Brain Sample Preparation After Performing in Utero Electroporation to Proliferating and Differentiated Cells in the Developing Mouse Neocortex.

In utero electroporation (IUE) enables labeling and manipulating specific types of cells by introducing DNA plasmids with desired promoters. After the surgery, mouse brains are fixed at any stage and analyzed after staining using specific antibodies. Here, we describe the flow of the IUE experiment from the preparation to microscopic observations.

Transcriptional control of motor pool formation and motor circuit connectivity by the LIM-HD protein Isl2.

The fidelity of motor control requires the precise positional arrangement of motor pools and the establishment of synaptic connections between them. During neural development in the spinal cord, motor nerves project to specific target muscles and receive proprioceptive input from these muscles via the sensorimotor circuit. LIM-homeodomain transcription factors are known to play a crucial role in successively restricting specific motor neuronal fates. However, their exact contribution to limb-based motor pools and locomotor circuits has not been fully understood. To address this, we conducted an investigation into the role of Isl2, a LIM-homeodomain transcription factor, in motor pool organization. We found that deletion of Isl2 led to the dispersion of motor pools, primarily affecting the median motor column (MMC) and lateral motor column (LMC) populations. Additionally, hindlimb motor pools lacked Etv4 expression, and we observed reduced terminal axon branching and disorganized neuromuscular junctions in Isl2-deficient mice. Furthermore, we performed transcriptomic analysis on the spinal cords of Isl2-deficient mice and identified a variety of downregulated genes associated with motor neuron (MN) differentiation, axon development, and synapse organization in hindlimb motor pools. As a consequence of these disruptions, sensorimotor connectivity and hindlimb locomotion were impaired in Isl2-deficient mice. Taken together, our findings highlight the critical role of Isl2 in organizing motor pool position and sensorimotor circuits in hindlimb motor pools. This research provides valuable insights into the molecular mechanisms governing motor control and its potential implications for understanding motor-related disorders in humans.

Lamin B1 as a key modulator of the developing and aging brain.

Lamin B1 is an essential protein of the nuclear lamina that plays a crucial role in nuclear function and organization. It has been demonstrated that lamin B1 is essential for organogenesis and particularly brain development. The important role of lamin B1 in physiological brain development and aging has only recently been at the epicenter of attention and is yet to be fully elucidated. Regarding the development of brain, glial cells that have long been considered as supporting cells to neurons have overturned this representation and current findings have displayed their active roles in neurogenesis and cerebral development. Although lamin B1 has increased levels during the differentiation of the brain cells, during aging these levels drop leading to senescent phenotypes and inciting neurodegenerative disorders such as Alzheimer's and Parkinson's disease. On the other hand, overexpression of lamin B1 leads to the adult-onset neurodegenerative disease known as Autosomal Dominant Leukodystrophy. This review aims at highlighting the importance of balancing lamin B1 levels in glial cells and neurons from brain development to aging.

Direct visualization of the transition status during neural differentiation by dual-fluorescent reporter human pluripotent stem cells.

Human induced pluripotent stem cells (hiPSCs) can differentiate into neurons and glia via neural progenitor cells and are widely used for neurogenic studies. However, directly visualizing the transition status during the neural differentiation of live cells is difficult. Here, targeting NEUROG2 (NGN2) and TUBB3 as markers of neurogenic cells and neurons, respectively, we established TUBB3EGFP/NGN2TagRFP dual-reporter hiPSCs using CRISPR-Cas9 technology. We induced the differentiation of cortical neurons from dual-reporter hiPSCs, successfully visualizing cell-fate conversion in two-dimensional (2D) culture and 3D organoids. The reporter cells were used to monitor drug effects to enhance neural induction, responses to gene knockdown, transplantation to the embryonic mouse brain, and live imaging at single-cell resolution. Notably, the earliest REELIN-positive neurons showed a distinctive migration pattern, and their production was accelerated by HES1-function loss. Together, these results demonstrate the potential for dual-reporter hiPSCs in therapeutic neural regeneration strategies and studies on human cortical development.

A Shift in Tissue Stiffness During Hippocampal Maturation Correlates to the Pattern of Neurogenesis and Composition of the Extracellular Matrix.

Aging changes the mechanical properties of brain tissue, such as stiffness. It has been proposed that the maintenance and differentiation of neural stem cells (NSCs) are regulated in accordance with extracellular stiffness. Neurogenesis is observed in restricted niches, including the dentate gyrus (DG) of the hippocampus, throughout mammalian lifetimes. However, profiles of tissue stiffness in the DG in comparison with the activity of NSCs from the neonatal to the matured brain have rarely been addressed so far. Here, we first applied ultrasound-based shear-wave elasticity imaging (SWEI) in living animals to assess shear modulus as in vivo brain stiffness. To complement the assay, atomic force microscopy (AFM) was utilized to determine the Young's modulus in the hippocampus as region-specific stiffness in the brain slice. The results revealed that stiffness in the granule cell layer (GCL) and the hilus, including the subgranular zone (SGZ), increased during hippocampal maturation. We then quantified NSCs and immature neural cells in the DG with differentiation markers, and verified an overall decrease of NSCs and proliferative/immature neural cells along stages, showing that a specific profile is dependent on the subregion. Subsequently, we evaluated the amount of chondroitin sulfate proteoglycans (CSPGs), the major extracellular matrix (ECM) components in the premature brain by CS-56 immunoreactivity. We observed differential signal levels of CSPGs by hippocampal subregions, which became weaker during maturation. To address the contribution of the ECM in determining tissue stiffness, we manipulated the function of CSPGs by enzymatic digestion or supplementation with chondroitin sulfate, which resulted in an increase or decrease of stiffness in the DG, respectively. Our results illustrate that stiffness in the hippocampus shifts due to the composition of ECM, which may affect postnatal neurogenesis by altering the mechanical environment of the NSC niche.

Comparative Analysis of Brain Stiffness Among Amniotes Using Glyoxal Fixation and Atomic Force Microscopy.

Brain structures are diverse among species despite the essential molecular machinery of neurogenesis being common. Recent studies have indicated that differences in the mechanical properties of tissue may result in the dynamic deformation of brain structure, such as folding. However, little is known about the correlation between mechanical properties and species-specific brain structures. To address this point, a comparative analysis of mechanical properties using several animals is required. For a systematic measurement of the brain stiffness of remotely maintained animals, we developed a novel strategy of tissue-stiffness measurement using glyoxal as a fixative combined with atomic force microscopy. A comparison of embryonic and juvenile mouse and songbird brain tissue revealed that glyoxal fixation can maintain brain structure as well as paraformaldehyde (PFA) fixation. Notably, brain tissue fixed by glyoxal remained much softer than PFA-fixed brains, and it can maintain the relative stiffness profiles of various brain regions. Based on this method, we found that the homologous brain regions between mice and songbirds exhibited different stiffness patterns. We also measured brain stiffness in other amniotes (chick, turtle, and ferret) following glyoxal fixation. We found stage-dependent and species-specific stiffness in pallia among amniotes. The embryonic chick and matured turtle pallia showed gradually increasing stiffness along the apico-basal tissue axis, the lowest region at the most apical region, while the ferret pallium exhibited a catenary pattern, that is, higher in the ventricular zone, the inner subventricular zone, and the cortical plate and the lowest in the outer subventricular zone. These results indicate that species-specific microenvironments with distinct mechanical properties emerging during development might contribute to the formation of brain structures with unique morphology.

Brain-stiffness-mimicking tilapia collagen gel promotes the induction of dorsal cortical neurons from human pluripotent stem cells.

The mechanical properties of the extracellular microenvironment, including its stiffness, play a crucial role in stem cell fate determination. Although previous studies have demonstrated that the developing brain exhibits spatiotemporal diversity in stiffness, it remains unclear how stiffness regulates stem cell fate towards specific neural lineages. Here, we established a culture substrate that reproduces the stiffness of brain tissue using tilapia collagen for in vitro reconstitution assays. By adding crosslinkers, we obtained gels that are similar in stiffness to living brain tissue (150-1500 Pa). We further examined the capability of the gels serving as a substrate for stem cell culture and the effect of stiffness on neural lineage differentiation using human iPS cells. Surprisingly, exposure to gels with a stiffness of approximately 1500 Pa during the early period of neural induction promoted the production of dorsal cortical neurons. These findings suggest that brain-stiffness-mimicking gel has the potential to determine the terminal neural subtype. Taken together, the crosslinked tilapia collagen gel is expected to be useful in various reconstitution assays that can be used to explore the role of stiffness in neurogenesis and neural functions. The enhanced production of dorsal cortical neurons may also provide considerable advantages for neural regenerative applications.

Systematic time-dependent visualization and quantitation of the neurogenic rate in brain organoids.

Organoids mimicking the formation of the brain cortex have been demonstrated to be powerful tools for developmental studies as well as pathological investigations of brain malformations. Here, we report an integrated approach for the quantification of temporal neural production (neurogenic rate) in organoids derived from embryonic brains. Spherical tissue fragments with polarized cytoarchitectures were incubated in multiple cavities arranged in a polymethylmethacrylate chip. The time-dependent neurogenic rate in the organoids was monitored by the level of EGFP under the promoter of Tbr2, a transcription factor that is transiently expressed in neural fate-committed progenitors during corticogenesis. Importantly, our monitoring system exhibited a quick response to DAPT, a drug that promotes neural differentiation. Furthermore, we successfully quantified the temporal neurogenic rate in a large number of organoids by applying image processing that semi-automatically recognized the positions of organoids and measured their signal intensities from sequential images. Taken together, we provide a strategy to quantitate the neurogenic rate in brain organoids in a time-dependent manner, which will also be a potent method for monitoring organoid formation and drug activity in other tissue types.

Deubiquitinating enzymes regulate Hes1 stability and neuronal differentiation.

Hairy and enhancer of split 1 (Hes1), a basic helix-loop-helix transcriptional repressor protein, regulates the maintenance of neural stem/progenitor cells by repressing proneural gene expression via Notch signaling. Previous studies showed that Hes1 expression oscillates in both mouse embryonic stem cells and neural stem cells, and that the oscillation contributes to their potency and differentiation fates. This oscillatory expression depends on the stability of Hes1, which is rapidly degraded by the ubiquitin/proteasome pathway. However, the detailed molecular mechanisms governing Hes1 stability remain unknown. We analyzed Hes1-interacting deubiquitinases purified from mouse embryonic stem cells using an Hes1-specific antibody, and identified the ubiquitin-specific protease 27x (Usp27x) as a new regulator of Hes1. We found that Hes1 was deubiquitinated and stabilized by Usp27x and its homologs ubiquitin-specific protease 22 (Usp22) and ubiquitin-specific protease 51 (Usp51). Knockdown of Usp22 shortened the half-life of Hes1, delayed its oscillation, and enhanced neuronal differentiation in mouse developing brain, whereas mis-expression of Usp27x reduced neuronal differentiation. These results suggest that these deubiquitinases modulate Hes1 protein dynamics by removing ubiquitin molecules, and thereby regulate neuronal differentiation of stem cells.

Systematic profiling of spatiotemporal tissue and cellular stiffness in the developing brain.

Accumulating evidence implicates the significance of the physical properties of the niche in influencing the behavior, growth and differentiation of stem cells. Among the physical properties, extracellular stiffness has been shown to have direct effects on fate determination in several cell types in vitro. However, little evidence exists concerning whether shifts in stiffness occur in vivo during tissue development. To address this question, we present a systematic strategy to evaluate the shift in stiffness in a developing tissue using the mouse embryonic cerebral cortex as an experimental model. We combined atomic force microscopy measurements of tissue and cellular stiffness with immunostaining of specific markers of neural differentiation to correlate the value of stiffness with the characteristic features of tissues and cells in the developing brain. We found that the stiffness of the ventricular and subventricular zones increases gradually during development. Furthermore, a peak in tissue stiffness appeared in the intermediate zone at E16.5. The stiffness of the cortical plate showed an initial increase but decreased at E18.5, although the cellular stiffness of neurons monotonically increased in association with the maturation of the microtubule cytoskeleton. These results indicate that tissue stiffness cannot be solely determined by the stiffness of the cells that constitute the tissue. Taken together, our method profiles the stiffness of living tissue and cells with defined characteristics and can therefore be utilized to further understand the role of stiffness as a physical factor that determines cell fate during the formation of the cerebral cortex and other tissues.

Novel and robust transplantation reveals the acquisition of polarized processes by cortical cells derived from mouse and human pluripotent stem cells.

Current stem cell technologies have enabled the induction of cortical progenitors and neurons from embryonic stem cells (ESCs) and induced pluripotent stem cells in vitro. To understand the mechanisms underlying the acquisition of apico-basal polarity and the formation of processes associated with the stemness of cortical cells generated in monolayer culture, here, we developed a novel in utero transplantation system based on the moderate dissociation of adherens junctions in neuroepithelial tissue. This method enables (1) the incorporation of remarkably higher numbers of grafted cells and (2) quantitative morphological analyses at single-cell resolution, including time-lapse recording analyses. We then grafted cortical progenitors induced from mouse ESCs into the developing brain. Importantly, we revealed that the mode of process extension depends on the extrinsic apico-basal polarity of the host epithelial tissue, as well as on the intrinsic differentiation state of the grafted cells. Further, we successfully transplanted cortical progenitors induced from human ESCs, showing that our strategy enables investigation of the neurogenesis of human neural progenitors within the developing mouse cortex. Specifically, human cortical cells exhibit multiple features of radial migration. The robust transplantation method established here could be utilized both to uncover the missing gap between neurogenesis from ESCs and the tissue environment and as an in vivo model of normal and pathological human corticogenesis.

Extra-cell cycle regulatory functions of cyclin-dependent kinases (CDK) and CDK inhibitor proteins contribute to brain development and neurological disorders.

In developing brains, neural progenitors exhibit cell cycle-dependent nuclear movement within the ventricular zone [interkinetic nuclear migration (INM)] and actively proliferate to produce daughter progenitors and/or neurons, whereas newly generated neurons exit from the cell cycle and begin pial surface-directed migration and maturation. Dysregulation of the balance between the proliferation and the cell cycle exit in neural progenitors is one of the major causes of microcephaly (small brain). Recent studies indicate that cell cycle machinery influences not only the proliferation but also INM in neural progenitors. Furthermore, several cell cycle-related proteins, including p27(kip1) , p57(kip2) , Cdk5, and Rb, regulate the migration of neurons in the postmitotic state, suggesting that the growth arrest confers dual functions on cell cycle regulators. Consistently, several types of microcephaly occur in conjunction with neuronal migration disorders, such as periventricular heterotopia and lissencephaly. However, cell cycle re-entry by disturbance of growth arrest in mature neurons is thought to trigger neuronal cell death in Alzheimer's disease. In this review, we introduce the cell cycle protein-mediated regulation of two types of nuclear movement, INM and neuronal migration, during cerebral cortical development, and discuss the roles of growth arrest in cortical development and neurological disorders.

Interkinetic nuclear migration: beyond a hallmark of neurogenesis.

Interkinetic nuclear migration (INM) is an oscillatory nuclear movement that is synchronized with the progression of the cell cycle. The efforts of several researchers, following the first report of INM in 1935, have revealed many of the molecular mechanisms of this fascinating phenomenon linking the timing of the cell cycle and nuclear positioning in tissue. Researchers are now faced with a more fundamental question: is INM important for tissue, particularly brain, development? In this review, I summarize the current understanding of the regulatory mechanisms governing INM, investigations involving several different tissues and species, and possible explanations for how nuclear movement affects cell-fate determination and tissue formation.

Regulation of interkinetic nuclear migration by cell cycle-coupled active and passive mechanisms in the developing brain.

A hallmark of neurogenesis in the vertebrate brain is the apical-basal nuclear oscillation in polarized neural progenitor cells. Known as interkinetic nuclear migration (INM), these movements are synchronized with the cell cycle such that nuclei move basally during G1-phase and apically during G2-phase. However, it is unknown how the direction of movement and the cell cycle are tightly coupled. Here, we show that INM proceeds through the cell cycle-dependent linkage of cell-autonomous and non-autonomous mechanisms. During S to G2 progression, the microtubule-associated protein Tpx2 redistributes from the nucleus to the apical process, and promotes nuclear migration during G2-phase by altering microtubule organization. Thus, Tpx2 links cell-cycle progression and autonomous apical nuclear migration. In contrast, in vivo observations of implanted microbeads, acute S-phase arrest of surrounding cells and computational modelling suggest that the basal migration of G1-phase nuclei depends on a displacement effect by G2-phase nuclei migrating apically. Our model for INM explains how the dynamics of neural progenitors harmonize their extensive proliferation with the epithelial architecture in the developing brain.

Lunatic fringe potentiates Notch signaling in the developing brain.

Notch signaling is essential for the self-renewal of mammalian neural progenitor cells. A variety of mechanisms modulate Notch signaling to balance the self-renewal and differentiation of progenitor cells. Fringe is a major Notch regulator and promotes or suppresses Notch signaling, depending on the Notch ligands. In the developing brain, Lunatic fringe (Lfng) is expressed in self-renewing progenitors, but its roles are unknown. In this study, in vivo mosaic analyses using in utero electroporation were developed to investigate the roles of Lfng in neural progenitor cells. We found that Lfng potentiates Notch signaling cell-autonomously. Its depletion did not affect the balance between neuronally committed cells and self-renewing progenitors, however, irrespective of the cell density of Lfng-depleted cells, and caused no obvious defects in brain development. In vivo overexpression experiments with Notch ligands suggest that Lfng strongly augments Notch signaling mediated by Delta-like 1 but not Jagged 1.

Basal process and cell divisions of neural progenitors in the developing brain.

The basal process is an extension of certain types of neural progenitors during brain development; that is, the neuroepithelial and radial glial cells, which show radial orientation, emanating from their cell body. Originally, the basal process was considered to serve as a scaffold for the migration of newborn neurons, but recent observations obtained by advanced genetic manipulations and microscopic methods show that the basal process has additional roles. In this review, we first summarize the role of the radial glial basal process for neuronal migration and signaling and for the proper organization of the developing brain. We then focus on the emerging roles of the basal process during the division of neural progenitor cells, specifically the various modes of division of neuroepithelial and radial glial cells.

Cytokinesis of neuroepithelial cells can divide their basal process before anaphase.

Neuroepithelial (NE) cells, the primary stem and progenitor cells of the vertebrate central nervous system, are highly polarized and elongated. They retain a basal process extending to the basal lamina, while undergoing mitosis at the apical side of the ventricular zone. By studying NE cells in the embryonic mouse, chick and zebrafish central nervous system using confocal microscopy, electron microscopy and time-lapse imaging, we show here that the basal process of these cells can split during M phase. Splitting occurred in the basal-to-apical direction and was followed by inheritance of the processes by either one or both daughter cells. A cluster of anillin, an essential component of the cytokinesis machinery, appeared at the distal end of the basal process in prophase and was found to colocalize with F-actin at bifurcation sites, in both proliferative and neurogenic NE cells. GFP-anillin in the basal process moved apically to the cell body prior to anaphase onset, followed by basal-to-apical ingression of the cleavage furrow in telophase. The splitting of the basal process of M-phase NE cells has implications for cleavage plane orientation and the relationship between mitosis and cytokinesis.

Aspm specifically maintains symmetric proliferative divisions of neuroepithelial cells.

The ASPM (abnormal spindle-like microcephaly-associated) protein has previously been implicated in the determination of human cerebral cortical size, but the cell biological basis of this regulation has not been studied. Here we investigate the role of Aspm in mouse embryonic neuroepithelial (NE) cells, the primary stem and progenitor cells of the mammalian brain. Aspm was found to be concentrated at mitotic spindle poles of NE cells and to be down-regulated with their switch from proliferative to neurogenic divisions. Upon RNA interference in telencephalic NE cells, Aspm mRNA is reduced, mitotic spindle poles lack Aspm protein, and the cleavage plane of NE cells is less frequently oriented perpendicular to the ventricular surface of the neuroepithelium. The alteration in the cleavage plane orientation of NE cells increases the probability that these highly polarized cells undergo asymmetric division, i.e., that apical plasma membrane is inherited by only one of the daughter cells. Concomitant with the resulting increase in abventricular cells in the ventricular zone, a larger proportion of NE cell progeny is found in the neuronal layer, implying a reduction in the number of NE progenitor cells upon Aspm knock-down relative to control. Our results demonstrate that Aspm is crucial for maintaining a cleavage plane orientation that allows symmetric, proliferative divisions of NE cells during brain development. These data provide a cell biological explanation of the primary microcephaly observed in humans with mutations in ASPM, which also has implications for the evolution of mammalian brains.

Symmetric versus asymmetric cell division during neurogenesis in the developing vertebrate central nervous system.

The type and number of cell divisions of neuronal progenitors determine the number of neurons generated during the development of the vertebrate central nervous system. Over the past several years, there has been substantial progress in characterizing the various kinds of neuronal progenitors and the types of symmetric and asymmetric divisions they undergo. The understanding of the cell-biological basis of symmetric versus asymmetric progenitor cell division has been consolidated, and the molecular machinery controlling these divisions is beginning to be unravelled. Other recent advances include comparative studies of brain development in rodents and primates, as well as the identification of gene mutations in humans that affect the balance between the various types of cell division of neuronal progenitors.

Asymmetric distribution of the apical plasma membrane during neurogenic divisions of mammalian neuroepithelial cells.

At the onset of neurogenesis in the mammalian central nervous system, neuroepithelial cells switch from symmetric, proliferative to asymmetric, neurogenic divisions. In analogy to the asymmetric division of Drosophila neuroblasts, this switch of mammalian neuroepithelial cells is thought to involve a change in cleavage plane orientation from perpendicular (vertical cleavage) to parallel (horizontal cleavage) relative to the apical surface of the neuroepithelium. Here, we report, using TIS21-GFP knock-in mouse embryos to identify neurogenic neuroepithelial cells, that at the onset as well as advanced stages of neurogenesis the vast majority of neurogenic divisions, like proliferative divisions, show vertical cleavage planes. Remarkably, however, neurogenic divisions of neuroepithelial cells, but not proliferative ones, involve an asymmetric distribution to the daughter cells of the apical plasma membrane, which constitutes only a minute fraction (1-2%) of the entire neuroepithelial cell plasma membrane. Our results support a novel concept for the cell biological basis of asymmetric, neurogenic divisions of neuroepithelial cells in the mammalian central nervous system.