Embryonic Mouse Brain Research Articles

The atypical chemokine receptor 3 interacts with Connexin 43 inhibiting astrocytic gap junctional intercellular communication

The atypical chemokine receptor 3 (ACKR3) plays a pivotal role in directing the migration of various cellular populations and its over-expression in tumors promotes cell proliferation and invasiveness. The intracellular signaling pathways transducing ACKR3-dependent effects remain poorly characterized, an issue we addressed by identifying the interactome of ACKR3. Here, we report that recombinant ACKR3 expressed in HEK293T cells recruits the gap junction protein Connexin 43 (Cx43). Cx43 and ACKR3 are co-expressed in mouse brain astrocytes and human glioblastoma cells and form a complex in embryonic mouse brain. Functional in vitro studies show enhanced ACKR3 interaction with Cx43 upon ACKR3 agonist stimulation. Furthermore, ACKR3 activation promotes β-arrestin2- and dynamin-dependent Cx43 internalization to inhibit gap junctional intercellular communication in primary astrocytes. These results demonstrate a functional link between ACKR3 and gap junctions that might be of pathophysiological relevance.

EMPAS: Electron Microscopy Screening for Endogenous Protein Architectures.

In cells, proteins form macromolecular complexes to execute their own unique roles in biological processes. Conventional structural biology methods adopt a bottom-up approach starting from defined sets of proteins to investigate the structures and interactions of protein complexes. However, this approach does not reflect the diverse and complex landscape of endogenous molecular architectures. Here, we introduce a top-down approach called Electron Microscopy screening for endogenous Protein ArchitectureS (EMPAS) to investigate the diverse and complex landscape of endogenous macromolecular architectures in an unbiased manner. By applying EMPAS, we discovered a spiral architecture and identified it as AdhE. Furthermore, we performed screening to examine endogenous molecular architectures of human embryonic stem cells (hESCs), mouse brains, cyanobacteria and plant leaves, revealing their diverse repertoires of molecular architectures. This study suggests that EMPAS may serve as a tool to investigate the moleculararchitectures of endogenous macromolecular proteins.

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.

High throughput error corrected Nanopore single cell transcriptome sequencing

Droplet-based high throughput single cell sequencing techniques tremendously advanced our insight into cell-to-cell heterogeneity. However, those approaches only allow analysis of one extremity of the transcript after short read sequencing. In consequence, information on splicing and sequence heterogeneity is lost. To overcome this limitation, several approaches that use long-read sequencing were introduced recently. Yet, those techniques are limited by low sequencing depth and/or lacking or inaccurate assignment of unique molecular identifiers (UMIs), which are critical for elimination of PCR bias and artifacts. We introduce ScNaUmi-seq, an approach that combines the high throughput of Oxford Nanopore sequencing with an accurate cell barcode and UMI assignment strategy. UMI guided error correction allows to generate high accuracy full length sequence information with the 10x Genomics single cell isolation system at high sequencing depths. We analyzed transcript isoform diversity in embryonic mouse brain and show that ScNaUmi-seq allows defining splicing and SNVs (RNA editing) at a single cell level.

Ancestral regulatory mechanisms specify conserved midbrain circuitry in arthropods and vertebrates

Corresponding attributes of neural development and function suggest arthropod and vertebrate brains may have an evolutionarily conserved organization. However, the underlying mechanisms have remained elusive. Here, we identify a gene regulatory and character identity network defining the deutocerebral-tritocerebral boundary (DTB) in Drosophila This network comprises genes homologous to those directing midbrain-hindbrain boundary (MHB) formation in vertebrates and their closest chordate relatives. Genetic tracing reveals that the embryonic DTB gives rise to adult midbrain circuits that in flies control auditory and vestibular information processing and motor coordination, as do MHB-derived circuits in vertebrates. DTB-specific gene expression and function are directed by cis-regulatory elements of developmental control genes that include homologs of mammalian Zinc finger of the cerebellum and Purkinje cell protein 4Drosophila DTB-specific cis-regulatory elements correspond to regulatory sequences of human ENGRAILED-2, PAX-2, and DACHSHUND-1 that direct MHB-specific expression in the embryonic mouse brain. We show that cis-regulatory elements and the gene networks they regulate direct the formation and function of midbrain circuits for balance and motor coordination in insects and mammals. Regulatory mechanisms mediating the genetic specification of cephalic neural circuits in arthropods correspond to those in chordates, thereby implying their origin before the divergence of deuterostomes and ecdysozoans.

Genome-Wide Analysis Identifies NURR1-Controlled Network of New Synapse Formation and Cell Cycle Arrest in Human Neural Stem Cells.

Nuclear receptor-related 1 (Nurr1) protein has been identified as an obligatory transcription factor in midbrain dopaminergic neurogenesis, but the global set of human NURR1 target genes remains unexplored. Here, we identified direct gene targets of NURR1 by analyzing genome-wide differential expression of NURR1 together with NURR1 consensus sites in three human neural stem cell (hNSC) lines. Microarray data were validated by quantitative PCR in hNSCs and mouse embryonic brains and through comparison to published human data, including genome-wide association study hits and the BioGPS gene expression atlas. Our analysis identified ~40 NURR1 direct target genes, many of them involved in essential protein modules such as synapse formation, neuronal cell migration during brain development, and cell cycle progression and DNA replication. Specifically, expression of genes related to synapse formation and neuronal cell migration correlated tightly with NURR1 expression, whereas cell cycle progression correlated negatively with it, precisely recapitulating midbrain dopaminergic development. Overall, this systematic examination of NURR1-controlled regulatory networks provides important insights into this protein’s biological functions in dopamine-based neurogenesis.

Cystatin B is essential for proliferation and interneuron migration in individuals with EPM1 epilepsy

Progressive myoclonus epilepsy (PME) of Unverricht–Lundborg type (EPM1) is an autosomal recessive neurodegenerative disorder with the highest incidence of PME worldwide. Mutations in the gene encoding cystatin B (CSTB) are the primary genetic cause of EPM1. Here, we investigate the role of CSTB during neurogenesis in vivo in the developing mouse brain and in vitro in human cerebral organoids (hCOs) derived from EPM1 patients. We find that CSTB (but not one of its pathological variants) is secreted into the mouse cerebral spinal fluid and the conditioned media from hCOs. In embryonic mouse brain, we find that functional CSTB influences progenitors’ proliferation and modulates neuronal distribution by attracting interneurons to the site of secretion via cell‐non‐autonomous mechanisms. Similarly, in patient‐derived hCOs, low levels of functional CSTB result in an alteration of progenitor's proliferation, premature differentiation, and changes in interneurons migration. Secretion and extracellular matrix organization are the biological processes particularly affected as suggested by a proteomic analysis in patients’ hCOs. Overall, our study sheds new light on the cellular mechanisms underlying the development of EPM1.

FAM19A5/TAFA5, a novel neurokine, plays a crucial role in depressive-like and spatial memory-related behaviors in mice.

FAM19A5/TAFA5 is a member of the family with sequence similarity 19 with unknown function in emotional and cognitive regulation. Here, we reported that FAM19A5 was highly expressed in the embryonic and postnatal mouse brain, especially in the hippocampus. Behaviorally, genetic deletion of Fam19a5 resulted in increased depressive-like behaviors and impaired hippocampus-dependent spatial memory. These behavioral alterations were associated with the decreased expression of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors and N-methyl-D-aspartic acid receptors, as well as significantly reduced glutamate release and neuronal activity in the hippocampus. Subsequently, these changes led to the decreased density of dendritic spines. In recent years, the roles of chronic stress participating in the development of depression have become increasingly clear, but the mechanism remains to be elucidated. We found that the levels of FAM19A5 in plasma and hippocampus of chronic stress-treated mice were significantly decreased whereas overexpression of human FAM19A5 selectively in the hippocampus could attenuate chronic stress-induced depressive-like behaviors. Taken together, our results revealed for the first time that FAM19A5 plays a key role in the regulation of depression and spatial cognition in the hippocampus. Furthermore, our study provided a new mechanism for chronic stress-induced depression, and also provided a potential biomarker for the diagnosis and a new strategy for the treatment of depression.

Disruption of the polyubiquitin gene Ubb reduces the self-renewal capacity of neural stem cells

Ubiquitin (Ub) is a highly conserved eukaryotic protein that plays pivotal roles in cellular signal transduction, differentiation, and proteolysis. Although we have previously reported that disruption of the polyubiquitin gene Ubb is associated with the dysregulated differentiation of neural stem cells (NSCs) into neurons, it is unclear how gene expression patterns are altered in Ubb knockout (KO) NSCs, and whether this altered gene expression contributes to Ubb KO neural phenotypes. To answer these questions, we used RNA-Seq to compare the transcriptomes of Ubb KO NSCs and Ubb heterozygous (HT) controls. We found that the expression levels of most proliferation markers were decreased in Ubb KO NSCs. To determine whether the reduced levels of proliferation markers were due to reduced self-renewal of NSCs, such as radial glia, we measured the levels of the radial glia marker, Pax6, in mouse embryonic brains at 14.5 dpc. We found that Pax6 levels were decreased and the ventricular zone was thinner in the embryonic brains of Ubb KO mice compared to those of wild-type (WT) control mice. To determine whether the decreased self-renewal of Ubb KO NSCs was caused by cell-autonomous defects and not due to their microenvironment, we transplanted NSCs into WT mouse brains using a cannula system. In mouse brain sections, immunoreactivity of the NSC marker, nestin, was much lower in Ubb KO NSCs than in Ubb HT controls. Therefore, our data suggest that cell-autonomous defects, due to the disruption of Ubb, lead to a decrease in the self-renewal capacity of NSCs and may contribute to their dysregulated differentiation into neurons.

Identification and prevention of heterotopias in mouse neocortical neural cell migration incurred by surgical damages during utero electroporation procedures

ABSTRACT In utero electroporation (IUE) is a useful technique for gene delivery in embryonic mouse brain. IUE technique is used to investigate the mammalian brain development in vivo. However, according to recent studies, IUE methodology has some limitations like the formation of artificial ectopias and heterotopias at the micro-injection site. Thus far, the artificial heterotopias generated by physical trauma during IUE are rarely reported. Here, we reported the artificial heterotopias and ectopias generated from surgical damages of micropipette in detail, and moreover, we described the protocol to avoid these phenotypes. For the experimental purpose, we transferred empty plasmids (pCAGIG-GFP) with green fluorescent-labelled protein into the cortical cortex by IUE and then compared the structure of the cortex region between the injected and un-injected cerebral hemispheres. The coronary section showed that ectopias and heterotopias were appeared on imperfect-injected brains, and layer maker staining, which including Ctip2 and TBR1 and laminin, can differentiate the physical damage, revealing the neurons in artificial ectopic and heterotopic area were not properly arranged. Moreover, premature differentiation of neurons in ectopias and heterotopias were observed. To avoid heterotopias and ectopias, we carefully manipulated the method of IUE application. Thus, this study might be helpful for the in utero electroporator to distinguish the artificial ectopias and heterotopias that caused by the physical injury by microneedle and the ways to avoid those undesirable circumstances.

Smad anchor for receptor activation nuclear localization during development identifies Layers V and VI of the neocortex.

Smad anchor for receptor activation (SARA, zfyve9) has been classically observed in early endosomes of different cells types where it regulates vesicular transport of proteins and membrane components. Very few other members of the zinc finger FYVE domain-containing family (zfyve) have different functions other than controlling membrane trafficking. By analyzing SARA localization throughout mouse embryonic brain development, we detected that besides the endosomal localization it also targets neuronal nuclei, specifically of the cortical layers V/VI. These findings were confirmed in human brain organoids. When evaluating neuronal cell lines, we found that SARA accumulates in nuclei of PC-12 cells, but not Neuro-2a, highlighting its specificity. SARA functions as a specific marker of the deep cortical layers until the first postnatal week. This temporal regulation corresponds with the final phases of neuron differentiation, such as soma ventral translocation and axonal targeting. In sum, here we report that SARA localization during brain development is temporarily regulated, and layer specific. This defined pattern helps in the identification of early born cortical neurons. We further show that other zfyve family members (FYCO1, WDFY3, Hrs) also distribute to nuclei of different cells in the brain cortex, which raises the possibility that this might be an extended feature within the protein family.

Subtle sex differences in vasopressin mRNA expression in the embryonic mouse brain.

Arginine vasopressin (AVP) is a neuropeptide which acts centrally to modulate numerous social behaviors. One receptor subtype through which these effects occur is the AVP 1a receptor (AVPR1A). The modulatory effects of Avp via the AVPR1A varies by species as well as sex, since both AVP and the AVPR1A tend to be expressed more prominently in males. Beyond these neuromodulatory effects there are also indications that the AVP system may play a role in early development to, in part, organize sex-specific neural circuitry that is important to sexually dimorphic social behaviors in adulthood. However, to date, AVP's role in early development is poorly understood, particularly with respect to its differential effect on males and females. In order to determine the timing and distribution of the AVP system in early brain development, we examined the brains of male and female C57BL/6J mice between embryonic day (E) 12.5 and postnatal day (P) 2 and quantified Avp and Avpr1a mRNA using qPCR and AVPR1A protein using receptor autoradiography. The mRNA for Avp was measurable in males and females starting at E14.5, with males producing more than females, while Avpr1a mRNA was found as early as E12.5, with no difference in expression between sexes. AVPR1A binding was observed in both sexes starting at E16.5, and while there were no observed sex differences, binding density and the number of neuroanatomical areas did increase over time. These data are significant as they provide the first whole-brain characterization of the vasopressin system in the embryonic mouse. Further, these findings are consistent with data from other species, that have documented a sex difference in the vasopressin system during early brain formation.

Abstract TMP101: Epigenetics in Blood Brain Barrier Formation and Maintenance

The blood brain barrier (BBB), unique to the central nervous system (CNS) vessels, tightly controls the passage of molecules between the blood and the brain. BBB breakdown is documented in many pathological conditions including, stroke, and contributes to the severity of disease and poor outcome. Currently, there is no efficient treatment strategy to prevent BBB breakdown or restore disrupted BBB . Despite BBB’s importance in the CNS function, t he genetic program and mechanism, that regulates BBB formation and maintenance is poorly characterized. To understand the molecular features underlying BBB gene regulation during development we utilize endothelial cell (EC) cultures derived from brain of both embryonic (E-13.5) and adult mice and investigated differences in BBB gene expression and epigenetic status using real time PCR analysis and ChIP-qPCR. We found that a distinct BBB profile exist in the CNS ECs during BBB formation and maintenance . When compared to embryonic ECs, adult ECs showed a significant down regulation in expression of tight junction (TJ) genes such as CLDN-1 , CLDN -11 , ZO-1 and OCLN . Conversely, another important TJ gene CLDN- 5 shows a significant increase in adult ECs. Further, we found that epigenetic-histone modifications are involved in the repression in the TJ gene in adult ECs. To more fully understand the role of Wnt/β-catenin in BBB formation and maintenance we block the downstream of the Wnt/β-catenin signaling pathway using LF3 (block the interaction of β-catenin and transcription factor TCF4) in developing ECs. This results in a adult BBB phenotype in developing ECs indicating, silencing of Wnt/β-catenin pathway is required for BBB maturation . Further, epigenetic component HDAC2 was increased significantly in developing ECs when Wnt/β-catenin signaling is blocked demonstrating this pathway can modulate the epigenetics of CNS ECs . In summary, we describe that BBB genes have distinguished expression pattern during BBB formation and maintenance, guided by the Wnt/β-catenin pathway through epigenetic modifications. Understanding the fundamental epigenetic and regulatory mechanisms of BBB formation and maintenance is critical for developing therapeutic interventions against BBB breakdown.

Persistent Cyfip1 Expression Is Required to Maintain the Adult Subventricular Zone Neurogenic Niche.

Neural stem cells (NSCs) persist throughout life in the subventricular zone (SVZ) neurogenic niche of the lateral ventricles as Type B1 cells in adult mice. Maintaining this population of NSCs depends on the balance between quiescence and self-renewing or self-depleting cell divisions. Interactions between B1 cells and the surrounding niche are important in regulating this balance, but the mechanisms governing these processes have not been fully elucidated. The cytoplasmic FMRP-interacting protein (Cyfip1) regulates apical-basal polarity in the embryonic brain. Loss of Cyfip1 during embryonic development in mice disrupts the embryonic niche and affects cortical neurogenesis. However, a direct role for Cyfip1 in the regulation of adult NSCs has not been established. Here, we demonstrate that Cyfip1 expression is preferentially localized to B1 cells in the adult mouse SVZ. Loss of Cyfip1 in the embryonic mouse brain results in altered adult SVZ architecture and expansion of the adult B1 cell population at the ventricular surface. Furthermore, acute deletion of Cyfip1 in adult NSCs results in a rapid change in adherens junction proteins as well as increased proliferation and number of B1 cells at the ventricular surface. Together, these data indicate that Cyfip1 plays a critical role in the formation and maintenance of the adult SVZ niche; furthermore, deletion of Cyfip1 unleashes the capacity of adult B1 cells for symmetric renewal to increase the adult NSC pool.SIGNIFICANCE STATEMENT Neural stem cells (NSCs) persist in the subventricular zone of the lateral ventricles in adult mammals, and the size of this population is determined by the balance between quiescence and self-depleting or renewing cell division. The mechanisms regulating these processes are not fully understood. This study establishes that the cytoplasmic FMRP interacting protein 1 (Cyfip1) regulates NSC fate decisions in the adult subventricular zone and adult NSCs that are quiescent or typically undergo self-depleting divisions retain the ability to self-renew. These results contribute to our understanding of how adult NSCs are regulated throughout life and has potential implications for human brain disorders.

Akhirin regulates the proliferation and differentiation of neural stem cells/progenitor cells at neurogenic niches in mouse brain

Specialized microenvironment, or neurogenic niche, in embryonic and postnatal mouse brain plays critical roles during neurogenesis throughout adulthood. The subventricular zone (SVZ) and the dentate gyrus (DG) of hippocampus in the mouse brain are two major neurogenic niches where neurogenesis is directed by numerous regulatory factors. Now, we report Akhirin (AKH), a stem cell maintenance factor in mouse spinal cord, plays a pivotal regulatory role in the SVZ and in the DG. AKH showed specific distribution during development in embryonic and postnatal neurogenic niches. Loss of AKH led to abnormal development of the ventricular zone and the DG along with reduction of cellular proliferation in both regions. In AKH knockout mice (AKH-/- ), quiescent neural stem cells (NSCs) increased, while proliferative NSCs or neural progenitor cells decreased at both neurogenic niches. In vitro NSC culture assay showed increased number of neurospheres and reduced neurogenesis in AKH-/- . These results indicate that AKH, at the neurogenic niche, exerts dynamic regulatory role on NSC self-renewal, proliferation and differentiation during SVZ and hippocampal neurogenesis.

Using a Primary Cell Culture Model to Study the Neural Extracellular Matrix

The extracellular matrix (ECM) provides structural support to cells, but also has key roles in mediating cell adhesion, cell signaling, and differentiation. While this topic is discussed in the lecture setting, it is not heavily studied in laboratory sessions. This lesson was created so that students can understand how the ECM is molecularly structured by using the ECM in the brain as a model. More specifically, the students study a particular structure in the neural ECM called the perineuronal net (PNN). In this exercise, students examine which cell type in the brain helps produce this structure and whether changes in activity affect PNN development. In this lesson students isolate and plate single cells derived from embryonic mouse brains; they then perform aspects of a standard immunofluorescence staining protocol. Lastly, the students will image the cultures using a fluorescence microscope. All of the aforementioned fundamental techniques are widely used by cell biologists. After completion, students hand in a formal lab report that is contextualized by primary literature. Students thoroughly enjoyed this exercise, and they found it challenging but rewarding and thought provoking. Read the Essay Article about how author KAG adapted this lesson for online in &quot;<a href="https://doi.org/10.24918/cs.2020.44">Going remote: An online adaptation to using a primary cell culture model to study the neural extracellular matrix.</a>&quot;

Spectrophotometric Method for Determining Glyoxalase 1 Activity in Cerebral Cavernous Malformation (CCM) Disease.

Glyoxalase 1 (Glo1) is a glutathione (GSH)-dependent enzyme that catalyzes the isomerization of the hemithioacetal formed non-enzymatically from methylglyoxal (MG) and GSH to S-D-lactoylglutathione (SLG). The activity of Glo1 is measured spectrophotometrically by following the increase of absorbance at 240nm and 25°C, attributable to the formation of SLG. The hemithioacetal is preformed by incubation of 2mM MG and 1mM GSH in 0.1M sodium phosphate buffer (PBS) pH7.2, at 25°C for 10min. The cell extract is then added, and the A240 is monitored over 5-min incubation against correction for blank. Glo1 activity is given in units per mg of protein where one unit activity is defined as 1 μmole of SLG produced per min under assay conditions. Here, we describe measurement of Glo1 activity in established cellular models of cerebral cavernous malformation (CCM) disease, including KRIT1-knockout mouse embryonic fibroblast (MEF) and KRIT1-silenced human brain microvascular endothelial (hBMEC) cells.

Organotypic Slice Culture of the Embryonic Mouse Brain.

Organotypic slice culture is a powerful technique for exploring the embryonic development of the mammalian brain. In this protocol we describe a basic slice culture technique we have used for two sets of experiments: axon guidance transplant assays and bead culture assays.

A Xenotransplant Model of Human Brain Tumors in Wild-Type Mice.

SummaryThe development of adequate model systems to study human malignancies is crucial for basic and preclinical research. Here, we exploit the “immune-privileged” developmental time window to achieve orthotopic xenotransplantation of human brain tumor cells in wild-type (WT) mice. We find that, when transplanted in utero, human glioblastoma (GBM) cells readily integrate in the embryonic mouse brain mirroring key tumor-associated pathological features such as infiltration, vascularization, and complex tumor microenvironment including reactive astrocytes and host immune cell infiltration. Remarkably, activation of the host IBA1 tumor-associated microglia/macrophages depends on the type of glioma cell transplanted, suggesting our approach allows one to study human GBM interactions with the immune system of WT host mice. The embryonic engraftment model complements existing ones, providing a rapid and valuable alternative to study fundamental biology of human brain tumors in immune competent mice.

Evaluation of Fluoro-Jade C Staining: Specificity and Application to Damaged Immature Neuronal Cells in the Normal and Injured Mouse Brain

Fluoro-Jade C (FJC) staining is widely used for the specific detection of all degenerating mature neurons, including apoptotic, necrotic, and autophagic cells. However, whether FJC staining can detect degenerating immature neurons and neural stem/precursor cells remains unclear. In addition, some conflicting studies have shown that FJC and its ancestral dyes, Fluoro-Jade (FJ) and FJB, can label resting/activated astrocytes and microglia. In the present study, we examined the validity of FJC staining for the detection of neuronal cells in adult and embryonic mouse brains under normal and injured conditions. In the adult rodent subventricular zone (SVZ)–rostral migratory stream (RMS)–olfactory bulb (OB) system, apoptosis associated with neurogenesis occurs under normal conditions. Using this system, we detected FCJ positive (+) cells, some of which were doublecortin (DCX)(+) neuroblasts, in addition to neuronal nuclei (NeuN)(+) mature neurons. FJC negative (−) apoptotic cells expressing activated Caspase 3 were also observed, and a small number of FJC(+)/ionized calcium-binding adaptor molecule 1 (Iba1)(+) microglia and FJC(+)/glial fibrillary acidic protein (GFAP)(+) astrocytes were observed in the normal brain. Next, we analyzed embryonic brains, in which the apoptosis of neural stem/precursor cells was induced by the administration of N-ethyl-N-nitrosourea (ENU) or ethanol at embryonic day 14 or 10, respectively. In those brains, FJC(+) neural stem/precursor cells and neuroepithelial cells expressing SRY-related HMG-box 2 (Sox2) were observed. Surprisingly degenerating mesenchymal cells were also FJC(+). The present study indicates that FJC is a reliable marker for degenerating neuronal cells during all differentiation stages. However, FJC could also label degenerating non-neuronal cells under some conditions.