–E18). All animal procedures complied with Chilean regulations and were approved by the Institutional Animal Care and Use Committee (CICUA) of the University of Chile (Verónica Palma: 22571-FCS-UCH-e1; María Jesús Garrido-Muñoz: 25885-FCS-UCH; Jesús Juárez-Balarezo: 25981-FCS-UCH). Gene expression analysis (qPCR) Total RNA was extracted from dissected OT at developmental stages HH32 to HH44 (n = 5 per stage) using Trizol/phenol - chloroform (15596026, Invitrogen). 1 µg of RNA was treated with DNase I (M6101, Promega, Madison, WI, USA). cDNA was synthesized (M-MLV reverse transcription kit (28025013, Invitrogen), and qPCR was performed using SyberGreen II (ST.600828, Agilent Technologies Thermocycler, Santa Clara, CA, USA). Data was analyzed by calculating the gene expression fold change via the 2 − ΔΔCt method, normalized to two reference genes ( ACTB and AARS1 ). Primer sequences are listed in Supplementary Table 1. Immunofluorescence and confocal microscopy Brain tissue was dissected on ice-cold PBS and fixed 4% in paraformaldehyde (PFA) overnight (ON). Following fixation, tissues were washed in PBS and equilibrated in 30% sucrose (w/v) ON. To preserve structural integrity, samples were embedded in gelatin (1.04070, Millipore), post-fixed for 2 hours and reequilibrated in 30% sucrose ON. Tissue blocks were then embedded in Tissue-Tek O.C.T. compound (4583, Sakura Finetek) and 50 µm sections were obtained using a cryostat. Sections were rinsed in PBS and blocked for 1 hour at room temperature (RT) under agitation in blocking solution (10% horse serum, 0.3% triton X-100 in PBS). Primary antibodies were diluted in blocking solution and applied ON at 4° C: anti-ZO-1 (1:100, 33-9100 Invitrogen) or anti-CLDN5 (1:50, 35-2500 Invitrogen). The samples were washed three times in PBS for 10 minutes each, and sections were incubated with goat anti-mouse Alexa Fluor 555 (A21424, Invitrogen) secondary antibody for 2 hours at RT under agitation. Finally, samples were counterstained with DAPI, mounted using FluorSave Reagent (345789, Millipore) and imaged on a Zeiss 710 confocal microscope using a C-Apochromat 40x/1.20 W Korr M27r water immersion objective (Unit of Advanced Microscopy, University of Chile). Tissue clearing and lightsheet microscopy Whole brains were dissected and fixed in 4% PFA ON. Brains were washed three times in PBS (1 hour each) and incubated in CUBIC-1 solution (25% urea, 5% Triton-X100, 10% Quadrol in PBS) at 37°C under constant shaking. After 5 days, brains were embedded in agarose 1% until solidified and transferred back to CUBIC-1 for additional 2–3 days. Once transparency was achieved, brains were incubated in CUBIC-2 solution 2 (10% Triethanolamine, 50% sucrose, 25% urea in double-distilled water) at 37°C for 2 days for refractive index matching. Images were acquired using a LS7 Zeiss lightsheet microscope (Unidad de Microscopía, Universidad Mayor), with a EC Plan-Neofluar 5x/0.16 foc objective, in CUBIC-2 solution (refractive index of 1.45). Image processing Confocal and lightsheet image data were processed using Fiji software. For confocal datasets, maximum intensity Z-projections were generated for each channel. Lightsheet data processing and stitching were performed using the BigStitcher plugin within Fiji. Three-dimensional reconstructions and volumetric rendering were conducted using Imaris 9.0.0 software; for lightsheet datasets, specific sub-volumes were extracted for detailed 3D analysis. Final figure panels and montages were assembled using Adobe Photoshop CC. Permeability assays Evans Blue (EB) (E2129, Sigma-Aldrich) was used to assess the changes in BBB permeability during chicken brain development. The protocol was adapted from Zhang et al., 2021 28 with slight modifications. Briefly, the embryos were injected intravascularly with 5% w/v of EB diluted in Ringer solution through CAM blood vessels. Volume of injection was calculated to achieve a final concentration of 0.2% w/v in chicken’s blood, according to the Chen et al. 2021 29 reported blood volume for each developmental stage. After 15 minutes, brain tissues were dissected, separating OT from the Forebrain. EB dye was dissolved using formamide. The samples were incubated at 55°C for 48h, followed by centrifugation at 14000g for 10 minutes at 4°C, then absorbance was determined at 620 nm using a Nano Quant Infinite 200 ProNano (Tecan, Männedorf, Switzerland). EB concentration was normalized to tissue weight. Blood vessels fluorescent labeling . Embryos were injected with wheat germ agglutinin (WGA) Lectin-FITC 30 and 10 kDa Dextran-Alexa Fluor 647 (D22914, Invitrogen) through the CAM blood vessels. Tissues were cleared and imaged via lightsheet microscopy to assess extravasation. Efflux transport assay (Rhodamine 123) P-glycoprotein (PGP) function assay was adapted from Koehn et al, 2021 31 by injecting Rhodamine-123 (#83702, Merck) through CAM blood vessels at a 2.5 ug/g concentration. After 1 hour, OT and forebrain were dissected and homogenized in HCl (0.1 M). Extracts were centrifuged at 10000g for 5 min and fluorescence in tissue homogenates was measured (Ex/Em: 485/530), using Infinite M PLEX (Tecan, Männedorf, Switzerland). Lower fluorescence indicates higher efflux activity. Rhodamine-123 concentration was normalized to tissue weight. Statistical analysis Data were analyzed using Graphpad Prism 9.0.2 (GraphPad Software Inc). Normality was assessed via the Shapiro-Wilk test. Comparisons were made using One-way ANOVA with Fisher’s LSD post hoc or Two-way RM ANOVA as appropriate. Statistical significance was set at p < 0.05. Results OT shows conserved Blood-Brain Barrier gene expression patterns that are acquired between HH36 and HH40 stages. Diverse studies have described the unique molecular composition of the BBB. However, a detailed transcriptional timeline of these patterns in the chicken embryo remains largely undefined 32 . Since the OT has been described as the first area to develop BBB properties 23 , 33 we profiled the expression of key genes related to angiogenesis, BBB signaling, tight junctions, and transport machinery from stage HH32 to HH44 (Fig. 1 A & S1). We observed distinct temporal patterns centered around the HH36-HH40 window. First, we analyzed signaling pathways known to regulate vascular and BBB development including SHH, Wnt and hypoxia inducible factor-1 (HIF1A). SHH expression in the OT showed a significant 2.03-fold decrease from HH32 to HH38, after which levels stabilized, consistent with a transition from angiogenic sprouting to barrier stabilization (Fig. 1 B). AXIN2 , a direct transcriptional target of canonical Wnt signalling 34 , exhibited a singular expression peak at HH36 (Figure S1 A). Regarding angiogenic drivers, hypoxia is known to activate HIF1A , which in turn upregulates angiogenic genes and has been associated with BBB disruption 35 – 37 . In our analysis, HIF1A displayed a significant 2.83-fold increase between HH32 and HH40, its only peak during development, before declining at HH42 (Fig. 1 C). Notable, VEGF significantly increased by 6.37-fold from HH32 to HH38, with a second surge at HH40 that was sustained until HH44 (Fig. 1 D), likely supporting vascular remodeling. Next, we assess tight junction components expression. The tight junction core protein Claudin-5 ( CLDN5) showed a 3.2-fold increase in expression from HH32 to HH36 (Fig. 1 E). Following a transient decrease in HH38, CLDN5 reached a second peak at HH44 (2.99-fold higher than HH32). while Occludin ( OCLN) , another known tight junction protein, displayed a similar bimodal pattern (Figure S1 B). We also profiled transporters that regulate molecular passage across the CNS endothelium 4 . The ATP-binding cassette efflux pump P-glycoprotein (encoded by the ABCB1 gene) exhibited a dramatic sustained upregulation throughout development. A statistically significant increase began at HH40, culminating in a 41.04- fold change between HH32 and HH44 (Fig. 1 F). In contrast, PLVAP , a component of endothelial fenestrations associated with transcytosis and high permeability 11 , 38 showed a high expression at HH32-34 followed by an abrupt 2.89- fold decline at HH36, which was sustained through HH44 (Fig. 1 G). Other BBB-associated genes described before mainly in rodent studies 39 – 41 showed fewer dynamic changes. Solute carrier transporters GLUT1 ( SLC2A1 ) and Large neutral Amino acid Transporter 1 or LAT1 ( SLC7A5 ) maintained high, stable expression throughout OT development with no significant fluctuations (Figure S1 C & D). Similarly, Basigin ( BSG ), a known avian BBB marker 42 – 44 , maintained steady expression with only minor increases at HH38 and HH42 (Figure S1 G). The lipid transporter Major Facilitator Superfamily Domain-Containing protein 2 or MFSD2A 40 , 41 showed a significant 1.62-fold increase at HH34, followed by a gradual decrease until HH44 (Figure S1 E). Collectively, these data demonstrate that classic BBB markers in the chicken OT display characteristic temporal patterns. Key genes such as CLDN5 and ABCB1 begin to surge around HH36, precisely correlating with the downregulation of the fenestration marker PLVAP . Furthermore, neuroangiogenic signaling molecules ( SHH and VEGF ) stabilize from HH38 onward. The convergence of these changes suggests the existence of a critical transcriptional window for barrier-genesis between HH36 and HH40. The tight junction protein ZO-1 distribution in OT blood vessels increases across developmental stages Since transcriptional changes were concentrated in the HH36-HH40 stages, we investigated whether these corresponded with changes in tight junction formation, a process essential for BBB integrity. We analyzed the distribution of Zonula Occludens-1 (ZO-1), which is fundamental for tight junction assembly and links transmembrane tight junction proteins to the actin cytoskeleton, providing structural integrity to the junctional complex 45 , 46 . Since chicken endothelial cells do not express the classic endothelial antigen CD31, we use lectins which are specific carbohydrate-binding proteins that have proven its utility for blood vessels visualization 30 . Using WGA-lectin to label the embryo's vasculature, we study ZO-1 distribution in the OT (Fig. 2 ). Confocal microscopy images of OT sections stained for tissue parenchyma (nuclear staining – gray), blood vessels (magenta) and ZO-1 (cyan). (A-B’’) At HH36, ZO-1 exhibits discontinuous labeling along the vessel walls. (C-D’’) By HH38, ZO-1 coverage increases but remains fragmented. (E-F’’) At HH40, ZO-1 displays continuous, linear distribution along the endothelial cell margins. Scale bar = 200 µm. Magnifications correspond to area delimited in white doted squares; Scale bar = 50 µm. Arrowheads indicate blood vessels regions with absence of ZO-1 signal. At HH36, ZO-1 immunoreactivity exhibited a discontinuous distribution along the vessels (Fig. 2 A-B’’’). By HH38 (Fig. 2 C-D’’’) ZO-1 coverage became broader, however, uncovered regions persisted. By HH40, ZO-1 appeared continuously distributed along the entirety of the vessel network (Fig. 2 E-F’’’). Three-dimensional reconstructions confirmed that ZO-1 labeling was specific to endothelial cells, strictly following the WGA-lectin vascular scaffold (Figure S3 ). We validated these findings by analyzing CLDN5 protein distribution across the same stages, which showed a consistent presence in blood vessels without major qualitative changes (Figure S4 ). These results indicate an active process of tight junction remodeling and expansion from HH36 onwards, correlating with the transcriptional pattern described above. BBB functional properties arise in a region-specific manner during OT development The first descriptions of chicken BBB development included the analysis of functional properties as permeability to vital tracers such as EB (960 Da) and HRP (40 kDa) in brain slices 20 , 23 , 33 . Dextrans are hydrophilic polysaccharides available in a wide range of molecular weights (from 3 kDa up to 2000 kDa). Fluorescent and biotinylated conjugated dextrans are often used as tracers, allowing the assessment of both small and large leakage of the BBB 47 . While 10 kDa dextran has been used in other models 40 , 48 its spatiotemporal restriction in the chick OT has not been described. To assess barrier function with spatial resolution, we performed 3D lightsheet microscopy of brains injected with 10 kDa fluorescent dextran and WGA-lectin (Fig. 3 ). At HH36 dextran signal is generally co-distributed with blood vessels (Fig. 3 A). However, detailed analysis of anterior, and latero-posterior regions (Fig. 3 B-D) revealed distinct sites of leakage, signal observed outside the vessel. At HH38, extravasation sites were markedly reduced (Fig. 3 E-I). By HH40, no observable dextran leakage was detected, with signal strictly confined to the vasculature across all regions (Figure J-N). Three-dimensional rendering confirmed these observations (Supplementary video 1–3). These data suggests that paracellular restriction to 10 kDa molecules is acquired in a region-dependent manner, starting in posterior zones and extending to the entire OT vasculature by HH40. OT and Forebrain decrease permeability and increase efflux activity in distinctive patterns Paracellular transport regulation appears at different time points depending on the brain area 20 , 23 , 33 . To quantify how the different barrier properties develop, we adapted the classic EB extravasation assay 28 and established a Rhodamine-123 (R123) efflux assay for the chick embryo. We compared the OT and the Forebrain to assess regional heterogeneity. Both the OT and Forebrain exhibited an initial increase in EB concentration between HH32 to HH34 (2.17-fold and 1.93-fold, respectively), likely reflecting active angiogenesis. This was followed by a significant decrease from HH34 to HH38 (Fig. 4 A-C). Interestingly, while both brain regions share a similar temporal profile, the forebrain shows significantly higher EB accumulations than the OT at the early stages (Figure S5A). From HH38 onwards, permeability continued to decline significantly relative to HH32 in both brain areas (Fig. 4 A) reaching its lowest point at HH44 (Figure S5B and C). The fact that ABCB1 mRNA expression showed a clear increase through OT development, led us to explore if this change could be related to an increase in the overall efflux activity of this transporter in the chick brain. We assessed P-glycoprotein (PGP) function by measuring the accumulation of its substrate, R123 31 . We determined an optimal concentration of 2.5 µg/g for the assay (Figure S6A & B). Our results showed an immediate, significant decrease in R123 accumulation (indicating increased efflux) starting at HH34 for the Forebrain and HH36 for the OT (Fig. 4 D). In both regions, the increase in efflux activity was statistically significant between HH34 and HH38 (Fig. 4 E and F). While R123 levels in the OT reached a minimum at HH40, accumulation in the Forebrain continued to decrease until HH42 (Figure S7). Notably, at late stages (HH40–HH44), R123 concentration was significantly higher in the OT than in the Forebrain (Figure S7A). Taken together, these results define a critical developmental window where gene expression, tight junction assembly, and functional properties appear all together shaping Gallus gallus BBB phenotype. Discussion Developmental stages HH36 to HH40 constitutes a critical time window for avian BBB development The endothelial BBB is a conserved feature of vertebrates, yet the precise timeline of its molecular and functional onset in non-mammalian models has remained unclear. To our knowledge, this is the first study to integrate transcriptional data, 3D structural analysis, and quantitative functional assays to study BBB development in the Gallus gallus OT. Our primary finding is the identification of a discrete "critical window" between HH36 and HH40 (E10–E14), during which the avian BBB undergoes a rapid, synchronized transition from a leaky vascular network to a highly selective barrier. This redefines the chicken embryo not just as a historical model, but as a precise tool for investigating the molecular regulation of barrier-genesis. Angiogenic Signaling and the Shift to BBB specialization Angiogenesis and barrier-genesis are often viewed as distinct sequential phases. Our transcriptional data supports this uncoupling in the avian OT. We observed that HIF1A and VEGF expression peaks around HH38–HH40, correlating with the intense vascular remodeling previously described in the OT 6, 26 . Wnt and SHH pathways have been described to regulate expression of tight junctions’ proteins and maintenance of BBB properties 9 , 10 , 12 , 13 . However, Wnt signaling also peaks early to drive angiogenesis in the mammalian cortex 9 . We found that the Wnt target AXIN2 and the barrier-promoting ligand SHH showed dynamic changes specifically preceding the HH36–HH40 window, around the same stages that CLDN5 and OCLN start to increase their expression. The decline in SHH expression after HH32 suggests that while it may promote angiogenesis, its downregulation coincides with the shift towards stabilization. This contrasts with mammalian models where SHH continues to promote immune quiescence in the adult BBB 13 . The synchronization of these signaling shifts with the sharp downregulation of the fenestration associated gene PLVAP at HH36 provides strong molecular evidence that the "vascular leakiness" observed in early stages is an actively regulated phenotype, not merely the absence of a barrier. This shift also aligns with changes in barrier permeability through development. Previously, Roncali et al 1985 described OT vasculogenesis process and found a clear increase of denseness of the radial vessels in the ventrolateral region of the tectum until HH34, which coincides with the considerable increment in EB permeability at the same stage found in our study. Then the percent of radial vessels is maintained from HH34 to HH40, but EB permeability keeps decreasing, showing the acquisition of BBB properties do not come only from lower angiogenic rates but the change towards an specialization of the existing vasculature. Temporal coordination of tight junction molecular assembly Our data suggests a complex interplay between transcriptional changes and protein distribution. While CLDN5 and OCLN mRNA levels rise starting at HH36, the distribution of ZO-1 protein reveals that structural sealing is a progressive process. The transition of ZO-1 from discontinuous foci at HH36 to linear, continuous strands by HH40 mirrors the decrease in extravasation of dextran we observed in our 3D lightsheet imaging, suggesting that the paracellular barrier formation has an inflection point between HH38 and HH40 stages for molecules about 10 kDa in size. Tight junction assembly involves recruitment of different proteins into the nascent junctional complex, being ZO-1 essential for this process 45 , 49 . ZO-1 proteins exist in phase separation compartments near the membrane, which sequester and locally concentrate tight junction proteins 50 . Deeper studies on the dynamic of tight junction assembly during BBB development are needed in order to establish more precisely the interdependence and temporality between gene expression, protein expression and protein interaction processes in Gallus gallus model. The developmental timeline found aligns remarkably well with historical dye-exclusion studies and previous reports about BBB markers such as alkaline phosphatase and transferrin receptor 20 – 22 , 33 but adds necessary molecular resolution. Importantly, we show that this window coincides with the onset of astrogenesis in the chicken brain 51 . Unlike mice, where the BBB forms prior to astrocyte differentiation 52 , the avian BBB properties appear concurrently with astrocyte differentiation, an idea that requires further investigation. This positions the chicken embryo as a unique model to dissect the specific contribution of astrocyte-endothelial interactions to barrier formation during embryonic development. Transport regulation across BBB develops gradually in a region-specific manner Beyond paracellular transport regulation, the fully developed BBB actively extrudes metabolic waste and xenobiotics via ABC transporters 53 , 54 . Studies in mammals reported expression and activity of ABCB1 to increase dramatically through development, reaching its highest capacity at adult stages 31 , 55 – 57 . We found a striking upregulation of ABCB1 mRNA (over 40-fold) starting at HH40 and peaking at HH44. However, our functional R123 assay revealed that significant efflux activity is detectable as early as HH36, reaching an apparent plateau at HH42. This could have profound implications for developmental toxicology, suggesting that the chicken embryo acquires protection against chemical insults gradually before the hatching. Regional heterogeneity was also evident. The forebrain showed higher basal permeability and earlier efflux activation compared to the OT. Our data also suggest that BBB low permeability is acquired at different levels of the vascular tree in different developmental time points. This rostro-caudal gradient supports classical observations and correlates with the findings where angiogenesis in the OT follows a defined pattern across spatial axes 6 , 20 , 33 , 58 and implies that barrier-genesis is not a uniform systemic event but is locally regulated by the specific neurogenic niche, a matter that deserves further investigation. Additionally, it is known that EB can bind to serum albumin, increasing its molecular size to 67 kDa, hence an important part of transport of this molecule is also regulated through transcytosis 59 . The role of MFSD2A and the diminution of PLVAP expression have been related to downregulation of transcytosis (Ben-Zvi et al 2014) 40 . In our study MFSD2A shows a significant increase while PLVAP decreases, around the same time where EB permeability declines. Still, it would be fundamental to explore other molecular changes associated and how this type of transport develops in future studies. Future Directions and Clinical Relevance By establishing HH36–HH40 as a highly relevant window for BBB development, our work opens several avenues for future research. First, the accessibility of the chicken embryo allows researchers to make use of the described time window, joining recent works that brought the chicken embryo to the spotlight of biomedical research 19 , 60 , 61 . Future studies can now investigate whether environmental stressors (e.g., hypoxia, hyperglycemia) applied during this specific window induce long-term barrier defects, providing a model for gestational neurovascular pathology. Second, the robust P-glycoprotein activity identified here validates the chick embryo as a cost-effective screening platform for CNS drug delivery. The clear separation between the “open” (pre-HH36) and "tight" (post-HH40) phases allows researchers to distinguish between passive diffusion and active transport mechanisms for novel therapeutics. Furthermore, while our transcriptional analysis identified key transporters, future proteomic characterization of the avian endothelial surface could uncover unique variants or evolutionarily conserved targets (such as the CD98 complex) that may be overlooked in rodent models 62 , 63 . Finally, our data demonstrates that the developing OT offers a new screening platform that complements the traditional CAM assay for neurovascular research. While the CAM assays is ubiquitous for evaluating anti-angiogenic drugs and tumor xenografts due to its accessibility and vascular density 29 , 64 , 65 , it remains an extra-embryonic tissue lacking the complex glial interplay that defines the NVU nor the specific barrier phenotype. Unlike the CAM, the embryonic brain between HH36 and HH40 actively recruits astrocytes, forming a true NVU with the microenvironment encountered by CNS-targeting therapeutics. By shifting focus from the CAM to the embryo, researchers can now screen compounds not just for vascular toxicity, but for specific BBB penetrance and efflux pump evasion in a physiologically intact CNS microenvironment. Conclusions In summary, we demonstrate that Gallus gallus BBB development occurs during an inflection point between stages HH36 and HH40. This window represents the convergence of molecular patterns, tight junction assembly, and the onset of active efflux. These findings not only update the biology of the avian BBB but also establish the chicken embryo as a rigorously validated model for investigating neurovascular development, disease, and CNS drug transport. Abbreviations AARS1 Alanyl-tRNA Synthetase 1 ABCB1 ATP binding cassette subfamily B member 1 ACTB Actin beta ATP Adenosine Triphosphate AXIN2 Axis Inhibition protein 2 BBB Blood Brain Barrier BSG Basigin CAM Chorioallantoic Membrane cDNA complementary Deoxyribonucleic acid CUBIC Clear Unobstructed Brain Imaging Cocktails CLDN5 Claudin 5 CNS Central Nervous System EB Evans Blue FITC Fluorescein IsoTioCyanate GLUT-1 Glucose Transporter 1 HH Hamburger-Hamilton stage HIF Hypoxia-Inducible Factor HIF1A Hypoxia-Inducible Factor 1 HRP Horseradish Peroxidase LAT1 Large neutral Amino acid Transporter 1 MFSD2A Major Facilitator Superfamily Domain-Containing protein 2 NVU Neurovascular Unit OCLN Occludin ON Overnight OT Optic Tectum PBS Phosphate-Buffered Saline PFA Paraformaldehyde PGP P-glycoprotein PLVAP Plasmalemma Vesicle-Associated Protein qPCR quantitative Polymerase Chain Reaction R123 Rhodamine 123 RNA Ribonucleic acid RT Room Temperature SHH Sonic Hedgehog SLC Solute Carrier Transporter VEGF Vascular Endothelial Growth Factor WGA Wheat Germ Agglutinin ZO-1 Zonula Occludens 1 Declarations Ethics approval and consent to participate Protocols for animal experiments were approved by the Animal Experimental Ethics Committee of Faculty of Sciences, Universidad de Chile and ANID (Certificate no. 22571–FCS–UCH-e1) on January 27, 2021, in compliance with the Bioethics committee of Universidad de Chile and ANID, for the care and use of laboratory animals. Consent for publication Not applicable Competing interests The author(s) declare no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. Funding Funding from ANID Fondecyt Regular #1221522 (VP), ANID MSc scholarship #22240623 (MJG-M) and ANID PhD scholarship #21242090 (JJ-B). Supplementary Figures. Additional Figures referenced in the main manuscript. Author Contribution JJ-B, MJG-M and VP conceived and designed the experiments. JJ-B, MJG-M, BR-B and IC-M performed the experiments and/or contributed to data acquisition. JJ-B, MJG-M and VP analyzed and interpreted the data. JJ-B and MJG-M wrote the original article. BR-B and IC-M critically revised and edited the article. VP provided resources and was responsible for general supervision, review, and editing. All authors approved the final version of the article. Acknowledgement We thank Dr. Joao Bothelo for kindly donating WGA-lectin and to give access to cryostat equipment. We are grateful to Dr. Aníbal Vargas and David Arancibia from the LiSIUM project for technical assistance with lightsheet images acquisition. Data Availability The datasets supporting the conclusions of this article are available in the Zenodo repository: https://zenodo.org/records/18672360 References Iadecola C. The Neurovascular Unit Coming of Age: A Journey through Neurovascular Coupling in Health and Disease. Neuron. 2017;96:17–42. Obermeier B, Daneman R, Ransohoff RM. Development, maintenance and disruption of the blood-brain barrier. Nat Med. 2013;19:1584–96. Daneman R, Prat A. The Blood–Brain Barrier. Cold Spring Harb Perspect Biol. 2015;7:a020412. Langen UH, Ayloo S, Gu C. Development and Cell Biology of the Blood-Brain Barrier. Annu Rev Cell Dev Biol. 2019;35:591–613. Dunton AD, Göpel T, Ho DH, Burggren W. Form and Function of the Vertebrate and Invertebrate Blood-Brain Barriers. Int J Mol Sci. 2021;22:12111. 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Barres BA Pericytes are required for blood–brain barrier integrity during embryogenesis. Nature. 2010;468:562–6. Schinkel AH, Wagenaar E, Mol CA, Van Deemter. L P-glycoprotein in the blood-brain barrier of mice influences the brain penetration and pharmacological activity of many drugs. J Clin Invest. 1996;97:2517–24. Lam FC, Liu R, Lu P, Shapiro AB, Sharom FJ. Reiner PB b-Amyloid efflux mediated by p-glycoprotein. J Neurochem. 2001;76:1121–8. Virgintino D, Robertson D, Errede M, Benagiano V, Girolamo F, Maiorano E, Roncali L, Bertossi M. Expression of P-Glycoprotein in Human Cerebral Cortex Microvessels. J Histochem Cytochem. 2002;50:1671–6. Daood M, Tsai C, Ahdab-Barmada M, Watchko JABC. Transporter (P-gp/ABCB1, MRP1/ABCC1, BCRP/ABCG2) Expression in the Developing Human CNS. Neuropediatr. 2008;39:211–8. Ek CJ, Wong A, Liddelow SA, Johansson PA, Dziegielewska KM. Saunders NR Efflux mechanisms at the developing brain barriers: ABC-transporters in the fetal and postnatal rat. Toxicol Lett. 2010;197:51–9. Delorme P, Gayet J, Grignon G. Ultrastructural study on transcapillary exchanges in the developing telencephalon of the chicken. Brain Res. 1970;22:269–83. Wolman M, Klatzo I, Chui E, Wilmes F, Nishimoto K, Fujiwara K. Spatz M Evaluation of the dye-protein tracers in pathophysiology of the blood-brain barrier. Acta Neuropathol. 1981;54:55–61. Sarnella A, Ferrara Y, Terlizzi C, Albanese S, Monti S, Licenziato L, Mancini M. The Chicken Embryo: An Old but Promising Model for In Vivo Preclinical Research. Biomedicines. 2024;12:2835. Bernardi DL, Den Ouden M, Nieuwenhuijzen-Van De Kaa K, Nguyen MVT, Schutgens V, Verhaar FC, Rookmaaker MB. Van Balkom B The chicken embryo model as a tool for investigating drug-induced acute kidney injury. J Pharmacol Toxicol Methods. 2026;137:108410. Singh N, Ecker GF. Insights into the Structure, Function, and Ligand Discovery of the Large Neutral Amino Acid Transporter 1, LAT1. Int J Mol Sci. 2018;19:1278. 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Supplementary Files SupplementaryFigures.docx Supplementaryvideo1.mp4 Supplementaryvideo2.mp4 Supplementaryvideo3.mp4 Cite Share Download PDF Status: Under Revision Version 1 posted Editorial decision: Revision requested 12 May, 2026 Reviews received at journal 12 May, 2026 Reviews received at journal 14 Apr, 2026 Reviewers agreed at journal 14 Apr, 2026 Reviewers agreed at journal 07 Apr, 2026 Reviewers invited by journal 24 Feb, 2026 Editor assigned by journal 22 Feb, 2026 Submission checks completed at journal 21 Feb, 2026 First submitted to journal 18 Feb, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8911082","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":596405267,"identity":"4edbbc6d-de10-44d4-8be4-0dd7a0ce6fd2","order_by":0,"name":"Jesús Juárez-Balarezo","email":"","orcid":"","institution":"Universidad de Chile","correspondingAuthor":false,"prefix":"","firstName":"Jesús","middleName":"","lastName":"Juárez-Balarezo","suffix":""},{"id":596405270,"identity":"b2aea755-91d3-4e74-a853-faa6b530a5a4","order_by":1,"name":"María Jesús Garrido-Muñoz","email":"","orcid":"","institution":"Universidad de Chile","correspondingAuthor":false,"prefix":"","firstName":"María","middleName":"Jesús","lastName":"Garrido-Muñoz","suffix":""},{"id":596405272,"identity":"14536e57-db6a-428c-a61c-5bb89063d7dc","order_by":2,"name":"Benjamín Reuse-Benavente","email":"","orcid":"","institution":"Universidad de Chile","correspondingAuthor":false,"prefix":"","firstName":"Benjamín","middleName":"","lastName":"Reuse-Benavente","suffix":""},{"id":596405274,"identity":"7a0dd451-33d2-4377-acc6-3a2d258dbef4","order_by":3,"name":"Ignacio Casanova-Maldonado","email":"","orcid":"","institution":"Universidad de Chile","correspondingAuthor":false,"prefix":"","firstName":"Ignacio","middleName":"","lastName":"Casanova-Maldonado","suffix":""},{"id":596405276,"identity":"f541a1ee-0db4-48da-a03c-9ecc9678715f","order_by":4,"name":"Verónica Palma","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAwUlEQVRIiWNgGAWjYHACxgMMDDYMDBKMjUAGgwxReoAq00BaGkBaeIjVchioBcwgQovB7eYHh27UnE/sn93ccOADgx0RWu4cMzicc+x24ow7BxsOzmBIJqxFckYCUAvb7cQNEokNh3kYDhCjJf3D4Zx/5yBa/hCjhV8ix+BwbtsBiBYGorTInCk4nNuXbDzjRmLDwR4DIvzCJt2+8XHONzvZ/hnpDx/8qLCTI6gFFB1IwICwBnQto2AUjIJRMAqwAAD4ikO9+DSJcwAAAABJRU5ErkJggg==","orcid":"","institution":"Universidad de Chile","correspondingAuthor":true,"prefix":"","firstName":"Verónica","middleName":"","lastName":"Palma","suffix":""}],"badges":[],"createdAt":"2026-02-18 16:54:28","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8911082/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8911082/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":103728504,"identity":"72c0db6a-567e-4246-87c0-77a1009a6501","added_by":"auto","created_at":"2026-03-02 08:43:17","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":279636,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTranscriptional profile of BBB-associated genes during optic tectum development.\u003c/strong\u003e (A) Schematic color-code representation of the experimental timeline according to Hamburger \u0026amp; Hamilton (HH) developmental stages, created with biorender.com. Optic tecta (OT) were dissected from embryos at stages HH32 to HH44 for mRNA expression analysis. (B-G) Relative mRNA expression of (B) \u003cem\u003eSHH\u003c/em\u003e, (C) \u003cem\u003eHIF1A\u003c/em\u003e, (D)\u003cem\u003e VEGF \u003c/em\u003e(E) \u003cem\u003eCLDN5\u003c/em\u003e(F) \u003cem\u003eABCB1\u003c/em\u003e and (G) \u003cem\u003ePLVAP\u003c/em\u003e. Expression levels were normalized to the reference genes \u003cem\u003eBACTIN\u003c/em\u003e and \u003cem\u003eAARS1\u003c/em\u003e and calculated relative to HH38. Data are presented as mean fold change ± SEM (n=5 per stage). Statistical significance was determined using one-way ANOVA with Fisher’s LSD post-hoc test. *p \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8911082/v1/66d2bf2d9375692c698794cb.jpeg"},{"id":103728506,"identity":"30794778-219c-4cfb-90ee-2fee6ec8df51","added_by":"auto","created_at":"2026-03-02 08:43:18","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":383786,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSpatiotemporal redistribution of ZO-1 tight junction protein in OT vasculature.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConfocal microscopy images of OT sections stained for tissue parenchyma (nuclear staining – gray), blood vessels (magenta) and ZO-1 (cyan). (A-B’’) At HH36, ZO-1 exhibits discontinuous labeling along the vessel walls. (C-D’’) By HH38, ZO-1 coverage increases but remains fragmented. (E-F’’) At HH40, ZO-1 displays continuous, linear distribution along the endothelial cell margins. Scale bar = 200 µm. Magnifications correspond to area delimited in white doted squares; Scale bar = 50 µm. Arrowheads indicate blood vessels regions with absence of ZO-1 signal.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8911082/v1/7cfbe4c0838960eb95ed6ea1.jpeg"},{"id":103728507,"identity":"b83045f4-060b-4002-957c-8e0b8feca8d8","added_by":"auto","created_at":"2026-03-02 08:43:18","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":485801,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRegion-specific restriction of paracellular permeability in the developing OT.\u003c/strong\u003e 3D Lightsheet microscopy reconstructions of whole OT hemispheres injected with WGA-lectin (red) and 10 kDa Dextran (cyan). \u003cstrong\u003e(A)\u003c/strong\u003e Overview of an HH36 OT showing widespread extravasation of dextran. \u003cstrong\u003e(B-D)\u003c/strong\u003e Higher magnification of anterior and latero-posterior regions at HH36, revealing a gradient of permeability with significant leakage in anterior/lateral zones. \u003cstrong\u003e(E-H)\u003c/strong\u003e At HH38, leakage is reduced but persists in discrete foci. \u003cstrong\u003e(I-M)\u003c/strong\u003e By HH40, dextran is strictly confined within the vascular lumen across all regions. Scale bar = 700 μm. Magnifications correspond to areas indicated in white dotted rectangles in the anterior region of the OT \u003cstrong\u003e(C, G K) \u003c/strong\u003eand latero-posterior region \u003cstrong\u003e(D, H, M)\u003c/strong\u003e,\u003cstrong\u003e \u003c/strong\u003edisplaying both channels. Scale bar = 200 µm.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8911082/v1/9850197313b216b96bdfb90c.jpeg"},{"id":103728509,"identity":"64e99e06-1c0b-4339-916e-476c6f2dbdfb","added_by":"auto","created_at":"2026-03-02 08:43:18","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":216884,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOT and Forebrain decrease permeability and increase efflux activity in distinctive patterns. \u003c/strong\u003e(A-C) Quantification of Evans Blue (EB) extravasation in the OT (red) and Forebrain (black). (A) EB concentration between HH32 and HH44 stages (normalized to tissue weight). (B-C) Comparison of EB leakage between HH34 and HH38 in OT and Forebrain. Data are presented as mean ± SD (n = 9). *p \u0026lt; 0.05, ****p \u0026lt; 0.0001 (Two-way RM ANOVA test for A, Unpaired t-test for B,C). (D-F) Functional assessment of P-glycoprotein (P-gp) efflux activity using Rhodamine-123 (R123). (D) R123 accumulation in OT and Forebrain between HH32 and HH44 stages. (E-F) Significant reduction in R123 accumulation between HH34 and HH38 in both regions. Data are presented as mean ± SD (n = 10). *p \u0026lt; 0.05, ****p \u0026lt; 0.0001 (Two-way RM ANOVA test for D, Unpaired t-test for E,F).\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8911082/v1/53d86680e8d58f4493cd03c0.jpeg"},{"id":104412828,"identity":"83fcc0cb-7b09-4fcf-80be-7e3d67948c26","added_by":"auto","created_at":"2026-03-11 13:01:18","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2557653,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8911082/v1/3bcd5524-c4bb-4e49-bf7e-90c7c4c40fce.pdf"},{"id":104407928,"identity":"2cd0072b-4583-4f72-b8f7-4afed69a6e13","added_by":"auto","created_at":"2026-03-11 12:40:50","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":2973555,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigures.docx","url":"https://assets-eu.researchsquare.com/files/rs-8911082/v1/8b61296bf669d42ef7cc5d62.docx"},{"id":103728584,"identity":"5ca758db-2caa-491b-a43c-4657e5f6ab92","added_by":"auto","created_at":"2026-03-02 08:43:25","extension":"mp4","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":43210484,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryvideo1.mp4","url":"https://assets-eu.researchsquare.com/files/rs-8911082/v1/6411a882b0896c54b08657d8.mp4"},{"id":103728547,"identity":"b7c12f93-731a-4830-9aba-bfec9f3a0260","added_by":"auto","created_at":"2026-03-02 08:43:20","extension":"mp4","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":29913463,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryvideo2.mp4","url":"https://assets-eu.researchsquare.com/files/rs-8911082/v1/5d30b65b751c2d96812365bf.mp4"},{"id":104406666,"identity":"d548280d-6317-4dd3-866f-310440426243","added_by":"auto","created_at":"2026-03-11 12:29:40","extension":"mp4","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":47568930,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryvideo3.mp4","url":"https://assets-eu.researchsquare.com/files/rs-8911082/v1/3bd4cec31457903aff2704b1.mp4"}],"financialInterests":"No competing interests reported.","formattedTitle":"Spatiotemporal arising of molecular and functional Blood-Brain Barrier properties in the developing Gallus gallus optic tectum","fulltext":[{"header":"Background","content":"\u003cp\u003eThe Blood Brain Barrier (BBB) is a set of properties that strictly regulates the movement of substances between the circulating blood and the brain parenchyma, ensuring central nervous system (CNS) homeostasis. Formed by specialized endothelial cells, the BBB is maintained by complex interactions within the neurovascular unit (NVU), which includes pericytes, astrocytes, microglia and neurons \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. This unique endothelial phenotype is characterized by the presence of continuous tight junctions, the absence of fenestrations, suppressed rates of transcytosis, the expression of specific influx and efflux transporters, and the low expression of adhesion molecules that restrict the entry of immune cells into the brain \u003csup\u003e\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn vertebrates, the development of the BBB proceeds through distinct phases: angiogenesis, differentiation and maturation \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Angiogenesis initiates when endothelial cells from the perineural vascular plexus invade the neuroectoderm \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. The onset of anti-angiogenic signaling, combined with the recruitment of pericytes and subsequent astrocyte interaction, drives the transition from sprouting angiogenesis to barrier differentiation. Finally, the maturation phase is reached with the persistent expression and redistribution of tight junction proteins in BBB vessels and the downregulation of transcytosis, while the close interaction with the other NVU components sustains its integrity and function \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eWhile general CNS angiogenesis is driven by conserved signaling programs, including Hypoxia-Inducible Factor (HIF), Vascular Endothelial Growth Factor (VEGF), and Notch signaling, the specific acquisition and maintenance of barrier properties is primarily orchestrated by the Wnt/beta-catenin pathway \u003csup\u003e\u003cspan additionalcitationids=\"CR10 CR11\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Canonical Wnt signaling strictly regulates the expression of essential transporters (e.g., Glucose transporter 1 or GLUT-1) and tight junction proteins (Claudin-3, Claudin-5), while simultaneously repressing the fenestration marker plasmalemma vesicle-associated protein (PLVAP) \u003csup\u003e\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Complementing Wnt, the Hedgehog pathway, specifically Sonic Hedgehog (SHH), has also been described as a critical modulator of barrier integrity \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Secreted largely by astrocytes, SHH promotes BBB formation by inducing Claudin-5 and Occludin expression and maintaining endothelial immune quiescence \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. However, while SHH is well-documented in embryonic morphogenesis and neural guidance, its precise contribution to the developmental timeline of the BBB remains barely explored.\u003c/p\u003e \u003cp\u003eAlthough the acquisition of BBB properties has been mostly characterized in mammals, the existence of a barrier that protects the CNS is found even in invertebrates, with variations in some structural and functional components \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Among vertebrates the avian embryo, particularly \u003cem\u003eGallus gallus\u003c/em\u003e, offers a powerful, accessible model for vascular research due to its external development and amenability to real-time imaging. The aforementioned Wnt/beta-catenin and SHH pathways are both conserved in avians and mammals in the context of neural and vascular development \u003csup\u003e\u003cspan additionalcitationids=\"CR18\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e, suggesting a conserved role in BBB development and homeostasis. However, our current understanding of the avian BBB relies largely on electron microscopy and qualitative dye-injection studies \u003csup\u003e\u003cspan additionalcitationids=\"CR21 CR22 CR23\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. While these early works established that barrier functions develop prior to hatching, they lacked the resolution to integrate signaling dynamics with molecular data and modern functional assays. Consequently, there is a critical gap in our knowledge regarding the molecular mechanisms that drive avian BBB development.\u003c/p\u003e \u003cp\u003eIn this study, we bridge this gap by defining a particular developmental period where the molecular and functional properties of the chicken BBB are developed. Focusing on the optic tectum (OT), a bilateral mesencephalic structure that shows a cortical architecture organized in 15 layers \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e with a well-characterized angiogenic pattern \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e, we combined transcriptional analysis, confocal and lightsheet microscopy, and quantitative permeability assays to study the chicken BBB development. Here, we show that the \u003cem\u003eGallus gallus\u003c/em\u003e BBB acquires its properties during a specific, narrow developmental window between HH36 and HH40. Complementing the widely established utility of the chicken chorioallantoic membrane (CAM) for general vascular studies, our work establishes a new framework for using the avian embryo to investigate neurovascular development and disease.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eEmbryos\u003c/h2\u003e \u003cp\u003eFertilized eggs of \u003cem\u003eGallus gallus domesticus\u003c/em\u003e were obtained from Agricola Chorombo S.A. (Chile) and maintained under controlled temperature conditions (15\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C) at the facilities of the Faculty of Sciences, University of Chile.\u003c/p\u003e \u003cp\u003ePrior to incubation, eggs were equilibrated at room temperature (\u0026plusmn;\u0026thinsp;22\u0026deg;C) for one hour. Incubation was conducted in experimental egg incubators (GQF 1520) at 37\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C with 70% relative humidity, in darkness, without rotation. Embryos were staged according to Hamburger and Hamilton 1951\u003csup\u003e27\u003c/sup\u003e and collected between stages HH32 and HH44 (E7\u0026ndash;E18).\u003c/p\u003e \u003cp\u003eAll animal procedures complied with Chilean regulations and were approved by the Institutional Animal Care and Use Committee (CICUA) of the University of Chile (Ver\u0026oacute;nica Palma: 22571-FCS-UCH-e1; Mar\u0026iacute;a Jes\u0026uacute;s Garrido-Mu\u0026ntilde;oz: 25885-FCS-UCH; Jes\u0026uacute;s Ju\u0026aacute;rez-Balarezo: 25981-FCS-UCH).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eGene expression analysis (qPCR)\u003c/h3\u003e\n\u003cp\u003eTotal RNA was extracted from dissected OT at developmental stages HH32 to HH44 (n\u0026thinsp;=\u0026thinsp;5 per stage) using Trizol/phenol - chloroform (15596026, Invitrogen). 1 \u0026micro;g of RNA was treated with DNase I (M6101, Promega, Madison, WI, USA). cDNA was synthesized (M-MLV reverse transcription kit (28025013, Invitrogen), and qPCR was performed using SyberGreen II (ST.600828, Agilent Technologies Thermocycler, Santa Clara, CA, USA). Data was analyzed by calculating the gene expression fold change via the 2\u003csup\u003e\u0026minus; ΔΔCt\u003c/sup\u003e method, normalized to two reference genes (\u003cem\u003eACTB\u003c/em\u003e and \u003cem\u003eAARS1\u003c/em\u003e). Primer sequences are listed in Supplementary Table\u0026nbsp;1.\u003c/p\u003e\n\u003ch3\u003eImmunofluorescence and confocal microscopy\u003c/h3\u003e\n\u003cp\u003eBrain tissue was dissected on ice-cold PBS and fixed 4% in paraformaldehyde (PFA) overnight (ON). Following fixation, tissues were washed in PBS and equilibrated in 30% sucrose (w/v) ON. To preserve structural integrity, samples were embedded in gelatin (1.04070, Millipore), post-fixed for 2 hours and reequilibrated in 30% sucrose ON. Tissue blocks were then embedded in Tissue-Tek O.C.T. compound (4583, Sakura Finetek) and 50 \u0026micro;m sections were obtained using a cryostat. Sections were rinsed in PBS and blocked for 1 hour at room temperature (RT) under agitation in blocking solution (10% horse serum, 0.3% triton X-100 in PBS). Primary antibodies were diluted in blocking solution and applied ON at 4\u0026deg; C: anti-ZO-1 (1:100, 33-9100 Invitrogen) or anti-CLDN5 (1:50, 35-2500 Invitrogen). The samples were washed three times in PBS for 10 minutes each, and sections were incubated with goat anti-mouse Alexa Fluor 555 (A21424, Invitrogen) secondary antibody for 2 hours at RT under agitation. Finally, samples were counterstained with DAPI, mounted using FluorSave Reagent (345789, Millipore) and imaged on a Zeiss 710 confocal microscope using a C-Apochromat 40x/1.20 W Korr M27r water immersion objective (Unit of Advanced Microscopy, University of Chile).\u003c/p\u003e\n\u003ch3\u003eTissue clearing and lightsheet microscopy\u003c/h3\u003e\n\u003cp\u003eWhole brains were dissected and fixed in 4% PFA ON. Brains were washed three times in PBS (1 hour each) and incubated in CUBIC-1 solution (25% urea, 5% Triton-X100, 10% Quadrol in PBS) at 37\u0026deg;C under constant shaking. After 5 days, brains were embedded in agarose 1% until solidified and transferred back to CUBIC-1 for additional 2\u0026ndash;3 days. Once transparency was achieved, brains were incubated in CUBIC-2 solution 2 (10% Triethanolamine, 50% sucrose, 25% urea in double-distilled water) at 37\u0026deg;C for 2 days for refractive index matching. Images were acquired using a LS7 Zeiss lightsheet microscope (Unidad de Microscop\u0026iacute;a, Universidad Mayor), with a EC Plan-Neofluar 5x/0.16 foc objective, in CUBIC-2 solution (refractive index of 1.45).\u003c/p\u003e\n\u003ch3\u003eImage processing\u003c/h3\u003e\n\u003cp\u003eConfocal and lightsheet image data were processed using Fiji software. For confocal datasets, maximum intensity Z-projections were generated for each channel. Lightsheet data processing and stitching were performed using the BigStitcher plugin within Fiji. Three-dimensional reconstructions and volumetric rendering were conducted using Imaris 9.0.0 software; for lightsheet datasets, specific sub-volumes were extracted for detailed 3D analysis. Final figure panels and montages were assembled using Adobe Photoshop CC.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003ePermeability assays\u003c/h2\u003e \u003cp\u003eEvans Blue (EB) (E2129, Sigma-Aldrich) was used to assess the changes in BBB permeability during chicken brain development. The protocol was adapted from Zhang et al., 2021 \u003csup\u003e28\u003c/sup\u003e with slight modifications. Briefly, the embryos were injected intravascularly with 5% w/v of EB diluted in Ringer solution through CAM blood vessels. Volume of injection was calculated to achieve a final concentration of 0.2% w/v in chicken\u0026rsquo;s blood, according to the Chen et al. 2021 \u003csup\u003e29\u003c/sup\u003e reported blood volume for each developmental stage. After 15 minutes, brain tissues were dissected, separating OT from the Forebrain. EB dye was dissolved using formamide. The samples were incubated at 55\u0026deg;C for 48h, followed by centrifugation at 14000g for 10 minutes at 4\u0026deg;C, then absorbance was determined at 620 nm using a Nano Quant Infinite 200 ProNano (Tecan, M\u0026auml;nnedorf, Switzerland). EB concentration was normalized to tissue weight.\u003c/p\u003e \u003cp\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eBlood vessels fluorescent labeling\u003c/span\u003e.\u003c/p\u003e \u003cp\u003eEmbryos were injected with wheat germ agglutinin (WGA) Lectin-FITC \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e and 10 kDa Dextran-Alexa Fluor 647 (D22914, Invitrogen) through the CAM blood vessels. Tissues were cleared and imaged via lightsheet microscopy to assess extravasation.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eEfflux transport assay (Rhodamine 123)\u003c/h3\u003e\n\u003cp\u003eP-glycoprotein (PGP) function assay was adapted from Koehn et al, 2021 \u003csup\u003e31\u003c/sup\u003e by injecting Rhodamine-123 (#83702, Merck) through CAM blood vessels at a 2.5 ug/g concentration. After 1 hour, OT and forebrain were dissected and homogenized in HCl (0.1 M). Extracts were centrifuged at 10000g for 5 min and fluorescence in tissue homogenates was measured (Ex/Em: 485/530), using Infinite M PLEX (Tecan, M\u0026auml;nnedorf, Switzerland). Lower fluorescence indicates higher efflux activity. Rhodamine-123 concentration was normalized to tissue weight.\u003c/p\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eData were analyzed using Graphpad Prism 9.0.2 (GraphPad Software Inc). Normality was assessed via the Shapiro-Wilk test. Comparisons were made using One-way ANOVA with Fisher\u0026rsquo;s LSD post hoc or Two-way RM ANOVA as appropriate. Statistical significance was set at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eOT shows conserved Blood-Brain Barrier gene expression patterns that are acquired between HH36 and HH40 stages.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eDiverse studies have described the unique molecular composition of the BBB. However, a detailed transcriptional timeline of these patterns in the chicken embryo remains largely undefined \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. Since the OT has been described as the first area to develop BBB properties \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e we profiled the expression of key genes related to angiogenesis, BBB signaling, tight junctions, and transport machinery from stage HH32 to HH44 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA \u0026amp; S1). We observed distinct temporal patterns centered around the HH36-HH40 window.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFirst, we analyzed signaling pathways known to regulate vascular and BBB development including SHH, Wnt and hypoxia inducible factor-1 (HIF1A). \u003cem\u003eSHH\u003c/em\u003e expression in the OT showed a significant 2.03-fold decrease from HH32 to HH38, after which levels stabilized, consistent with a transition from angiogenic sprouting to barrier stabilization (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). \u003cem\u003eAXIN2\u003c/em\u003e, a direct transcriptional target of canonical Wnt signalling \u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e, exhibited a singular expression peak at HH36 (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003eRegarding angiogenic drivers, hypoxia is known to activate \u003cem\u003eHIF1A\u003c/em\u003e, which in turn upregulates angiogenic genes and has been associated with BBB disruption \u003csup\u003e\u003cspan additionalcitationids=\"CR36\" citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. In our analysis, \u003cem\u003eHIF1A\u003c/em\u003e displayed a significant 2.83-fold increase between HH32 and HH40, its only peak during development, before declining at HH42 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Notable, \u003cem\u003eVEGF\u003c/em\u003e significantly increased by 6.37-fold from HH32 to HH38, with a second surge at HH40 that was sustained until HH44 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD), likely supporting vascular remodeling.\u003c/p\u003e \u003cp\u003eNext, we assess tight junction components expression. The tight junction core protein Claudin-5 (\u003cem\u003eCLDN5)\u003c/em\u003e showed a 3.2-fold increase in expression from HH32 to HH36 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). Following a transient decrease in HH38, \u003cem\u003eCLDN5\u003c/em\u003e reached a second peak at HH44 (2.99-fold higher than HH32). while Occludin (\u003cem\u003eOCLN)\u003c/em\u003e, another known tight junction protein, displayed a similar bimodal pattern (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eWe also profiled transporters that regulate molecular passage across the CNS endothelium \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. The ATP-binding cassette efflux pump P-glycoprotein (encoded by the \u003cem\u003eABCB1\u003c/em\u003e gene) exhibited a dramatic sustained upregulation throughout development. A statistically significant increase began at HH40, culminating in a 41.04- fold change between HH32 and HH44 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). In contrast, \u003cem\u003ePLVAP\u003c/em\u003e, a component of endothelial fenestrations associated with transcytosis and high permeability \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e showed a high expression at HH32-34 followed by an abrupt 2.89- fold decline at HH36, which was sustained through HH44 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG).\u003c/p\u003e \u003cp\u003eOther BBB-associated genes described before mainly in rodent studies \u003csup\u003e\u003cspan additionalcitationids=\"CR40\" citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e showed fewer dynamic changes. Solute carrier transporters GLUT1 (\u003cem\u003eSLC2A1\u003c/em\u003e) and Large neutral Amino acid Transporter 1 or LAT1 (\u003cem\u003eSLC7A5\u003c/em\u003e) maintained high, stable expression throughout OT development with no significant fluctuations (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eC \u0026amp; D). Similarly, Basigin (\u003cem\u003eBSG\u003c/em\u003e), a known avian BBB marker \u003csup\u003e\u003cspan additionalcitationids=\"CR43\" citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e, maintained steady expression with only minor increases at HH38 and HH42 (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eG). The lipid transporter Major Facilitator Superfamily Domain-Containing protein 2 or \u003cem\u003eMFSD2A\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e showed a significant 1.62-fold increase at HH34, followed by a gradual decrease until HH44 (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eE).\u003c/p\u003e \u003cp\u003eCollectively, these data demonstrate that classic BBB markers in the chicken OT display characteristic temporal patterns. Key genes such as \u003cem\u003eCLDN5\u003c/em\u003e and \u003cem\u003eABCB1\u003c/em\u003e begin to surge around HH36, precisely correlating with the downregulation of the fenestration marker \u003cem\u003ePLVAP\u003c/em\u003e. Furthermore, neuroangiogenic signaling molecules (\u003cem\u003eSHH\u003c/em\u003e and \u003cem\u003eVEGF\u003c/em\u003e) stabilize from HH38 onward. The convergence of these changes suggests the existence of a critical transcriptional window for barrier-genesis between HH36 and HH40.\u003c/p\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eThe tight junction protein ZO-1 distribution in OT blood vessels increases across developmental stages\u003c/h2\u003e \u003cp\u003eSince transcriptional changes were concentrated in the HH36-HH40 stages, we investigated whether these corresponded with changes in tight junction formation, a process essential for BBB integrity. We analyzed the distribution of Zonula Occludens-1 (ZO-1), which is fundamental for tight junction assembly and links transmembrane tight junction proteins to the actin cytoskeleton, providing structural integrity to the junctional complex \u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. Since chicken endothelial cells do not express the classic endothelial antigen CD31, we use lectins which are specific carbohydrate-binding proteins that have proven its utility for blood vessels visualization \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. Using WGA-lectin to label the embryo's vasculature, we study ZO-1 distribution in the OT (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eConfocal microscopy images of OT sections stained for tissue parenchyma (nuclear staining \u0026ndash; gray), blood vessels (magenta) and ZO-1 (cyan). (A-B\u0026rsquo;\u0026rsquo;) At HH36, ZO-1 exhibits discontinuous labeling along the vessel walls. (C-D\u0026rsquo;\u0026rsquo;) By HH38, ZO-1 coverage increases but remains fragmented. (E-F\u0026rsquo;\u0026rsquo;) At HH40, ZO-1 displays continuous, linear distribution along the endothelial cell margins. Scale bar =\u0026thinsp;200 \u0026micro;m. Magnifications correspond to area delimited in white doted squares; Scale bar =\u0026thinsp;50 \u0026micro;m. Arrowheads indicate blood vessels regions with absence of ZO-1 signal.\u003c/p\u003e \u003cp\u003eAt HH36, ZO-1 immunoreactivity exhibited a discontinuous distribution along the vessels (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-B\u0026rsquo;\u0026rsquo;\u0026rsquo;). By HH38 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC-D\u0026rsquo;\u0026rsquo;\u0026rsquo;) ZO-1 coverage became broader, however, uncovered regions persisted. By HH40, ZO-1 appeared continuously distributed along the entirety of the vessel network (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE-F\u0026rsquo;\u0026rsquo;\u0026rsquo;). Three-dimensional reconstructions confirmed that ZO-1 labeling was specific to endothelial cells, strictly following the WGA-lectin vascular scaffold (Figure \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e). We validated these findings by analyzing CLDN5 protein distribution across the same stages, which showed a consistent presence in blood vessels without major qualitative changes (Figure \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThese results indicate an active process of tight junction remodeling and expansion from HH36 onwards, correlating with the transcriptional pattern described above.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eBBB functional properties arise in a region-specific manner during OT development\u003c/h2\u003e \u003cp\u003eThe first descriptions of chicken BBB development included the analysis of functional properties as permeability to vital tracers such as EB (960 Da) and HRP (40 kDa) in brain slices \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Dextrans are hydrophilic polysaccharides available in a wide range of molecular weights (from 3 kDa up to 2000 kDa). Fluorescent and biotinylated conjugated dextrans are often used as tracers, allowing the assessment of both small and large leakage of the BBB \u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. While 10 kDa dextran has been used in other models \u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e its spatiotemporal restriction in the chick OT has not been described. To assess barrier function with spatial resolution, we performed 3D lightsheet microscopy of brains injected with 10 kDa fluorescent dextran and WGA-lectin (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAt HH36 dextran signal is generally co-distributed with blood vessels (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). However, detailed analysis of anterior, and latero-posterior regions (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB-D) revealed distinct sites of leakage, signal observed outside the vessel. At HH38, extravasation sites were markedly reduced (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE-I). By HH40, no observable dextran leakage was detected, with signal strictly confined to the vasculature across all regions (Figure J-N). Three-dimensional rendering confirmed these observations (Supplementary video 1\u0026ndash;3).\u003c/p\u003e \u003cp\u003eThese data suggests that paracellular restriction to 10 kDa molecules is acquired in a region-dependent manner, starting in posterior zones and extending to the entire OT vasculature by HH40.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eOT and Forebrain decrease permeability and increase efflux activity in distinctive patterns\u003c/h2\u003e \u003cp\u003eParacellular transport regulation appears at different time points depending on the brain area \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. To quantify how the different barrier properties develop, we adapted the classic EB extravasation assay \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e and established a Rhodamine-123 (R123) efflux assay for the chick embryo. We compared the OT and the Forebrain to assess regional heterogeneity.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eBoth the OT and Forebrain exhibited an initial increase in EB concentration between HH32 to HH34 (2.17-fold and 1.93-fold, respectively), likely reflecting active angiogenesis. This was followed by a significant decrease from HH34 to HH38 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-C). Interestingly, while both brain regions share a similar temporal profile, the forebrain shows significantly higher EB accumulations than the OT at the early stages (Figure S5A). From HH38 onwards, permeability continued to decline significantly relative to HH32 in both brain areas (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA) reaching its lowest point at HH44 (Figure S5B and C).\u003c/p\u003e \u003cp\u003eThe fact that \u003cem\u003eABCB1\u003c/em\u003e mRNA expression showed a clear increase through OT development, led us to explore if this change could be related to an increase in the overall efflux activity of this transporter in the chick brain. We assessed P-glycoprotein (PGP) function by measuring the accumulation of its substrate, R123 \u003csup\u003e31\u003c/sup\u003e. We determined an optimal concentration of 2.5 \u0026micro;g/g for the assay (Figure S6A \u0026amp; B). Our results showed an immediate, significant decrease in R123 accumulation (indicating increased efflux) starting at HH34 for the Forebrain and HH36 for the OT (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). In both regions, the increase in efflux activity was statistically significant between HH34 and HH38 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE and F). While R123 levels in the OT reached a minimum at HH40, accumulation in the Forebrain continued to decrease until HH42 (Figure S7). Notably, at late stages (HH40\u0026ndash;HH44), R123 concentration was significantly higher in the OT than in the Forebrain (Figure S7A).\u003c/p\u003e \u003cp\u003eTaken together, these results define a critical developmental window where gene expression, tight junction assembly, and functional properties appear all together shaping \u003cem\u003eGallus gallus\u003c/em\u003e BBB phenotype.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eDevelopmental stages HH36 to HH40 constitutes a critical time window for avian BBB development\u003c/h2\u003e \u003cp\u003eThe endothelial BBB is a conserved feature of vertebrates, yet the precise timeline of its molecular and functional onset in non-mammalian models has remained unclear. To our knowledge, this is the first study to integrate transcriptional data, 3D structural analysis, and quantitative functional assays to study BBB development in the \u003cem\u003eGallus gallus\u003c/em\u003e OT. Our primary finding is the identification of a discrete \"critical window\" between HH36 and HH40 (E10\u0026ndash;E14), during which the avian BBB undergoes a rapid, synchronized transition from a leaky vascular network to a highly selective barrier. This redefines the chicken embryo not just as a historical model, but as a precise tool for investigating the molecular regulation of barrier-genesis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eAngiogenic Signaling and the Shift to BBB specialization\u003c/h2\u003e \u003cp\u003eAngiogenesis and barrier-genesis are often viewed as distinct sequential phases. Our transcriptional data supports this uncoupling in the avian OT. We observed that \u003cem\u003eHIF1A\u003c/em\u003e and \u003cem\u003eVEGF\u003c/em\u003e expression peaks around HH38\u0026ndash;HH40, correlating with the intense vascular remodeling previously described in the OT \u003csup\u003e6, 26\u003c/sup\u003e. Wnt and SHH pathways have been described to regulate expression of tight junctions\u0026rsquo; proteins and maintenance of BBB properties \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. However, Wnt signaling also peaks early to drive angiogenesis in the mammalian cortex \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. We found that the Wnt target \u003cem\u003eAXIN2\u003c/em\u003e and the barrier-promoting ligand \u003cem\u003eSHH\u003c/em\u003e showed dynamic changes specifically preceding the HH36\u0026ndash;HH40 window, around the same stages that \u003cem\u003eCLDN5\u003c/em\u003e and \u003cem\u003eOCLN\u003c/em\u003e start to increase their expression.\u003c/p\u003e \u003cp\u003eThe decline in SHH expression after HH32 suggests that while it may promote angiogenesis, its downregulation coincides with the shift towards stabilization. This contrasts with mammalian models where SHH continues to promote immune quiescence in the adult BBB \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. The synchronization of these signaling shifts with the sharp downregulation of the fenestration associated gene \u003cem\u003ePLVAP\u003c/em\u003e at HH36 provides strong molecular evidence that the \"vascular leakiness\" observed in early stages is an actively regulated phenotype, not merely the absence of a barrier.\u003c/p\u003e \u003cp\u003eThis shift also aligns with changes in barrier permeability through development. Previously, Roncali et al 1985 described OT vasculogenesis process and found a clear increase of denseness of the radial vessels in the ventrolateral region of the tectum until HH34, which coincides with the considerable increment in EB permeability at the same stage found in our study. Then the percent of radial vessels is maintained from HH34 to HH40, but EB permeability keeps decreasing, showing the acquisition of BBB properties do not come only from lower angiogenic rates but the change towards an specialization of the existing vasculature.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eTemporal coordination of tight junction molecular assembly\u003c/h2\u003e \u003cp\u003eOur data suggests a complex interplay between transcriptional changes and protein distribution. While \u003cem\u003eCLDN5\u003c/em\u003e and \u003cem\u003eOCLN\u003c/em\u003e mRNA levels rise starting at HH36, the distribution of ZO-1 protein reveals that structural sealing is a progressive process. The transition of ZO-1 from discontinuous foci at HH36 to linear, continuous strands by HH40 mirrors the decrease in extravasation of dextran we observed in our 3D lightsheet imaging, suggesting that the paracellular barrier formation has an inflection point between HH38 and HH40 stages for molecules about 10 kDa in size.\u003c/p\u003e \u003cp\u003eTight junction assembly involves recruitment of different proteins into the nascent junctional complex, being ZO-1 essential for this process \u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. ZO-1 proteins exist in phase separation compartments near the membrane, which sequester and locally concentrate tight junction proteins \u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. Deeper studies on the dynamic of tight junction assembly during BBB development are needed in order to establish more precisely the interdependence and temporality between gene expression, protein expression and protein interaction processes in \u003cem\u003eGallus gallus\u003c/em\u003e model.\u003c/p\u003e \u003cp\u003eThe developmental timeline found aligns remarkably well with historical dye-exclusion studies and previous reports about BBB markers such as alkaline phosphatase and transferrin receptor \u003csup\u003e\u003cspan additionalcitationids=\"CR21\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e but adds necessary molecular resolution. Importantly, we show that this window coincides with the onset of astrogenesis in the chicken brain \u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e. Unlike mice, where the BBB forms prior to astrocyte differentiation \u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e, the avian BBB properties appear concurrently with astrocyte differentiation, an idea that requires further investigation. This positions the chicken embryo as a unique model to dissect the specific contribution of astrocyte-endothelial interactions to barrier formation during embryonic development.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eTransport regulation across BBB develops gradually in a region-specific manner\u003c/h2\u003e \u003cp\u003eBeyond paracellular transport regulation, the fully developed BBB actively extrudes metabolic waste and xenobiotics via ABC transporters \u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e. Studies in mammals reported expression and activity of ABCB1 to increase dramatically through development, reaching its highest capacity at adult stages \u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan additionalcitationids=\"CR56\" citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e. We found a striking upregulation of \u003cem\u003eABCB1\u003c/em\u003e mRNA (over 40-fold) starting at HH40 and peaking at HH44. However, our functional R123 assay revealed that significant efflux activity is detectable as early as HH36, reaching an apparent plateau at HH42. This could have profound implications for developmental toxicology, suggesting that the chicken embryo acquires protection against chemical insults gradually before the hatching.\u003c/p\u003e \u003cp\u003eRegional heterogeneity was also evident. The forebrain showed higher basal permeability and earlier efflux activation compared to the OT. Our data also suggest that BBB low permeability is acquired at different levels of the vascular tree in different developmental time points. This rostro-caudal gradient supports classical observations and correlates with the findings where angiogenesis in the OT follows a defined pattern across spatial axes \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e and implies that barrier-genesis is not a uniform systemic event but is locally regulated by the specific neurogenic niche, a matter that deserves further investigation.\u003c/p\u003e \u003cp\u003eAdditionally, it is known that EB can bind to serum albumin, increasing its molecular size to 67 kDa, hence an important part of transport of this molecule is also regulated through transcytosis \u003csup\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e. The role of MFSD2A and the diminution of PLVAP expression have been related to downregulation of transcytosis (Ben-Zvi et al 2014) \u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. In our study \u003cem\u003eMFSD2A\u003c/em\u003e shows a significant increase while \u003cem\u003ePLVAP\u003c/em\u003e decreases, around the same time where EB permeability declines. Still, it would be fundamental to explore other molecular changes associated and how this type of transport develops in future studies.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eFuture Directions and Clinical Relevance\u003c/h2\u003e \u003cp\u003eBy establishing HH36\u0026ndash;HH40 as a highly relevant window for BBB development, our work opens several avenues for future research. First, the accessibility of the chicken embryo allows researchers to make use of the described time window, joining recent works that brought the chicken embryo to the spotlight of biomedical research \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e, \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e. Future studies can now investigate whether environmental stressors (e.g., hypoxia, hyperglycemia) applied during this specific window induce long-term barrier defects, providing a model for gestational neurovascular pathology.\u003c/p\u003e \u003cp\u003eSecond, the robust P-glycoprotein activity identified here validates the chick embryo as a cost-effective screening platform for CNS drug delivery. The clear separation between the \u0026ldquo;open\u0026rdquo; (pre-HH36) and \"tight\" (post-HH40) phases allows researchers to distinguish between passive diffusion and active transport mechanisms for novel therapeutics. Furthermore, while our transcriptional analysis identified key transporters, future proteomic characterization of the avian endothelial surface could uncover unique variants or evolutionarily conserved targets (such as the CD98 complex) that may be overlooked in rodent models \u003csup\u003e\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e, \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eFinally, our data demonstrates that the developing OT offers a new screening platform that complements the traditional CAM assay for neurovascular research. While the CAM assays is ubiquitous for evaluating anti-angiogenic drugs and tumor xenografts due to its accessibility and vascular density \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e, \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e\u003c/sup\u003e, it remains an extra-embryonic tissue lacking the complex glial interplay that defines the NVU nor the specific barrier phenotype. Unlike the CAM, the embryonic brain between HH36 and HH40 actively recruits astrocytes, forming a true NVU with the microenvironment encountered by CNS-targeting therapeutics. By shifting focus from the CAM to the embryo, researchers can now screen compounds not just for vascular toxicity, but for specific BBB penetrance and efflux pump evasion in a physiologically intact CNS microenvironment.\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn summary, we demonstrate that \u003cem\u003eGallus gallus\u003c/em\u003e BBB development occurs during an inflection point between stages HH36 and HH40. This window represents the convergence of molecular patterns, tight junction assembly, and the onset of active efflux. These findings not only update the biology of the avian BBB but also establish the chicken embryo as a rigorously validated model for investigating neurovascular development, disease, and CNS drug transport.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eAARS1\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eAlanyl-tRNA Synthetase 1\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eABCB1\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eATP binding cassette subfamily B member 1\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eACTB\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eActin beta\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eATP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eAdenosine Triphosphate\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eAXIN2\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eAxis Inhibition protein 2\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eBBB\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eBlood Brain Barrier\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eBSG\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eBasigin\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCAM\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eChorioallantoic Membrane\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ecDNA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ecomplementary Deoxyribonucleic acid\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCUBIC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eClear Unobstructed Brain Imaging Cocktails\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCLDN5\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eClaudin 5\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCNS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eCentral Nervous System\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eEB\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eEvans Blue\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eFITC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eFluorescein IsoTioCyanate\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eGLUT-1\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eGlucose Transporter 1\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHH\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eHamburger-Hamilton stage\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHIF\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eHypoxia-Inducible Factor\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHIF1A\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eHypoxia-Inducible Factor 1\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHRP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eHorseradish Peroxidase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eLAT1\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eLarge neutral Amino acid Transporter 1\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMFSD2A\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMajor Facilitator Superfamily Domain-Containing protein 2\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNVU\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eNeurovascular Unit\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eOCLN\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eOccludin\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eON\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eOvernight\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eOT\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eOptic Tectum\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePBS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePhosphate-Buffered Saline\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePFA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eParaformaldehyde\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePGP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eP-glycoprotein\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePLVAP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePlasmalemma Vesicle-Associated Protein\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eqPCR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003equantitative Polymerase Chain Reaction\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eR123\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eRhodamine 123\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eRNA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eRibonucleic acid\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eRT\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eRoom Temperature\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSHH\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eSonic Hedgehog\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSLC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eSolute Carrier Transporter\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eVEGF\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eVascular Endothelial Growth Factor\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eWGA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eWheat Germ Agglutinin\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eZO-1\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eZonula Occludens 1\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":" \u003cp\u003e \u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e \u003cp\u003eProtocols for animal experiments were approved by the Animal Experimental Ethics Committee of Faculty of Sciences, Universidad de Chile and ANID (Certificate no. 22571\u0026ndash;FCS\u0026ndash;UCH-e1) on January 27, 2021, in compliance with the Bioethics committee of Universidad de Chile and ANID, for the care and use of laboratory animals.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConsent for publication\u003c/strong\u003e \u003cp\u003eNot applicable\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eCompeting interests\u003c/h2\u003e \u003cp\u003eThe author(s) declare no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eFunding from ANID Fondecyt Regular #1221522 (VP), ANID MSc scholarship #22240623 (MJG-M) and ANID PhD scholarship #21242090 (JJ-B).\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eSupplementary Figures.\u003c/h2\u003e \u003cp\u003eAdditional Figures referenced in the main manuscript.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eJJ-B, MJG-M and VP conceived and designed the experiments. JJ-B, MJG-M, BR-B and IC-M performed the experiments and/or contributed to data acquisition. JJ-B, MJG-M and VP analyzed and interpreted the data. JJ-B and MJG-M wrote the original article. BR-B and IC-M critically revised and edited the article. VP provided resources and was responsible for general supervision, review, and editing. All authors approved the final version of the article.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eWe thank Dr. Joao Bothelo for kindly donating WGA-lectin and to give access to cryostat equipment. We are grateful to Dr. An\u0026iacute;bal Vargas and David Arancibia from the LiSIUM project for technical assistance with lightsheet images acquisition.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe datasets supporting the conclusions of this article are available in the Zenodo repository: https://zenodo.org/records/18672360\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eIadecola C. The Neurovascular Unit Coming of Age: A Journey through Neurovascular Coupling in Health and Disease. Neuron. 2017;96:17\u0026ndash;42.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eObermeier B, Daneman R, Ransohoff RM. Development, maintenance and disruption of the blood-brain barrier. Nat Med. 2013;19:1584\u0026ndash;96.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDaneman R, Prat A. The Blood\u0026ndash;Brain Barrier. Cold Spring Harb Perspect Biol. 2015;7:a020412.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLangen UH, Ayloo S, Gu C. Development and Cell Biology of the Blood-Brain Barrier. Annu Rev Cell Dev Biol. 2019;35:591\u0026ndash;613.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDunton AD, G\u0026ouml;pel T, Ho DH, Burggren W. Form and Function of the Vertebrate and Invertebrate Blood-Brain Barriers. Int J Mol Sci. 2021;22:12111.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCelin AR, Rapacioli M, Gonzalez MA, Ballarin VL, De Plazas SF, L\u0026oacute;pez-Costa JJ, Flores V. Temporal-Spatial Correlation between Angiogenesis and Corticogenesis in the Developing Chick Optic Tectum. PLoS ONE. 2015;10:e0116343.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHaddad-T\u0026oacute;volli R, Dragano NRV, Ramalho AFS, Velloso LA. Development and Function of the Blood-Brain Barrier in the Context of Metabolic Control. Front Neurosci. 2017;11:224.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHagan N, Ben-Zvi A. The molecular, cellular, and morphological components of blood\u0026ndash;brain barrier development during embryogenesis. Semin Cell Dev Biol. 2015;38:7\u0026ndash;15.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiebner S, Corada M, Bangsow T, Babbage J, Taddei A, Czupalla CJ, et al. Wnt/β-catenin signaling controls development of the blood\u0026ndash;brain barrier. J Cell Biol. 2008;183:409\u0026ndash;17.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDaneman R, Agalliu D, Zhou L, Kuhnert F, Kuo CJ, Barres BA, Kuo CJ, Barres BA. Wnt/b-catenin signaling is required for CNS, but not non-CNS, angiogenesis. Proc Natl Acad Sci USA. 2009;106:641\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhou Y, Wang Y, Tischfield M, Williams J, Smallwood PM, Rattner A, Taketo MM, Nathans J. 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Proc Natl Acad Sci USA. 2021;118:e2017779118.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCserr HF, Bundgaard M. Blood-brain interfaces in vertebrates: a comparative approach. Am J Physiol Regul Integr Comp Physiol. 1984;246:R277\u0026ndash;88.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRibatti D, Nico B, Crivellato E, Artico M. Development of the blood-brain barrier: A historical point of view. Anat Rec B New Anat. 2006;289B:3\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRapacioli M, Palma V, Flores V. Morphogenetic and Histogenetic Roles of the Temporal-Spatial Organization of Cell Proliferation in the Vertebrate Corticogenesis as Revealed by Inter-specific Analyses of the Optic Tectum Cortex Development. Front Cell Neurosci. 2016;10:67.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHerbert SP, Stainier DYR. Molecular control of endothelial cell behaviour during blood vessel morphogenesis. Nat Rev Mol Cell Biol. 2011;12:551\u0026ndash;64.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePouncey L, Mok GF. Unravelling early hematoendothelial development through the chick model: Insights and future perspectives. Dev Bio. 2025;523:20\u0026ndash;31.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWakai S, Hirokawa N. Development of the blood-brain barrier to horseradish peroxidase in the chick embryo. Cell Tissue Res. 1978;195(2):195\u0026ndash;203.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eStewart PA, Wiley MJ. Structural and histochemical features of the avian blood-brain barrier. J Comp Neurol. 1981;202:157\u0026ndash;67.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRisau W, Hallmann R, Albrecht U. Differentiation-dependent expression of proteins in brain endothelium during development of the blood-brain barrier. Dev Bio. 1986;117:537\u0026ndash;45.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRoncali L, Nico B, Ribatti D, Bertossi M, Mancini L. Microscopical and ultrastructural investigations on the development of the blood-brain barrier in the chick embryo optic tectum. Acta Neuropathol. 1986;70:193\u0026ndash;201.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNico B, Quondamatteo F, Ribatti D, Bertossi M, Russo G, Herken R. Roncali L Ultrastructural localization of lectin binding sites in the developing brain microvasculature. Anat Embryol (Berl). 1998;197:305\u0026ndash;15.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLaVail JH, Maxwell Cowan W. The development of the chick optic tectum. I. normal morphology and cytoarchitectonic development. Brain Res. 1971;28:391\u0026ndash;419.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRoncali L, Ribatti D, Ambrosi G. Vasculogenesis in the Chick Embryo Optic Tectum. Cells Tissues Organs. 1985;122:229\u0026ndash;34.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHamburger V, Hamilton. HL A series of normal stages in the development of the chick embryo. J Morphol. 1951;88:49\u0026ndash;92.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang Q, Zheng M, Betancourt CE, Liu L, Sitikov A, Sladojevic N, Zhao Q, Zhang JH, Liao JK, Wu R. Increase in Blood-Brain Barrier (BBB) Permeability Is Regulated by MMP3 via the ERK Signaling Pathway. Oxid Med Cell Longev. 2021; 2021:6655122.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen L, Wang S, Feng Y, Zhang J, Du Y, Zhang J, Ongeval CV, Ni Y, Li Y. Utilisation of Chick Embryo Chorioallantoic Membrane as a Model Platform for Imaging-Navigated Biomedical Research. Cells. 2021;10:463.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJilani SM, Murphy TJ, Thai SNM, Eichmann A, Alva JA. 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Ann Anat. 1993;175:85\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJho E, Zhang T, Domon C, Joo C-K, Freund J-N, Costantini F. -Catenin/Tcf Signaling Induces the Transcription of Axin2, a Negative Regulator of the Signaling Pathway. Mol Cell Biol. 2002;22(4):1172\u0026ndash;83.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePugh CW, Ratcliffe PJ. Regulation of angiogenesis by hypoxia: role of the HIF system. Nat Med. 2003;9:677\u0026ndash;84.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFan X, Heijnen CJ, Van Der Kooij MA, Groenendaal F, Van Bel F. The role and regulation of hypoxia-inducible factor-1α expression in brain development and neonatal hypoxic\u0026ndash;ischemic brain injury. Brain Res Rev. 2009;62:99\u0026ndash;108.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eArgaw AT, Gurfein BT, Zhang Y, Zameer A. John GR VEGF-mediated disruption of endothelial CLN-5 promotes blood-brain barrier breakdown. 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J Pharmacol Toxicol Methods. 2026;137:108410.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSingh N, Ecker GF. Insights into the Structure, Function, and Ligand Discovery of the Large Neutral Amino Acid Transporter 1, LAT1. Int J Mol Sci. 2018;19:1278.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChew KS, Wells RC, Moshkforoush A, Chan D, Lechtenberg KJ, Tran HL, et al. CD98hc is a target for brain delivery of biotherapeutics. Nat Commun. 2023;14:5053.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYuan Y-J, Xu K, Wu W, Luo Q, Yu J-L. Application of the Chick Embryo Chorioallantoic Membrane in Neurosurgery Disease. Int J Med Sci. 2014;11:1275\u0026ndash;81.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRibatti D. The chick embryo chorioallantoic membrane (CAM) assay. Reprod Toxicol. 2017;70:97\u0026ndash;101.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"fluids-and-barriers-of-the-cns","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"fbcn","sideBox":"Learn more about [Fluids and Barriers of the CNS](http://fluidsbarrierscns.biomedcentral.com/)","snPcode":"12987","submissionUrl":"https://submission.nature.com/new-submission/12987/3","title":"Fluids and Barriers of the CNS","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Blood-Brain Barrier, Chicken embryo, Barrier-genesis, Optic tectum, Permeability, ABC Transporters, Tight junctions","lastPublishedDoi":"10.21203/rs.3.rs-8911082/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8911082/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eThe cerebrovascular endothelium possesses unique properties that strictly regulate the neural microenvironment. While mammalian Blood Brain Barrier (BBB) development is well-characterized, the avian embryo (\u003cem\u003eGallus gallus\u003c/em\u003e) offers a unique, highly accessible model for real-time developmental imaging and manipulation. However, the precise molecular and functional timeline of BBB maturation in the avian brain remains poorly explored. Here, we provide the first comprehensive spatiotemporal characterization of BBB development in the \u003cem\u003eGallus gallus\u003c/em\u003e optic tectum (OT).\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eWe performed a multi-modal analysis between developmental stages HH32 and HH44, combining transcriptional analysis of BBB-associated genes, confocal and lightsheet microscopy, and quantitative functional assays for permeability and efflux activity.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eWe identified a critical developmental window between stages HH36 and HH40 that marks the transition to a specialized barrier. This period is characterized by the significant upregulation of tight junction proteins (\u003cem\u003eCLDN5, OCLN\u003c/em\u003e) and efflux transporters (\u003cem\u003eABCB1\u003c/em\u003e), accompanied by the downregulation of fenestration markers (\u003cem\u003ePLVAP\u003c/em\u003e). Also, tight junction assembly protein ZO-1 undergoes a progressive redistribution from a discontinuous pattern to continuous linear expression along the vessel walls during this period. Functionally, we demonstrate that paracellular restriction to 10 kDa dextran develops in a region-dependent manner, while quantitative assays reveal a sharp reduction in Evans Blue permeability starting at HH38. Furthermore, we detected the onset of P-glycoprotein efflux activity from HH36 onwards.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eAltogether, our findings redefine the chicken embryo as a tractable model for barrier genesis research, pinpointing the HH36-40 developmental window as the discrete period where the molecular and functional BBB properties are acquired. This establishes a robust baseline for future mechanistic investigations into neurovascular development and pathology.\u003c/p\u003e","manuscriptTitle":"Spatiotemporal arising of molecular and functional Blood-Brain Barrier properties in the developing Gallus gallus optic tectum","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-02 08:43:07","doi":"10.21203/rs.3.rs-8911082/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-05-12T19:03:02+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-12T18:45:55+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-14T15:19:21+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"314053620673254142511975747556985595809","date":"2026-04-14T10:38:02+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"296978039006919599341274562021242881122","date":"2026-04-07T10:39:36+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-02-24T15:17:35+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-02-22T13:55:57+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-02-21T07:16:02+00:00","index":"","fulltext":""},{"type":"submitted","content":"Fluids and Barriers of the CNS","date":"2026-02-18T16:41:23+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"fluids-and-barriers-of-the-cns","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"fbcn","sideBox":"Learn more about [Fluids and Barriers of the CNS](http://fluidsbarrierscns.biomedcentral.com/)","snPcode":"12987","submissionUrl":"https://submission.nature.com/new-submission/12987/3","title":"Fluids and Barriers of the CNS","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"f459f31e-018a-4eb5-aa4f-659c56221b35","owner":[],"postedDate":"March 2nd, 2026","published":true,"recentEditorialEvents":[{"type":"decision","content":"Revision requested","date":"2026-05-12T19:03:02+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-12T18:45:55+00:00","index":22,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[],"tags":[],"updatedAt":"2026-05-12T19:09:06+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-02 08:43:07","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8911082","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8911082","identity":"rs-8911082","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}