A new method for obtaining bankable and expandable adult-like microglia in mice

Background The emerging role of microglia in neurological disorders requires a novel method for obtaining massive amounts of adult microglia. We aim to develop a new method for obtaining bankable and expandable adult-like microglia in mice. Methods The head neuroepithelial layer (NEL) that composed of microglial progenitor and neuroepithelial cells at mouse E13.5 was dissected and then cultured or banked. Microglia (MG) isolated from the cultured NEL by magnetic-activated cell sorting system were obtained and named NEL-MG. Results The NEL included microglia progenitors that proliferate and ramify over time with neuroepithelial cells as feeder. In functional analysis, NEL-MG exhibited microglial functions, such as phagocytosis (microbeads, amyloid β, synaptosome), migration, and inflammatory response following lipopolysaccharide (LPS) stimulation. NEL was passage cultured and the NEL-MG exhibited a higher expression of microglia signature genes than the neonatal microglia, a widely used in vitro surrogate. Banking or long-term passage culture of NEL did not affect NEL-MG characteristics. Transcriptome analysis revealed that NEL-MG exhibited better conservation of microglia signature genes with a closer fidelity to freshly isolated adult microglia than neonatal microglia. NEL-MG could be re-expandable when they were plated again on neuroepithelial cells. Conclusions This new method effectively contributes to obtaining sufficient matured form of microglia (adult-like microglia), even when only a small number of experimental animals are available, leading to a broad application in the field of neuroscience.

A lateral protrusion latticework connects neuroepithelial cells and is regulated during neurogenesis

Dynamic contacts between cells within the developing neuroepithelium are poorly understood but play important roles in cell and tissue morphology and cell signalling. Here, using live-cell imaging and electron microscopy we reveal multiple distinct protrusive structures in chicken neuroepithelial apical endfeet, including sub-apical protrusions that extend laterally within the tissue, and observe similar structures in human neuroepithelium. We characterise the dynamics, shape, and cytoskeleton of these lateral protrusions and distinguish these structures from cytonemes/filopodia and tunnelling nanotubes. We demonstrate that lateral protrusions form a latticework of membrane contacts between non-adjacent cells, depend on actin but not microtubule dynamics and provide a lamellipodial-like platform for further extending fine actin-dependent filipodia. We find that lateral protrusions depend on the actin-binding protein WAVE1: mutant-WAVE1 misexpression attenuated protrusion and generated a round-ended apical endfoot morphology. However, this did not alter apico-basal cell polarity nor reduce tissue integrity. During normal neuronal delamination sub-apical protrusions were withdrawn, but mutant-WAVE1-induced precocious protrusion loss was insufficient to trigger neurogenesis. This study uncovers a new form of cell-cell contact within the developing neuroepithelium regulation of which prefigures neuronal delamination.

Valproic acid induces p19Arf-mediated cellular senescence to cause neurodevelopmental defects

Valproic acid (VPA) is widely prescribed to treat epilepsy, bipolar disorder and migraine. However, if taken during pregnancy, exposure to the developing embryo can cause birth defects, cognitive impairment and Autism-Spectrum Disorder. How VPA causes these developmental defects remains unclear. Here, we used embryonic mice and human organoids to model key features of drug exposure, including exencephaly, microcephaly and spinal defects. In the malformed tissues, in which neurogenesis is defective, we find that induction of cellular senescence in neuroepithelial cells is a core feature. Through genetic and functional studies, we identified p19Arf as the instrumental mediator of senescence and microcephaly, but not exencephaly and spinal defects. These findings identify VPA-induced ectopic senescence as a causative mechanism disrupting normal neurodevelopment, illuminating how VPA-exposure during embryonic development can lead to cognitive defects and Autism-Spectrum Disorder.

A new method for obtaining bankable and expandable adult-like microglial cells

Background The emerging role of microglia in neurological disorders requires a novel method for obtaining massive amounts of adult microglia. We aim to develop a new method for obtaining bankable and expandable adult-like microglial cells.Methods The head neuroepithelial layer (NEL) that composed of microglial progenitor and neuroepithelial cells at mouse E13.5 was dissected and then cultured or banked. CD11b-positive cells (NEL-MG) were isolated from the cultured NEL by magnetic-activated cell sorting system (MACS).Results The NEL included microglia progenitors that proliferate and ramify over time with neuroepithelial cells as feeder. Functional validation with a MACS using the NEL showed that the NEL-MG exhibited microglial functions, such as phagocytosis (microbeads, amyloid β, synaptosome), migration, and inflammatory changes following lipopolysaccharide (LPS) stimulation. NEL was subcultured and the NEL-MG exhibited a higher expression of microglia signature genes than the neonatal microglia, a widely used in vitro surrogate. Banking or long-term subculture of NEL did not affect NEL-MG characteristics. Transcriptome analysis revealed that NEL-MG exhibited better conservation of microglia signature genes with a closer fidelity to freshly isolated adult microglia than neonatal microglia. NEL-MG could be re-expandable when they were plated again on neuroepithelial cellsConclusions This new method effectively contributes to obtaining adult-like microglial cells, even when only a small number of experimental animals are available, leading to a broad application in neuroscience-associated fields.

Single-cell analysis of early chick hypothalamic development reveals that hypothalamic cells are induced from prethalamic-like progenitors.

The hypothalamus is an evolutionarily ancient brain region that regulates many innate behaviors, but its development is still poorly understood. To identify molecular mechanisms controlling hypothalamic specification and patterning, we used single-cell RNA-Seq to profile multiple stages of early hypothalamic development in the chick. We observe that hypothalamic neuroepithelial cells are initially induced from prethalamic-like cells. Two distinct hypothalamic progenitor populations emerge later, which give rise to paraventricular/mammillary and tuberal hypothalamus, respectively. At later developmental stages, the regional organization of the chick and mouse hypothalamus closely resembles one another. This study identifies selective markers for major subdivisions of the developing chick hypothalamus and many uncharacterized candidate regulators of hypothalamic patterning and neurogenesis. As proof of concept for the utility of the dataset, we demonstrate that prethalamic progenitor-derived follistatin inhibits hypothalamic induction. This study both clarifies the organization of the early developing hypothalamus and identifies novel molecular mechanisms controlling hypothalamic induction, regionalization, and neurogenesis.

Single-cell atlas of early human brain development highlights heterogeneity of human neuroepithelial cells and early radial glia

The human cortex comprises diverse cell types that emerge from an initially uniform neuroepithelium that gives rise to radial glia, the neural stem cells of the cortex. To characterize the earliest stages of human brain development, we performed single-cell RNA-sequencing across regions of the developing human brain, including the telencephalon, diencephalon, midbrain, hindbrain and cerebellum. We identify nine progenitor populations physically proximal to the telencephalon, suggesting more heterogeneity than previously described, including a highly prevalent mesenchymal-like population that disappears once neurogenesis begins. Comparison of human and mouse progenitor populations at corresponding stages identifies two progenitor clusters that are enriched in the early stages of human cortical development. We also find that organoid systems display low fidelity to neuroepithelial and early radial glia cell types, but improve as neurogenesis progresses. Overall, we provide a comprehensive molecular and spatial atlas of early stages of human brain and cortical development.

Cannabis Use Induces Distinctive Proteomic Alterations in Olfactory Neuroepithelial Cells of Schizophrenia Patients

A close epidemiological link has been reported between cannabis use and schizophrenia (SCZ). However, biochemical markers in living humans related to the impact of cannabis in this disease are still missing. Olfactory neuroepithelium (ON) cells express neural features and offer a unique advantage to study biomarkers of psychiatric diseases. The aim of our study was to find exclusively deregulated proteins in ON cells of SCZ patients with and without a history of cannabis use. Thus, we compared the proteomic profiles of SCZ non-cannabis users (SCZ/nc) and SCZ cannabis users (SCZ/c) with control subjects non-cannabis users (C/nc) and control cannabis users (C/c). The results revealed that the main cascades affected in SCZ/nc were cell cycle, DNA replication, signal transduction and protein localization. Conversely, cannabis use in SCZ patients induced specific alterations in metabolism of RNA and metabolism of proteins. The levels of targeted proteins in each population were then correlated with cognitive performance and clinical scores. In SCZ/c, the expression levels of 2 proteins involved in the metabolism of RNA (MTREX and ZNF326) correlated with several cognitive markers and clinical signs. Moreover, use duration of cannabis negatively correlated with ZNF326 expression. These findings indicate that RNA-related proteins might be relevant to understand the influence of cannabis use on SCZ.

Cell non-autonomy amplifies disruption of neurulation by mosaic Vangl2 deletion in mice

Post-zygotic mutations that generate tissue mosaicism are increasingly associated with severe congenital defects, including those arising from failed neural tube closure. Here we report that neural fold elevation during mouse spinal neurulation is vulnerable to deletion of the VANGL planar cell polarity protein 2 (Vangl2) gene in as few as 16% of neuroepithelial cells. Vangl2-deleted cells are typically dispersed throughout the neuroepithelium, and each non-autonomously prevents apical constriction by an average of five Vangl2-replete neighbours. This inhibition of apical constriction involves diminished myosin-II localisation on neighbour cell borders and shortening of basally-extending microtubule tails, which are known to facilitate apical constriction. Vangl2-deleted neuroepithelial cells themselves continue to apically constrict and preferentially recruit myosin-II to their apical cell cortex rather than to apical cap localisations. Such non-autonomous effects can explain how post-zygotic mutations affecting a minority of cells can cause catastrophic failure of morphogenesis leading to clinically important birth defects.