A novel preparation for histological analyses of intraventricular macrophages in the embryonic brain.

Microglia colonize the brain starting on embryonic day (E) 9.5 in mice, and their population increases with development. We have previously demonstrated that some microglia are derived from intraventricular macrophages, which frequently infiltrate the pallium at E12.5. To address how the infiltration of intraventricular macrophages is spatiotemporally regulated, histological analyses detecting how these cells associate with the surrounding cells at the site of infiltration into the pallial surface are essential. Using two-photon microscopy-based in vivo imaging, we demonstrated that most intraventricular macrophages adhere to the ventricular surface. This is a useful tool for imaging intraventricular macrophages maintaining their original position, but this method cannot be used for observing deeper brain regions. Meanwhile, we found that conventional cryosection-based and naked pallial slice-based observation resulted in unexpected detachment from the ventricular surface of intraventricular macrophages and their mislocation, suggesting that previous histological analyses might have failed to determine their physiological number and location in the ventricular space. To address this, we sought to establish a methodological preparation that enables us to delineate the structure and cellular interactions when intraventricular macrophages infiltrate the pallium. Here, we report that brain slices pretreated with agarose-embedding maintained adequate density and proper positioning of intraventricular macrophages on the ventricular surface. This method also enabled us to perform the immunostaining. We believe that this is helpful for conducting histological analyses to elucidate the mechanisms underlying intraventricular macrophage infiltration into the pallium and their cellular properties, leading to further understanding of the process of microglial colonization into the developing brain.

Isolation of ependymal cilia from mouse brain.

BACKGROUND: Ependymal cilia play a major role in the circulation of cerebrospinal fluid. Although isolation of cilia is an essential technique for investigating ciliary structure, to the best of our knowledge, no report on the isolation and structural analysis of ependymal cilia from mouse brain is available. NEW METHOD: We developed a novel method for isolating ependymal cilia from mouse brain ventricles. We isolated ependymal cilia by partially opening the lateral ventricles and gently applying shear stress, followed by pipetting and ultracentrifugation. RESULTS: Using this new method, we were able to observe cilia separately. The results demonstrated that our method successfully isolated intact ependymal cilia with preserved morphology and ultrastructure. In this procedure, the ventricular ependymal cell layer was partially detached. COMPARISON WITH EXISTING METHODS: Compared to existing methods for isolating cilia from other tissues, our method is meticulously tailored for extracting ependymal cilia from the mouse brain. Designed with a keen understanding of the fragility of the ventricular ependyma, our method prioritizes minimizing tissue damage during the isolation procedure. CONCLUSIONS: We isolated ependymal cilia from mouse brain by applying shear stress selectively to the ventricles. Our method can be used to conduct more detailed studies on the structure of ependymal cilia.

Protocol for electroporating and isolating murine (sub)ventricular zone cells for single-nuclei omics.

In vivo genetic modification of neural stem cells is necessary to model the origins and pathogenesis of neurological disorders. Electroporation is a technique that applies a transient electrical field to direct charged molecules into living cells to genetically modify the mouse brain. Here, we provide a protocol to electroporate the neural stem cells surrounding the neonatal ventricles. We describe subsequent steps to isolate and prepare nuclei from the cells and their cellular progeny for single-nuclei omics. For complete details on the use and execution of this protocol, please refer to Riley et al.1.

The laminar position, morphology, and gene expression profiles of cortical astrocytes are influenced by time of birth from ventricular/subventricular progenitors.

Astrocytes that reside in superficial (SL) and deep cortical layers have distinct molecular profiles and morphologies, which may underlie specific functions. Here, we demonstrate that the production of SL and deep layer (DL) astrocyte populations from neural progenitor cells in the mouse is temporally regulated. Lineage tracking following in utero and postnatal electroporation with PiggyBac (PB) EGFP and birth dating with EdU and FlashTag, showed that apical progenitors produce astrocytes during late embryogenesis (E16.5) that are biased to the SL, while postnatally labeled (P0) astrocytes are biased to the DL. In contrast, astrocytes born during the predominantly neurogenic window (E14.5) showed a random distribution in the SL and DL. Of interest, E13.5 astrocytes birth dated at E13.5 with EdU showed a lower layer bias, while FT labeling of apical progenitors showed no bias. Finally, examination of the morphologies of "biased" E16.5- and P0-labeled astrocytes demonstrated that E16.5-labeled astrocytes exhibit different morphologies in different layers, while P0-labeled astrocytes do not. Differences based on time of birth are also observed in the molecular profiles of E16.5 versus P0-labeled astrocytes. Altogether, these results suggest that the morphological, molecular, and positional diversity of cortical astrocytes is related to their time of birth from ventricular/subventricular zone progenitors.

The localization of amyloid precursor protein to ependymal cilia in vertebrates and its role in ciliogenesis and brain development in zebrafish.

Amyloid precursor protein (APP) is expressed in many tissues in human, mice and in zebrafish. In zebrafish, there are two orthologues, Appa and Appb. Interestingly, some cellular processes associated with APP overlap with cilia-mediated functions. Whereas the localization of APP to primary cilia of in vitro-cultured cells has been reported, we addressed the presence of APP in motile and in non-motile sensory cilia and its potential implication for ciliogenesis using zebrafish, mouse, and human samples. We report that Appa and Appb are expressed by ciliated cells and become localized at the membrane of cilia in the olfactory epithelium, otic vesicle and in the brain ventricles of zebrafish embryos. App in ependymal cilia persisted in adult zebrafish and was also detected in mouse and human brain. Finally, we found morphologically abnormal ependymal cilia and smaller brain ventricles in appa-/-appb-/- mutant zebrafish. Our findings demonstrate an evolutionary conserved localisation of APP to cilia and suggest a role of App in ciliogenesis and cilia-related functions.

The regulatory roles of motile cilia in CSF circulation and hydrocephalus.

BACKGROUND: Cerebrospinal fluid (CSF) is an ultra-filtrated colorless brain fluid that circulates within brain spaces like the ventricular cavities, subarachnoid space, and the spine. Its continuous flow serves many primary functions, including nourishment, brain protection, and waste removal. MAIN BODY: The abnormal accumulation of CSF in brain cavities triggers severe hydrocephalus. Accumulating evidence had indicated that synchronized beats of motile cilia (cilia from multiciliated cells or the ependymal lining in brain ventricles) provide forceful pressure to generate and restrain CSF flow and maintain overall CSF circulation within brain spaces. In humans, the disorders caused by defective primary and/or motile cilia are generally referred to as ciliopathies. The key role of CSF circulation in brain development and its functioning has not been fully elucidated. CONCLUSIONS: In this review, we briefly discuss the underlying role of motile cilia in CSF circulation and hydrocephalus. We have reviewed cilia and ciliated cells in the brain and the existing evidence for the regulatory role of functional cilia in CSF circulation in the brain. We further discuss the findings obtained for defective cilia and their potential involvement in hydrocephalus. Furthermore, this review will reinforce the idea of motile cilia as master regulators of CSF movements, brain development, and neuronal diseases.

Release of stem cells from quiescence reveals gliogenic domains in the adult mouse brain.

Quiescent neural stem cells (NSCs) in the adult mouse ventricular-subventricular zone (V-SVZ) undergo activation to generate neurons and some glia. Here we show that platelet-derived growth factor receptor beta (PDGFRß) is expressed by adult V-SVZ NSCs that generate olfactory bulb interneurons and glia. Selective deletion of PDGFRß in adult V-SVZ NSCs leads to their release from quiescence, uncovering gliogenic domains for different glial cell types. These domains are also recruited upon injury. We identify an intraventricular oligodendrocyte progenitor derived from NSCs inside the brain ventricles that contacts supraependymal axons. Together, our findings reveal that the adult V-SVZ contains spatial domains for gliogenesis, in addition to those for neurogenesis. These gliogenic NSC domains tend to be quiescent under homeostasis and may contribute to brain plasticity.

Progressions on the Coexistence of Neuronal and Glial Precursor Cells in the Cerebral Ventricular Zone.

Heterogeneity is defined as the quality or state of being diverse in character or content. This article summarizes the natural progression from my studies, reported in the first issue of the Journal of Neuroscience, that identified molecular heterogeneity in precursor cells of the developing primate cerebral cortex to the current state in which differences defined at the molecular, cellular, circuit, and systems levels are building data encyclopedias. The emphasis on heterogeneity has impacted many contributors in the field of developmental neuroscience, who have led a quest to determine the extent to which there is diversity, when it appears developmentally, and what heritable and nonheritable factors mediate nervous system assembly and function. Since the appearance of the article on progenitor cell heterogeneity in the inaugural issue of the Journal of Neuroscience, there have been continuous advances in technologies and data analytics that are contributing to a much better understanding of the origins of neurobiological and behavioral heterogeneity.

Development of the Neuro-Immune-Vascular Plexus in the Ventricular Zone of the Prenatal Rat Neocortex.

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.

Altered cleavage plane orientation with increased genomic aneuploidy produced by receptor-mediated lysophosphatidic acid (LPA) signaling in mouse cerebral cortical neural progenitor cells.

The brain is composed of cells having distinct genomic DNA sequences that arise post-zygotically, known as somatic genomic mosaicism (SGM). One form of SGM is aneuploidy-the gain and/or loss of chromosomes-which is associated with mitotic spindle defects. The mitotic spindle orientation determines cleavage plane positioning and, therefore, neural progenitor cell (NPC) fate during cerebral cortical development. Here we report receptor-mediated signaling by lysophosphatidic acid (LPA) as a novel extracellular signal that influences cleavage plane orientation and produces alterations in SGM by inducing aneuploidy during murine cortical neurogenesis. LPA is a bioactive lipid whose actions are mediated by six G protein-coupled receptors, LPA1-LPA6. RNAscope and qPCR assessment of all six LPA receptor genes, and exogenous LPA exposure in LPA receptor (Lpar)-null mice, revealed involvement of Lpar1 and Lpar2 in the orientation of the mitotic spindle. Lpar1 signaling increased non-vertical cleavage in vivo by disrupting cell-cell adhesion, leading to breakdown of the ependymal cell layer. In addition, genomic alterations were significantly increased after LPA exposure, through production of chromosomal aneuploidy in NPCs. These results identify LPA as a receptor-mediated signal that alters both NPC fate and genomes during cortical neurogenesis, thus representing an extracellular signaling mechanism that can produce stable genomic changes in NPCs and their progeny. Normal LPA signaling in early life could therefore influence both the developing and adult brain, whereas its pathological disruption could contribute to a range of neurological and psychiatric diseases, via long-lasting somatic genomic alterations.

Dynamic Changes in the Neurogenic Potential in the Ventricular-Subventricular Zone of Common Marmoset during Postnatal Brain Development.

Even after birth, neuronal production continues in the ventricular-subventricular zone (V-SVZ) and hippocampus in many mammals. The immature new neurons ("neuroblasts") migrate and then mature at their final destination. In humans, neuroblast production and migration toward the neocortex and the olfactory bulb (OB) occur actively only for a few months after birth and then sharply decline with age. However, the precise spatiotemporal profiles and fates of postnatally born neurons remain unclear due to methodological limitations. We previously found that common marmosets, small nonhuman primates, share many features of V-SVZ organization with humans. Here, using marmosets injected with thymidine analogue(s) during various postnatal periods, we demonstrated spatiotemporal changes in neurogenesis during development. V-SVZ progenitor proliferation and neuroblast migration toward the OB and neocortex sharply decreased by 4 months, most strikingly in a V-SVZ subregion from which neuroblasts migrated toward the neocortex. Postnatally born neurons matured within a few months in the OB and hippocampus but remained immature until 6 months in the neocortex. While neurogenic activity was sustained for a month after birth, the distribution and/or differentiation diversity was more restricted in 1-month-born cells than in the neonatal-born population. These findings shed light on distinctive features of postnatal neurogenesis in primates.

Brain Ventricular System and Cerebrospinal Fluid Development and Function: Light at the End of the Tube: A Primer with Latest Insights.

The brain ventricular system is a series of connected cavities, filled with cerebrospinal fluid (CSF), that forms within the vertebrate central nervous system (CNS). The hollow neural tube is a hallmark of the chordate CNS, and a closed neural tube is essential for normal development. Development and function of the ventricular system is examined, emphasizing three interdigitating components that form a functional system: ventricle walls, CSF fluid properties, and activity of CSF constituent factors. The cellular lining of the ventricle both can produce and is responsive to CSF. Fluid properties and conserved CSF components contribute to normal CNS development. Anomalies of the CSF/ventricular system serve as diagnostics and may cause CNS disorders, further highlighting their importance. This review focuses on the evolution and development of the brain ventricular system, associated function, and connected pathologies. It is geared as an introduction for scholars with little background in the field.

Galectin-3 modulates postnatal subventricular zone gliogenesis.

Postnatal subventricular zone (SVZ) neural stem cells generate forebrain glia, namely astrocytes and oligodendrocytes. The cues necessary for this process are unclear, despite this phase of brain development being pivotal in forebrain gliogenesis. Galectin-3 (Gal-3) is increased in multiple brain pathologies and thereby regulates astrocyte proliferation and inflammation in injury. To study the function of Gal-3 in inflammation and gliogenesis, we carried out functional studies in mouse. We overexpressed Gal-3 with electroporation and using immunohistochemistry surprisingly found no inflammation in the healthy postnatal SVZ. This allowed investigation of inflammation-independent effects of Gal-3 on gliogenesis. Loss of Gal-3 function via knockdown or conditional knockout reduced gliogenesis, whereas Gal-3 overexpression increased it. Gal-3 overexpression also increased the percentage of striatal astrocytes generated by the SVZ but decreased the percentage of oligodendrocytes. These novel findings were further elaborated with multiple analyses demonstrating that Gal-3 binds to the bone morphogenetic protein receptor one alpha (BMPR1α) and increases bone morphogenetic protein (BMP) signaling. Conditional knockout of BMPR1α abolished the effect of Gal-3 overexpression on gliogenesis. Gain-of-function of Gal-3 is relevant in pathological conditions involving the human forebrain, which is particularly vulnerable to hypoxia/ischemia during perinatal gliogenesis. Hypoxic/ischemic injury induces astrogliosis, inflammation and cell death. We show that Gal-3 immunoreactivity was increased in the perinatal human SVZ and striatum after hypoxia/ischemia. Our findings thus show a novel inflammation-independent function for Gal-3; it is necessary for gliogenesis and when increased in expression can induce astrogenesis via BMP signaling.

Multiple steps characterise ventricular layer attrition to form the ependymal cell lining of the adult mouse spinal cord central canal.

The ventricular layer of the spinal cord is remodelled during embryonic development and ultimately forms the ependymal cell lining of the adult central canal, which retains neural stem cell potential. This anatomical transformation involves the process of dorsal collapse; however, accompanying changes in tissue organisation and cell behaviour as well as the precise origin of cells contributing to the central canal are not well understood. Here, we describe sequential localised cell rearrangements which accompany the gradual attrition of the spinal cord ventricular layer during development. This includes local breakdown of the pseudostratified organisation of the dorsal ventricular layer prefiguring dorsal collapse and evidence for a new phenomenon, ventral dissociation, during which the ventral-most floor plate cells separate from a subset that are retained around the central canal. Using cell proliferation markers and cell-cycle reporter mice, we further show that following dorsal collapse, ventricular layer attrition involves an overall reduction in cell proliferation, characterised by an intriguing increase in the percentage of cells in G1/S. In contrast, programmed cell death does not contribute to ventricular layer remodelling. By analysing transcript and protein expression patterns associated with key signalling pathways, we provide evidence for a gradual decline in ventral sonic hedgehog activity and an accompanying ventral expansion of initial dorsal bone morphogenetic protein signalling, which comes to dominate the forming the central canal lining. This study identifies multiple steps that may contribute to spinal cord ventricular layer attrition and adds to increasing evidence for the heterogeneous origin of the spinal cord ependymal cell population, which includes cells from the floor plate and the roof plate as well as ventral progenitor domains.

Intraventricular Transplantation of Engineered Neuronal Precursors for In Vivo Neuroarchitecture Studies.

Gene control of neuronal cytoarchitecture is currently the subject of intensive investigation. Described here is a simple method developed to study in vivo gene control of neocortical projection neuron morphology. This method is based on (1) in vitro lentiviral engineering of neuronal precursors as "test" and "control" cells, (2) their co-transplantation into wild-type brains, and (3) paired morphometric evaluation of their neuronal derivatives. Specifically, E12.5 pallial precursors from panneuronal, genetically labeled donors, are employed for this purpose. They are engineered to take advantage of selected promoters and tetON/OFF technology, and they are free-hand transplanted into neonatal lateral ventricles. Later, upon immunofluorescence profiling of recipient brains, silhouettes of transplanted neurons are fed into NeurphologyJ open source software, their morphometric parameters are extracted, and average length and branching index are calculated. Compared to other methods, this one offers three main advantages: it permits achieving of fine control of transgene expression at affordable costs, it only requires basic surgical skills, and it provides statistically reliable results upon analysis of a limited number of animals. Because of its design, however, it is not adequate to address non cell-autonomous control of neuroarchitecture. Moreover, it should be preferably used to investigate neurite morphology control after completion of neuronal migration. In its present formulation, this method is exquisitely tuned to investigate gene control of glutamatergic neocortical neuron architecture. Taking advantage of transgenic lines expressing EGFP in other specific neural cell types, it can be re-purposed to address gene control of their architecture.

Generation of the squamous epithelial roof of the 4th ventricle.

We use the transparency of zebrafish embryos to reveal the de novo generation of a simple squamous epithelium and identify the cellular architecture in the epithelial transition zone that ties this squamous epithelium to the columnar neuroepithelium within the embryo's brain. The simple squamous epithelium of the rhombencephalic roof plate is pioneered by distinct mesenchymal cells at the dorsal midline of the neural tube. Subsequently, a progenitor zone is established at the interface between columnar epithelium of the rhombic lip and the expanding squamous epithelium of the roof plate. Surprisingly, this interface consists of a single progenitor cell type that we have named the veil cell. Veil cells express gdf6a and constitute a lineage restricted stem zone that generates the squamous roof plate by direct transformation and asymmetrically fated divisions. Experimental restriction of roof plate expansion leads to extrusion of veil cell daughters and squamous cells, suggesting veil cell fate is regulated by the space available for roof plate growth.

Biodistribution of Glial Progenitors in a Three Dimensional-Printed Model of the Piglet Cerebral Ventricular System.

White matter damage persists in hypoxic-ischemic newborns even when treated with hypothermia. We have previously shown that intraventricular delivery of human glial progenitors (GPs) at the neonatal stage is capable of replacing abnormal host glia and rescuing the lifespan of dysmyelinated mice. However, such transplantation in the human brain poses significant challenges as related to high-volume ventricles and long cell migration distances. These challenges can only be studied in large animal model systems. In this study, we developed a three dimensional (3D)-printed model of the ventricular system sized to a newborn pig to investigate the parameters that can maximize a global biodistribution of injected GPs within the ventricular system, while minimizing outflow to the subarachnoid space. Bioluminescent imaging and magnetic resonance imaging were used to image the biodistribution of luciferase-transduced GPs in simple fluid containers and a custom-designed, 3D-printed model of the piglet ventricular system. Seven independent variables were investigated. The results demonstrated that a low volume (0.1 mL) of cell suspension is essential to keep cells within the ventricular system. If higher volumes (1 mL) are needed, a very slow infusion speed (0.01 mL/min) is necessary. Real-time magnetic resonance imaging demonstrated that superparamagnetic iron oxide (SPIO) labeling significantly alters the rheological properties of the GP suspension, such that, even at high speeds and high volumes, the outflow to the subarachnoid space is reduced. Several other factors, including GP species (human vs. mouse), type of catheter tip (end hole vs. side hole), catheter length (0.3 vs. 7.62 m), and cell concentration, had less effect on the overall distribution of GPs. We conclude that the use of a 3D-printed phantom model represents a robust, reproducible, and cost-saving alternative to in vivo large animal studies for determining optimal injection parameters.

Synergistic Activity of Floor-Plate- and Ventricular-Zone-Derived Netrin-1 in Spinal Cord Commissural Axon Guidance.

In vertebrates, commissural axons extend ventrally toward the floor plate in the spinal cord and hindbrain. Netrin-1, secreted by floor plate cells, was proposed to attract commissural axons at a distance. However, recent genetic studies in mice have shown that netrin-1 is also produced by ventricular zone (VZ) progenitors and that in the hindbrain, it represents the main source of netrin-1 for commissural axons. Here, we show that genetically deleting netrin-1 either from the VZ or the floor plate does not prevent midline crossing in the spinal cord, although axon pathfinding and fasciculation are perturbed. Strikingly, the VZ and floor plate act synergistically, as the simultaneous ablation of netrin-1 from these two sources severely impedes crossing. These results suggest that floor-plate-derived netrin-1 has a distinct impact on commissural axons in the spinal cord and hindbrain.

Long-Range Guidance of Spinal Commissural Axons by Netrin1 and Sonic Hedgehog from Midline Floor Plate Cells.

An important model for axon pathfinding is provided by guidance of embryonic commissural axons from dorsal spinal cord to ventral midline floor plate (FP). FP cells produce a chemoattractive activity, comprised largely of netrin1 (FP-netrin1) and Sonic hedgehog (Shh), that can attract the axons at a distance in vitro. netrin1 is also produced by ventricular zone (VZ) progenitors along the axons' route (VZ-netrin1). Recent studies using region-specific netrin1 deletion suggested that FP-netrin1 is dispensable and VZ-netrin1 sufficient for netrin guidance activity in vivo. We show that removing FP-netrin1 actually causes guidance defects in spinal cord consistent with long-range action (i.e., over hundreds of micrometers), and double mutant analysis supports that FP-netrin1 and Shh collaborate to attract at long range. We further provide evidence that netrin1 may guide via chemotaxis or haptotaxis. These results support the model that netrin1 signals at both short and long range to guide commissural axons in spinal cord.