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Confocal images of human cerebral cortex at midgestation double labeled with Glut-1/GFAP, occludin/P-glycoprotein, and occludin/claudin-5. (A–C) A radial microvessel surrounded by GFAP-reactive RG fibers is revealed by the Glut-1 reactive endothelial cells. (D–F) On a stem vessel and its collateral (arrow) endothelial P-glycoprotein colocalize with occludin reactivity; note in (E) the lower levels of P-glycoprotein expression on the newly formed vessel branch (arrow) and in (D) and (F) the junctional linear pattern of occludin (arrowhead). (G–I) Occludin appears arranged according to a typical junctional pattern and colocalizes at points with claudin-5 (arrowheads). Bars: A–C and D–F 25 μm; G–I 30 μm.
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This study was conducted on human developing brain by laser confocal and transmission electron microscopy (TEM) to make a detailed analysis of important features of blood-brain barrier (BBB) microvessels and possible control mechanisms of vessel growth and differentiation during cerebral cortex vascularization. The BBB status of cortex microvessels...
Citations
... Interestingly, in the progenitor cytoplasm, the vesicles clustering around tip cell processes were not delimited by a surrounding membrane, as is the case for multivesicular bodies, but were a loose aggregation of vesicles of slightly heterogeneous size. It has previously been reported (Errede et al., 2014) that the CXCL12-positive vesicle cluster accumulates at contact points between neural progenitor cells and blood vessels and co-localizes with connexin 43. The direct contact of the vessel wall may have a similar vesicle-inducing effect as the tip cell processes. ...
The development of the neocortex involves an interplay between neural cells and the vasculature. However, little is known about this interplay at the ultrastructural level. To gain a 3D insight into the ultrastructure of the developing neocortex, we have analyzed the embryonic mouse neocortex by serial block-face scanning electron microscopy (SBF-SEM). In this study, we report a first set of findings that focus on the interaction of blood vessels, notably endothelial tip cells (ETCs), and the neural cells in this tissue. A key observation was that the processes of ETCs, located either in the ventricular zone (VZ) or subventricular zone (SVZ)/intermediate zone (IZ), can enter, traverse the cytoplasm, and even exit via deep plasma membrane invaginations of the host cells, including apical progenitors (APs), basal progenitors (BPs), and newborn neurons. More than half of the ETC processes were found to enter the neural cells. Striking examples of this ETC process “invasion” were (i) protrusions of apical progenitors or newborn basal progenitors into the ventricular lumen that contained an ETC process inside and (ii) ETC process-containing protrusions of neurons that penetrated other neurons. Our observations reveal a — so far unknown — complexity of the ETC–neural cell interaction.
... This finding underscores the essential role NSC expression of VEGF has in neurovascular development. In 22 week old human fetal cortical samples, NSCs expressing CXCl2 were shown to have basal processes that exerted a pro-angiogenic effect via direct contact with blood vessels (67). This further highlights the importance of NSC's role in neurovasculature development and patterning. ...
Cancer stem cells are thought to be the main drivers of tumorigenesis for malignancies such as glioblastoma (GBM). They are maintained through a close relationship with the tumor vasculature. Previous literature has well-characterized the components and signaling pathways for maintenance of this stem cell niche, but details on how the niche initially forms are limited. This review discusses development of the nonmalignant neural and hematopoietic stem cell niches in order to draw important parallels to the malignant environment. We then discuss what is known about the cancer stem cell niche, its relationship with angiogenesis, and provide a hypothesis for its development in GBM. A better understanding of the mechanisms of development of the tumor stem cell niche may provide new insights to potentially therapeutically exploit.
... targe tscan. org/ fish_ 62) that has been shown to be expressed in radial glia [66] and RPE cells [67], and mediates microglia activity and migration through the CXCR4 receptor. CXCR4 is also a predicted regulatory target of miR-18a (http:// www. ...
In mammals, photoreceptor loss causes permanent blindness, but in zebrafish (Danio rerio), photoreceptor loss reprograms Müller glia to function as stem cells, producing progenitors that regenerate photoreceptors. MicroRNAs (miRNAs) regulate CNS neurogenesis, but the roles of miRNAs in injury-induced neuronal regeneration are largely unknown. In the embryonic zebrafish retina, miR-18a regulates photoreceptor differentiation. The purpose of the current study was to determine, in zebrafish, the function of miR-18a during injury-induced photoreceptor regeneration. RT-qPCR, in situ hybridization, and immunohistochemistry showed that miR-18a expression increases throughout the retina between 1 and 5 days post-injury (dpi). To test miR-18a function during photoreceptor regeneration, we used homozygous miR-18a mutants (miR-18ami5012), and knocked down miR-18a with morpholino oligonucleotides. During photoreceptor regeneration, miR-18ami5012 retinas have fewer mature photoreceptors than WT at 7 and 10 dpi, but there is no difference at 14 dpi, indicating that photoreceptor regeneration is delayed. Labeling dividing cells with 5-bromo-2′-deoxyuridine (BrdU) showed that at 7 and 10 dpi, there are excess dividing progenitors in both mutants and morphants, indicating that miR-18a negatively regulates injury-induced proliferation. Tracing 5-ethynyl-2′-deoxyuridine (EdU) and BrdU-labeled cells showed that in miR-18ami5012 retinas excess progenitors migrate to other retinal layers in addition to the photoreceptor layer. Inflammation is critical for photoreceptor regeneration, and RT-qPCR showed that in miR-18ami5012 retinas, inflammatory gene expression and microglia activation are prolonged. Suppressing inflammation with dexamethasone rescues the miR-18ami5012 phenotype. Together, these data show that in the injured zebrafish retina, disruption of miR-18a alters proliferation, inflammation, the microglia/macrophage response, and the timing of photoreceptor regeneration.
... While many cell types express TJs, claudin-5 is specific to BMEC and particularly enriched in cerebrovasculature [68,78]. In addition, the localization of claudin-5, occludin, JAMs, and ZO-1 is often a criterion for validating cell sources for in vitro models [68,77,[79][80][81][82]. The general method for visualizing these proteins at cell-cell junctions is immunocytochemistry. ...
... The general method for visualizing these proteins at cell-cell junctions is immunocytochemistry. In physiological environments, TJ strands should be seen flocculently, whereas in pathological or nonphysiological conditions, they are discontinuous or show intracellular localization [68,[82][83][84][85]. ...
... The expression of GLUT1 is critical for the transport of glucose to fulfill the high metabolic requirements of the brain. The other appropriate markers for the BBB are the primary efflux transporters P-gp and breast cancer resistance protein (BCRP) [21,82]. These efflux systems have overlapping substrate affinities that actively pump compounds from endothelial cells into the bloodstream [21]. ...
The blood-brain barrier (BBB) functions as a highly selective border of endothelial cells, protecting the central nervous system from potentially harmful substances by selectively controlling the entry of cells and molecules, including components of the immune system. To study the BBB properties, find suitable therapies, and identify new drug targets, there is a need to develop representative in vitro BBB models. In this article, we describe the astrocyte roles in the BBB functioning and human in vitro BBB models.
... At this time, typical endothelial sprouts coexist with a variety of forebrain PC-driven angiogenesis-associated structures [84] and, together with the classical signaling systems [127], alternative pathways, such as the CXCL12/CXCR4/CXCR7 ligand receptors systems, are involved in radial glia-like stem cells-microvessel and endothelial-pericyte interactions that are also seen to include pericyte TNT/MT structures (Fig. 9). In particular, in the developing cerebral cortex, chemokine CXCL12 is highly expressed by radial glia-like stem cells, immature radial astrocytes, perivascular astrocyte endfeet, and activated, CD105 + endothelial tip cells, while CXCR4 appears to be specifically expressed by sprout-associated PCs and migrating neuroblasts [180,181] (Figs. 9, 10). ...
Central nervous system diseases involving the parenchymal microvessels are frequently associated with a ‘microvasculopathy’, which includes different levels of neurovascular unit (NVU) dysfunction, including blood–brain barrier alterations. To contribute to the understanding of NVU responses to pathological noxae, we have focused on one of its cellular components, the microvascular pericytes, highlighting unique features of brain pericytes with the aid of the analyses carried out during vascularization of human developing neocortex and in human gliomas. Thanks to their position, centred within the endothelial/glial partition of the vessel basal lamina and therefore inserted between endothelial cells and the perivascular and vessel-associated components (astrocytes, oligodendrocyte precursor cells (OPCs)/NG2-glia, microglia, macrophages, nerve terminals), pericytes fulfil a central role within the microvessel NVU. Indeed, at this critical site, pericytes have a number of direct and extracellular matrix molecule- and soluble factor-mediated functions, displaying marked phenotypical and functional heterogeneity and carrying out multitasking services. This pericytes heterogeneity is primarily linked to their position in specific tissue and organ microenvironments and, most importantly, to their ontogeny. During ontogenesis, pericyte subtypes belong to two main embryonic germ layers, mesoderm and (neuro)ectoderm, and are therefore expected to be found in organs ontogenetically different, nonetheless, pericytes of different origin may converge and colonize neighbouring areas of the same organ/apparatus. Here, we provide a brief overview of the unusual roles played by forebrain pericytes in the processes of angiogenesis and barriergenesis by virtue of their origin from midbrain neural crest stem cells. A better knowledge of the ontogenetic subpopulations may support the understanding of specific interactions and mechanisms involved in pericyte function/dysfunction, including normal and pathological angiogenesis, thereby offering an alternative perspective on cell subtype-specific therapeutic approaches.
... On the contrary, conditioned media from RGs decreases brain ECs proliferation (da Silva et al., 2019), promotes migration and formation of vessel-like structures in vitro (Siqueira et al., 2018). Also, in an autopsy of telencephalon from 22-week old human embryo, a defined Gfap + Cx43 + CXCL12 + RG population appeared to establish physical contact and interaction with angiogenic-activated (CD105 + ) ECs (Errede et al., 2014). These specialized contacts, recognizable on both perforating radial vessels and growing collaterals, appeared as CXCL12-reactive. ...
In this review, we discuss the state of our knowledge as it relates to embryonic brain vascular patterning in model systems zebrafish and mouse. We focus on the origins of endothelial cell and the distinguishing features of brain endothelial cells compared to non-brain endothelial cells, which is revealed by single cell RNA-sequencing methodologies. We also discuss the cross talk between brain endothelial cells and neural stem cells, and their effect on each other. In terms of mechanisms, we focus exclusively on Wnt signaling and the recent developments associated with this signaling network in brain vascular patterning, and the benefits and challenges associated with strategies for targeting the brain vasculature. We end the review with a discussion on the emerging areas of meningeal lymphatics, endothelial cilia biology and novel cerebrovascular structures identified in vertebrates.
... Cx43 is also expressed by migrating neurons (Nadarajah et al. 1997;Cina et al. 2007), serving to regulate neuronal migration in the developing brain (Elias et al. 2007;Cina et al. 2009;Elias et al. 2010). Published reports also support expression of Cx43 by endothelial cells (Okamoto et al. 2019), specifically in rat brain (De Bock et al. 2011;Belousov et al. 2017), in fetal human telencephalon (Errede et al. 2002), and by radial glial NPCs at their interface with endothelial cells in the human brain (Errede et al. 2014). Cx43 regulates essential endothelial cell functions including angiogenesis (Chen et al. 2015), migration (Kameritsch et al. 2019;Okamoto et al. 2019), and formation of the blood-brain barrier Figure 10. ...
... Our results indicate this signaling could occur at close range. Additional explanations for contacts between vasculature and NPCs include feed-forward signaling in which endothelial cells induce specific NPC behavior or influence daughter cell fate decisions, as well as feedback signaling from NPCs to regulate key functions identified for endothelial cells, such as vasculogenesis, stabilization of cortical vessels (Errede et al. 2014), or regulation of blood flow in proliferative regions with high metabolic demands. The data we present here suggest that endothelial cells communicate via gap junctions, hemichannels, or release of secretable factors with neighboring cells including NPCs. ...
Microglial cells make extensive contacts with neural precursor cells (NPCs) and affiliate with vasculature in the developing cerebral cortex. But how vasculature contributes to cortical histogenesis is not yet fully understood. To better understand functional roles of developing vasculature in the embryonic rat cerebral cortex, we investigated the temporal and spatial relationships between vessels, microglia, and NPCs in the ventricular zone. Our results show that endothelial cells in developing cortical vessels extend numerous fine processes that directly contact mitotic NPCs and microglia; that these processes protrude from vessel walls and are distinct from tip cell processes; and that microglia, NPCs, and vessels are highly interconnected near the ventricle. These findings demonstrate the complex environment in which NPCs are embedded in cortical proliferative zones and suggest that developing vasculature represents a source of signaling with the potential to broadly influence cortical development. In summary, cortical histogenesis arises from the interplay among NPCs, microglia, and developing vasculature. Thus, factors that impinge on any single component have the potential to change the trajectory of cortical development and increase susceptibility for altered neurodevelopmental outcomes.
... Radial glia (RG) cells are the major multipotent neural stem cell population during the embryonic cerebral cortex development period and originate most of the neuronal and glial cell types found in neural tissue, by activation of multiple signaling pathways (Gotz and Barde, 2005;Kriegstein and Alvarez-Buylla, 2009;Stipursky et al., 2012;Stipursky et al., 2014). Besides its well-known role as neural stem cells, RG have recently been demonstrated to directly control vascular development and blood brain barrier (BBB) formation in the embryonic cerebral cortex (Ma et al., 2013;Errede et al., 2014;Hirota et al., 2015;Siqueira et al., 2018). ...
... Although RG physiology greatly determines the correct formation of the cerebral cortex, including its vascularization (Ma et al., 2013;Errede et al., 2014;Hirota et al., 2015;Siqueira et al., 2018), the understanding of the impact of T. gondii infection on RG-endothelial interactions in the embryonic CNS has never been addressed. ...
... Vascular development by angiogenesis results from a fine-tuned control of pro-and anti-angiogenic molecules produced by endothelial and neighboring cells, as well as environmental cues (Carmeliet and Jain, 2000). In the last few years, RG has been pointed out as an essential cellular and molecular scaffold for blood vessel formation and vascular stability acquisition during cerebral cortex development (Ma et al., 2013;Errede et al., 2014;Hirota et al., 2015;Silva Siqueira et al., 2019;Siqueira et al., 2018). Herein, we demonstrate that RG-CM treatment of endothelial cells increases tight junction ZO-1 protein levels and organization, suggesting that RG-secreted factors promote microvascular barrier formation. ...
Congenital toxoplasmosis is a parasitic disease that occurs due vertical transmission of the protozoan Toxoplasma gondii (T. gondii) during pregnancy. The parasite crosses the placental barrier and reaches the developing brain, infecting progenitor, glial, neuronal and vascular cell types. Although the role of Radial glia (RG) neural stem cells in the development of the brain vasculature has been recently investigated, the impact of T. gondii infection in these events is not yet understood. Herein, we studied the role of T. gondii infection on RG cell function and its interaction with endothelial cells. By infecting isolated RG cultures with T. gondii tachyzoites, we observed a cytotoxic effect with reduced numbers of RG populations together with decrease neuronal and oligodendrocyte progenitor populations. Conditioned medium (CM) from RG control cultures increased ZO-1 protein levels and organization on endothelial bEnd.3 cells membranes, which was impaired by CM from infected RG, accompanied by decreased trans-endothelial electrical resistance (TEER). ELISA assays revealed reduced levels of anti-inflammatory cytokine TGF-β1 in CM from T. gondii-infected RG cells. Treatment with recombinant TGF-β1 concomitantly with CM from infected RG cultures led to restoration of ZO-1 staining in bEnd.3 cells. Congenital infection in Swiss Webster mice led to abnormalities in the cortical microvasculature in comparison to uninfected embryos. Our results suggest that infection of RG cells by T. gondii modulate cytokine secretion, which might contribute to endothelial loss of barrier properties, thus leading to impairment of neurovascular interaction establishment.
... In the mature BBB, capillary ECs interact with neighboring astrocytes, pericytes, microglia and neurons, constituting the neurovascular unit of the CNS that dynamically controls BBB function [1,9]. RG cells are the major neural stem cells population in the embryonic cerebral cortex since they generate neurons, astrocytes [10][11][12][13][14][15][16][17][18][19][20], and adult neural progenitor cells and their progeny [21]. Astrocytes interact with neighboring capillary ECs by extending their endfeet cellular processes that ensheath blood vessels to control BBB-ECs features and functions by cytokine secretion, such as TGF-β1, glial-derived neurotrophic factor (GDNF), basic fibroblast growth factor (bFGF) and Angiopoietin 1. ...
... Astrocyte differentiation from RG cells involves a decrease in neural stem cell expression markers, such as Nestin, brain lipid-binding protein (BLBP), and Pax6 and enhancement of the expression of astrocyte-specific genes such as glial fibrillary acidic protein (GFAP) [11,[15][16][17]. Astrocyte generation is an essential step for BBB formation, maturation and function [28]. ...
... Blood vessels and astrocytes endfeet maintain molecular and physical interactions throughout the life of the individual, providing a stable BBB function [36]. Thus, our data reinforce the idea that, in addition to the role of RG cells in vascular development, their differentiation into astrocytes [15,17] that eventually become associated with nascent vasculature, may contribute to the precocious stage of BBB development. ...
Background:
In the developing cerebral cortex, radial glia (RG) multipotent neural stem, among other functions, differentiate into astrocytes and serve as a scaffold for blood vessel development. Later on, blood vessel endothelial cells (ECs) associate with astrocytes to form the neurovascular blood brain barrier (BBB) unit.
Objective:
Since little is known about the mechanisms underlying bidirectional RG-ECs interactions in both vascular development and astrocyte differentiation, this study investigated the impact of interactions between RG and ECs mediated by secreted factors on EC maturation and gliogenesis control.
Method:
First, we demonstrated that immature vasculature in the murine embryonic cerebral cortex physically interacts with Nestin positive RG neural stem cells in vivo. Isolated microcapillary brain endothelial cells (MBEC) treated with the conditioned medium from RG cultures (RG-CM) displayed decreased proliferation, reduction in the protein levels of the endothelial tip cell marker Delta-like 4 (Dll4), and decreased expression levels of the vascular permeability associated gene, plasmalemma vesicle associated protein-1 (PLVAP1). These events were also accompanied by increased levels of the tight junction protein expression, zonula occludens-1 (ZO-1).
Result:
Finally, we demonstrated that isolated RG cells cultures treated with MBEC conditioned medium promoted astrocyte differentiation into astrocytes in a vascular endothelial growth factor – A (VEGF-A) dependent manner
Conclusion:
These results suggest that the bidirectional interaction between RG and ECs is essential to induce vascular maturation and astrocyte generation, which may be an essential cell-cell communication mechanism to promote BBB establishment.
... However, recent evidence suggests the importance of contributions from pericytes and radial glia cells in this process. 38,71,72 Pericytes are now thought to play a critical role in the early embryonic endothelial differentiation before astrocytes are present. 39,40,46 This concept is supported by in vivo studies in rodents demonstrating pericyte recruitment by PDGF signaling in E12 mice and that signaling molecules such as TGF-β and angiopoietin promote tight junction acquisition and barrier maturation. ...
... 39,40,46 Moreover, a cooperative effect has been suggested during human brain development between Wnt/β-catenin signaling and the CXCL12/CXCR4 chemokyne axis in neurovascular patterning and barrier differentiation that is mediated by radial glia cells. 72 An important concept is that BBB does not "switch" to a tight barrier at a specific time during angiogenesis, but rather the tightness of the barrier gradually increases in a region-dependent manner at the same time that angiogenesis is ongoing. 73 Wnt 7a and Wnt 7b appear to mediate the regulation of vascular development in the embryonic mouse brain. ...
