isolation-and-culture-of-adult-zebrafish-brain-derived-neurospheres

Isolation and Culture of Adult Zebrafish Brain-derived Neurospheres

The zebrafish is a highly relevant model organism for understanding the cellular and molecular mechanisms involved in neurogenesis and brain regeneration ...

murine-model-cerebral-venous-outflow-occlusion-through-bilateral

Murine Model of Cerebral Venous Outflow Occlusion Through Bilateral Ligation of Jugular Veins

Lymphatic vessels play a key role in maintaining fluid homeostasis and immune surveillance within tissues. At the brain's border, the dural meninges ...

Vascular endothelial growth factors (VEGFs) and their high-affinity tyrosine kinase VEGF receptors (VEGFRs) are key regulators of both angiogenesis and neurogenesis. The current issue of CMLS discusses recent literature and work implementing these signals in nervous system development, maintenance and disease pathology.

The power of VEGF (vascular endothelial growth factor) family molecules.

Notch cell interaction mechanism governs cell fate decisions in many different cell contexts throughout the lifetime of all Metazoan species. It links the fate of one cell to that of its neighbors through cell-to-cell contacts, and binding of Notch receptors expressed on one cell to their membrane bound ligands on an adjacent cell. Environmental cues, such as growth factors and extracellular matrix molecules, superimpose a dynamic regulation on this canonical Notch signaling pathway. In this review, we will focus on Notch signaling in the vertebrate vascular and nervous systems and examine its role in angiogenesis, neurogenesis, and neurovascular interactions. We will also highlight the molecular relationships of the Notch pathway with vascular endothelial growth factors (VEGFs) and their high-affinity tyrosine kinase VEGF receptors, key regulators of both angiogenesis and neurogenesis.

Interactions between VEGFR and Notch signaling pathways in endothelial and neural cells.

Neural stem cells (NSCs) continuously produce new neurons within the adult mammalian hippocampus. NSCs are typically quiescent but activated to self-renew or differentiate into neural progenitor cells. The molecular mechanisms of NSC activation remain poorly understood. Here, we show that adult hippocampal NSCs express vascular endothelial growth factor receptor (VEGFR) 3 and its ligand VEGF-C, which activates quiescent NSCs to enter the cell cycle and generate progenitor cells. Hippocampal NSC activation and neurogenesis are impaired by conditional deletion of Vegfr3 in NSCs. Functionally, this is associated with compromised NSC activation in response to VEGF-C and physical activity. In NSCs derived from human embryonic stem cells (hESCs), VEGF-C/VEGFR3 mediates intracellular activation of AKT and ERK pathways that control cell fate and proliferation. These findings identify VEGF-C/VEGFR3 signaling as a specific regulator of NSC activation and neurogenesis in mammals.

Vascular endothelial growth factor receptor 3 controls neural stem cell activation in mice and humans.

In multiple sclerosis, development of screening tools for remyelination-promoting molecules is timely.

The Xenopus tadpole: An in vivo model to screen drugs favoring remyelination.

The recent discovery of meningeal lymphatic vessels (LVs) has raised interest in their possible involvement in neuropathological processes, yet little is known about their development or maintenance. We show here that meningeal LVs develop postnatally, appearing first around the foramina in the basal parts of the skull and spinal canal, sprouting along the blood vessels and cranial and spinal nerves to various parts of the meninges surrounding the central nervous system (CNS). VEGF-C, expressed mainly in vascular smooth muscle cells, and VEGFR3 in lymphatic endothelial cells were essential for their development, whereas VEGF-D deletion had no effect. Surprisingly, in adult mice, the LVs showed regression after VEGF-C or VEGFR3 deletion, administration of the tyrosine kinase inhibitor sunitinib, or expression of VEGF-C/D trap, which also compromised the lymphatic drainage function. Conversely, an excess of VEGF-C induced meningeal lymphangiogenesis. The plasticity and regenerative potential of meningeal LVs should allow manipulation of cerebrospinal fluid drainage and neuropathological processes in the CNS.

Development and plasticity of meningeal lymphatic vessels.

In this issue of Cerebral Cortex, Cipriani et al. are following up on the recent report of Sorrels et al. to add novel immunohistological observations indicating that, unlike rodents, adult and aging humans do not acquire new neurons in the hippocampus. The common finding emerging from these 2 different, but almost simultaneous studies is highly significant because the dentate gyrus of the hippocampus was, until recently, considered as the only structure in the human brain that may continue neurogenesis throughout the full life span.

Adult Human Hippocampus: No New Neurons in Sight.

Macrophages are well characterized as immune cells. However, in recent years, a multitude of non-immune functions have emerged many of which play essential roles in a variety of developmental processes (Wynn et al., 2013; DeFalco et al., 2014). In adult animals, macrophages are derived from circulating monocytes originating in the bone marrow, but much of the tissue-resident population arise from erythro-myeloid progenitors (EMPs) in the extra-embryonic yolk sac, appearing around the same time as primitive erythroblasts (Schulz et al., 2012; Kierdorf et al., 2013; McGrath et al., 2015; Gomez Perdiguero et al., 2015; Mass et al., 2016). Of particular interest to our group, macrophages have been shown to act as pro-angiogenic regulators during development (Wynn et al., 2013; DeFalco et al., 2014; Hsu et al., 2015), but there is still much to learn about these early cells. The goal of the present study was to isolate and expand progenitors of yolk-sac-derived Embryonic Macrophages (EMs) in vitro to generate a new platform for mechanistic studies of EM differentiation. To accomplish this goal, we isolated pure (>98%) EGFP populations by flow cytometry from embryonic day 9.5 (E9.5) Csf1r-EGFP mice, then evaluated the angiogenic potential of EMs relative to Bone Marrow-Derived Macrophages (BMDMs). We found that EMs expressed more pro-angiogenic and less pro-inflammatory macrophage markers than BMDMs. EMs also promoted more endothelial cell (EC) cord formation in vitro, as compared to BMDMs in a manner that required direct cell-to-cell contact. Importantly, EMs preferentially matured into microglia when co-cultured with mouse Neural Stem/Progenitor Cells (NSPCs). In conclusion, we have established a protocol to isolate and propagate EMs in vitro, have further defined specialized properties of yolk-sac-derived macrophages, and have identified EM-EC and EM-NSPC interactions as key inducers of EC tube formation and microglial cell maturation, respectively.

The phenotypic and functional properties of mouse yolk-sac-derived embryonic macrophages.

Oligodendrocyte precursor cells (OPCs) constitute the main proliferative cells in the adult brain, and deregulation of OPC proliferation-differentiation balance results in either glioma formation or defective adaptive (re)myelination. OPC differentiation requires significant genetic reprogramming, implicating chromatin remodeling. Mounting evidence indicates that chromatin remodelers play important roles during normal development and their mutations are associated with neurodevelopmental defects, with CHD7 haploinsuficiency being the cause of CHARGE syndrome and CHD8 being one of the strongest autism spectrum disorder (ASD) high-risk-associated genes. Herein, we report on uncharacterized functions of the chromatin remodelers  and  in OPCs. Their OPC-chromatin binding profile, combined with transcriptome and chromatin accessibility analyses of -deleted OPCs, demonstrates that Chd7 protects nonproliferative OPCs from apoptosis by chromatin closing and transcriptional repression of  Furthermore, Chd7 controls OPC differentiation through chromatin opening and transcriptional activation of key regulators, including , , and  However, Chd7 is dispensable for oligodendrocyte stage progression, consistent with Chd8 compensatory function, as suggested by their common chromatin-binding profiles and genetic interaction. Finally, CHD7 and CHD8 bind in OPCs to a majority of ASD risk-associated genes, suggesting an implication of oligodendrocyte lineage cells in ASD neurological defects. Our results thus offer new avenues to understand and modulate the  and  functions in normal development and disease.

Oligodendrocyte precursor survival and differentiation requires chromatin remodeling by Chd7 and Chd8.

Orchestrating cortical brain development.

In this issue of Developmental Cell, Anbalagan et al. demonstrate that pituicytes, a subtype of astroglia, drive endothelial cell permeability in the zebrafish embryo neurohypophysis. This occurs via secretion of Vegfa/Tgfβ3 permeability factors that promote formation of fenestrae and the repression of anti-inflammatory retinoic acid signaling that induces tight cell junctions.

To BBB or Not to BBB?

The considerable metabolic activity of the central nervous system (CNS) requires an efficient system of tissue drainage and detoxification. The CNS is however devoid of lymphatic vessels, a vasculature ensuring interstitial fluid drainage and immune survey in other organs. A unique system of drainage has recently been identified between the cerebrospinal fluid (CSF), brain interstitial fluids and meningeal lymphatic vessels. This system is coupling a cerebral "glymphatic" flow with a meningeal lymphatic vasculature. The "glymphatic" system includes perivascular spaces and astrocytes, and drains interstitial fluids, from and towards the CSF. Meningeal lymphatic vessels are functionally linked to the cerebral "glymphatic" efflux by clearing intracerebral macromolecules and antigens towards the peripheral lymphatic system. The "glymphatic"-"meningeal lymphatics" system is potentially offering new therapeutic targets to improve cerebral drainage and immune survey in human CNS diseases.

[Lymphatic system in central nervous system].

Anamniotes, rodents, and young humans maintain neural stem cells in the ependymal zone (EZ) around the central canal of the spinal cord, representing a possible endogenous source for repair in mammalian lesions. Cell diversity and genes specific for this region are ill defined. A cellular and molecular resource is provided here for the mouse and human EZ based on RNA profiling, immunostaining, and fluorescent transgenic mice. This uncovered the conserved expression of 1,200 genes including 120 transcription factors. Unexpectedly the EZ maintains an embryonic-like dorsal-ventral pattern of expression of spinal cord developmental transcription factors (ARX, FOXA2, MSX1, and PAX6). In mice, dorsal and ventral EZ cells express Vegfr3 and are derived from the embryonic roof and floor plates. The dorsal EZ expresses a high level of Bmp6 and Gdf10 genes and harbors a subpopulation of radial quiescent cells expressing MSX1 and ID4 transcription factors.

RNA Profiling of the Human and Mouse Spinal Cord Stem Cell Niches Reveals an Embryonic-like Regionalization with MSX1 Roof-Plate-Derived Cells.

Endothelial cell migration, proliferation and survival are triggered by VEGF-A activation of VEGFR2. However, how these cell behaviors are regulated individually is still unknown. Here we identify Endophilin-A2 (ENDOA2), a BAR-domain protein that orchestrates CLATHRIN-independent internalization, as a critical mediator of endothelial cell migration and sprouting angiogenesis. We show that EndoA2 knockout mice exhibit postnatal angiogenesis defects and impaired front-rear polarization of sprouting tip cells. ENDOA2 deficiency reduces VEGFR2 internalization and inhibits downstream activation of the signaling effector PAK but not ERK, thereby affecting front-rear polarity and migration but not proliferation or survival. Mechanistically, VEGFR2 is directed towards ENDOA2-mediated endocytosis by the SLIT2-ROBO pathway via SLIT-ROBO-GAP1 bridging of ENDOA2 and ROBO1. Blocking ENDOA2-mediated endothelial cell migration attenuates pathological angiogenesis in oxygen-induced retinopathy models. This work identifies a specific endocytic pathway controlling a subset of VEGFR2 mediated responses that could be targeted to prevent excessive sprouting angiogenesis in pathological conditions.

Endophilin-A2 dependent VEGFR2 endocytosis promotes sprouting angiogenesis.

Cranial lymphatic vessels (LVs) are involved in the transport of fluids, macromolecules and central nervous system (CNS) immune responses. Little information about spinal LVs is available, because these delicate structures are embedded within vertebral tissues and difficult to visualize using traditional histology. Here we show an extended vertebral column LV network using three-dimensional imaging of decalcified iDISCO-clarified spine segments. Vertebral LVs connect to peripheral sensory and sympathetic ganglia and form metameric vertebral circuits connecting to lymph nodes and the thoracic duct. They drain the epidural space and the dura mater around the spinal cord and associate with leukocytes. Vertebral LVs remodel extensively after spinal cord injury and VEGF-C-induced vertebral lymphangiogenesis exacerbates the inflammatory responses, T cell infiltration and demyelination following focal spinal cord lesion. Therefore, vertebral LVs add to skull meningeal LVs as gatekeepers of CNS immunity and may be potential targets to improve the maintenance and repair of spinal tissues.

Anatomy and function of the vertebral column lymphatic network in mice.

Stem cell therapies demonstrate promising results as treatment for neurological disease and injury, owing to their innate ability to enhance endogenous neural tissue repair and promote functional recovery. However, delivery of undifferentiated and viable neuronal stem cells requires an engineered delivery system that promotes integration of transplanted cells into the inflamed and cytotoxic region of damaged tissue. Within the brain, endothelial cells (EC) of the subventricular zone play a critical role in neural stem cell (NSC) maintenance, quiescence and survival. Therefore, here, we describe the use of polyethylene glycol microbeads for the coincident delivery of EC and NSC as a means of enhancing appropriate NSC quiescence and survival during transplantation into the mouse brain. We demonstrate that EC and NSC co-encapsulation maintained NSC quiescence, enhanced NSC viability, and facilitated NSC extravasation in vitro, as compared to NSC encapsulated alone. In addition, co-encapsulated cells delivered to an in vivo non-injury model reduced inflammatory response compared to freely injected NSC. These results suggest the strong potential of a biomimetic engineered niche for NSC delivery into the brain following neurological injury.

Minimally Invasive Delivery of Microbeads with Encapsulated, Viable and Quiescent Neural Stem Cells to the Adult Subventricular Zone.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Author Correction: Minimally Invasive Delivery of Microbeads with Encapsulated, Viable and Quiescent Neural Stem Cells to the Adult Subventricular Zone.

Immune surveillance against pathogens and tumours in the central nervous system is thought to be limited owing to the lack of lymphatic drainage. However, the characterization of the meningeal lymphatic network has shed light on previously unappreciated ways that an immune response can be elicited to antigens that are expressed in the brain. Despite progress in our understanding of the development and structure of the meningeal lymphatic system, the contribution of this network in evoking a protective antigen-specific immune response in the brain remains unclear. Here, using a mouse model of glioblastoma, we show that the meningeal lymphatic vasculature can be manipulated to mount better immune responses against brain tumours. The immunity that is mediated by CD8 T cells to the glioblastoma antigen is very limited when the tumour is confined to the central nervous system, resulting in uncontrolled tumour growth. However, ectopic expression of vascular endothelial growth factor C (VEGF-C) promotes enhanced priming of CD8 T cells in the draining deep cervical lymph nodes, migration of CD8 T cells into the tumour, rapid clearance of the glioblastoma and a long-lasting antitumour memory response. Furthermore, transfection of an mRNA construct that expresses VEGF-C works synergistically with checkpoint blockade therapy to eradicate existing glioblastoma. These results reveal the capacity of VEGF-C to promote immune surveillance of tumours, and suggest a new therapeutic approach to treat brain tumours.

VEGF-C-driven lymphatic drainage enables immunosurveillance of brain tumours.

Gene expression levels vary across developmental stage, cell type, and region in the brain. Genomic variants also contribute to the variation in expression, and some neuropsychiatric disorder loci may exert their effects through this mechanism. To investigate these relationships, we present BrainVar, a unique resource of paired whole-genome and bulk tissue RNA sequencing from the dorsolateral prefrontal cortex of 176 individuals across prenatal and postnatal development. Here we identify common variants that alter gene expression (expression quantitative trait loci [eQTLs]) constantly across development or predominantly during prenatal or postnatal stages. Both "constant" and "temporal-predominant" eQTLs are enriched for loci associated with neuropsychiatric traits and disorders and colocalize with specific variants. Expression levels of more than 12,000 genes rise or fall in a concerted late-fetal transition, with the transitional genes enriched for cell-type-specific genes and neuropsychiatric risk loci, underscoring the importance of cataloging developmental trajectories in understanding cortical physiology and pathology.

ArticleTotal : 2	Year
Publication title Cited by 28	2016
Publication title	2025

Article	Year
The power of VEGF (vascular endothelial growth factor) family molecules. Cellular and molecular life sciences : CMLS\| PubMed ID: 23475064	2013
Interactions between VEGFR and Notch signaling pathways in endothelial and neural cells. Cellular and molecular life sciences : CMLS\| PubMed ID: 23479133	2013
Vascular endothelial growth factor receptor 3 controls neural stem cell activation in mice and humans. Cell reports\| PubMed ID: 25704818	2015
The Xenopus tadpole: An in vivo model to screen drugs favoring remyelination. Multiple sclerosis (Houndmills, Basingstoke, England)\| PubMed ID: 28752787	2018
Development and plasticity of meningeal lymphatic vessels. The Journal of experimental medicine\| PubMed ID: 29141865	2017
Adult Human Hippocampus: No New Neurons in Sight. Cerebral cortex (New York, N.Y. : 1991)\| PubMed ID: 29746611	2018
The phenotypic and functional properties of mouse yolk-sac-derived embryonic macrophages. Developmental biology\| PubMed ID: 30016639	2018
Oligodendrocyte precursor survival and differentiation requires chromatin remodeling by Chd7 and Chd8. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 30108144	2018
Orchestrating cortical brain development. Science (New York, N.Y.)\| PubMed ID: 30139860	2018
To BBB or Not to BBB? Developmental cell\| PubMed ID: 30562511	2018
[Lymphatic system in central nervous system]. Medecine sciences : M/S\| PubMed ID: 30672459	2019
RNA Profiling of the Human and Mouse Spinal Cord Stem Cell Niches Reveals an Embryonic-like Regionalization with MSX1 Roof-Plate-Derived Cells. Stem cell reports\| PubMed ID: 31031189	2019
Endophilin-A2 dependent VEGFR2 endocytosis promotes sprouting angiogenesis. Nature communications\| PubMed ID: 31138815	2019
Anatomy and function of the vertebral column lymphatic network in mice. Nature communications\| PubMed ID: 31597914	2019
Minimally Invasive Delivery of Microbeads with Encapsulated, Viable and Quiescent Neural Stem Cells to the Adult Subventricular Zone. Scientific reports\| PubMed ID: 31780709	2019
Author Correction: Minimally Invasive Delivery of Microbeads with Encapsulated, Viable and Quiescent Neural Stem Cells to the Adult Subventricular Zone. Scientific reports\| PubMed ID: 31932638	2020
VEGF-C-driven lymphatic drainage enables immunosurveillance of brain tumours. Nature\| PubMed ID: 31942068	2020
Whole-Genome and RNA Sequencing Reveal Variation and Transcriptomic Coordination in the Developing Human Prefrontal Cortex. Cell reports\| PubMed ID: 32268104	2020

Jean-Léon Thomas

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