Compartmentalized VEGF Receptor Expression in Hypothalamic Tanycytes Reveals a Novel Non- Endothelial Axis of VEGF Signaling (Tanycytes as a Novel Non-Endothelial Target of VEGF Signaling)

Compartmentalized VEGF Receptor Expression in Hypothalamic Tanycytes Reveals a Novel Non- Endothelial Axis of VEGF Signaling (Tanycytes as a Novel Non-Endothelial Target of VEGF Signaling) | 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 Compartmentalized VEGF Receptor Expression in Hypothalamic Tanycytes Reveals a Novel Non- Endothelial Axis of VEGF Signaling (Tanycytes as a Novel Non-Endothelial Target of VEGF Signaling) Ombeline Desruelle, Manon Leclerc, Sreekala Nampoothiri, Daniela Fernandois, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7878186/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 14 Feb, 2026 Read the published version in Fluids and Barriers of the CNS → Version 1 posted 9 You are reading this latest preprint version Abstract Background: Vascular endothelial growth factors (VEGFs) and their receptors (VEGFRs) are critical regulators of angiogenesis and vascular homeostasis. While VEGF signaling has been extensively studied in endothelial cells, emerging evidence suggests it also plays roles in non-endothelial brain cells. However, its spatial and cell-type-specific function within the hypothalamus, and more specifically at the level of the blood/CSF barrier remains poorly defined. In particular, little is known about VEGF receptor expression in tanycytes, a specialized glial population that lines the third ventricle and regulates body-brain communication within the median eminence (ME), a key neurovascular interface located at the tuberal region of the hypothalamus. Methods: We used a multi-modal approach including single-cell RNA sequencing (scRNA-seq) reanalysis, RNAscope in situ hybridization, immunohistochemistry, FACS-isolated qPCR in male and female mice, and human spatial transcriptomics to map the expression of VEGFR1 ( Flt1 ), VEGFR2 ( Kdr ), and VEGF ligands in hypothalamic tanycytes across gender, development and aging. Results: Our data reveal a striking spatial compartmentalization of VEGFR expression in tanycytes within the ME and the arcuate (ARH), ventromedial (VMH) and dorsomedial (DMH) hypothalamus. VEGFR2 is selectively expressed in ARH-tanycytes, while VEGFR1 is confined to VMH/DMH-tanycytes; and none of these receptors are expressed in ME-tanycytes. This pattern is unique to the ME and not observed in other circumventricular organs. VEGFR1 expression is established neonatally in mice (P0) and remains stable throughout life, whereas VEGFR2 expression becomes progressively refined postnatally, localizing to ARH-tanycytes in adulthood and showing a significant decline with aging. VEGFA is broadly expressed in all hypothalamic tanycytes, including ME-tanycytes, supporting a paracrine model of signaling. Importantly, in human hypothalamic tissue, VEGFR2, but not VEGFR1, is expressed in tanycytes, suggesting a partial evolutionary conservation. Conclusion: Our findings unveil for the first time, a non-endothelial VEGF signaling system in hypothalamic tanycytes that is spatially compartmentalized, developmentally programmed and age-dependant. These insights reveal new roles for VEGF signaling in neurovascular and neuroendocrine function, raising important considerations for central effects of VEGF-targeted therapies in aging and disease. Tanycytes VEGF signaling Median eminence Neurovascular interface Hypothalamus Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 INTRODUCTION Vascular endothelial growth factor receptors (VEGFRs) are membrane-bound receptor tyrosine kinases essential for angiogenesis, vascular permeability, lymphangiogenesis, and the regulation of cell proliferation and differentiation. The VEGFR family comprises three main receptors—VEGFR1 ( Flt-1 ), VEGFR2 ( Kdr ), and VEGFR3 ( Flt-4 )—which interact with five structurally related VEGF ligands (VEGFA, VEGFB, VEGFC, VEGFD, and placental growth factor [PlGF]). In addition, VEGFs bind with high affinity to neuropilin co-receptors (NRP1 and NRP2) and to heparan sulfate proteoglycans (HSPGs) 1 . Each ligand–receptor interaction activates distinct signaling pathways that are essential for vascular and lymphatic system development and maintenance 1 , 2 . However, although historically associated with vascular biology, VEGF/VEGFR signaling also plays diverse roles within the central nervous system (CNS), contributing to blood–brain barrier (BBB) regulation, neurogenesis, synaptic plasticity, and astrocyte function 2 – 4 . Among the VEGFRs, VEGFR1 has an affinity for VEGFA and is involved in monocyte migration and hematopoiesis. This receptor also acts as a decoy receptor to control the availability of VEGF ligands and is expressed on the surface of monocytes, macrophages, and endothelial cells. VEGFR2 is a critical regulator of VEGF induced angiogenesis, and it is predominantly localized on the surface of endothelial cells, facilitating key signaling processes essential for vascular development. This receptor has a strong affinity for VEGFA and promotes endothelial cell migration, proliferation, differentiation, and survival. On the other hand, VEGFR3 is mainly expressed in lymphatic endothelial cells exhibiting a strong affinity for VEGFC and VEGFD 1 . Importantly, both VEGFR1 and VEGFR2 are also expressed in non-endothelial cells 4 – 6 including neurons and glia, which can produce and respond to VEGFs 6 – 8 . Despite their increasing non-endothelial central roles in the brain, the spatial and cell-type-specific expression of VEGFRs in the healthy brain remains poorly characterized. One region of particular interest in the study of VEGFRs is the median eminence (ME) at the tuberal hypothalamus, a circumventricular organ (CVO) that serves as a critical interface for neuroendocrine regulation. Specifically, the pituitary portal blood vessels in the ME contain endothelial cells that are fenestrated and lack blood-brain barrier properties, allowing circulating molecules to freely diffuse into the brain parenchyma. At this specific location, the blood-barrier functions are instead mediated by tanycytes, which are specialised ependymoglial cells lining the walls and floor of the third ventricle 9 , 10 . Tanycytes form a tight junction-based blood–cerebrospinal fluid (CSF) barrier, also known as the tanycytic barrier, and extend long processes that contact both fenestrated vessels in the ME and BBB-protected vessels of adjacent hypothalamic nuclei 11 , 12 . Their strategic positioning and contact with different nuclei of the hypothalamus enable them to regulate neurovascular exchange, sense peripheral metabolic cues, and control the entry of circulating signals into hypothalamic neural circuits 13 – 17 (Fig. 1 A). Tanycytes are a heterogeneous population; they have been historically classified into distinct subtypes (α1, α2, β1, and β2) based on their dorsoventral location, molecular identity, and anatomical projections 9 . These different subtypes are believed to play specialized roles in barrier regulation, hormone transport, neurogenesis, and metabolic signaling 9 , 18 . Despite this functional heterogeneity, our understanding of how VEGF signaling pathways are differentially distributed across tanycyte subtypes remains limited. A few studies from us and others have reported that VEGF expression in the hypothalamus could change in response to metabolic changes 6 , 7 , increasing ME’s permeability and access of circulating cues in the area. However, it remains unclear whether and which tanycytic populations express VEGFRs. Additionally, the role of VEGF signaling in tanycyte physiology or neuroendocrine function is unknown. This knowledge gap is of particularly relevant in light of the widespread clinical use of anti-angiogenic therapies, including VEGF-neutralizing antibodies and VEGFR2 inhibitors, in conditions ranging from cancer to age-related macular degeneration 19 – 21 . While these drugs are designed to target peripheral vasculature, their potential effects on VEGF-sensitive glial cells in the brain have not been systematically addressed. In this study, we combined single-cell RNA sequencing (scRNA-seq), fluorescent in situ hybridization, and quantitative PCR to map the expression of VEGFR1, VEGFR2, and VEGF ligands across tanycyte subtypes in the ME of mouse and human brain. Our findings uncover a non-endothelial VEGF signaling axis in hypothalamic tanycytes, with distinct spatial and temporal regulation. These insights not only broaden novel roles of VEGF signaling in non-endothelial cells in the brain but also raise far-reaching considerations for the central effects of systemic VEGF-targeted therapies, particularly in the context of aging and metabolic disease in neuroendocrine regulation. RESULTS Spatially distinct VEGFR1/VEGFR2 expression in tanycyte subtypes in males and females To assess the cell-type specificity of Vegfr2 and Vegfr1 expression within the ME, we re-analysed publicly available single-cell RNA sequencing (scRNA-seq) datasets from the adult male mouse hypothalamus 18 . Clustering analysis identified distinct tanycyte subtypes, including β1/β2- and α1/α2-tanycytes, that will be referred to hereafter by their anatomical localization as ME-tanycytes (β2), arcuate nucleus (ARH)-tanycytes (β1, α2), ventromedial hypothalamic (VMH)-tanycytes (α2, α1), and dorsomedial hypothalamic (DMH)-tanycytes (α1) 17 (Fig. 1 and Supp Fig. 1 ). Our re-analysis revealed that both VEGFRs, Vegfr2 (Kdr ) and Vegfr1 (Flt1 ), are differentially enriched across tanycyte subtypes. Vegfr2 expression is primarily restricted to ARH-tanycytes, with minimal to no expression in ME-tanycytes and low expression in VMH and DMH-tanycytes. In contrast, Vegfr1 exhibits a complementary expression pattern, being selectively expressed in VMH/DMH-tanycytes, while largely absent in ME- and ARH-tanycytes. This mutually exclusive expression of Vegfr1 and Vegfr2 was confirmed using a dot plot (Fig. 1 D). Interestingly, the third member of the VEGFR family, Vegfr3 (Flt4 ), was not detected in any tanycytic subpopulations (Fig. 1 B-D and Supp Fig. 1 ). We also examined expression of co-receptors Nrp1 and Nrp2 . Both were expressed across all tanycyte subtypes, although Nrp2 showed relatively lower expression in ME-tanycytes than Nrp1 (Fig. 1 B-D and Supp Fig. 1 ). In parallel, to investigate the potential source and anatomical distribution of VEGF signaling components within the ME, we analysed the expression patterns of VEGF family ligands. VEGFA mRNA was robustly expressed across all tanycytic populations, with particularly high levels in ME-tanycytes (Fig. 1 C-D), which notably lack Vegfr1 and Vegfr2 , and in DMH-tanycytes, which express high levels of Vegfr1 (Fig. 1 C-D). Other VEGF ligands were also evaluated. Vegfb was expressed in all tanycytic populations, while Vegfc showed only low-level expression if any (Fig. 1 C). We validated the scRNA-seq results by performing in-situ-hybridization (RNAscope) analysis of male coronal brain slices. Our results confirm the differential expression of Vegfr1/Vegfr2 and Vegfa across the various tanycytic populations. Vegfr2 mRNA was prominently localized to tanycytes facing the ARH, while Vegfr1 mRNA was restricted to the VMH/DMH-tanycytes along the ventricle wall (Fig. 2 A, Sup Fig. 2A ). Notably, Vegfr1 expression and compartmentalization appeared stronger than that of Vegfr2. Furthermore, we observed overlapping expression of Vegfa and Vegfr1 in regions where Vegfr1 was highly expressed (Fig. 2 B, Supp Fig. 2 B). As expected, based on the sc-RNAseq results, Vegfr3 was not detected in tanycytes ( Supp Fig. 2 C). These spatial patterns were consistent across biological replicates. Signals for Vegfr1, Vegfr2 and Vegfr3 were also detected, as expected according to the single cell sequencing data, in endothelial cells of both fenestrated (very strong signal) and non-fenestrated blood–brain barrier vessels (weaker signal) (Fig. 2 C, Sup Fig. 2A ). These results suggest that VEGFRs and ligand expression are spatially compartmentalized among tanycyte subtypes, with ME-tanycytes potentially acting as signaling hubs despite lacking VEGFR expression themselves. This spatial architecture supports a model of directional, inter-subtype communication and suggests a sophisticated VEGF signaling landscape within the ME. To further characterize the spatial architecture of VEGFR expression along the antero-posterior axis of the ME, which extends over 1.2mm, we performed RNAscope across serial coronal sections spanning from the anterior to posterior ME (Bregma − 1.34 to Bregma − 2.54), creating a detailed anatomical expression atlas ( Supp Fig. 3 – 4 ). This analysis revealed that the compartmentalization of Vegfr1 and Vegfr2 in tanycytes is most clearly defined in the central portion of the ME, where the spatial segregation between ARH- and VMH/DMH-tanycytes is sharply maintained. In contrast, the anterior and posterior ME regions displayed more overlapping expression domains, with some co-localization of Vegfr1 and Vegfr2 mRNA in tanycytes. This rostro-caudal gradient highlights additional heterogeneity within the ME and suggests that VEGFR-mediated signaling may be differentially modulated along its longitudinal axis ( Supp Fig. 3 – 4 ). Protein expression was further confirmed to complete our characterization using immunofluorescence. VEGFR1 and VEGFR2 proteins were detected at the level of the tanycytic cell body, facing the ventricular lumen, but not in the processes. VEGFR2 protein is localized to ARH tanycytes and VEGFR1 protein is detected mainly at the level of VMH/DMH tanycytes (Fig. 2 D). However, their expression levels were substantially lower in tanycytes compared to the strong vascular expression observed in the surrounding vasculature (Fig. 2 C, Supp Fig. 2 D). Considering that the VEGFR family can be modulated by estrogens 22 , 23 , and estradiol can influence VEGFR2 expression by stimulating angiogenesis through VEGFR2 upregulation 23 , we next investigated whether the observed compartmentalization pattern is conserved in female mice (Fig. 3 ). In situ hybridization (RNAscope) on female mice brain sections at estrus and proestrus confirmed the receptor compartmentalization observed in males ( Vegfr2 at the level of the ARH and Vegfr1 in VMH/DMH tanycytes) (Fig. 3 A, Supp Fig. 4 ). We observed a reduced spatial overlap between Vegfr1 and Vegfa expression domains in females, suggesting sex-specific differences in ligand-receptor interactions or signaling microenvironments. To better analyse the levels of Vegfr2 in female tanycytes at the different stages of the estrus cycle we used a qPCR-based approach in isolated tanycytes. R26-tdTomato female mice were stereotaxically injected with AAV1/2-Dio2-Cre into the lateral ventricle to drive Cre-dependent tdTomato expression in tanycytes 24 (Fig. 3 B). Two weeks post-injection, animals were sacrificed at defined stages of the estrous cycle (estrus, proestrus and diestrus), and Tomato + tanycytes were isolated by fluorescence-activated cell sorting (FACS). qPCR analysis was then performed to assess Vegfr2 mRNA expression. The qPCR results revealed no significant fluctuations in Vegfr2 expression in tanycytes during the estrous cycle (Fig. 3 B) VEGF receptor expression patterns are specific to median eminence tanycytes All the circumventricular organs (CVOs) but one, the subcommisural organ (SCO), are characterized by the presence of a fenestrated endothelium, and tanycyte-like cells forming a barrier (blood-cerebrospinal fluid barrier) 13 , 25 . Each CVO is specialized in distinct physiological functions, raising the important question of whether the VEGFR expression patterns identified in the ME are conserved across other CVOs containing tanycyte-like cells. We extended our analysis to include the organum vasculosum of the lamina terminalis (OVLT), subfornical organ (SFO), area postrema (AP), and subcommissural organ (SCO). We also included the choroid plexus (CP) in our analysis, given its epithelial specialization and high secretory activity, despite it not being a CVO in the strict sense 26 . Using in situ hybridization, we evaluated the expression on coronal brain sections, of Vegfr1 (Flt1), Vegfr2 (Kdr) , and Vegfa , alongside collagen IV immunostaining as a marker of vascular extracellular matrix to distinguish vascular from extravascular expression. Surprisingly, neither Vegfr1 nor Vegfr2 was detected in tanycyte-like cells of the OVLT, SFO, SCO, or AP. As expected, endothelial expression of both receptors was evident in blood vessels across all CVOs confirmed by co-localization with collagen IV, validating the specificity of the RNAscope probes (Fig. 4 A-B). We observed, however, low Vegfr2 expression in epithelial cells of the choroid plexus (Fig. 4 A-B) On the other hand, Vegfa was robustly expressed in ventricular and parenchymal cells of both the SFO and OVLT, suggesting that ligand availability alone is not sufficient to drive receptor expression in tanycytes. Additionally, we observed strong Vegfa expression in the CP, consistent with previous reports 27 , 28 (Fig. 5 A-B). These findings suggest that the coordinated, subtype-specific expression of VEGFA, VEGFR1, and VEGFR2 in tanycytes is a unique feature of the ME, likely reflecting the specialized neurovascular and neuroendocrine functions of this region. This regional specificity supports the notion that tanycyte-like populations are molecularly and functionally heterogeneous across CVOs, and that VEGF signaling plays a particularly specialized role in ME tanycyte-mediated vascular and endocrine regulation. Developmental refinement of VEGF receptor compartmentalization Electron microscopy studies from the 1970s demonstrated that the external zone of the ME begins to form during late embryogenesis and continues to mature throughout the first postnatal weeks 29 – 31 In mice, capillary loops of the fenestrated vessels only penetrate the ME after birth, first appearing towards the end of the first postnatal week and becoming more numerous during the second week 31 . In parallel, tanycytes undergo progressive postnatal differentiation starting around postnatal day 4 (P4) and continuing through P14, with full transcriptional maturity typically reached between P14 and P20 32,33 . Given this temporal window of structural and cellular maturation, we wondered whether VEGFR expression is also acquired postnatally. To address this, we investigated the developmental timing of VEGFR expression and compartmentalization within the ME. Specifically we performed and quantified RNAscope for Vegfr1, Vegfr2 , and Vegfa in coronal brain sections at different mouse postnatal stages (P0, P4, P8, P12, P16, and P21 just before puberty onset), and compared them to the adult mouse brain (3 months old). (Figs. 6 and 7 ). We observed that both, Vegfr1 and Vegfr2 are already excluded from ME tanycytes at P0. Moreover, Vegfr1 mRNA was already robustly expressed in VMH/DMH-tanycytes at P0, and this expression remained spatially restricted throughout postnatal development, indicating early and stable compartmentalization of Vegfr1 . In contrast, the developmental trajectory of Vegfr2 was slightly different; Vegfr2 is expressed in ARH and VMH/DMH tanycytes at P0, indicating an early-established exclusion from the ME tanycyte subtype, however, the preferential expression of Vegfr2 in ARH-tanycytes became more evident in adulthood. This coincided with a slight downregulation of its expression in VMH/DMH-tanycytes of adult mice (Fig. 7 B). Thus, the full spatial segregation of Vegfr2 among tanycytes subtypes appears to be a postnatal refinement process. As expected, Vegfr1 and Vegfr2 expression in vasculature was detectable from P0, as confirmed by their presence in vascular structures throughout development (Fig. 6 , white arrows). Regarding the ligand, Vegfa mRNA was present in all tanycytes from P0, with a higher expression in ME and VMH/DMH tanycytes starting from P4 (Fig. 6 , blue arrows). Notably, in P8 mice, we identified discrete clusters of cells expressing high levels of Vegfa in the ME parenchyma, suggesting either local signaling shifts or cellular recruitment/remodelling events during this critical period (indicated in Fig. 6 with red arrows). Our findings suggest that Vegfa expression is established early during postnatal development, preceding the full maturation of VEGFR2 compartmentalization. This temporal sequence supports a model in which VEGF ligand availability primes the microenvironment, while receptor expression is dynamically regulated to fine-tune signaling specificity during early postnatal remodelling of the ME. To contextualize these transcriptional changes with morphological development, we performed vimentin immunofluorescence to visualize tanycyte architecture 12 at the same developmental stages. We observed that at early postnatal timepoints (P0–P4), tanycytes in the ME lacked the characteristic branched structure shown in adult animals. Beginning at P8, we observed the presence of branched tanycytic end feet in close apposition to the fenestrated capillaries of the ME, while a single basal process became evident in other tanycyte populations ( Supp Fig. 6 ). This morphological maturation aligns with previous observations 33 , and supports the idea that VEGFR dynamics may be linked to tanycyte specialization and vascular interaction. Age-dependent regulation of VEGF receptor expression The VEGF system undergoes an age-related decline in signaling efficiency, resulting in reduced angiogenesis, impaired tissue regeneration, and increased susceptibility to age-associated diseases 34 . Dysregulation of this pathway has also been implicated in neurodegenerative disorders such as Alzheimer’s disease, where altered VEGFA signaling contributes to neuroinflammation, vascular dysfunction, and cognitive decline 35 – 37 . In light of these findings, we sought to explore whether VEGFR signaling in tanycytes is similarly regulated across lifespan. To this end, we conducted qPCR analysis on FACS-isolated tomato⁺ tanycytes from microdissected ME explants from male mice at 3, 6, 12, and 18 months of age. Using our AAV approach, we specifically label cells lining the 3V, including all tanycytic subtypes 14 ( Supp Fig. 7 ). Our qPCR results revealed that, as in blood vessels, Vegfr2 (Kdr ) expression is aged-regulated, with the highest levels at 3 months of age, corresponding to early adulthood, followed by an abrupt decline starting from 6 months of age (Fig. 8 A, Supp Fig. 7 ). This age-related decrease suggests that VEGFR2-mediated signaling becomes attenuated in tanycytes during aging, which could impact their interactions with the vasculature or influence their neuroendocrine regulatory functions. In contrast, Vegfr1 (Flt1) expression remained stable throughout aging, with a slight increase by 18 months of age, indicating that VEGFR1 may serve a more constitutive or maintenance-related function in tanycytes that is less sensitive to age-related cues. As in previous experiments, Vegfr3 (Flt4) was barely detected in tanycytes at any age, reinforcing the conclusion that VEGFR3 is not an essential component of tanycyte-VEGF signaling, even under potential age-induced stress or remodelling. We next assessed expression of VEGF ligands (Fig. 8 B), which are critical for autocrine and paracrine signaling. Vegfa exhibited relatively stable expression across all time points, with a modest increase in older mice, potentially reflecting a compensatory response to declining receptor levels or vascular alterations with age. Vegfc , the main ligand for VEGFR3 , remained largely unchanged. Strikingly, Vegfb expression showed a robust and significant increase from 3 to 18 months, suggesting that VEGFB–VEGFR1 signaling may become more prominent in the ventricular wall of aged mice, possibly as a mechanism to maintain vascular or metabolic homeostasis in the face of Vegfr2 decline. Finally, we evaluated the expression of VEGF co-receptors Nrp1 and Nrp2 . Nrp2 , which modulates VEGF signaling 38 , exhibited a clear age-dependent downregulation, paralleling the decline in Vegfr2 itself, and reinforcing the idea that Vegfr2/Nrp2 -dependent pathways are progressively silenced with age. In contrast, Nrp1 expression remained unchanged, suggesting differential regulatory control of neuropilin family members in tanycytes (Fig. 8 C). VEGFR2, but not VEGFR1 is expressed in human median eminence tanycytes Tanycytes are also found in humans, with similar characteristics to the ones present in mice 39 , 40 .Thus we wanted to assess if VEGFR expression is conserved in human ME-tanycytes. To that end we decided to analyse the human spatial transcriptomics data from 41 for the different members of the VEGF family (Fig. 9 ). We observed that indeed, VEGFA and VEGFR2 are highly expressed in the human ME, while VEGFB and VEGFR1 show a more dispersed expression. As in mice, almost no expression was detected for VEGFR3 (Fig. 9 A). We further confirmed our observations using RNAscope in situ hybridization in post-mortem hypothalamic tissue from healthy individuals. After validating the specificity of the technique ( Supp Fig. 8 ), tissue samples from the region surrounding the third ventricle were examined for VEGFR1 and VEGFR2 expression. Consistent with our findings in mice, VEGFR2 mRNA was detected in vimentin-positive cells lining the third ventricle, at the level of the ME (Fig. 9 B). In contrast, VEGFR1 mRNA was not detected in this vimentin-positive population (at the level of the ME or dorsally in the ventricle), suggesting a potential species-specific difference in receptor expression or regulatory control. Importantly, both VEGFR1 and VEGFR2 mRNA were readily detected in vascular endothelial cells, confirming the specificity and functionality of the probes used for this analysis. These results indicate that VEGFR2 expression in ME tanycytes is conserved between mice and humans, while VEGFR1 expression appears absent or restricted in adult human tanycytes. This divergence suggests evolutionary differences in VEGFR utilization, which may reflect species-specific adaptations in tanycyte function, vascular interaction, or neuroendocrine signaling at the brain’s interface with the bloodstream. DISCUSSION Here, we identify a compartmentalized VEGF signaling axis across distinct tanycyte subtypes in the median eminence (ME). VEGFR2 is enriched in β1-tanycytes that interface with the arcuate nucleus (ARH), whereas VEGFR1 is selectively expressed in α-tanycytes projecting toward dorsomedial and ventromedial hypothalamic regions. In contrast, β2 tanycytes, which directly contact the fenestrated vasculature in the ME, show the highest VEGFA expression yet lack detectable VEGFRs. This ligand–receptor partitioning reveals an unexpectedly complex arrangement of autocrine and paracrine signaling within the tanycyte population and underscores the functional specialization of ME tanycytes relative to other CVOs. Our intriguing findings suggest that VEGF signaling in tanycytes may extend beyond its well-established endothelial roles in angiogenesis, vascular remodeling, and permeability. One possibility is that VEGFR1, enriched in α-tanycytes at the upper ventricular wall, limits the availability of VEGFA in the CSF, thereby indirectly shaping VEGFR2-dependent responses in ARH-facing tanycytes. Alternatively, VEGFR1⁺ or VEGFR2⁺ tanycytes may respond to VEGFA secreted locally by neurons projecting from the lateral area of the hypothalamus (notably MCH-neurons 6 ) or astrocytes 8 . These models remain speculative but underscore the potential for region- and subtype-specific VEGF signaling to regulate hypothalamic neurovascular communication. Notably, we observed co-expression of VEGFA and VEGFR1 within α-tanycytes, a configuration that could bias VEGF signaling toward VEGFR1-specific functions or act in parallel to mechanisms described in angiogenic endothelial cells. Indeed, the exclusive expression of VEGFR2 in ARH-tanycytes and VEGFR1 in VMH/DMH-tanycytes is reminiscent of tip- and stalk-cell dynamics during sprouting angiogenesis, where VEGFR2 drives sprouting responses to VEGFA in tip cells, while VEGFR1 in stalk cells functions as a decoy receptor to limit VEGFR2 activation 42 . By analogy, VEGFR1⁺ tanycytes may serve to buffer VEGFA availability in the CSF, whereas VEGFR2⁺ tanycytes could help maintain the barrier properties of the ventricular wall. Although functional studies are required to test these hypotheses, our observations suggest the possibility that VEGFR1 and VEGFR2 contribute to distinct and spatially segregated roles of tanycyte subtypes, that may from include region-specific vascular interactions, regulation of barrier permeability to modulation of endocrine access to hypothalamic circuits. Importantly, the strict confinement of VEGFR1 expression to α- but not β- tanycytes, further emphasizes that VEGF signaling is tightly compartmentalized, perhaps reflecting the unique physiological demands of different hypothalamic regions. From a translational perspective, uncovering a pathway that regulates tanycytic–vascular interactions at both fenestrated and BBB-protected vessels could provide new opportunities to modulate barrier permeability, for example to facilitate drug delivery. At the same time, recognizing new non-endothelial functions of this pathway may help avoid off-target effects of anti-angiogenic therapies while also opening the possibility to exploit tanycytic VEGF signaling for CNS-directed interventions. Another striking observation that makes tanycytic VEGF/VEGFR signaling even more interesting is that VEGFR expression pattern is unique to the ME and absent in other CVOs that contain tanycyte-like cells, such as the OVLT, SFO, SCO, and AP 13 . While these regions also contain fenestrated and BBB-protected vessels, their tanycytic interfaces appear molecularly distinct. The high VEGFA expression in OVLT and SCO, but not in the AP, suggests that each CVO employs tailored VEGF-based mechanisms adapted to its physiological roles. The exclusivity of VEGFR1/2 expression to ME tanycytes may therefore reflect the central role of this structure in metabolic sensing and hypothalamic regulation. It is tempting to speculate that specific metabolic cues trigger VEGF release from hypothalamic cells, which then act in a paracrine manner on neighbouring tanycytes to regulate vascular exchange or other, as yet unidentified, tanycytic functions. Tanycytes express estrogen receptor-α, and cyclical changes in estrogen have been shown to remodel ME barrier properties 43 . Estradiol regulates communication between tanycytes and endothelial cells, while also modulating VEGF expression and VEGFR2 signaling in the brain vasculature 44 , 23 . Given these established interactions, we anticipated sex differences in VEGFR expression. Although our RNAscope analysis showed reduced spatial overlap between Vegfr1 and Vegfa females when compared to males, qPCR suggested modest, non-significant trends that merit deeper investigation using more selective approaches. Even if preliminary, our observations raise intriguing questions about how hormonal states may intersect with VEGF signaling in tanycytes. In particular, the dramatic decline in circulating estrogen during menopause could influence tanycytic VEGFR function and thereby impact hypothalamic barrier regulation. Because women spend nearly one-third of their lives in a post-menopausal state, which is characterized by the loss of ovarian steroids, understanding whether this dramatic and permanent decrease in circulating estrogens reshapes tanycytic VEGF signaling has broad physiological and clinical implications. Future studies in ovariectomized mouse models, or in hormonally manipulated animals, could directly test whether estradiol regulates VEGFR1/2 expression or function in tanycytes. Such work would clarify whether sex hormones act as modulators of this newly identified VEGF signaling axis and whether menopause represents a critical window for altered tanycytic–vascular communication Tanycytes derive from radial glia–like progenitors and mature postnatally into subtype-specific identities 33 . Our data acquired during postnatal development reveal distinct temporal trajectories of VEGFR expression. VEGFR1 is present from birth (P0) and remains confined to VMH/DMH-tanycytes, suggesting early specification of this subtype. In contrast, the restriction of VEGFR2 expression to ARH-tanycytes only becomes obvious in adulthood, consistent with later maturation of this domain. Both receptors are excluded from ME-tanycytes from the earliest stages, indicating that compartmentalization is not a consequence of postnatal differentiation. These temporal patterns support a paracrine model in which ligand and receptor are spatially segregated. Of interest is that clusters of high Vegfa -expressing cells appeared around P8 in the ME parenchyma, coinciding with the first vascular loops described in the ME, raising the possibility that local VEGF signaling contributes to vascular remodeling. Notably, this time window also corresponds to morphological maturation of tanycytes at around P8. Our findings suggest a coordinated program in which VEGF signaling integrates with tanycyte differentiation and vascular development to establish specialized hypothalamic barriers during early postnatal life, remarkable coordination for a small region with such essential roles in metabolism and reproduction. It will also be important to determine whether other barrier systems in the ME, such as the lateral diffusion barrier or perineuronal net barrier 45 , participate in this coordinated maturation. Systemic studies have placed VEGF deficiency at the center of multiorgan aging, and sustained VEGF activity has been shown to promote healthier lifespan 34 . In line with this, we observed a progressive decline in VEGFR2 expression in tanycytes beginning at 6 months, accompanied by a parallel decrease in its co-receptor Nrp2. This mirrors endothelial cell aging, where reduced VEGF signaling contributes to vascular dysfunction 34 . By contrast, VEGFR1 expression remained stable suggesting a shift from a VEGFR2- to a VEGFR1-dominant axis in tanycytes over time. Whether this transition is adaptive, helping to preserve barrier integrity or tanycytic function, or maladaptive, contributing to impaired neuroendocrine communication, remains an open question. Given that VEGF dysregulation has been linked to Alzheimer’s disease and other disorders of vascular and metabolic dysfunction 35 – 37 . tanycytic VEGFR2 downregulation could represent one mechanism linking hypothalamic aging to systemic pathology. These observations highlight tanycytic VEGFR as a potential therapeutic target: Enhancing VEGFR2 activity could improve barrier properties or metabolic resilience, whereas systemic anti-angiogenic therapies 46 , 47 may inadvertently impair tanycytic function. Indeed, we observed tanycyte fragmentation in Alzheimer’s disease patients 48 , consistent with emerging evidence implicating VEGFA–VEGFR2 in neurodegeneration 36 , 49 . Importantly we confirmed VEGFR2 expression in human tanycyte-like cells lining the floor of the third ventricle. However, VEGFR2 and VEGFA were co-expressed in the same tanycytic population, while we could not detect VEGFR1. The conserved VEGFR2 expression highlights a fundamental role for this pathway in tanycytes across mammals. These findings emphasize the need for deeper functional characterization of VEGFRs in tanycytes and their potential role in controlling vascular–tanycytic communication. In conclusion , beyond their well-established roles in vascular biology, our findings demonstrate that VEGFRs are expressed in a spatially organized manner in the distinct hypothalamic tanycytes populations, revealing a previously underappreciated non-endothelial site of VEGF signaling in the brain. This organization adds a new layer of complexity to how systemic VEGF cues may be interpreted at the brain–body interface and underscores the importance of considering central nervous system targets, such as tanycytes, when developing or administering VEGF-targeted therapies. METHODS Animals R26-tdTomato loxP−STOP−loxP reporter mice (JAX#007914) were obtained from Jackson Laboratory. Wild-type C57BL/6J mice were purchased from Charles River Laboratories. Age details for each experimental group is provided in the corresponding figure legend. Mice were housed in a temperature-controlled facility (21°C) under a 12 h light/dark cycle, with ad libitum access to water and standard chow (20% of energy from protein, 67% from carbohydrates, and 12% from fat by dry weight). For developmental studies, pups were collected at postnatal day (P) 0, P4, P8, P12, P16, P21, and in adulthood (3 months of age). For aging experiments, adult male mice were sacrificed at 3, 6, 12, and 18 months. All experiments presented in the paper were performed using 3–6 animals per group depending on the experiment. All animal procedures were conducted in accordance with the European Union Directive 2010/63/EU for animal experiments and approved by the Institutional Ethics Committee for the Care and Use of Experimental Animals of the University of Lille and the French Ministry of National Education, Higher Education and Research APAFIS#29172-2020121811279767 v5 Single-cell RNA sequencing data reanalysis and human spatial transcriptomics Single-cell RNA sequencing data from the Gene Expression Omnibus (GEO; accession number GSE90806, PMID: 28166221) were retrieved to analyze expression profile of VEGF receptors and their ligands in cells of the ARH/ME region, particularly tanycytes. Pre-processed gene counts and metadata were obtained as a SingleCellExperiment object via the CampbellBrainData() function of the scRNAseq package and reanalyzed using Seurat v5.1.0, a toolkit for single-cell genomics in R v4.4.2. Data integration was performed with the Harmony workflow 51 within Seurat. Standard Seurat functions were applied for clustering, identification of cluster-specific markers, and visualization. Major clusters were annotated into distinct cell types using previously reported marker genes 18 . Tanycytes were subset, reclustered (dimensions = 1:16; resolution = 0.6) and annotated to define the four subpopulations based on established marker genes 18 . hUMAP_1 and hUMAP_2 represent the two-dimensional embedding coordinates of cells grouped by similarity in their gene expression profiles. The preprocessed spatial transcriptomics data was retrieved from human hypomap 41 to map the spatial localization of in human tanycytes. Feature plots were generated using scCustomize package (RRID:SCR_024675). Human samples Human brain tissue was obtained in accordance with French regulations (Good Practice Concerning the Conservation, Transformation and Transportation of Human Tissue to be Used Therapeutically, published on December 29, 1998). Authorization for the use of human tissue was granted by the French Agency for Biomedical Research (Agence de la Biomédecine, Saint-Denis la Plaine, France; protocol no. PFS16-002) and the Lille Neurobiobank. Ethical approval for this study was provided by the Comité de Protection des Personnes (CPP) SUD-EST II (BIOWATCH, protocol #2021-A00879-32). Dissected blocks of adult human brain tissue containing the hypothalamus were immersion-fixed in 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS), pH 7.4, at 4°C for 1 week. Tissues were then cryoprotected in 30% sucrose in PBS at 4°C until they sank, embedded in Tissue-Tek OCT compound (Sakura Finetek), frozen on dry ice, and stored at − 80°C until sectioning. Coronal cryosections were cut at 20 µm thickness for downstream analyses. Stereotaxic injection into the lateral ventricle Recombinant AAV1/2-Dio2-iCre (1.25×10⁹ genomic particles/µL) was produced as previously described 24 . For female analysis experiments, 2 µL of AAV1/2-Dio2-iCre were stereotaxically injected into the lateral ventricle (anteroposterior: −0.3 mm; mediolateral: ±1.0 mm; dorsoventral: −2.5 mm from bregma) of isoflurane-anesthetized R26-tdTomato mice. Three to four weeks post-injection, mice were sacrificed and the ME was micro-dissected for downstream cell dissociation and fluorescence-activated cell sorting (FACS) analysis. For aging studies, 2 µL of AAV1/2-CAG-TdTomat (VectorBuilder) were similarly infused into the lateral ventricle of wild-type C57BL/6J mice under isoflurane anesthesia, four weeks prior to the age of sacrifice. For all stereotaxic surgeries, mice were anesthetized with isoflurane and placed on a heating pad to maintain body temperature. Eyes were protected with ophthalmic gel (Ocrygel). Injections were performed using a Kopf 963/962 stereotaxic frame, following the coordinates − 0.3 mm posterior to bregma, ± 1.0 mm lateral, and − 2.5 mm ventral from the skull surface. A total volume of 2 µL was infused at a rate of 0.3 µL/min using a KD Scientific Legato 130 syringe pump and a 2 µL Neuro-Hamilton syringe. After injection, the skin was sutured and mice were allowed to recover on a heating pad before being returned to clean cages. Estrous cycle To determine the stage of the estrous cycle, vaginal cytology was performed by flushing the vaginal canal with a small volume of sterile saline using a pipette, every day at the same time of the day, for at least 2 weeks. The cell suspension was collected and a few drops were placed on a clean glass microscope slide for immediate examination under a light microscope. Estrous cycle phases were identified based on the relative proportions of leukocytes, cornified epithelial cells, and nucleated epithelial cells. The stages were categorized as proestrus, estrus, or diestrus. Tissue processing and immunostaining For immunostaining, brains were fixed in 4% PFA/PBS overnight at 4°C before cryoprotection in 30% sucrose in PBS overnight at 4°C. Cryoprotected brains were frozen in OCT on dry ice and stored at -80°C until analysis. For RNA in situ hybridization, fresh brains were immediately frozen in OCT after harvesting and stored at -20°C. Frozen brains were placed at -20°C overnight for equilibration and coronally cut on a Leica CM3050S cryostat at 20 µm (sections on slides). Slides were kept at -20°C until further processing. Selected sections were dried for 30 minutes at room temperature before fixing in cold acetone/Ethanol (50%v/v) for 1 minute. Then, after 3 washes of 5 minutes with PBS-Triton 0.1%, sections were blocked in incubation solution (ICS, 1% BSA in PBS-Triton 0.3% pH 7,4) for 1 hour. Blocking was followed with primary antibody incubation in ICS for 24-48h at 4°C. Primary antibodies were then rinsed out, before incubation in fluorophore-coupled secondary antibodies for 1h in ICS at room temperature. Secondary antibodies were washed and sections counterstained with DAPI (D9542, Sigma). Finally, after a last wash of 10 minutes in PBS 1X, sections were mounted on slides with Mowiol, left to dry on the bench and stored at 4C until images analysis. Target Concentration Reference Supplier Rabbit anti-vimentin [1:4000] AB92547 Abcam Goat anti-collagen IV [1:500] AB769 Abcam anti-m-VEGF R1 (Flt-1) [1:100] AF471 R&D System anti-mVEGF R2 (Flk-1) [1:100] AG644 R&D System Donkey anti-rabbit (488) [1:300] A21206 Invitrogen Donkey anti-goat (488) [1:300] A11055 Invitrogen FACs sorting and quantitative PCR For fluorescence-activated cell sorting (FACS) of hypothalamic tanycytes, mice were injected with either AAV-Dio2-iCre into R26-tdTomato mice or AAV-CAG-tomato into wild-type C57BL/6J mice. Three to four weeks post-injection, median eminences (MEs) were micro dissected and enzymatically dissociated using the Papain Dissociation System (Worthington Biochemical Corporation, Lakewood, NJ) to obtain single-cell suspensions. FACS was performed on a SONY SH800 Sorter Cytometer device using a 70 µm sorting chip. In tdTomato expressing mice, tanycytes were identified based on tdTomato fluorescence (excitation: 561 nm; detection: 675 ± 20 nm). A sample of the cortex (That does not contain any fluorescence) was used as negative control for gating. For each animal, between 4,000/8,000 fluorescent-positive and -negative cells were sorted directly into 10/20 µL of lysis buffer (0.1% Triton X-100, Sigma-Aldrich; 0.4 U/µL RNaseOUT, ThermoFisher Scientific). Starting with the same amount of sorted tanycytes, for gene expression analysis, total RNA from FACS-sorted cells was treated with DNase I (Invitrogen, ThermoFisher) to remove genomic DNA contamination and then reverse transcribed using SuperScript III Reverse Transcriptase (Invitrogen, ThermoFisher) according to the manufacturer's protocol. A linear preamplification step was performed using TaqMan PreAmp Master Mix (Applied Biosystems™, Cat. No. 4488593). Quantitative PCR (qPCR) was subsequently performed using the TaqMan® Universal Master Mix II (Applied Biosystems™, Cat. No. 4440049) on an Applied Biosystems QuantStudio 3 Real-Time PCR instrument (ref: A28131) and Applied Biosystems StepOnePlus Real-Time PCR using TaqMan Gene Expression Assays (Applied Biosystems, see list of probes below). Relative gene expression was normalized to housekeeping genes (18S and Actb) and analysed using the 2-ΔΔCt method. The purity of the sorted cells was confirmed with endothelial ( Pecam1 ), neuronal ( Elav4 ) and tanycytic markers ( GPR50 . Data are presented as mean ± SEM, and statistical tests are specified in the relevant figure legends. Gene Reference ActB Mm02619580_g1 Elavl4 Mm01263580_mH Gpr50 Mm00439147_m1 Nrp1 Mm01253208_m1 Nrp2 Mm00803099_m1 Pecam1 Mm01242576_m1 Rn18S Mm03928990_g1 VEGFa Mm00437306_m1 VEGFa Mm01281449_m1 VEGFb Mm00442102_m1 VEGFc Mm00437310_m1 VEGFR1 ( Flt1 ) Mm00438992_m1 VEGFR2 ( Kdr ) Mm01222421_m1 VEGFR3 ( Flt4 ) Mm01292604_m1 RNAscope RNAscope is performed by following the user manual RNAscope® Multiplex Fluorescent Reagents Kit v2 Assay (Advanced Cell Diagnostics - ACD) following the manufacturer's instructions. Briefly, fresh frozen brain sections were first fixed in PFA for 15 min at 4°C. Slides were dehydrated in different solutions of ethanol (50%, 70% and 100% ethanol). Hydrogen Peroxide was added on each slide in a humid chamber and rinsed. Protease IV was then added to each brain section in a humid chamber followed by a wash step. Selected probes were prepared and applied to the tissue, then incubated for 2 hours at 40°C in a HybEZ oven. Following hybridization, slides were washed in RNAscope wash buffer and subjected to signal amplification using AMP 1, AMP 2, and AMP 3 reagents, each incubated for 30 minutes at 40°C with washes in between. HRP-C1 and HRP-C2 channels were developed sequentially as per the kit instructions, with appropriate wash steps. For mouse, specific probes were used to detect Flt1 (415541-C1, NM_010228.3, target region 756–1663), Kdr (414811-C2, NM_010612.2, target region 1766–2673), and Vegfa (412261-C3, NM_001025257.3, target region 946–2156) mRNAs and Flt4 (481371-C1, NM_008029.3, target region 95–1058). For human, specific probes were used to detect Flt1 (560701-C2, NM_001160031.1, target region 817–1810) and Kdr (435371-C1, NM_002253.2, target region 1400–3515). Following RNAscope, immunofluorescence was performed on the same sections. sections were blocked for 1 hour in PBS 1X + 0.5% Triton X-100 + 1% BSA and then incubated with the primary antibody in the blocking solution overnight at 4°C. The following day, sections were rinsed 3x10 min in PBS 1X at room temperature. Sections were then incubated with secondary antibody in blocking solution for 2 hours at RT, and washed again 3x10 min in PBS 1X. Nuclear staining was performed with DAPI (1:10,000, 5 minutes), and slides were mounted using Mowiol mounting medium. Image analysis and quantification Images were acquired using the Leica Stellaris 5 inverted confocal microscope (Leica Microsystems), using the 20x/0.8 dry objective or the 40x/1.4 oil immersion objective. Z-stack were done on 0.6 to 1µm thick optical sections on the whole depth of the section. Tiled images were acquired and automatically stitched using LasX software (Leica Microsystems). Maximum intensity projections were systematically generated using the same software. Image processing was done using Fiji (ImageJ) software. Regions of interest (ROIs) corresponding to specific tanycyte subtypes were manually annotated based on anatomical landmarks and Vimentin expression. For RNAscope quantification, images were first converted to 8-bit and thresholded to the same value for each image of a same channel. The area and number of pixels of each region were taken to apply correction on the value, as the RNAscope method stipulates that the number of pixels is representative of the number of mRNA ( https://acdbio.com/ ). For immunofluorescence quantification of the intensity of fluorescence, images were acquired with the same settings during confocal microscopy. Mean intensity was measured on hand-drawn ROIs. Statistical analysis All data are presented as mean ± standard error of the mean (SEM). Statistical analyses were performed using GraphPad Prism 10, using Student’s t-test, one-way ANOVA test, or Kruskal Wallis test. P-values under 0.05 were considered to be statistically significant. All experiments were independently replicated at least three times. The number of mice used per experiment is reported in the figure legends. Declarations COMPETING FINANCIAL INTERESTS The authors declare no competing financial interests FUNDING This work was supported by a doctoral fellowship from the University of Lille to O.D. The ANR grant ANR-23-CE14-0006-01 to IMC and the European Research Council WATCH Synergy Grant (No 810331 to MS and VP). Author Contribution O.D, M.L and D.F planned and performed experiments. S.N performed the scRNAseq and transcriptomic data analysis. C.A and M.S provided materials. I.M-C, O.D, D.F and V.P. discussed and interpreted the results I.M-C conceived the project and wrote the manuscript with the input of all the authors. Acknowledgement The authors would like to thank PLBS UAR 2014 – US41 (https://ums-plbs.univ-lille.fr/) with its different platforms and staff for expert technical assistance: Meryem Tardivel and Antonino Bongiovanni (microscopy core facility, BICeL) and Julien Devassine (Animal facility, BICeL). The authors would like also to thank Dr Adrian Coutteau-Robles, Thomas Chretien and Amandine Legrand for technical assistance. References Simons, M., Gordon, E. & Claesson-Welsh, L. Mechanisms and regulation of endothelial VEGF receptor signalling. Nat. Rev. Mol. Cell Biol. 17 , 611–625 (2016). Lee, C. et al. Vascular endothelial growth factor signaling in health and disease: from molecular mechanisms to therapeutic perspectives. Signal Transduct. Target. Ther. 10 , 170 (2025). Lange, C., Storkebaum, E., de Almodóvar, C. R., Dewerchin, M. & Carmeliet, P. Vascular endothelial growth factor: a neurovascular target in neurological diseases. Nat. Rev. Neurol. 12 , 439–454 (2016). 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Supplementary Files Desruelleetal.Supplementarymaterial.docx Cite Share Download PDF Status: Published Journal Publication published 14 Feb, 2026 Read the published version in Fluids and Barriers of the CNS → Version 1 posted Editorial decision: Revision requested 13 Nov, 2025 Reviews received at journal 13 Nov, 2025 Reviews received at journal 06 Nov, 2025 Reviewers agreed at journal 23 Oct, 2025 Reviewers agreed at journal 23 Oct, 2025 Reviewers invited by journal 21 Oct, 2025 Editor assigned by journal 20 Oct, 2025 Submission checks completed at journal 18 Oct, 2025 First submitted to journal 16 Oct, 2025 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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On the right: Correlation between tanycyte subtypes and their corresponding hypothalamic nuclei (B) UMAP showing clusters representative of four main tanycytic subpopulations generated by the reanalysis of scRNA-seq ME dataset from \u003csup\u003e18\u003c/sup\u003e. (C) Feature plots showing VEGFRs (\u003cem\u003eKdr\u003c/em\u003e, Flt1 and \u003cem\u003eFlt4\u003c/em\u003e), ligands (Vegfa, Vegfb and Vegfc) and co-receptors (Nrp1 and Nrp2) in tanycytic subclusters. (E) Dot plot showing the expression of the different member of the family across the tanycytic subclusters.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7878186/v1/8cb81586345be2b2523093ca.jpg"},{"id":94907267,"identity":"303fd5e2-d26a-4228-8758-3707f7eac963","added_by":"auto","created_at":"2025-11-01 07:42:01","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1714054,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eVEGFR1 and VEGFR2 are differentially expressed in tanycytes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eA\u003c/strong\u003e) \u003cem\u003eIn situ\u003c/em\u003e hybridization images for \u003cem\u003eVegfr1\u003c/em\u003e (red) and \u003cem\u003eVegfr2\u003c/em\u003e (green) in coronal sections of the hypothalamic tuberal region of adult male mice. Vimentin immunoreactivity is shown in white and nuclei counterstaining (DAPI) in blue. Images are low-magnification views of the 3 V at the level of the ME. ME and VMH/DMH regions are magnified on the right. Blue arrows point to ME tanycytes showing no expression of \u003cem\u003eVegfr1\u003c/em\u003e and \u003cem\u003eVegfr2\u003c/em\u003e. Note that ARH tanycytes express only Vegfr2 while VMH/DMH tanycytes express high \u003cem\u003eVegfr1\u003c/em\u003e and low levels of \u003cem\u003eVegfr2\u003c/em\u003e. Blood vessels are indicated by asterisks. (\u003cstrong\u003eB\u003c/strong\u003e) \u003cem\u003eIn situ\u003c/em\u003e hybridization images for \u003cem\u003eVegfr1\u003c/em\u003e (red), \u003cem\u003eVegfr2\u003c/em\u003e (green) and \u003cem\u003eVegfa\u003c/em\u003e (yellow). High magnification of the boxed region is shown on the right. Individual channels are shown for \u003cem\u003eVegfr2/Vegfr1\u003c/em\u003e and \u003cem\u003eVegfa\u003c/em\u003e. Note that \u003cem\u003eVegfa\u003c/em\u003e is highly expressed at the level of the ME tanycytes (blue arrows) and DMH tanycytes (red arrows).\u003cstrong\u003e (C) \u003c/strong\u003eImmunofluorescence of ME coronal sections for \u003cem\u003eVegfr1\u003c/em\u003e (left panel, in red) and Vegfr2 (right panel, in green). Magnification of ME, ARH and VMH are shown for each immunoreactivity showing \u003cem\u003eVegfr1\u003c/em\u003e predominantly in VMH region while VEGFR2 in ARH region (white arrowheads). (\u003cstrong\u003eD\u003c/strong\u003e) Quantification is shown in for VEGFR1 and VEGFR2 protein expression in tanycytes. (\u003cstrong\u003eE\u003c/strong\u003e) Schematic image and representation of the ME illustrating the compartmentalization of the VEGFR1, VEGFR2 and VEGFA. Data is represented in percentage of intensity and corrected to background from ME tanycytes (n=3). Data are represented as mean ± standard error deviation (SEM). Statistical analysis was performed using ANOVA and Kruskal-wallis test. n = 3. * = p \u0026lt; 005. Scale bar = 100 μm in all panels. ARH, arcuate nucleus of the hypothalamus; ME, median eminence; VMH, ventromedial nucleus of the hypothalamus; DMH, dorsomedial nucleus of the hypothalamus; 3V, third ventricle.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7878186/v1/67f4cb67c0fe582fac001d02.jpg"},{"id":94907275,"identity":"48b12e12-c71e-4584-aa22-484a8fc379b5","added_by":"auto","created_at":"2025-11-01 07:42:02","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":408836,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTanycytic VEGFRs in female mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A) \u003c/strong\u003e\u003cem\u003eIn situ\u003c/em\u003e hybridization (RNAscope) images for \u003cem\u003eVegfr1\u003c/em\u003e (red), \u003cem\u003eVegfr2\u003c/em\u003e (green) and \u003cem\u003eVegfa\u003c/em\u003e(yellow) in coronal sections of the hypothalamic tuberal region of adult female mice. Boxed region is amplified on the right. Note that \u003cem\u003eVegfa\u003c/em\u003e is highly expressed at the level of the ME tanycytes (blue arrows) and VMH tanycytes (red arrows). (\u003cstrong\u003eB\u003c/strong\u003e) Left: Schematic representing qPCR sorting strategy. Right: qPCR analysis of Vegfr2 (\u003cem\u003ekdr\u003c/em\u003e) variation during the estrus cycle (n=4-8). Data are represented as mean ± standard error deviation (SEM). Statistical analysis was performed using ANOVA and Kruskal-wallis non-parametric test. n = 4-8. * = p \u0026lt; 005. Scale bar = 100 μm in all panels. ARH, arcuate nucleus of the hypothalamus; ME, median eminence; VMH, ventromedial nucleus of the hypothalamus; DMH, dorsomedial nucleus of the hypothalamus; 3V, third ventricle; E, estrus: D, diestrus; P, proestrus.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7878186/v1/cdd1d0b063c034f97d80257b.jpg"},{"id":94988227,"identity":"01fcdc63-77e8-44ce-b5d4-b7ac50c0b951","added_by":"auto","created_at":"2025-11-03 07:07:39","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1216467,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eVEGFRs expression is exclusive to ME tanycytes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Schematic representation of the localization of the different CVOs (\u003cstrong\u003eB\u003c/strong\u003e) \u003cem\u003eIn situ\u003c/em\u003e hybridization images for \u003cem\u003eVegfr1\u003c/em\u003e (red) and \u003cem\u003eVegfr2\u003c/em\u003e (yellow) in coronal sections of different circumventricular organs: Median eminence (ME), Subfornical organ (SFO), Subcommisural organ (SCO), Area postrema (AP) and Organum vasculosum of lamina terminalis (OVLT). The Choroid plexus (CP) is also shown. Collagen IV immunoreactivity is shown in green to identify blood vessels and nuclei counterstaining (DAPI) in blue. Scale bar\u0026nbsp;=\u0026nbsp;50 μm in all panels. Dotted line in white limits the region of the CVO. Red square marks the region magnified in (\u003cstrong\u003eC\u003c/strong\u003e). Note that only tanycytes in the ME express both receptors (white arrowheads). Low levels of \u003cem\u003eVegfr2\u003c/em\u003e can also be detected at the level of the SCO and the epithelial cells of the CP, not overlapping with blood vessels (orange arrowheads). Collagen IV deposits (fractones\u003csup\u003e50\u003c/sup\u003e) are marked with asterisks.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7878186/v1/0097a945e2d0ec512510bb7c.jpg"},{"id":94907273,"identity":"cb0dfc54-19ee-4cb0-91a0-e5683ce95b65","added_by":"auto","created_at":"2025-11-01 07:42:02","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1507529,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eVEGFA is expressed in all the circumventricular organs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eA–B\u003c/strong\u003e) Representative RNAscope \u003cem\u003ein situ\u003c/em\u003e hybridization images showing \u003cem\u003eVegfr1\u003c/em\u003e (red) and \u003cem\u003eVegfa\u003c/em\u003e(yellow) mRNA expression across coronal sections of several CVOs: the median eminence (ME), subfornical organ (SFO), subcommissural organ (SCO), and area postrema (AP). The choroid plexus (CP) is also included for comparison. Blood vessels are visualized by Collagen IV immunoreactivity (green), and nuclei are counterstained with DAPI (blue). White dotted lines delineate the boundaries of each CVO. Scale bar = 50 μm for all panels. (\u003cstrong\u003eB\u003c/strong\u003e) Higher magnification of boxed regions from (A) to highlight subcellular localization. Yellow arrowheads indicate strong \u003cem\u003eVegfa\u003c/em\u003e expression in ME tanycytes, the SFO, and the CP. Other CVOs (SCO, AP) also exhibit \u003cem\u003eVegfa\u003c/em\u003e signal, though at lower levels. Collagen IV deposits (fractones) are marked by asterisks.\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7878186/v1/4d92646c84902f64c087e3b8.jpg"},{"id":94907285,"identity":"10599f60-3908-4f1e-8a43-491b9a5301ef","added_by":"auto","created_at":"2025-11-01 07:42:02","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1889852,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDifferential expression of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eVegfr1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e, \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003evegfr2\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003evegfa\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e during postnatal development\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eIn situ\u003c/em\u003e hybridization images showing mRNA expression of \u003cem\u003eVegfr1\u003c/em\u003e (red), \u003cem\u003eVegfr2\u003c/em\u003e (yellow), and \u003cem\u003eVegfa\u003c/em\u003e(green) in coronal sections of the hypothalamic tuberal region from male mice at postnatal days P0, P4, P8, P12, P16, and P21, compared to adult (3-month-old) animals. The third ventricle (3V) is labeled in all the images for spatial orientation. Note the absence of \u003cem\u003eVegfr1\u003c/em\u003e and \u003cem\u003eVegfr2\u003c/em\u003eexpression in ME tanycytes (indicated by a white discontinuous line) at all time points, including at birth, and the emergence of high \u003cem\u003eVegfa\u003c/em\u003eexpression in non-tanycytic cells within the ME region beginning around P8 (red arrowheads). White arrowheads at P0 indicate blood vessels. Scale bar = 100 μm in all panels. n=3 animals per time point. ARH, arcuate nucleus of the hypothalamus; ME, median eminence; VMH, ventromedial nucleus of the hypothalamus; DMH, dorsomedial nucleus of the hypothalamus.\u003c/p\u003e","description":"","filename":"Figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7878186/v1/0c6a6af1a8eaf09bd03a5be1.jpg"},{"id":94988125,"identity":"ef0a9d9f-dc60-4905-a139-2ebf0f90728d","added_by":"auto","created_at":"2025-11-03 07:04:56","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":727855,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eQuantification of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eVegfr1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e, \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003evegfr2\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003evegf\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003ea during postnatal development\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Representative RNAscope \u003cem\u003ein situ\u003c/em\u003e hybridization images showing \u003cem\u003eVegfa\u003c/em\u003e (green), \u003cem\u003eVegfr1\u003c/em\u003e(red), and \u003cem\u003eVegfr2\u003c/em\u003e (yellow) mRNA expression in the tuberal hypothalamus. White dotted boxes indicate the regions selected for quantification: median eminence (ME), arcuate nucleus (ARH), and ventromedial/dorsomedial hypothalamic regions (VMH/DMH). Scale bar = 100 µm. (\u003cstrong\u003eB\u003c/strong\u003e) Quantification of Vegfr1 (red), Vegfr2 (yellow), and Vegfa (green) expression across developmental stages (P0, P4, P8, P12, P16, P21, and adult) in each of the three regions. Expression is represented as signal area (pixels/µm²). Red lines illustrate relative changes across regions for each time point, highlighting spatiotemporal compartmentalization. (\u003cstrong\u003eC\u003c/strong\u003e) Summary of expression levels at P0, P21, and adult stages across the three regions. Notably, \u003cem\u003eVegfa\u003c/em\u003e expression shows a marked increase in adult samples, particularly in the ME and VMH/DMH regions, suggesting enhanced ligand availability in mature hypothalamic tissue. n=3 animals per time point. Statistical analysis performed using two-way ANOVA followed by Fisher's LSD post hoc test. p \u0026lt; 0.05 considered significant.\u003c/p\u003e","description":"","filename":"Figure7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7878186/v1/8246f20ff0710b2efba7c675.jpg"},{"id":94907280,"identity":"689445f1-cbfe-4887-a020-3862d08a683c","added_by":"auto","created_at":"2025-11-01 07:42:02","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":402194,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eQuantification of Vegfr1, vegfr2 and vegf1 during postnatal development\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eA\u003c/strong\u003e) qPCR analysis of \u003cem\u003eVegfr1, Vegfr2\u003c/em\u003e, and \u003cem\u003eVegfr3\u003c/em\u003e mRNA expression levels in tanycytes isolated from male mice aged 3, 6, 9, 12, and 18 months. Notably, \u003cem\u003eVegfr2\u003c/em\u003e expression shows a progressive decline with age, while \u003cem\u003eVegfr1\u003c/em\u003e and \u003cem\u003eVegfr3\u003c/em\u003eremain stable. (\u003cstrong\u003eB\u003c/strong\u003e) qPCR analysis of VEGF ligands. \u003cem\u003eVegfa\u003c/em\u003e and \u003cem\u003eVegfc\u003c/em\u003eremain relatively stable across age, while \u003cem\u003eVegfb\u003c/em\u003e shows a modest upregulation in older mice. (\u003cstrong\u003eC\u003c/strong\u003e) Expression of VEGF co-receptors \u003cem\u003eNrp1\u003c/em\u003eand \u003cem\u003eNrp2 \u003c/em\u003ein sorted tanycytes. While \u003cem\u003eNrp1\u003c/em\u003e remains unchanged, \u003cem\u003eNrp2\u003c/em\u003eexpression declines with age, paralleling the pattern of \u003cem\u003eVegfr2\u003c/em\u003e. qPCR data are normalized to housekeeping genes \u003cem\u003e18S\u003c/em\u003e and \u003cem\u003eActb\u003c/em\u003e, and presented as mean ± SEM; n = 5–6 mice per time point. Statistical analysis was performed using one-way ANOVA with Fisher’s LSD post hoc test. p \u0026lt; 0.05 considered significant.\u003c/p\u003e","description":"","filename":"Figure8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7878186/v1/a89e7dd107fc4762158ef2e8.jpg"},{"id":94907274,"identity":"c65037d6-ceb3-45a0-b2f7-80d0fbb8dd1a","added_by":"auto","created_at":"2025-11-01 07:42:02","extension":"jpg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":1723958,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eVegfr2 is expressed in human tanycytes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u0026nbsp; \u003c/strong\u003eSpatial transcriptomics data from human hypomap\u003csup\u003e41\u003c/sup\u003e for VEGF receptors, co-receptors and ligands. Arrows point to regions with detected expression \u003cstrong\u003e(B)\u003c/strong\u003e\u0026nbsp;RNAscope \u003cem\u003ein situ\u003c/em\u003e hybridization for\u0026nbsp;\u003cem\u003eVEGFR1\u003c/em\u003e\u0026nbsp;(yellow) and\u0026nbsp;\u003cem\u003eVEGFR2\u003c/em\u003e\u0026nbsp;(red) in coronal sections of the hypothalamic tuberal region from an adult human male. Immunofluorescence for\u0026nbsp;vimentin\u0026nbsp;(green) was used to identify tanycyte-like cells along the third ventricle. Boxed regions are magnified on the right.\u0026nbsp;\u003cem\u003eVEGFR2\u003c/em\u003e\u0026nbsp;mRNA puncta were observed in\u0026nbsp;vimentin⁺\u0026nbsp;cells, consistent with tanycyte identity, whereas\u0026nbsp;\u003cem\u003eVEGFR1\u003c/em\u003e\u0026nbsp;expression was restricted to blood vessels (asterisks) and not detected in periventricular vimentin⁺ cells. White arrow points to ME tanycytes, where we observe clear \u003cem\u003emVegfr2\u003c/em\u003e puncta. Scale bar 100um for all panels.\u003c/p\u003e","description":"","filename":"Figure9.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7878186/v1/b76c286708501df8a09144c0.jpg"},{"id":102785190,"identity":"d4bad207-624e-4fe3-85cd-6842299555eb","added_by":"auto","created_at":"2026-02-16 16:02:10","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":11641076,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7878186/v1/c9f8633a-597d-4797-b4f8-e3087e2432e2.pdf"},{"id":94987068,"identity":"b1833db4-2b2b-4b68-ae41-9b9a6a5be366","added_by":"auto","created_at":"2025-11-03 07:01:11","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":10595099,"visible":true,"origin":"","legend":"","description":"","filename":"Desruelleetal.Supplementarymaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-7878186/v1/8d1c91712698138cb0d20907.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Compartmentalized VEGF Receptor Expression in Hypothalamic Tanycytes Reveals a Novel Non- Endothelial Axis of VEGF Signaling (Tanycytes as a Novel Non-Endothelial Target of VEGF Signaling)","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eVascular endothelial growth factor receptors (VEGFRs) are membrane-bound receptor tyrosine kinases essential for angiogenesis, vascular permeability, lymphangiogenesis, and the regulation of cell proliferation and differentiation. The VEGFR family comprises three main receptors\u0026mdash;VEGFR1 (\u003cem\u003eFlt-1\u003c/em\u003e), VEGFR2 (\u003cem\u003eKdr\u003c/em\u003e), and VEGFR3 (\u003cem\u003eFlt-4\u003c/em\u003e)\u0026mdash;which interact with five structurally related VEGF ligands (VEGFA, VEGFB, VEGFC, VEGFD, and placental growth factor [PlGF]). In addition, VEGFs bind with high affinity to neuropilin co-receptors (NRP1 and NRP2) and to heparan sulfate proteoglycans (HSPGs)\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Each ligand\u0026ndash;receptor interaction activates distinct signaling pathways that are essential for vascular and lymphatic system development and maintenance\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. However, although historically associated with vascular biology, VEGF/VEGFR signaling also plays diverse roles within the central nervous system (CNS), contributing to blood\u0026ndash;brain barrier (BBB) regulation, neurogenesis, synaptic plasticity, and astrocyte function\u003csup\u003e\u003cspan additionalcitationids=\"CR3\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eAmong the VEGFRs, VEGFR1 has an affinity for VEGFA and is involved in monocyte migration and hematopoiesis. This receptor also acts as a decoy receptor to control the availability of VEGF ligands and is expressed on the surface of monocytes, macrophages, and endothelial cells. VEGFR2 is a critical regulator of VEGF induced angiogenesis, and it is predominantly localized on the surface of endothelial cells, facilitating key signaling processes essential for vascular development. This receptor has a strong affinity for VEGFA and promotes endothelial cell migration, proliferation, differentiation, and survival. On the other hand, VEGFR3 is mainly expressed in lymphatic endothelial cells exhibiting a strong affinity for VEGFC and VEGFD\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Importantly, both VEGFR1 and VEGFR2 are also expressed in non-endothelial cells\u003csup\u003e\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e including neurons and glia, which can produce and respond to VEGFs\u003csup\u003e\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Despite their increasing non-endothelial central roles in the brain, the spatial and cell-type-specific expression of VEGFRs in the healthy brain remains poorly characterized.\u003c/p\u003e\u003cp\u003eOne region of particular interest in the study of VEGFRs is the median eminence (ME) at the tuberal hypothalamus, a circumventricular organ (CVO) that serves as a critical interface for neuroendocrine regulation. Specifically, the pituitary portal blood vessels in the ME contain endothelial cells that are fenestrated and lack blood-brain barrier properties, allowing circulating molecules to freely diffuse into the brain parenchyma. At this specific location, the blood-barrier functions are instead mediated by tanycytes, which are specialised ependymoglial cells lining the walls and floor of the third ventricle\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Tanycytes form a tight junction-based blood\u0026ndash;cerebrospinal fluid (CSF) barrier, also known as the tanycytic barrier, and extend long processes that contact both fenestrated vessels in the ME and BBB-protected vessels of adjacent hypothalamic nuclei\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Their strategic positioning and contact with different nuclei of the hypothalamus enable them to regulate neurovascular exchange, sense peripheral metabolic cues, and control the entry of circulating signals into hypothalamic neural circuits \u003csup\u003e\u003cspan additionalcitationids=\"CR14 CR15 CR16\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Tanycytes are a heterogeneous population; they have been historically classified into distinct subtypes (α1, α2, β1, and β2) based on their dorsoventral location, molecular identity, and anatomical projections\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. These different subtypes are believed to play specialized roles in barrier regulation, hormone transport, neurogenesis, and metabolic signaling\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Despite this functional heterogeneity, our understanding of how VEGF signaling pathways are differentially distributed across tanycyte subtypes remains limited. A few studies from us and others have reported that VEGF expression in the hypothalamus could change in response to metabolic changes \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e, increasing ME\u0026rsquo;s permeability and access of circulating cues in the area. However, it remains unclear whether and which tanycytic populations express VEGFRs. Additionally, the role of VEGF signaling in tanycyte physiology or neuroendocrine function is unknown.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThis knowledge gap is of particularly relevant in light of the widespread clinical use of anti-angiogenic therapies, including VEGF-neutralizing antibodies and VEGFR2 inhibitors, in conditions ranging from cancer to age-related macular degeneration\u003csup\u003e\u003cspan additionalcitationids=\"CR20\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. While these drugs are designed to target peripheral vasculature, their potential effects on VEGF-sensitive glial cells in the brain have not been systematically addressed. In this study, we combined single-cell RNA sequencing (scRNA-seq), fluorescent \u003cem\u003ein situ\u003c/em\u003e hybridization, and quantitative PCR to map the expression of VEGFR1, VEGFR2, and VEGF ligands across tanycyte subtypes in the ME of mouse and human brain. Our findings uncover a non-endothelial VEGF signaling axis in hypothalamic tanycytes, with distinct spatial and temporal regulation. These insights not only broaden novel roles of VEGF signaling in non-endothelial cells in the brain but also raise far-reaching considerations for the central effects of systemic VEGF-targeted therapies, particularly in the context of aging and metabolic disease in neuroendocrine regulation.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eSpatially distinct VEGFR1/VEGFR2 expression in tanycyte subtypes in males and females\u003c/h2\u003e\u003cp\u003eTo assess the cell-type specificity of \u003cem\u003eVegfr2\u003c/em\u003e and \u003cem\u003eVegfr1\u003c/em\u003e expression within the ME, we re-analysed publicly available single-cell RNA sequencing (scRNA-seq) datasets from the adult male mouse hypothalamus\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Clustering analysis identified distinct tanycyte subtypes, including β1/β2- and α1/α2-tanycytes, that will be referred to hereafter by their anatomical localization as ME-tanycytes (β2), arcuate nucleus (ARH)-tanycytes (β1, α2), ventromedial hypothalamic (VMH)-tanycytes (α2, α1), and dorsomedial hypothalamic (DMH)-tanycytes (α1)\u003csup\u003e17\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e \u003cb\u003eand Supp\u003c/b\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Our re-analysis revealed that both VEGFRs, \u003cem\u003eVegfr2 (Kdr\u003c/em\u003e) and \u003cem\u003eVegfr1 (Flt1\u003c/em\u003e), are differentially enriched across tanycyte subtypes. \u003cem\u003eVegfr2\u003c/em\u003e expression is primarily restricted to ARH-tanycytes, with minimal to no expression in ME-tanycytes and low expression in VMH and DMH-tanycytes. In contrast, \u003cem\u003eVegfr1\u003c/em\u003e exhibits a complementary expression pattern, being selectively expressed in VMH/DMH-tanycytes, while largely absent in ME- and ARH-tanycytes. This mutually exclusive expression of \u003cem\u003eVegfr1\u003c/em\u003e and \u003cem\u003eVegfr2\u003c/em\u003e was confirmed using a dot plot (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). Interestingly, the third member of the VEGFR family, \u003cem\u003eVegfr3 (Flt4\u003c/em\u003e), was not detected in any tanycytic subpopulations (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB-D \u003cb\u003eand Supp\u003c/b\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). We also examined expression of co-receptors \u003cem\u003eNrp1\u003c/em\u003e and \u003cem\u003eNrp2\u003c/em\u003e. Both were expressed across all tanycyte subtypes, although \u003cem\u003eNrp2\u003c/em\u003e showed relatively lower expression in ME-tanycytes than \u003cem\u003eNrp1\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB-D \u003cb\u003eand Supp\u003c/b\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn parallel, to investigate the potential source and anatomical distribution of VEGF signaling components within the ME, we analysed the expression patterns of VEGF family ligands. VEGFA mRNA was robustly expressed across all tanycytic populations, with particularly high levels in ME-tanycytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC-D), which notably lack \u003cem\u003eVegfr1\u003c/em\u003e and \u003cem\u003eVegfr2\u003c/em\u003e, and in DMH-tanycytes, which express high levels of \u003cem\u003eVegfr1\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC-D). Other VEGF ligands were also evaluated. \u003cem\u003eVegfb\u003c/em\u003e was expressed in all tanycytic populations, while \u003cem\u003eVegfc\u003c/em\u003e showed only low-level expression if any (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC).\u003c/p\u003e\u003cp\u003eWe validated the scRNA-seq results by performing in-situ-hybridization (RNAscope) analysis of male coronal brain slices. Our results confirm the differential expression of \u003cem\u003eVegfr1/Vegfr2\u003c/em\u003e and \u003cem\u003eVegfa\u003c/em\u003e across the various tanycytic populations. \u003cem\u003eVegfr2\u003c/em\u003e mRNA was prominently localized to tanycytes facing the ARH, while \u003cem\u003eVegfr1\u003c/em\u003e mRNA was restricted to the VMH/DMH-tanycytes along the ventricle wall (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, \u003cb\u003eSup Fig.\u0026nbsp;2A\u003c/b\u003e). Notably, \u003cem\u003eVegfr1\u003c/em\u003e expression and compartmentalization appeared stronger than that of \u003cem\u003eVegfr2.\u003c/em\u003e Furthermore, we observed overlapping expression of \u003cem\u003eVegfa\u003c/em\u003e and \u003cem\u003eVegfr1\u003c/em\u003e in regions where \u003cem\u003eVegfr1\u003c/em\u003e was highly expressed (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, \u003cb\u003eSupp\u003c/b\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). As expected, based on the sc-RNAseq results, \u003cem\u003eVegfr3\u003c/em\u003e was not detected in tanycytes (\u003cb\u003eSupp\u003c/b\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). These spatial patterns were consistent across biological replicates. Signals for \u003cem\u003eVegfr1, Vegfr2\u003c/em\u003e and \u003cem\u003eVegfr3\u003c/em\u003e were also detected, as expected according to the single cell sequencing data, in endothelial cells of both fenestrated (very strong signal) and non-fenestrated blood\u0026ndash;brain barrier vessels (weaker signal) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, \u003cb\u003eSup Fig.\u0026nbsp;2A\u003c/b\u003e). These results suggest that VEGFRs and ligand expression are spatially compartmentalized among tanycyte subtypes, with ME-tanycytes potentially acting as signaling hubs despite lacking VEGFR expression themselves. This spatial architecture supports a model of directional, inter-subtype communication and suggests a sophisticated VEGF signaling landscape within the ME.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo further characterize the spatial architecture of VEGFR expression along the antero-posterior axis of the ME, which extends over 1.2mm, we performed RNAscope across serial coronal sections spanning from the anterior to posterior ME (Bregma \u0026minus;\u0026thinsp;1.34 to Bregma \u0026minus;\u0026thinsp;2.54), creating a detailed anatomical expression atlas (\u003cb\u003eSupp\u003c/b\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). This analysis revealed that the compartmentalization of \u003cem\u003eVegfr1\u003c/em\u003e and \u003cem\u003eVegfr2\u003c/em\u003e in tanycytes is most clearly defined in the central portion of the ME, where the spatial segregation between ARH- and VMH/DMH-tanycytes is sharply maintained. In contrast, the anterior and posterior ME regions displayed more overlapping expression domains, with some co-localization of \u003cem\u003eVegfr1\u003c/em\u003e and \u003cem\u003eVegfr2\u003c/em\u003e mRNA in tanycytes. This rostro-caudal gradient highlights additional heterogeneity within the ME and suggests that VEGFR-mediated signaling may be differentially modulated along its longitudinal axis (\u003cb\u003eSupp\u003c/b\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eProtein expression was further confirmed to complete our characterization using immunofluorescence. VEGFR1 and VEGFR2 proteins were detected at the level of the tanycytic cell body, facing the ventricular lumen, but not in the processes. VEGFR2 protein is localized to ARH tanycytes and VEGFR1 protein is detected mainly at the level of VMH/DMH tanycytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). However, their expression levels were substantially lower in tanycytes compared to the strong vascular expression observed in the surrounding vasculature (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, \u003cb\u003eSupp\u003c/b\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD).\u003c/p\u003e\u003cp\u003eConsidering that the VEGFR family can be modulated by estrogens\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e, and estradiol can influence VEGFR2 expression by stimulating angiogenesis through VEGFR2 upregulation\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e, we next investigated whether the observed compartmentalization pattern is conserved in female mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). \u003cem\u003eIn situ\u003c/em\u003e hybridization (RNAscope) on female mice brain sections at estrus and proestrus confirmed the receptor compartmentalization observed in males (\u003cem\u003eVegfr2\u003c/em\u003e at the level of the ARH and \u003cem\u003eVegfr1\u003c/em\u003e in VMH/DMH tanycytes) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, \u003cb\u003eSupp\u003c/b\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). We observed a reduced spatial overlap between \u003cem\u003eVegfr1\u003c/em\u003e and \u003cem\u003eVegfa\u003c/em\u003e expression domains in females, suggesting sex-specific differences in ligand-receptor interactions or signaling microenvironments. To better analyse the levels of \u003cem\u003eVegfr2\u003c/em\u003e in female tanycytes at the different stages of the estrus cycle we used a qPCR-based approach in isolated tanycytes. \u003cem\u003eR26-tdTomato\u003c/em\u003e female mice were stereotaxically injected with AAV1/2-Dio2-Cre into the lateral ventricle to drive Cre-dependent \u003cem\u003etdTomato\u003c/em\u003e expression in tanycytes\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Two weeks post-injection, animals were sacrificed at defined stages of the estrous cycle (estrus, proestrus and diestrus), and Tomato\u003csup\u003e+\u003c/sup\u003e tanycytes were isolated by fluorescence-activated cell sorting (FACS). qPCR analysis was then performed to assess \u003cem\u003eVegfr2\u003c/em\u003e mRNA expression. The qPCR results revealed no significant fluctuations in \u003cem\u003eVegfr2\u003c/em\u003e expression in tanycytes during the estrous cycle (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB)\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eVEGF receptor expression patterns are specific to median eminence tanycytes\u003c/h3\u003e\n\u003cp\u003eAll the circumventricular organs (CVOs) but one, the subcommisural organ (SCO), are characterized by the presence of a fenestrated endothelium, and tanycyte-like cells forming a barrier (blood-cerebrospinal fluid barrier)\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Each CVO is specialized in distinct physiological functions, raising the important question of whether the VEGFR expression patterns identified in the ME are conserved across other CVOs containing tanycyte-like cells. We extended our analysis to include the organum vasculosum of the lamina terminalis (OVLT), subfornical organ (SFO), area postrema (AP), and subcommissural organ (SCO). We also included the choroid plexus (CP) in our analysis, given its epithelial specialization and high secretory activity, despite it not being a CVO in the strict sense\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eUsing \u003cem\u003ein situ\u003c/em\u003e hybridization, we evaluated the expression on coronal brain sections, of \u003cem\u003eVegfr1 (Flt1), Vegfr2 (Kdr)\u003c/em\u003e, and \u003cem\u003eVegfa\u003c/em\u003e, alongside \u003cem\u003ecollagen IV\u003c/em\u003e immunostaining as a marker of vascular extracellular matrix to distinguish vascular from extravascular expression. Surprisingly, neither \u003cem\u003eVegfr1\u003c/em\u003e nor \u003cem\u003eVegfr2\u003c/em\u003e was detected in tanycyte-like cells of the OVLT, SFO, SCO, or AP. As expected, endothelial expression of both receptors was evident in blood vessels across all CVOs confirmed by co-localization with collagen IV, validating the specificity of the RNAscope probes (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-B). We observed, however, low \u003cem\u003eVegfr2\u003c/em\u003e expression in epithelial cells of the choroid plexus (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-B)\u003c/p\u003e\u003cp\u003eOn the other hand, \u003cem\u003eVegfa\u003c/em\u003e was robustly expressed in ventricular and parenchymal cells of both the SFO and OVLT, suggesting that ligand availability alone is not sufficient to drive receptor expression in tanycytes. Additionally, we observed strong \u003cem\u003eVegfa\u003c/em\u003e expression in the CP, consistent with previous reports\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e,\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-B). These findings suggest that the coordinated, subtype-specific expression of VEGFA, VEGFR1, and VEGFR2 in tanycytes is a unique feature of the ME, likely reflecting the specialized neurovascular and neuroendocrine functions of this region. This regional specificity supports the notion that tanycyte-like populations are molecularly and functionally heterogeneous across CVOs, and that VEGF signaling plays a particularly specialized role in ME tanycyte-mediated vascular and endocrine regulation.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003eDevelopmental refinement of VEGF receptor compartmentalization\u003c/h3\u003e\n\u003cp\u003eElectron microscopy studies from the 1970s demonstrated that the external zone of the ME begins to form during late embryogenesis and continues to mature throughout the first postnatal weeks\u003csup\u003e\u003cspan additionalcitationids=\"CR30\" citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e In mice, capillary loops of the fenestrated vessels only penetrate the ME after birth, first appearing towards the end of the first postnatal week and becoming more numerous during the second week\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. In parallel, tanycytes undergo progressive postnatal differentiation starting around postnatal day 4 (P4) and continuing through P14, with full transcriptional maturity typically reached between P14 and P20\u003csup\u003e32,33\u003c/sup\u003e. Given this temporal window of structural and cellular maturation, we wondered whether VEGFR expression is also acquired postnatally. To address this, we investigated the developmental timing of VEGFR expression and compartmentalization within the ME. Specifically we performed and quantified RNAscope for \u003cem\u003eVegfr1, Vegfr2\u003c/em\u003e, and \u003cem\u003eVegfa\u003c/em\u003e in coronal brain sections at different mouse postnatal stages (P0, P4, P8, P12, P16, and P21 just before puberty onset), and compared them to the adult mouse brain (3 months old). (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e and \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). We observed that both, \u003cem\u003eVegfr1\u003c/em\u003e and \u003cem\u003eVegfr2\u003c/em\u003e are already excluded from ME tanycytes at P0. Moreover, \u003cem\u003eVegfr1\u003c/em\u003e mRNA was already robustly expressed in VMH/DMH-tanycytes at P0, and this expression remained spatially restricted throughout postnatal development, indicating early and stable compartmentalization of \u003cem\u003eVegfr1\u003c/em\u003e. In contrast, the developmental trajectory of \u003cem\u003eVegfr2\u003c/em\u003e was slightly different; \u003cem\u003eVegfr2\u003c/em\u003e is expressed in ARH and VMH/DMH tanycytes at P0, indicating an early-established exclusion from the ME tanycyte subtype, however, the preferential expression of \u003cem\u003eVegfr2\u003c/em\u003e in ARH-tanycytes became more evident in adulthood. This coincided with a slight downregulation of its expression in VMH/DMH-tanycytes of adult mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). Thus, the full spatial segregation of \u003cem\u003eVegfr2\u003c/em\u003e among tanycytes subtypes appears to be a postnatal refinement process. As expected, \u003cem\u003eVegfr1\u003c/em\u003e and \u003cem\u003eVegfr2\u003c/em\u003e expression in vasculature was detectable from P0, as confirmed by their presence in vascular structures throughout development (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, white arrows).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eRegarding the ligand, \u003cem\u003eVegfa\u003c/em\u003e mRNA was present in all tanycytes from P0, with a higher expression in ME and VMH/DMH tanycytes starting from P4 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, blue arrows). Notably, in P8 mice, we identified discrete clusters of cells expressing high levels of \u003cem\u003eVegfa\u003c/em\u003e in the ME parenchyma, suggesting either local signaling shifts or cellular recruitment/remodelling events during this critical period (indicated in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e with red arrows). Our findings suggest that \u003cem\u003eVegfa\u003c/em\u003e expression is established early during postnatal development, preceding the full maturation of VEGFR2 compartmentalization. This temporal sequence supports a model in which VEGF ligand availability primes the microenvironment, while receptor expression is dynamically regulated to fine-tune signaling specificity during early postnatal remodelling of the ME.\u003c/p\u003e\u003cp\u003eTo contextualize these transcriptional changes with morphological development, we performed vimentin immunofluorescence to visualize tanycyte architecture\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e at the same developmental stages. We observed that at early postnatal timepoints (P0\u0026ndash;P4), tanycytes in the ME lacked the characteristic branched structure shown in adult animals. Beginning at P8, we observed the presence of branched tanycytic end feet in close apposition to the fenestrated capillaries of the ME, while a single basal process became evident in other tanycyte populations (\u003cb\u003eSupp\u003c/b\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). This morphological maturation aligns with previous observations\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e, and supports the idea that VEGFR dynamics may be linked to tanycyte specialization and vascular interaction.\u003c/p\u003e\n\u003ch3\u003eAge-dependent regulation of VEGF receptor expression\u003c/h3\u003e\n\u003cp\u003eThe VEGF system undergoes an age-related decline in signaling efficiency, resulting in reduced angiogenesis, impaired tissue regeneration, and increased susceptibility to age-associated diseases\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. Dysregulation of this pathway has also been implicated in neurodegenerative disorders such as Alzheimer\u0026rsquo;s disease, where altered VEGFA signaling contributes to neuroinflammation, vascular dysfunction, and cognitive decline\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 light of these findings, we sought to explore whether VEGFR signaling in tanycytes is similarly regulated across lifespan. To this end, we conducted qPCR analysis on FACS-isolated tomato⁺ tanycytes from microdissected ME explants from male mice at 3, 6, 12, and 18 months of age. Using our AAV approach, we specifically label cells lining the 3V, including all tanycytic subtypes\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e (\u003cb\u003eSupp\u003c/b\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). Our qPCR results revealed that, as in blood vessels, \u003cem\u003eVegfr2 (Kdr\u003c/em\u003e) expression is aged-regulated, with the highest levels at 3 months of age, corresponding to early adulthood, followed by an abrupt decline starting from 6 months of age (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA, \u003cb\u003eSupp\u003c/b\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). This age-related decrease suggests that VEGFR2-mediated signaling becomes attenuated in tanycytes during aging, which could impact their interactions with the vasculature or influence their neuroendocrine regulatory functions. In contrast, \u003cem\u003eVegfr1 (Flt1)\u003c/em\u003e expression remained stable throughout aging, with a slight increase by 18 months of age, indicating that VEGFR1 may serve a more constitutive or maintenance-related function in tanycytes that is less sensitive to age-related cues. As in previous experiments, \u003cem\u003eVegfr3 (Flt4)\u003c/em\u003e was barely detected in tanycytes at any age, reinforcing the conclusion that VEGFR3 is not an essential component of tanycyte-VEGF signaling, even under potential age-induced stress or remodelling.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eWe next assessed expression of VEGF ligands (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB), which are critical for autocrine and paracrine signaling. \u003cem\u003eVegfa\u003c/em\u003e exhibited relatively stable expression across all time points, with a modest increase in older mice, potentially reflecting a compensatory response to declining receptor levels or vascular alterations with age. \u003cem\u003eVegfc\u003c/em\u003e, the main ligand for \u003cem\u003eVEGFR3\u003c/em\u003e, remained largely unchanged. Strikingly, \u003cem\u003eVegfb\u003c/em\u003e expression showed a robust and significant increase from 3 to 18 months, suggesting that VEGFB\u0026ndash;VEGFR1 signaling may become more prominent in the ventricular wall of aged mice, possibly as a mechanism to maintain vascular or metabolic homeostasis in the face of \u003cem\u003eVegfr2\u003c/em\u003e decline. Finally, we evaluated the expression of VEGF co-receptors \u003cem\u003eNrp1\u003c/em\u003e and \u003cem\u003eNrp2\u003c/em\u003e. \u003cem\u003eNrp2\u003c/em\u003e, which modulates VEGF signaling\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e, exhibited a clear age-dependent downregulation, paralleling the decline in \u003cem\u003eVegfr2\u003c/em\u003e itself, and reinforcing the idea that \u003cem\u003eVegfr2/Nrp2\u003c/em\u003e-dependent pathways are progressively silenced with age. In contrast, \u003cem\u003eNrp1\u003c/em\u003e expression remained unchanged, suggesting differential regulatory control of neuropilin family members in tanycytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC).\u003c/p\u003e\n\u003ch3\u003eVEGFR2, but not VEGFR1 is expressed in human median eminence tanycytes\u003c/h3\u003e\n\u003cp\u003eTanycytes are also found in humans, with similar characteristics to the ones present in mice\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e,\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e.Thus we wanted to assess if VEGFR expression is conserved in human ME-tanycytes. To that end we decided to analyse the human spatial transcriptomics data from \u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e for the different members of the VEGF family (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e). We observed that indeed, VEGFA and VEGFR2 are highly expressed in the human ME, while VEGFB and VEGFR1 show a more dispersed expression. As in mice, almost no expression was detected for VEGFR3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eA). We further confirmed our observations using RNAscope \u003cem\u003ein situ\u003c/em\u003e hybridization in post-mortem hypothalamic tissue from healthy individuals. After validating the specificity of the technique (\u003cb\u003eSupp\u003c/b\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e), tissue samples from the region surrounding the third ventricle were examined for VEGFR1 and VEGFR2 expression. Consistent with our findings in mice, VEGFR2 mRNA was detected in vimentin-positive cells lining the third ventricle, at the level of the ME (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eB). In contrast, VEGFR1 mRNA was not detected in this vimentin-positive population (at the level of the ME or dorsally in the ventricle), suggesting a potential species-specific difference in receptor expression or regulatory control. Importantly, both VEGFR1 and VEGFR2 mRNA were readily detected in vascular endothelial cells, confirming the specificity and functionality of the probes used for this analysis.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThese results indicate that VEGFR2 expression in ME tanycytes is conserved between mice and humans, while VEGFR1 expression appears absent or restricted in adult human tanycytes. This divergence suggests evolutionary differences in VEGFR utilization, which may reflect species-specific adaptations in tanycyte function, vascular interaction, or neuroendocrine signaling at the brain\u0026rsquo;s interface with the bloodstream.\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eHere, we identify a compartmentalized VEGF signaling axis across distinct tanycyte subtypes in the median eminence (ME). VEGFR2 is enriched in β1-tanycytes that interface with the arcuate nucleus (ARH), whereas VEGFR1 is selectively expressed in α-tanycytes projecting toward dorsomedial and ventromedial hypothalamic regions. In contrast, β2 tanycytes, which directly contact the fenestrated vasculature in the ME, show the highest VEGFA expression yet lack detectable VEGFRs. This ligand\u0026ndash;receptor partitioning reveals an unexpectedly complex arrangement of autocrine and paracrine signaling within the tanycyte population and underscores the functional specialization of ME tanycytes relative to other CVOs.\u003c/p\u003e\u003cp\u003eOur intriguing findings suggest that VEGF signaling in tanycytes may extend beyond its well-established endothelial roles in angiogenesis, vascular remodeling, and permeability. One possibility is that VEGFR1, enriched in α-tanycytes at the upper ventricular wall, limits the availability of VEGFA in the CSF, thereby indirectly shaping VEGFR2-dependent responses in ARH-facing tanycytes. Alternatively, VEGFR1⁺ or VEGFR2⁺ tanycytes may respond to VEGFA secreted locally by neurons projecting from the lateral area of the hypothalamus (notably MCH-neurons\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e) or astrocytes\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. These models remain speculative but underscore the potential for region- and subtype-specific VEGF signaling to regulate hypothalamic neurovascular communication.\u003c/p\u003e\u003cp\u003eNotably, we observed co-expression of VEGFA and VEGFR1 within α-tanycytes, a configuration that could bias VEGF signaling toward VEGFR1-specific functions or act in parallel to mechanisms described in angiogenic endothelial cells. Indeed, the exclusive expression of VEGFR2 in ARH-tanycytes and VEGFR1 in VMH/DMH-tanycytes is reminiscent of tip- and stalk-cell dynamics during sprouting angiogenesis, where VEGFR2 drives sprouting responses to VEGFA in tip cells, while VEGFR1 in stalk cells functions as a decoy receptor to limit VEGFR2 activation\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. By analogy, VEGFR1⁺ tanycytes may serve to buffer VEGFA availability in the CSF, whereas VEGFR2⁺ tanycytes could help maintain the barrier properties of the ventricular wall.\u003c/p\u003e\u003cp\u003eAlthough functional studies are required to test these hypotheses, our observations suggest the possibility that VEGFR1 and VEGFR2 contribute to distinct and spatially segregated roles of tanycyte subtypes, that may from include region-specific vascular interactions, regulation of barrier permeability to modulation of endocrine access to hypothalamic circuits. Importantly, the strict confinement of VEGFR1 expression to α- but not β- tanycytes, further emphasizes that VEGF signaling is tightly compartmentalized, perhaps reflecting the unique physiological demands of different hypothalamic regions. From a translational perspective, uncovering a pathway that regulates tanycytic\u0026ndash;vascular interactions at both fenestrated and BBB-protected vessels could provide new opportunities to modulate barrier permeability, for example to facilitate drug delivery. At the same time, recognizing new non-endothelial functions of this pathway may help avoid off-target effects of anti-angiogenic therapies while also opening the possibility to exploit tanycytic VEGF signaling for CNS-directed interventions.\u003c/p\u003e\u003cp\u003eAnother striking observation that makes tanycytic VEGF/VEGFR signaling even more interesting is that VEGFR expression pattern is unique to the ME and absent in other CVOs that contain tanycyte-like cells, such as the OVLT, SFO, SCO, and AP\u003csup\u003e13\u003c/sup\u003e. While these regions also contain fenestrated and BBB-protected vessels, their tanycytic interfaces appear molecularly distinct. The high VEGFA expression in OVLT and SCO, but not in the AP, suggests that each CVO employs tailored VEGF-based mechanisms adapted to its physiological roles. The exclusivity of VEGFR1/2 expression to ME tanycytes may therefore reflect the central role of this structure in metabolic sensing and hypothalamic regulation. It is tempting to speculate that specific metabolic cues trigger VEGF release from hypothalamic cells, which then act in a paracrine manner on neighbouring tanycytes to regulate vascular exchange or other, as yet unidentified, tanycytic functions.\u003c/p\u003e\u003cp\u003eTanycytes express estrogen receptor-α, and cyclical changes in estrogen have been shown to remodel ME barrier properties\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. Estradiol regulates communication between tanycytes and endothelial cells, while also modulating VEGF expression and VEGFR2 signaling in the brain vasculature\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e,\u003csup\u003e23\u003c/sup\u003e. Given these established interactions, we anticipated sex differences in VEGFR expression. Although our RNAscope analysis showed reduced spatial overlap between \u003cem\u003eVegfr1\u003c/em\u003e and \u003cem\u003eVegfa\u003c/em\u003e females when compared to males, qPCR suggested modest, non-significant trends that merit deeper investigation using more selective approaches. Even if preliminary, our observations raise intriguing questions about how hormonal states may intersect with VEGF signaling in tanycytes. In particular, the dramatic decline in circulating estrogen during menopause could influence tanycytic VEGFR function and thereby impact hypothalamic barrier regulation. Because women spend nearly one-third of their lives in a post-menopausal state, which is characterized by the loss of ovarian steroids, understanding whether this dramatic and permanent decrease in circulating estrogens reshapes tanycytic VEGF signaling has broad physiological and clinical implications. Future studies in ovariectomized mouse models, or in hormonally manipulated animals, could directly test whether estradiol regulates VEGFR1/2 expression or function in tanycytes. Such work would clarify whether sex hormones act as modulators of this newly identified VEGF signaling axis and whether menopause represents a critical window for altered tanycytic\u0026ndash;vascular communication\u003c/p\u003e\u003cp\u003eTanycytes derive from radial glia\u0026ndash;like progenitors and mature postnatally into subtype-specific identities\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Our data acquired during postnatal development reveal distinct temporal trajectories of VEGFR expression. VEGFR1 is present from birth (P0) and remains confined to VMH/DMH-tanycytes, suggesting early specification of this subtype. In contrast, the restriction of VEGFR2 expression to ARH-tanycytes only becomes obvious in adulthood, consistent with later maturation of this domain. Both receptors are excluded from ME-tanycytes from the earliest stages, indicating that compartmentalization is not a consequence of postnatal differentiation. These temporal patterns support a paracrine model in which ligand and receptor are spatially segregated. Of interest is that clusters of high \u003cem\u003eVegfa\u003c/em\u003e-expressing cells appeared around P8 in the ME parenchyma, coinciding with the first vascular loops described in the ME, raising the possibility that local VEGF signaling contributes to vascular remodeling. Notably, this time window also corresponds to morphological maturation of tanycytes at around P8. Our findings suggest a coordinated program in which VEGF signaling integrates with tanycyte differentiation and vascular development to establish specialized hypothalamic barriers during early postnatal life, remarkable coordination for a small region with such essential roles in metabolism and reproduction. It will also be important to determine whether other barrier systems in the ME, such as the lateral diffusion barrier or perineuronal net barrier\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e, participate in this coordinated maturation.\u003c/p\u003e\u003cp\u003eSystemic studies have placed VEGF deficiency at the center of multiorgan aging, and sustained VEGF activity has been shown to promote healthier lifespan\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. In line with this, we observed a progressive decline in VEGFR2 expression in tanycytes beginning at 6 months, accompanied by a parallel decrease in its co-receptor Nrp2. This mirrors endothelial cell aging, where reduced VEGF signaling contributes to vascular dysfunction\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. By contrast, VEGFR1 expression remained stable suggesting a shift from a VEGFR2- to a VEGFR1-dominant axis in tanycytes over time. Whether this transition is adaptive, helping to preserve barrier integrity or tanycytic function, or maladaptive, contributing to impaired neuroendocrine communication, remains an open question. Given that VEGF dysregulation has been linked to Alzheimer\u0026rsquo;s disease and other disorders of vascular and metabolic dysfunction\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. tanycytic VEGFR2 downregulation could represent one mechanism linking hypothalamic aging to systemic pathology. These observations highlight tanycytic VEGFR as a potential therapeutic target: Enhancing VEGFR2 activity could improve barrier properties or metabolic resilience, whereas systemic anti-angiogenic therapies\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e,\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e may inadvertently impair tanycytic function. Indeed, we observed tanycyte fragmentation in Alzheimer\u0026rsquo;s disease patients\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e, consistent with emerging evidence implicating VEGFA\u0026ndash;VEGFR2 in neurodegeneration\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e,\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eImportantly we confirmed VEGFR2 expression in human tanycyte-like cells lining the floor of the third ventricle. However, VEGFR2 and VEGFA were co-expressed in the same tanycytic population, while we could not detect VEGFR1. The conserved VEGFR2 expression highlights a fundamental role for this pathway in tanycytes across mammals. These findings emphasize the need for deeper functional characterization of VEGFRs in tanycytes and their potential role in controlling vascular\u0026ndash;tanycytic communication.\u003c/p\u003e\u003cp\u003e\u003cb\u003eIn conclusion\u003c/b\u003e, beyond their well-established roles in vascular biology, our findings demonstrate that VEGFRs are expressed in a spatially organized manner in the distinct hypothalamic tanycytes populations, revealing a previously underappreciated non-endothelial site of VEGF signaling in the brain. This organization adds a new layer of complexity to how systemic VEGF cues may be interpreted at the brain\u0026ndash;body interface and underscores the importance of considering central nervous system targets, such as tanycytes, when developing or administering VEGF-targeted therapies.\u003c/p\u003e"},{"header":"METHODS","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003eAnimals\u003c/h2\u003e\u003cp\u003e\u003cem\u003eR26-tdTomato\u003c/em\u003e\u003csup\u003e\u003cem\u003eloxP\u0026minus;STOP\u0026minus;loxP\u003c/em\u003e\u003c/sup\u003ereporter mice (JAX#007914) were obtained from Jackson Laboratory. Wild-type C57BL/6J mice were purchased from Charles River Laboratories. Age details for each experimental group is provided in the corresponding figure legend. Mice were housed in a temperature-controlled facility (21\u0026deg;C) under a 12 h light/dark cycle, with ad libitum access to water and standard chow (20% of energy from protein, 67% from carbohydrates, and 12% from fat by dry weight). For developmental studies, pups were collected at postnatal day (P) 0, P4, P8, P12, P16, P21, and in adulthood (3 months of age). For aging experiments, adult male mice were sacrificed at 3, 6, 12, and 18 months. All experiments presented in the paper were performed using 3\u0026ndash;6 animals per group depending on the experiment. All animal procedures were conducted in accordance with the European Union Directive 2010/63/EU for animal experiments and approved by the Institutional Ethics Committee for the Care and Use of Experimental Animals of the University of Lille and the French Ministry of National Education, Higher Education and Research APAFIS#29172-2020121811279767 v5\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eSingle-cell RNA sequencing data reanalysis and human spatial transcriptomics\u003c/h2\u003e\u003cp\u003eSingle-cell RNA sequencing data from the Gene Expression Omnibus (GEO; accession number GSE90806, PMID: 28166221) were retrieved to analyze expression profile of VEGF receptors and their ligands in cells of the ARH/ME region, particularly tanycytes. Pre-processed gene counts and metadata were obtained as a SingleCellExperiment object via the CampbellBrainData() function of the scRNAseq package and reanalyzed using Seurat v5.1.0, a toolkit for single-cell genomics in R v4.4.2. Data integration was performed with the Harmony workflow\u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e within Seurat. Standard Seurat functions were applied for clustering, identification of cluster-specific markers, and visualization. Major clusters were annotated into distinct cell types using previously reported marker genes\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Tanycytes were subset, reclustered (dimensions\u0026thinsp;=\u0026thinsp;1:16; resolution\u0026thinsp;=\u0026thinsp;0.6) and annotated to define the four subpopulations based on established marker genes\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. hUMAP_1 and hUMAP_2 represent the two-dimensional embedding coordinates of cells grouped by similarity in their gene expression profiles.\u003c/p\u003e\u003cp\u003eThe preprocessed spatial transcriptomics data was retrieved from human hypomap\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e to map the spatial localization of \u0026lt;\u0026thinsp;list of genes\u0026thinsp;\u0026gt;\u0026thinsp;in human tanycytes. Feature plots were generated using scCustomize package (RRID:SCR_024675).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eHuman samples\u003c/h2\u003e\u003cp\u003eHuman brain tissue was obtained in accordance with French regulations (Good Practice Concerning the Conservation, Transformation and Transportation of Human Tissue to be Used Therapeutically, published on December 29, 1998). Authorization for the use of human tissue was granted by the French Agency for Biomedical Research (Agence de la Biom\u0026eacute;decine, Saint-Denis la Plaine, France; protocol no. PFS16-002) and the Lille Neurobiobank. Ethical approval for this study was provided by the Comit\u0026eacute; de Protection des Personnes (CPP) SUD-EST II (BIOWATCH, protocol #2021-A00879-32). Dissected blocks of adult human brain tissue containing the hypothalamus were immersion-fixed in 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS), pH 7.4, at 4\u0026deg;C for 1 week. Tissues were then cryoprotected in 30% sucrose in PBS at 4\u0026deg;C until they sank, embedded in Tissue-Tek OCT compound (Sakura Finetek), frozen on dry ice, and stored at \u0026minus;\u0026thinsp;80\u0026deg;C until sectioning. Coronal cryosections were cut at 20 \u0026micro;m thickness for downstream analyses.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eStereotaxic injection into the lateral ventricle\u003c/h2\u003e\u003cp\u003eRecombinant \u003cem\u003eAAV1/2-Dio2-iCre\u003c/em\u003e (1.25\u0026times;10⁹ genomic particles/\u0026micro;L) was produced as previously described\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. For female analysis experiments, 2 \u0026micro;L of AAV1/2-Dio2-iCre were stereotaxically injected into the lateral ventricle (anteroposterior: \u0026minus;0.3 mm; mediolateral: \u0026plusmn;1.0 mm; dorsoventral: \u0026minus;2.5 mm from bregma) of isoflurane-anesthetized R26-tdTomato mice. Three to four weeks post-injection, mice were sacrificed and the ME was micro-dissected for downstream cell dissociation and fluorescence-activated cell sorting (FACS) analysis. For aging studies, 2 \u0026micro;L of \u003cem\u003eAAV1/2-CAG-TdTomat\u003c/em\u003e (VectorBuilder) were similarly infused into the lateral ventricle of wild-type C57BL/6J mice under isoflurane anesthesia, four weeks prior to the age of sacrifice. For all stereotaxic surgeries, mice were anesthetized with isoflurane and placed on a heating pad to maintain body temperature. Eyes were protected with ophthalmic gel (Ocrygel). Injections were performed using a Kopf 963/962 stereotaxic frame, following the coordinates \u0026minus;\u0026thinsp;0.3 mm posterior to bregma, \u0026plusmn;\u0026thinsp;1.0 mm lateral, and \u0026minus;\u0026thinsp;2.5 mm ventral from the skull surface. A total volume of 2 \u0026micro;L was infused at a rate of 0.3 \u0026micro;L/min using a KD Scientific Legato 130 syringe pump and a 2 \u0026micro;L Neuro-Hamilton syringe. After injection, the skin was sutured and mice were allowed to recover on a heating pad before being returned to clean cages.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eEstrous cycle\u003c/h2\u003e\u003cp\u003eTo determine the stage of the estrous cycle, vaginal cytology was performed by flushing the vaginal canal with a small volume of sterile saline using a pipette, every day at the same time of the day, for at least 2 weeks. The cell suspension was collected and a few drops were placed on a clean glass microscope slide for immediate examination under a light microscope. Estrous cycle phases were identified based on the relative proportions of leukocytes, cornified epithelial cells, and nucleated epithelial cells. The stages were categorized as proestrus, estrus, or diestrus.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eTissue processing and immunostaining\u003c/h2\u003e\u003cp\u003eFor immunostaining, brains were fixed in 4% PFA/PBS overnight at 4\u0026deg;C before cryoprotection in 30% sucrose in PBS overnight at 4\u0026deg;C. Cryoprotected brains were frozen in OCT on dry ice and stored at -80\u0026deg;C until analysis. For RNA \u003cem\u003ein situ\u003c/em\u003e hybridization, fresh brains were immediately frozen in OCT after harvesting and stored at -20\u0026deg;C. Frozen brains were placed at -20\u0026deg;C overnight for equilibration and coronally cut on a Leica CM3050S cryostat at 20 \u0026micro;m (sections on slides). Slides were kept at -20\u0026deg;C until further processing.\u003c/p\u003e\u003cp\u003eSelected sections were dried for 30 minutes at room temperature before fixing in cold acetone/Ethanol (50%v/v) for 1 minute. Then, after 3 washes of 5 minutes with PBS-Triton 0.1%, sections were blocked in incubation solution (ICS, 1% BSA in PBS-Triton 0.3% pH 7,4) for 1 hour. Blocking was followed with primary antibody incubation in ICS for 24-48h at 4\u0026deg;C. Primary antibodies were then rinsed out, before incubation in fluorophore-coupled secondary antibodies for 1h in ICS at room temperature. Secondary antibodies were washed and sections counterstained with DAPI (D9542, Sigma). Finally, after a last wash of 10 minutes in PBS 1X, sections were mounted on slides with Mowiol, left to dry on the bench and stored at 4C until images analysis.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"No\" id=\"Taba\" border=\"1\"\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTarget\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eConcentration\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eReference\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eSupplier\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eRabbit anti-vimentin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e[1:4000]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eAB92547\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eAbcam\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGoat anti-collagen IV\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e[1:500]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eAB769\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eAbcam\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eanti-m-VEGF R1 (Flt-1)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e[1:100]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eAF471\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eR\u0026amp;D System\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eanti-mVEGF R2 (Flk-1)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e[1:100]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eAG644\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eR\u0026amp;D System\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDonkey anti-rabbit (488)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e[1:300]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eA21206\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eInvitrogen\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDonkey anti-goat (488)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e[1:300]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eA11055\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eInvitrogen\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eFACs sorting and quantitative PCR\u003c/h2\u003e\u003cp\u003eFor fluorescence-activated cell sorting (FACS) of hypothalamic tanycytes, mice were injected with either \u003cem\u003eAAV-Dio2-iCre\u003c/em\u003e into \u003cem\u003eR26-tdTomato\u003c/em\u003e mice or \u003cem\u003eAAV-CAG-tomato\u003c/em\u003e into wild-type C57BL/6J mice. Three to four weeks post-injection, median eminences (MEs) were micro dissected and enzymatically dissociated using the Papain Dissociation System (Worthington Biochemical Corporation, Lakewood, NJ) to obtain single-cell suspensions.\u003c/p\u003e\u003cp\u003eFACS was performed on a SONY SH800 Sorter Cytometer device using a 70 \u0026micro;m sorting chip. In \u003cem\u003etdTomato\u003c/em\u003e expressing mice, tanycytes were identified based on \u003cem\u003etdTomato\u003c/em\u003e fluorescence (excitation: 561 nm; detection: 675\u0026thinsp;\u0026plusmn;\u0026thinsp;20 nm). A sample of the cortex (That does not contain any fluorescence) was used as negative control for gating. For each animal, between 4,000/8,000 fluorescent-positive and -negative cells were sorted directly into 10/20 \u0026micro;L of lysis buffer (0.1% Triton X-100, Sigma-Aldrich; 0.4 U/\u0026micro;L RNaseOUT, ThermoFisher Scientific).\u003c/p\u003e\u003cp\u003e Starting with the same amount of sorted tanycytes, for gene expression analysis, total RNA from FACS-sorted cells was treated with DNase I (Invitrogen, ThermoFisher) to remove genomic DNA contamination and then reverse transcribed using SuperScript III Reverse Transcriptase (Invitrogen, ThermoFisher) according to the manufacturer's protocol. A linear preamplification step was performed using TaqMan PreAmp Master Mix (Applied Biosystems\u0026trade;, Cat. No. 4488593). Quantitative PCR (qPCR) was subsequently performed using the TaqMan\u0026reg; Universal Master Mix II (Applied Biosystems\u0026trade;, Cat. No. 4440049) on an Applied Biosystems QuantStudio 3 Real-Time PCR instrument (ref: A28131) and Applied Biosystems StepOnePlus Real-Time PCR using TaqMan Gene Expression Assays (Applied Biosystems, see list of probes below). Relative gene expression was normalized to housekeeping genes (18S and Actb) and analysed using the 2-ΔΔCt method. The purity of the sorted cells was confirmed with endothelial (\u003cem\u003ePecam1\u003c/em\u003e), neuronal (\u003cem\u003eElav4\u003c/em\u003e) and tanycytic markers (\u003cem\u003eGPR50\u003c/em\u003e. Data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM, and statistical tests are specified in the relevant figure legends.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"No\" id=\"Tabb\" border=\"1\"\u003e\u003ccolgroup cols=\"2\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGene\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eReference\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eActB\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMm02619580_g1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eElavl4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMm01263580_mH\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGpr50\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMm00439147_m1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNrp1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMm01253208_m1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNrp2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMm00803099_m1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePecam1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMm01242576_m1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eRn18S\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMm03928990_g1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eVEGFa\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMm00437306_m1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eVEGFa\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMm01281449_m1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eVEGFb\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMm00442102_m1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eVEGFc\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMm00437310_m1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eVEGFR1 (\u003cem\u003eFlt1\u003c/em\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMm00438992_m1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eVEGFR2 (\u003cem\u003eKdr\u003c/em\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMm01222421_m1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eVEGFR3 (\u003cem\u003eFlt4\u003c/em\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMm01292604_m1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eRNAscope\u003c/h2\u003e\u003cp\u003eRNAscope is performed by following the user manual RNAscope\u0026reg; Multiplex Fluorescent Reagents Kit v2 Assay (Advanced Cell Diagnostics - ACD) following the manufacturer's instructions. Briefly, fresh frozen brain sections were first fixed in PFA for 15 min at 4\u0026deg;C. Slides were dehydrated in different solutions of ethanol (50%, 70% and 100% ethanol). Hydrogen Peroxide was added on each slide in a humid chamber and rinsed. Protease IV was then added to each brain section in a humid chamber followed by a wash step. Selected probes were prepared and applied to the tissue, then incubated for 2 hours at 40\u0026deg;C in a HybEZ oven. Following hybridization, slides were washed in RNAscope wash buffer and subjected to signal amplification using AMP 1, AMP 2, and AMP 3 reagents, each incubated for 30 minutes at 40\u0026deg;C with washes in between. HRP-C1 and HRP-C2 channels were developed sequentially as per the kit instructions, with appropriate wash steps. For mouse, specific probes were used to detect \u003cem\u003eFlt1\u003c/em\u003e (415541-C1, NM_010228.3, target region 756\u0026ndash;1663), \u003cem\u003eKdr\u003c/em\u003e (414811-C2, NM_010612.2, target region 1766\u0026ndash;2673), and \u003cem\u003eVegfa\u003c/em\u003e (412261-C3, NM_001025257.3, target region 946\u0026ndash;2156) mRNAs and \u003cem\u003eFlt4\u003c/em\u003e (481371-C1, NM_008029.3, target region 95\u0026ndash;1058). For human, specific probes were used to detect \u003cem\u003eFlt1\u003c/em\u003e (560701-C2, NM_001160031.1, target region 817\u0026ndash;1810) and \u003cem\u003eKdr\u003c/em\u003e (435371-C1, NM_002253.2, target region 1400\u0026ndash;3515).\u003c/p\u003e\u003cp\u003eFollowing RNAscope, immunofluorescence was performed on the same sections. sections were blocked for 1 hour in PBS 1X\u0026thinsp;+\u0026thinsp;0.5% Triton X-100\u0026thinsp;+\u0026thinsp;1% BSA and then incubated with the primary antibody in the blocking solution overnight at 4\u0026deg;C. The following day, sections were rinsed 3x10 min in PBS 1X at room temperature. Sections were then incubated with secondary antibody in blocking solution for 2 hours at RT, and washed again 3x10 min in PBS 1X. Nuclear staining was performed with DAPI (1:10,000, 5 minutes), and slides were mounted using Mowiol mounting medium.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003eImage analysis and quantification\u003c/h2\u003e\u003cp\u003eImages were acquired using the Leica Stellaris 5 inverted confocal microscope (Leica Microsystems), using the 20x/0.8 dry objective or the 40x/1.4 oil immersion objective. Z-stack were done on 0.6 to 1\u0026micro;m thick optical sections on the whole depth of the section. Tiled images were acquired and automatically stitched using LasX software (Leica Microsystems). Maximum intensity projections were systematically generated using the same software.\u003c/p\u003e\u003cp\u003eImage processing was done using Fiji (ImageJ) software. Regions of interest (ROIs) corresponding to specific tanycyte subtypes were manually annotated based on anatomical landmarks and Vimentin expression. For RNAscope quantification, images were first converted to 8-bit and thresholded to the same value for each image of a same channel. The area and number of pixels of each region were taken to apply correction on the value, as the RNAscope method stipulates that the number of pixels is representative of the number of mRNA (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://acdbio.com/\u003c/span\u003e\u003cspan address=\"https://acdbio.com/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). For immunofluorescence quantification of the intensity of fluorescence, images were acquired with the same settings during confocal microscopy. Mean intensity was measured on hand-drawn ROIs.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eAll data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (SEM). Statistical analyses were performed using GraphPad Prism 10, using Student\u0026rsquo;s t-test, one-way ANOVA test, or Kruskal Wallis test. P-values under 0.05 were considered to be statistically significant. All experiments were independently replicated at least three times. The number of mice used per experiment is reported in the figure legends.\u003c/p\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eCOMPETING FINANCIAL INTERESTS\u003c/h2\u003e\u003cp\u003eThe authors declare no competing financial interests\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFUNDING\u003c/h2\u003e\u003cp\u003eThis work was supported by a doctoral fellowship from the University of Lille to O.D. The ANR grant ANR-23-CE14-0006-01 to IMC and the European Research Council WATCH Synergy Grant (No 810331 to MS and VP).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eO.D, M.L and D.F planned and performed experiments. S.N performed the scRNAseq and transcriptomic data analysis. C.A and M.S provided materials. I.M-C, O.D, D.F and V.P. discussed and interpreted the results I.M-C conceived the project and wrote the manuscript with the input of all the authors.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThe authors would like to thank PLBS UAR 2014 \u0026ndash; US41 (https://ums-plbs.univ-lille.fr/) with its different platforms and staff for expert technical assistance: Meryem Tardivel and Antonino Bongiovanni (microscopy core facility, BICeL) and Julien Devassine (Animal facility, BICeL). The authors would like also to thank Dr Adrian Coutteau-Robles, Thomas Chretien and Amandine Legrand for technical assistance.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSimons, M., Gordon, E. \u0026amp; Claesson-Welsh, L. Mechanisms and regulation of endothelial VEGF receptor signalling. \u003cem\u003eNat. Rev. Mol. 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Methods\u003c/em\u003e \u003cstrong\u003e16\u003c/strong\u003e, 1289\u0026ndash;1296 (2019).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"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":"Tanycytes, VEGF signaling, Median eminence, Neurovascular interface, Hypothalamus","lastPublishedDoi":"10.21203/rs.3.rs-7878186/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7878186/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eVascular endothelial growth factors (VEGFs) and their receptors (VEGFRs) are critical regulators of angiogenesis and vascular homeostasis. While VEGF signaling has been extensively studied in endothelial cells, emerging evidence suggests it also plays roles in non-endothelial brain cells. However, its spatial and cell-type-specific function within the hypothalamus, and more specifically at the level of the blood/CSF barrier remains poorly defined. In particular, little is known about VEGF receptor expression in tanycytes, a specialized glial population that lines the third ventricle and regulates body-brain communication within the median eminence (ME), a key neurovascular interface located at the tuberal region of the hypothalamus.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe used a multi-modal approach including single-cell RNA sequencing (scRNA-seq) reanalysis, RNAscope \u003cem\u003ein situ\u003c/em\u003e hybridization, immunohistochemistry, FACS-isolated qPCR in male and female mice, and human spatial transcriptomics to map the expression of VEGFR1 (\u003cem\u003eFlt1\u003c/em\u003e), VEGFR2 (\u003cem\u003eKdr\u003c/em\u003e), and VEGF ligands in hypothalamic tanycytes across gender, development and aging.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOur data reveal a striking spatial compartmentalization of VEGFR expression in tanycytes within the ME and the arcuate (ARH), ventromedial (VMH) and dorsomedial (DMH) hypothalamus. VEGFR2 is selectively expressed in ARH-tanycytes, while VEGFR1 is confined to VMH/DMH-tanycytes; and none of these receptors are expressed in ME-tanycytes. This pattern is unique to the ME and not observed in other circumventricular organs. VEGFR1 expression is established neonatally in mice (P0) and remains stable throughout life, whereas VEGFR2 expression becomes progressively refined postnatally, localizing to ARH-tanycytes in adulthood and showing a significant decline with aging. VEGFA is broadly expressed in all hypothalamic tanycytes, including ME-tanycytes, supporting a paracrine model of signaling. Importantly, in human hypothalamic tissue, VEGFR2, but not VEGFR1, is expressed in tanycytes, suggesting a partial evolutionary conservation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOur findings unveil for the first time, a non-endothelial VEGF signaling system in hypothalamic tanycytes that is spatially compartmentalized, developmentally programmed and age-dependant. These insights reveal new roles for VEGF signaling in neurovascular and neuroendocrine function, raising important considerations for central effects of VEGF-targeted therapies in aging and disease.\u003c/p\u003e","manuscriptTitle":"Compartmentalized VEGF Receptor Expression in Hypothalamic Tanycytes Reveals a Novel Non- Endothelial Axis of VEGF Signaling (Tanycytes as a Novel Non-Endothelial Target of VEGF Signaling)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-01 07:41:56","doi":"10.21203/rs.3.rs-7878186/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-11-13T20:59:37+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-13T14:31:48+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-06T07:39:47+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"126349858036798153958490986687331952047","date":"2025-10-23T16:57:23+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"5312450937139091380052771604001844055","date":"2025-10-23T14:31:47+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-10-21T14:26:29+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-10-20T13:44:55+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-10-18T06:21:39+00:00","index":"","fulltext":""},{"type":"submitted","content":"Fluids and Barriers of the CNS","date":"2025-10-16T13:14:41+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":"2d0f57ec-cb6f-4e18-bb23-e09f16ac67a7","owner":[],"postedDate":"November 1st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-02-16T16:00:28+00:00","versionOfRecord":{"articleIdentity":"rs-7878186","link":"https://doi.org/10.1186/s12987-026-00774-w","journal":{"identity":"fluids-and-barriers-of-the-cns","isVorOnly":false,"title":"Fluids and Barriers of the CNS"},"publishedOn":"2026-02-14 15:57:04","publishedOnDateReadable":"February 14th, 2026"},"versionCreatedAt":"2025-11-01 07:41:56","video":"","vorDoi":"10.1186/s12987-026-00774-w","vorDoiUrl":"https://doi.org/10.1186/s12987-026-00774-w","workflowStages":[]},"version":"v1","identity":"rs-7878186","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7878186","identity":"rs-7878186","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}