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Folic acid (FA) is well known to prevent neural tube defects (NTDs), but we do not know why many human NTD cases still remain refractory to FA supplementation. Here, we investigate how the DNA demethylase TET1 interacts with maternal FA status to regulate mouse embryonic brain development. We determined that cranial NTDs display higher penetrance in non-inbred than in inbred Tet1−/− embryos and are resistant to FA supplementation across strains. Maternal diets that are either too rich or deficient in FA are linked to an increased incidence of cranial deformities in wild type and Tet1+/− offspring and to altered DNA hypermethylation in Tet1−/− embryos, primarily at neurodevelopmental loci. Excess FA in Tet1−/− embryos results in phospholipid metabolite loss and reduced expression of multiple membrane solute carriers, including a FA transporter gene that exhibits increased promoter DNA methylation and thereby mimics FA deficiency. Moreover, FA deficiency reveals that Tet1 haploinsufficiency can contribute to DNA hypermethylation and susceptibility to NTDs. Overall, our study suggests that epigenetic dysregulation may underlie NTD development despite FA supplementation. Synopsis: Interactions between modified maternal folic acid intake and Tet1 gene dosage affect embryonic phenotypes, metabolism, and DNA methylation post neural tube closure. Epigenetic dysregulation may underlie neural tube defects refractory to folic acid supplementation. Neural tube defects (NTDs) resulting from loss of Tet1 are refractory to rescue by folic acid (FA) supplementation across strains. Both excess and deprivation of FA alter DNA hypermethylation in Tet1 null embryos at neurodevelopmental loci. Excess FA coupled with loss of Tet1 mimics FA deficiency by downregulating membrane transporters. FA deficiency reveals DNA hypermethylation and NTD susceptibility due to haploinsufficiency. Interactions between modified maternal folic acid intake and Tet1 gene dosage affect embryonic phenotypes, metabolism, and DNA methylation post neural tube closure. Epigenetic dysregulation may underlie neural tube defects refractory to folic acid supplementation. [ABSTRACT FROM AUTHOR]

Abstract Background Tibetan chickens (Gallus gallus; TBCs), an indigenous breed distributed in the Qinghai-Tibet Plateau, are well adapted to the hypoxic environment. Currently, the molecular genetic basis of hypoxia adaptation in TBCs remains unclear. This study investigated hypoxia adaptation patterns of embryonic brain at different development stages by integrating analysis of the transcriptome with our previously published metabolome data in TBCs and Dwarf Laying Chickens (DLCs), a lowland chicken breed. Results During hypoxia, the results revealed that 1334, 578, and 417 differentially expressed genes (DEGs) (|log2 fold change|>1, p-value

Tibetan chickens (Gallus gallus; TBCs) are a good model for studying hypoxia-related challenges. However, lipid composition in TBC embryonic brains has not been elucidated. In this study, we characterized brain lipid profiles of embryonic day 18 TBCs and dwarf laying chickens (DLCs) during hypoxia (13% O 2 , HTBC18, and HDLC18) and normoxia (21% O 2 , NTBC18, and NDLC18) by using lipidomics. A total of 50 lipid classes, including 3540 lipid molecular species, were identified and grouped into glycerophospholipids, sphingolipids, glycerolipids, sterols, prenols, and fatty acyls. Of these lipids, 67 and 97 were expressed at different levels in the NTBC18 and NDLC18, and HTBC18 and HDLC18 samples, respectively. Several lipid species, including phosphatidylethanolamines (PEs), hexosylceramides, phosphatidylcholines (PCs), and phospha-tidylserines (PSs), were highly expressed in HTBC18. These results suggest that TBCs adapt bet-ter to hypoxia than DLCs and may have distinct cell membrane composition and nervous system development, at least partly owing to differential expression of several lipid species. One tri-glyceride, one PC, one PS, and three PE lipids were identified as potential markers that discrim-inated between lipid profiles of the HTBC18 and HDLC18 samples. The present study provides valuable information about the dynamic composition of lipids in TBCs that may explain the adaptation of this species to hypoxia. • Lipid profile characterization of TBCs embryonic brain has been little examined. • Hypoxia leads to an altered lipid profile between TBCs and lowland chicken. • TBCs have better nervous system development regulated by different lipids. • We provides basic data for exploring hypoxia-induced problems associated with lipid. [ABSTRACT FROM AUTHOR]

Abstract Background Prenatal stress (PS) is considered as a risk factor for many mental disorders. PS-induced transcriptomic alterations may contribute to the functional dysregulation during brain development. Here, we used RNA-seq to explore changes of gene expression in the mouse fetal brain after prenatal exposure to chronic unpredictable mild stress (CUMS). Results We compared the stressed brains to the controls and identified groups of significantly differentially expressed genes (DEGs). GO analysis on up-regulated DEGs revealed enrichment for the cell cycle pathways, while down-regulated DEGs were mostly enriched in the neuronal pathways related to synaptic transmission. We further performed cell-type enrichment analysis using published scRNA-seq data from the fetal mouse brain and revealed cell-type-specificity for up- and down-regulated DEGs, respectively. The up-regulated DEGs were highly enriched in the radial glia, while down-regulated DEGs were enriched in different types of neurons. Cell deconvolution analysis further showed altered cell fractions in the stressed brain, indicating accumulation of neuroblast and impaired neurogenesis. Moreover, we also observed distinct brain-region expression pattern when mapping DEGs onto the developing Allen brain atlas. The up-regulated DEGs were primarily enriched in the dorsal forebrain regions including the cortical plate and hippocampal formation. Surprisingly, down-regulated DEGs were found excluded from the cortical region, but highly expressed on various regions in the ventral forebrain, midbrain and hindbrain. Conclusion Taken together, we provided an unbiased data source for transcriptomic alterations of the whole fetal brain after chronic PS, and reported differential cell-type and brain-region vulnerability of the developing brain in response to environmental insults during the pregnancy.

Background: Tibetan chickens (Gallus gallus; TBCs), an indigenous breed distributed in the Qinghai-Tibet Plateau, are well adapted to the hypoxic environment. Currently, the molecular genetic basis of hypoxia adaptation in TBCs remains unclear. This study investigated hypoxia adaptation patterns of embryonic brain at different development stages by integrating analysis of the transcriptome with our previously published metabolome data in TBCs and Dwarf Laying Chickens (DLCs), a lowland chicken breed. Results: During hypoxia, the results revealed that 1334, 578, and 417 differentially expressed genes (DEGs) (|log2 fold change|>1, p-value < 0.05) on days 8, 12, and 18 of development, respectively between TBCs and DLCs. Gene Ontology (GO) and pathway analyses revealed that DEGs are mainly related to metabolic pathways, vessel development, and immune response under hypoxia. This is consistent with our metabolome data that TBCs have higher energy metabolism than DLCs during hypoxia. Some vital DEGs between TBCs and DLCs, such as EPAS1, VEGFD, FBP1, FBLN5, LDHA, and IL-6 which are involved in the HIF pathway and hypoxia regulation. Conclusion: These results suggest varied adaptation patterns between TBCs and DLCs under hypoxia. Our study provides a basis for uncovering the molecular regulation mechanism of hypoxia adaptation in TBCs and a potential application of hypoxia adaptation research for other animals living on the Qinghai-Tibet Plateau, and may even contribute to the study of brain diseases caused by hypoxia. [ABSTRACT FROM AUTHOR]

Abstract Background The chicken in ovo model is an attractive system to explore underlying mechanisms of neural and brain development, and it is important to develop effective genetic modification techniques that permit analyses of gene functions in vivo. Although electroporation and viral vector-mediated gene delivery techniques have been used to introduce exogenous DNA into chicken embryonic cells, transducing neurons efficiently and specifically remains challenging. Methods In the present study, we performed a comparative study of the ubiquitous CMV promoter and three neuron-specific promoters, chicken Ca2+/calmodulin-dependent kinase (cCaMKII), chicken Nestin (cNestin), and human synapsin I. We explored the possibility of manipulating gene expression in chicken embryonic brain cells using in ovo electroporation with the selected promoters. Results Transgene expression by two neuron-specific promoters (cCaMKII and cNestin) was preliminarily verified in vitro in cultured brain cells, and in vivo, expression levels of an EGFP transgene in brain cells by neuron-specific promoters were comparable to or higher than those of the ubiquitous CMV promoter. Overexpression of the FOXP2 gene driven by the cNestin promoter in brain cells significantly affected expression levels of target genes, CNTNAP2 and ELAVL4. Conclusion We demonstrated that exogenous DNA can be effectively introduced into neuronal cells in living embryos by in ovo electroporation with constructs containing neuron-specific promoters. In ovo electroporation offers an easier and more efficient way to manipulate gene expression during embryonic development, and this technique will be useful for neuron-targeted transgene expression.

Background: Prenatal stress (PS) is considered as a risk factor for many mental disorders. PS-induced transcriptomic alterations may contribute to the functional dysregulation during brain development. Here, we used RNA-seq to explore changes of gene expression in the mouse fetal brain after prenatal exposure to chronic unpredictable mild stress (CUMS). Results: We compared the stressed brains to the controls and identified groups of significantly differentially expressed genes (DEGs). GO analysis on up-regulated DEGs revealed enrichment for the cell cycle pathways, while down-regulated DEGs were mostly enriched in the neuronal pathways related to synaptic transmission. We further performed cell-type enrichment analysis using published scRNA-seq data from the fetal mouse brain and revealed cell-type-specificity for up- and down-regulated DEGs, respectively. The up-regulated DEGs were highly enriched in the radial glia, while down-regulated DEGs were enriched in different types of neurons. Cell deconvolution analysis further showed altered cell fractions in the stressed brain, indicating accumulation of neuroblast and impaired neurogenesis. Moreover, we also observed distinct brain-region expression pattern when mapping DEGs onto the developing Allen brain atlas. The up-regulated DEGs were primarily enriched in the dorsal forebrain regions including the cortical plate and hippocampal formation. Surprisingly, down-regulated DEGs were found excluded from the cortical region, but highly expressed on various regions in the ventral forebrain, midbrain and hindbrain. Conclusion: Taken together, we provided an unbiased data source for transcriptomic alterations of the whole fetal brain after chronic PS, and reported differential cell-type and brain-region vulnerability of the developing brain in response to environmental insults during the pregnancy. [ABSTRACT FROM AUTHOR]

Background: The chicken in ovo model is an attractive system to explore underlying mechanisms of neural and brain development, and it is important to develop effective genetic modification techniques that permit analyses of gene functions in vivo. Although electroporation and viral vector-mediated gene delivery techniques have been used to introduce exogenous DNA into chicken embryonic cells, transducing neurons efficiently and specifically remains challenging. Methods: In the present study, we performed a comparative study of the ubiquitous CMV promoter and three neuron-specific promoters, chicken Ca2+/calmodulin-dependent kinase (cCaMKII), chicken Nestin (cNestin), and human synapsin I. We explored the possibility of manipulating gene expression in chicken embryonic brain cells using in ovo electroporation with the selected promoters. Results: Transgene expression by two neuron-specific promoters (cCaMKII and cNestin) was preliminarily verified in vitro in cultured brain cells, and in vivo, expression levels of an EGFP transgene in brain cells by neuron-specific promoters were comparable to or higher than those of the ubiquitous CMV promoter. Overexpression of the FOXP2 gene driven by the cNestin promoter in brain cells significantly affected expression levels of target genes, CNTNAP2 and ELAVL4. Conclusion: We demonstrated that exogenous DNA can be effectively introduced into neuronal cells in living embryos by in ovo electroporation with constructs containing neuron-specific promoters. In ovo electroporation offers an easier and more efficient way to manipulate gene expression during embryonic development, and this technique will be useful for neuron-targeted transgene expression. [ABSTRACT FROM AUTHOR]

In the mammalian central nervous system, nerve-glia antigen 2 (NG2) glia are considered the fourth glial population in addition to astrocytes, oligodendrocytes and microglia. The fate of NG2 glia in vivo has been carefully studied in several transgenic mouse models using the Cre/loxP strategy. There is a clear agreement that NG2 glia mainly serve as progenitors for oligodendrocytes and a subpopulation of astrocytes mainly in the ventral forebrain, whereas the existence of a neurogenic potential of NG2 glia is lack of adequate evidence. This mini review summarizes the findings from recent studies regarding the fate of NG2 glia during development. We will highlight the age-and-region-dependent heterogeneity of the NG2 glia differentiation potential. We will also discuss putative reasons for inconsistent findings in various transgenic mouse lines of previous studies.

The purinergic system is one of the oldest cell-to-cell communication mechanisms and exhibits relevant functions in the regulation of the central nervous system (CNS) development. Amongst the components of the purinergic system, the ionotropic P2X7 receptor (P2X7R) stands out as a potential regulator of brain pathology and physiology. Thus, P2X7R is known to regulate crucial aspects of neuronal cell biology, including axonal elongation, path-finding, synapse formation and neuroprotection. Moreover, P2X7R modulates neuroinflammation and is posed as a therapeutic target in inflammatory, oncogenic and degenerative disorders. However, the lack of reliable technical and pharmacological approaches to detect this receptor represents a major hurdle in its study. Here, we took advantage of the P2rx7-EGFP reporter mouse, which expresses enhanced green fluorescence protein (EGFP) immediately downstream of the P2rx7 proximal promoter, to conduct a detailed study of its distribution. We performed a comprehensive analysis of the pattern of P2X7R expression in the brain of E18.5 mouse embryos revealing interesting areas within the CNS. Particularly, strong labelling was found in the septum, as well as along the entire neural roof plate zone of the brain, except chorioidal roof areas, but including specialized circumventricular roof formations, such as the subfornical and subcommissural organs (SFO; SCO). Moreover, our results reveal what seems a novel circumventricular organ, named by us postarcuate organ (PArcO). Furthermore, this study sheds light on the ongoing debate regarding the specific presence of P2X7R in neurons and may be of interest for the elucidation of additional roles of P2X7R in the idiosyncratic histologic development of the CNS and related systemic functions. [ABSTRACT FROM AUTHOR]

Abstract Maternal stress during pregnancy is associated with an increased risk of psychopathology in offspring. Resident immune cells of the brain, microglia, may be mediators of prenatal stress and altered neurodevelopment. Here, we demonstrate that neither the exogenous pro-inflammatory cytokine, interleukin-1β (IL-1β), nor the glucocorticoid hormone, corticosterone, recapitulated the full effects of prenatal stress on the morphology of microglial cells in the cortical plate of embryonic mice; IL-1β effects showed greater similarity to prenatal stress effects on microglia. Unexpectedly, oil vehicle alone, which has antioxidant properties, moderated the effects of prenatal stress on microglia. Microglia changes with prenatal stress were also sensitive to the antioxidant, N-acetylcysteine, suggesting redox dysregulation as a mechanism of prenatal stress.

Abstract Background MicroRNAs, small non-coding RNAs, are highly expressed in the mammalian brain, and the dysregulation of microRNA levels may be involved in neurodevelopmental disorders such as autism spectrum disorder (ASD). In the present study, we examined whether prenatal valproic acid (VPA) exposure affects levels of microRNAs, especially the brain specific and enriched microRNAs, in the mouse embryonic brain. Results Prenatal exposure to VPA at E12.5 immediately increased miR-132 levels, but not miR-9 or miR-124 levels, in the male embryonic brain. Prenatal exposure to VPA at E12.5 also increased miR-132 levels in the female embryonic brain. We further found that the prenatal exposure to VPA at E12.5 increased mRNA levels of Arc, c-Fos and brain-derived neurotrophic factor in both male and female embryonic brains, prior to miR-132 expression. In contrast, prenatal exposure to VPA at E14.5 did not affect miR-132 levels in either male or female embryonic brain. The prenatal VPA exposure at E12.5 also decreased mRNA levels of methyl-CpG-binding protein 2 and Rho GTPase-activating protein p250GAP, both of which are molecular targets of miR-132. Furthermore, RNA sequence analysis revealed that prenatal VPA exposure caused changes in several microRNA levels other than miR-132 in the embryonic whole brain. Conclusions These findings suggest that the alterations in neuronal activity-dependent microRNAs levels, including an increased level of miR-132, in the embryonic period, at least in part, underlie the ASD-like behaviors and cortical pathology produced by prenatal VPA exposure.

• Fluoro-Jade C can label degenerating immature neurons during adult neurogenesis under normal conditions. • Fluoro-Jade C can label neural stem/precursor cells in apoptosis-induced embryonic brains. • Some degenerating microglia and astrocytes can be Fluoro-Jade C positive under some conditions. • Apoptotic mesenchymal cells induced by the administration of a toxic dose of ethanol were also Fluoro-Jade C positive. 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. [ABSTRACT FROM AUTHOR]

Primary neuronal culture is a valuable in vitro model for analyzing the molecular mechanisms underlying the development and function of neural circuits. In contrast to neurons in vivo, primary cultured neurons can easily be transfected with genes of interest or treated with chemicals such as agonists and inhibitors of a specific target molecule. Furthermore, time-dependent morphological changes, such as the acquisition of neuronal polarity, axon elongation, and dendrite branch formation, can be analyzed by using primary neuronal cultures. Here, we describe a method for preparing a primary culture of neurons from the developing cerebral cortex, together with a method for gene transfer to primary cultured cortical neurons., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Summary: Lowicryl resins enable processing of biological material for electron microscopy at the lowest temperatures compatible with resin embedding. When combined with high‐pressure freezing and freeze‐substitution, Lowicryl embedding supports preservation of fine structural details and fluorescent markers. Here, we analysed the applicability of Lowicryl HM20 embedding for focused ion beam (FIB) scanning electron microscopy (SEM) tomography of Drosophila melanogaster embryonic and larval model systems. We show that the freeze‐substitution with per‐mill concentrations of uranyl acetate provided sufficient contrast and an image quality of SEM imaging in the range of similar samples analysed by transmission electron microscopy (TEM). Preservation of genetically encoded fluorescent proteins allowed correlative localization of regions of interest (ROI) within the embedded tissue block. TEM on sections cut from the block face enabled evaluation of structural preservation to allow ROI ranking and thus targeted, time‐efficient FIB‐SEM tomography data collection. The versatility of Lowicryl embedding opens new perspectives for designing hybrid SEM‐TEM workflows to comprehensively analyse biological structures. Lay Description: Focused ion beam scanning electron microscopy is becoming a widely used technique for the three‐dimensional analysis of biological samples at fine structural details beyond levels feasible for light microscopy. To withstand the abrasion of material by the ion beam and the imaging by the scanning electron beam, biological samples have to be embedded into resins, most commonly these are very dense epoxy‐based plastics. However, dense resins generate electron scattering which interferes with the signal from the biological specimen. Furthermore, to improve the imaging contrast, epoxy embedding requires chemical treatments with e.g. heavy metals, which deteriorate the ultrastructure of the biological specimen. In this study we explored the applicability of an electron lucent resin, Lowicryl HM 20, for focused ion beam scanning electron microscopy. The Lowicryl embedding workflow operates at milder chemical treatments and lower temperatures, thus preserving the sub‐cellular and sub‐organellar organization, as well as fluorescent markers visible by light microscopy. Here we show that focus ion beam scanning electron microscopy of Lowicryl‐embedded fruit flies tissues provides reliable imaging revealing fine structural details. Our workflow benefited from use of transmission electron microscopy for the quality control of the ultrastructural preservation and fluorescent light microscopy for localization of regions of interest. The versatility of Lowicryl embedding opens up new perspectives for designing hybrid workflows combining fluorescent light, scanning, and transmission electron microscopy techniques to comprehensively analyze biological structures. [ABSTRACT FROM AUTHOR]

Ionizing radiation is a potent activator of the tumor suppressor gene p53, which itself regulates the transcription of genes involved in canonical pathways such as the cell cycle, DNA repair and apoptosis as well as other biological processes like metabolism, autophagy, differentiation and development. In this study, we performed a meta-analysis on gene expression data from different in vivo and in vitro experiments to identify a signature of early radiation-responsive genes which were predicted to be predominantly regulated by p53. Moreover, we found that several genes expressed different transcript isoforms after irradiation in a p53-dependent manner. Among this gene signature, we identified novel p53 targets, some of which have not yet been functionally characterized. Surprisingly, in contrast to genes from the canonical p53-regulated pathways, our gene signature was found to be highly enriched during embryonic and post-natal brain development and during in vitro neuronal differentiation. Furthermore, we could show that for a number of genes, radiation-responsive transcript variants were upregulated during development and differentiation, while radiation non-responsive variants were not. This suggests that radiation exposure of the developing brain and immature cortical neurons results in the p53-mediated activation of a neuronal differentiation program. Overall, our results further increase the knowledge of the radiation-induced p53 network of the embryonic brain and provide more evidence concerning the importance of p53 and its transcriptional targets during mouse brain development.

The exposure of mouse embryos in utero and primary cortical neurons to ionizing radiation results in the P53-dependent activation of a subset of genes that is highly induced during brain development and neuronal maturation, a feature that these genes reportedly share with circular RNAs (circRNAs). Interestingly, some of these genes are predicted to express circular transcripts. In this study, we validated the abundance of the circular transcript variants of four P53 target genes (Pvt1, Ano3, Sec14l5, and Rnf169). These circular variants were overall more stable than their linear counterparts. They were furthermore highly enriched in the brain and their transcript levels continuously increase during subsequent developmental stages (from embryonic day 12 until adulthood), while no further increase could be observed for linear mRNAs beyond post-natal day 30. Finally, whereas radiation-induced expression of P53 target mRNAs peaks early after exposure, several of the circRNAs showed prolonged induction in irradiated embryonic mouse brain, primary mouse cortical neurons, and mouse blood. Together, our results indicate that the circRNAs from these P53 target genes are induced in response to radiation and they corroborate the findings that circRNAs may represent biomarkers of brain age. We also propose that they may be superior to mRNA as long-term biomarkers for radiation exposure.

Summary In the developing brain, neurons are produced from neural stem cells termed radial glia [ 1, 2 ]. Radial glial progenitors span the neuroepithelium, extending long basal processes to form endfeet hundreds of micrometers away from the soma. Basal structures influence neuronal migration, tissue integrity, and proliferation [ 3–7 ]. Yet, despite the significance of these distal structures, their cell biology remains poorly characterized, impeding our understanding of how basal processes and endfeet influence neurogenesis. Here we use live imaging of embryonic brain tissue to visualize, for the first time, rapid mRNA transport in radial glia, revealing that the basal process is a highway for directed molecular transport. RNA- and mRNA-binding proteins, including the syndromic autism protein FMRP, move in basal processes at velocities consistent with microtubule-based transport, accumulating in endfeet. We develop an ex vivo tissue preparation to mechanically isolate radial glia endfeet from the soma, and we use photoconvertible proteins to demonstrate that mRNA is locally translated. Using RNA immunoprecipitation and microarray analyses of endfeet, we discover FMRP-bound transcripts, which encode signaling and cytoskeletal regulators, including many implicated in autism and neurogenesis. We show FMRP controls transport and localization of one target, Kif26a . These discoveries reveal a rich, regulated local transcriptome in radial glia, far from the soma, and establish a tractable mammalian model for studying mRNA transport and local translation in vivo. We conclude that cytoskeletal and signaling events at endfeet may be controlled through translation of specific mRNAs transported from the soma, exposing new mechanistic layers within stem cells of the developing brain. [ABSTRACT FROM AUTHOR]

Quantitative methods for detection of biological molecules are needed more than ever before in the emerging age of 'omics' and 'big data.' Here, we provide an integrated approach for systematic analysis of the 'lipidome' in tissue. To test our approach in a biological context, we utilized brain tissue selectively deficient for the transcription factor Specificity Protein 2 (Sp2). Conditional deletion of Sp2 in the mouse cerebral cortex results in developmental deficiencies including disruption of lipid metabolism. Silver (Ag) cationization was implemented for infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) to enhance the ion abundances for olefinic lipids, as these have been linked to regulation by Sp2. Combining Ag-doped and conventional IR-MALDESI imaging, this approach was extended to IR-MALDESI imaging of embryonic mouse brains. Further, our imaging technique was combined with bottom-up shotgun proteomic LC-MS/MS analysis and western blot for comparing Sp2 conditional knockout (Sp2-cKO) and wild-type (WT) cortices of tissue sections. This provided an integrated omics dataset which revealed many specific changes to fundamental cellular processes and biosynthetic pathways. In particular, step-specific altered abundances of nucleotides, lipids, and associated proteins were observed in the cerebral cortices of Sp2-cKO embryos. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

Human fetuses with Down syndrome demonstrate abnormal brain growth and reduced neurogenesis. Despite the prenatal onset of the phenotype, most therapeutic trials have been conducted in adults. Here, we present evidence for fetal brain molecular and neonatal behavioral alterations in the Ts1Cje mouse model of Down syndrome. Embryonic day 15.5 brain hemisphere RNA from Ts1Cje embryos (n = 5) and wild type littermates (n = 5) was processed and hybridized to mouse gene 1.0 ST arrays. Bioinformatic analyses were implemented to identify differential gene and pathway regulation during Ts1Cje fetal brain development. In separate experiments, the Fox scale, ultrasonic vocalization and homing tests were used to investigate behavioral deficits in Ts1Cje pups (n = 29) versus WT littermates (n = 64) at postnatal days 3-21. Ts1Cje fetal brains displayed more differentially regulated genes (n = 71) than adult (n = 31) when compared to their age-matched euploid brains. Ts1Cje embryonic brains showed up-regulation of cell cycle markers and down-regulation of the solute-carrier amino acid transporters. Several cellular processes were dysregulated at both stages, including apoptosis, inflammation, Jak/Stat signaling, G-protein signaling, and oxidoreductase activity. In addition, early behavioral deficits in surface righting, cliff aversion, negative geotaxis, forelimb grasp, ultrasonic vocalization, and the homing tests were observed. The Ts1Cje mouse model exhibits abnormal gene expression during fetal brain development, and significant neonatal behavioral deficits in the pre-weaning period. In combination with human studies, this suggests that the Down syndrome phenotype manifests prenatally and provides a rationale for prenatal therapy to improve perinatal brain development and postnatal neurocognition. © 2015 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

The formation of the embryonic brain and spinal cord begins as the neural plate bends to form the neural folds, which meet and adhere to close the neural tube. The neural ectoderm and surrounding tissues also coordinate proliferation, differentiation, and patterning. This highly orchestrated process is susceptible to disruption, leading to neural tube defects (NTDs), a common birth defect. Here, we highlight genetic and epigenetic contributions to neural tube closure. We describe an online database we created as a resource for researchers, geneticists, and clinicians. Neural tube closure is sensitive to environmental influences, and we discuss disruptive causes, preventative measures, and possible mechanisms. New technologies will move beyond candidate genes in small cohort studies toward unbiased discoveries in sporadic NTD cases. This will uncover the genetic complexity of NTDs and critical gene-gene interactions. Animal models can reveal the causative nature of genetic variants, the genetic interrelationships, and the mechanisms underlying environmental influences. [ABSTRACT FROM AUTHOR]

Background Immunohistochemistry (IHC) studies in paraffin-embedded sections can be challenging due to epitope masking. To counteract this problem the microwave oven is widely used for heat-induced epitope retrieval (HIER). However, with this method important parameters cannot be specifically controlled, such as the intensity and duration of heating. New method We describe here a consistent and sensitive HIER method that uses a device for epitope retrieval in tissue sections, the decloaking chamber NxGen. With this method, heat temperature and time can be accurately set. Both qualitative (sensitivity and specificity of positive immunostaining) and quantitative (amount of positive-stained cells) analyses were compared between the microwave oven approach and the decloaking chamber, in paraffin-embedded sections from embryonic mouse brain. Results We found that the decloaking chamber-based HIER method was preferable to the commonly used microwave oven for the immunodetection and discrimination in mouse brain sections of single Olig2-positive cells, a marker of oligodendrocyte precursor cells. Both automated (ICY software) and manual counting (Adobe Photoshop software) showed a significant underestimation of Olig2-positive cells in microwave oven-treated sections compared to decloaking chamber-treatment. Comparison with existing methods Compared to other IHC procedures for cell automated quantification, the presently established protocol is easy to use, fast, and effective for the immunodetection and quantification of Olig2 in the developing mouse telencephalon. Conclusions We conclude that the combination of decloaking chamber-based HIER method and spot detector in ICY software is a reliable and valuable tool, suited to basic research and clinical studies. [ABSTRACT FROM AUTHOR]

Background IGFBP-4 has been considered as a factor involving in development of the central nevous system (CNS), but its role needs to be further clarified. In present study, the localization of IGFBP-4 expression in the embryonic forebrain, midbrain and hindbrain was determined using immunohistochemistry, and the levels of IGFBP-4 protein and mRNA were semiquantified using RT-PCR and Western blot in the embryonic (forebrain, midbrain and hindbrain) and postnatal brain (cerebral cortex, cerebellum and midbrain). Results A clear immunoreactivity of IGFBP-4 covered almost the entire embryonic brain (forebrain, midbrain, hindbrain) from E10.5 to E18.5, except for the area near the ventricle from E14.5. The change of IGFBP-4 mRNA level was regularly from E10.5 to E18.5: its expression peaked at E13.5 and E14.5, followed by gradual decreasing from E15.5. The expression of IGFBP-4 protein was similar to that of mRNA in embryonic stage. After birth, the pattern of IGFBP-4 expression was shown to be rather divergent in different brain areas. In the cerebral cortex, the IGFBP-4 mRNA increased gradually after birth (P0), while the protein showed little changes from P0 to P28, but decreased significantly at P70. In the cerebellum, the IGFBP-4 mRNA decreased gradually from P0, reached the lowest level at P21, and then increased again. However, its protein level gradually increased from P0 to P70. In the midbrain, the IGFBP-4 mRNA first decreased and reached its lowest level at P28 before it increased, while the protein remained constant from P0 to P70. At P7, P14, P21, P28 and P70, the levels of IGFBP-4 mRNA in the cerebral cortex were significantly higher than that in the cerebellum or in the midbrain. Differently, the protein levels in the cerebellum were significantly higher than that either in the cerebral cortex or in the midbrain at P14, P21, P28 and P70. Conclusions The temporal expression pattern of IGFBP-4 in the embryonic brain from E10.5 to E18.5 was consistent with the course of neurogenesis in the ventricular zone, suggesting an important role of IGFBP-4 in regulating differentiation of neural stem cells. A strikingly higher abundance of the IGFBP-4 protein observed in the cerebellum from P14 to P70 suggests that IGFBP-4 may participate in the maintenance of cerebellar plasticity. [ABSTRACT FROM AUTHOR]

The transcription factor Pax6 has been reported to specify neural progenitor cell fates during development and maintain neuronal commitments in the adult. The spatiotemporal patterns of Pax6 expression were examined in sagittal and horizontal sections of the embryonic, postnatal, and adult brains using immunohistochemistry and double immunolabeling. The proportion of Pax6-immunopositive cells in various parts of the adult brain was estimated using the isotropic fractionator methodology. It was shown that at embryonic day 11 (E11) Pax6 was robustly expressed in the proliferative neuroepithelia of the ventricular zone in the forebrain and hindbrain, and in the floor and the mesencephalic reticular formation (mRt) in the midbrain. At E12, its expression emerged in the nucleus of the lateral lemniscus in the rhombencephalon and disappeared from the floor of the midbrain. As neurodevelopment proceeds, the expression pattern of Pax6 changes from the mitotic germinal zone in the ventricular zone to become extensively distributed in cell groups in the forebrain and hindbrain, and the expression persisted in the mRt. The majority of Pax6-positive cell groups were maintained until adult life, but the intensity of Pax6 expression became much weaker. Pax6 expression was maintained in the mitotic subventricular zone in the adult brain, but not in the germinal region dentate gyrus in the adult hippocampus. There was no obvious colocalization of Pax6 and NeuN during embryonic development, suggesting Pax6 is found primarily in developing progenitor cells. In the adult brain, however, Pax6 maintains neuronal features of some subtypes of neurons, as indicated by 97.1% of Pax6-positive cells co-expressing NeuN in the cerebellum, 40.7% in the olfactory bulb, 38.3% in the cerebrum, and 73.9% in the remaining brain except the hippocampus. Differentiated tyrosine hydroxylase (TH) neurons were observed in the floor of the E11 midbrain where Pax6 was also expressed, but no obvious colocaliztion of TH and Pax6 was detected. No Pax6 expression was observed in TH-expressing areas in the midbrain at E12, E14, and postnatal day 1. These results support the notion that Pax6 plays pivotal roles in specifying neural progenitor cell commitments and maintaining certain mature neuronal fates. [ABSTRACT FROM AUTHOR]

The generation of diverse neuronal types and subtypes from multipotent progenitors during development is crucial for assembling functional neural circuits in the adult central nervous system. It is well known that the Notch signalling pathway through the inhibition of proneural genes is a key regulator of neurogenesis in the vertebrate central nervous system. However, the role of Notch during hypothalamus formation along with its downstream effectors remains poorly defined. Results Here, we have transiently blocked Notch activity in chick embryos and used global gene expression analysis to provide evidence that Notch signalling modulates the generation of neurons in the early developing hypothalamus by lateral inhibition. Most importantly, we have taken advantage of this model to identify novel targets of Notch signalling, such as Tagln3 and Chga, which were expressed in hypothalamic neuronal nuclei. Conclusions These data give essential advances into the early generation of neurons in the hypothalamus. We demonstrate that inhibition of Notch signalling during early development of the hypothalamus enhances expression of several new markers. These genes must be considered as important new targets of the Notch/proneural network. [ABSTRACT FROM AUTHOR]

Purpose: Exposure to heavy-ion radiation is considered a critical health risk on long-term space missions. The developing central nervous system (CNS) is a highly radiosensitive tissue; however, the biological effects of heavy-ion radiation, which are greater than those of low-linear energy transfer (LET) radiation, are not well studied, especially in vivo in intact organisms. Here, we examined the effects of iron-ions on the developing CNS using vertebrate organism, fish embryos of medaka ( Oryzias latipes). Materials and methods: Medaka embryos at developmental stage 28 were irradiated with iron-ions at various doses of 0-1.5 Gy. At 24 h after irradiation, radiation-induced apoptosis was examined using an acridine orange (AO) assay and histologically. To estimate the relative biological effectiveness (RBE), we quantified only characteristic AO-stained rosette-shaped apoptosis in the developing optic tectum (OT). At the time of hatching, morphological abnormalities in the irradiated brain were examined histologically. Results: The dose-response curve utilizing an apoptotic index for the iron-ion irradiated embryos was much steeper than that for X-ray irradiated embryos, with RBE values of 3.7-4.2. Histological examinations of irradiated medaka brain at 24 h after irradiation showed AO-positive rosette-shaped clusters as aggregates of condensed nuclei, exhibiting a circular hole, mainly in the marginal area of the OT and in the retina. However, all of the irradiated embryos hatched normally without apparent histological abnormalities in their brains. Conclusion: Our present study indicates that the medaka embryo is a useful model for evaluating neurocytotoxic effects on the developing CNS induced by exposure to heavy iron-ions relevant to the aerospace radiation environment. [ABSTRACT FROM AUTHOR]