Embryo Brain Research Articles

Compensatory gene expression potentially rescues impaired brain development in Kit mutant mice

While loss-of-function mutations in the murine dominant white spotting/Kit (W) locus affect a diverse array of cell lineages and organs, the brain, organ with the highest expression show the least number of defective phenotypes. We performed transcriptome analysis of the brains of KitW embryos and found prominent gene expression changes specifically in the E12.5 KitW/W homozygous mutant. Although other potentially effective changes in gene expression were observed, uniform downregulation of ribosomal protein genes and oxidative phosphorylation pathway genes specifically observed in the E12.5 brain may comprise a genetic compensation system exerting protective metabolic effects against the deleterious effect of KitW/W mutation in the developing brain.

Biological properties of domestic strain vRub-Ant of rubella virus

Introduction. Rubella is a mild infectious disease affecting mainly children and is caused by the rubella virus, part of the Matonoviridae family, genus Rubivirus. Rubella causes congenital rubella syndrome (CRS) and is the main cause of developmental abnormalities, especially blindness and deafness.
 There is no specific treatment for rubella and CRS. In order to avoid possible complications from rubella infection, a live attenuated rubella vaccine based on the foreign strain of Wistar RA 27/3 rubella virus is used. However, the actual, more effective and preferred vaccine strain the rubella virus for the Russian Federation is considered to be a viral strain of rubella circulating on its territory.
 The aim of the study was to study the biological properties of the developed domestic cold-adapted strain vRub-Ant circulating in the territory of the Russian Federation.
 Materials and methods. Following cell cultures were used in the study human embryo lung diploid cell strain LECH-3, transferable cell line from embryonic kidney cells of green monkeys Vero CCL-81 and Vero ECC, human mesenchymal stem cells, human peripheral blood mononuclear cells (PBMC). Cell cultures were grown on a DMEM/F12 nutrient medium with the addition of 5% fetal bovine serum. Swabs from the pharynx and nasal passages from a child with rubella were used as clinical virus-containing material. Monoclonal anti-idiotypic antibodies m(anti-ID)Ab were used to assess the expression level of alpha/beta and gamma interferon receptors (/ and IFN-R)Ab, imitating the biological effects of alpha/beta and gamma interferons (/ and IFN) of humans. The cultural, virological, immunochemical and serological research methods were applied in the study.
 Results. Attenuation of the vRub-Ant clinical isolate of rubella virus was carried out for 20 consecutive passages on LECH-3 diploid cells at a reduced temperature of 30C. The main biological markers of attenuation were determined to be ts and ca phenotypes. The avirulence of the attenuated viral strain (att-phenotype) was assessed by the level of expression of / and IFN-R. A lower level of / and IFN-R expression was found on the membranes of human PBMC induced by the vaccine strain vRub-Ant in comparison with the parent wild variant of the rubella virus. This trait,the att phenotype, is characteristic of attenuated viral strains. It has been shown that the vaccine strain vRub-Ant has lost neurotropism and was unable to bind to the membrane receptors of the brain (MRB) of guinea pig embryos, unlike its parent rubella virus strain. The high immunogenicity of the domestic cold-adapted strain vRub-Ant was confirmed by high titers of neutralizing rubella antibodies observed in guinea pigs immunized subcutaneously with one vaccination dose of the virus.
 Conclusion. A domestic attenuated vaccine strain vRub-Ant of the rubella virus that has the main biological markers of attenuation (ts-ca and att phenotypes) has been developed. The vaccine strain vRub-Ant induces a high levels of neutralizing antibodies in guinea pigs following the immunization with a single vaccination dose of the vaccine. The viral strain vRub-Ant has lost its tropism to the MRB of guinea pig embryos, unlike its parent variant.

Stem cell ethics and policy: What’s old is new again

Stem cells are increasingly being used to model human development and disease in the form of self-organizing embryo models, brain organoids, and neurological chimeras. These new research directions are resurrecting old embryo debates around moral status and personhood. Hyun considers how these old questions are tackled in these new contexts.

Three-Dimensional Ultrasound Localization Microscopy with Bipartite Graph-Based Microbubble Pairing and Kalman-Filtering-Based Tracking on a 256-Channel Verasonics Ultrasound System with a 32 × 32 Matrix Array

PurposeThree-dimensional (3D) ultrasound localization microscopy (ULM) using a 2-D matrix probe and microbubbles (MBs) has recently been proposed to visualize microvasculature in three spatial dimensions beyond the ultrasound diffraction limit. However, 3D ULM has several limitations, including: (1) high system complexity, (2) complex MB flow dynamics in 3D, and (3) extremely long acquisition time that had to be addressed.MethodTo reduce the system complexity while maintaining high image quality, we used a sub-aperture process to reduce received channel counts. To address the second issue, a 3D bipartite graph-based method with Kalman filtering-based tracking was used in this study for MB tracking. An MB separation approach was incorporated to separate high concentration MB data into multiple, sparser MB datasets, allowing better MB localization and tracking for a limited acquisition time.ResultsThe proposed method was first validated in a flow channel phantom, showing improved spatial resolutions compared with the contrasted enhanced power Doppler image. Then the proposed method was evaluated with an in vivo chicken embryo brain dataset. Results showed that the reconstructed 3D super-resolution image achieved a spatial resolution of around 52 μm (smaller than the wavelength of around 200 μm).ConclusionA lower system complexity of 3D ULM has been proposed. In addition, our proposed 3D ULM provided the capability of 3D motion compensation and MB tracking. Microvessels that cannot be resolved clearly using localization only, can be well identified with the proposed method.

Fenpropathrin exposure induces neurotoxicity in zebrafish embryos.

Fenpropathrin has been a commonly used insecticide to control agricultural and household insects over a few decades. Up to now, fenpropathrin residue in soil and water has been often determined due to its widespread use, which poses serious threat to environment and aquatic organisms. The potential of fenpropathrin to affect aquatic lives is still poorly understood. In this study, we used zebrafish (Danio rerio) embryo as an experimental model system to evaluate the toxicity of fenpropathrin to the development of zebrafish nervous system. Zebrafish embryos were separately exposed to fenpropathrin at the dose of 0.016mg/L, 0.032mg/L, 0.064mg/L, starting at 6h post-fertilizationhpf (hpf) up to 96 hpf. The results showed that fenpropathrin exposure gives rise to physiological, behavioral, and neurodevelopmental impairments in zebrafish embryos, including enhanced acetylcholinesterase (AChE) activity, abnormal swimming behavior, karyopyknosis in brain cells, increased intercellular space, and uneven migration of neuron in brain area. In addition, the expressions of genes concerning neurodevelopment and neurotransmitter system were inhibited following fenpropathrin exposure. We also found that fenpropathrin exposure distinctly induced oxidative stress by increasing reactive oxygen species (ROS) generation and inhibiting the production of antioxidant enzymes catalase (CAT) and superoxide dismutase (SOD). Expectedly, some apoptosis-associated genes were induced and the apoptosis appeared in the brain and heart cells of zebrafish embryos. Moreover, fenpropathrin exposure also inhibited the expressions of genes in Nrf2 signaling pathway, such as heme oxygenase-1 (HO-1) and SOD. In summary, the results of this study indicate that oxidative stress-triggered apoptosis may be an underlying fundamental of fenpropathrin-induced neurotoxicity in zebrafish embryos.

Left-right asymmetric expression of the Nodal-Lefty-Pitx2 module in developing turtle forebrain.

The epithalamus of zebrafish shows morphological and molecular left-right (L-R) asymmetry, but such asymmetry is not apparent in tetrapods. To provide further insight into the evolutionary diversity of brain L-R asymmetry, we have now examined the developing brains of reptile embryos for expression of Nodal, Lefty, and Pitx2. Two turtle species, the Chinese softshell turtle and the red-eared slider turtle, showed left-sided expression of these three genes in the developing forebrain, with this expression occurring after Nodal expression at the lateral plate and the L-R organizer has disappeared. Nodal activity, as revealed by the detection of phosphorylated Smad2/3, was also apparent in the neural epithelium on the left side in both turtle species. In the Chinese softshell turtle, the habenula did not show apparent asymmetry in size and the parapineal organ was absent, but the expression of Kctd12 in the habenula showed a small yet reproducible asymmetry. In contrast to the turtles, L-R asymmetric expression of Nodal, Lefty, Pitx2, or Kctd12 was not detected in the developing brain of the Madagascar ground gecko. The transcriptional enhancer (ASE) responsible for the asymmetric expression of Nodal, Lefty, and Pitx2 was conserved among reptiles, including the Chinese softshell turtle and Madagascar ground gecko. Our findings suggest that Nodal, Lefty, and Pitx2 have the potential to be asymmetrically expressed in the developing brain of vertebrates, but that their expression varies even among reptiles.

Abstracts from the NIH Office of Research on Women's Health 2022 Annual BIRCWH Meeting:Building Interdisciplinary Research Careers in Women's Health November 2, 2022

Abstracts from the NIH Office of Research on Women's Health 2022 Annual BIRCWH Meeting:Building Interdisciplinary Research Careers in Women's Health November 2, 2022

Ethalfluralin induces developmental toxicity in zebrafish via oxidative stress and inflammation

Ethalfluralin, of dinitroaniline herbicide family, is an effective weed controller. Following residue detection in herbicide-treated fields, ethalfluralin was reported to interfere with early stages of implantation in some vertebrate species. However, the role of ethalfluralin in the development of zebrafish embryos has not been elucidated yet. Therefore, in the present study, we investigated the morphological and physiological changes that occur in the embryonic development of zebrafish due to ethalfluralin exposure. Results indicated that ethalfluralin decreased survival rate along with reduction in the hatching ratio and heartbeat. It was observed to cause edema in the heart and yolk sac, and apoptosis in the anterior region of the developing zebrafish larvae; as visualized through acridine orange and TUNEL staining. In addition, ethalfluralin increased the expression of the apoptosis-associated genes including tp53, cyc1, casp8, casp9, and casp3. The Seahorse Mito Stress analysis revealed that ethalfluralin slightly reduced mitochondrial respiration in live zebrafish embryos. Reactive oxygen species (ROS) production was also observed to be elevated in zebrafish larvae in response to ethalfluralin. Treatment with ethalfluralin decreased blood vessel formation in brain and intestine in flk1 transgenic zebrafish embryos. The decrease in angiogenesis related gene expression was specifically observed in vegfc, flt1, and kdrl, and in the intestinal vasculature related genes apoa4a, aqp3, fabp2, and vil1. Moreover, an increase in inflammatory genes such as cox2a, cox2b, cxcl-c1c, il8, mcl1a, mcl1b, and nf-κb was observed using real-time PCR analysis. Collectively, these results indicate that oxidative stress generated by exposure to ethalfluralin induced ROS generation, apoptosis, inflammation and anti-angiogenic effects, and therefore, ethalfluralin may be toxic to the development of zebrafish embryos.

Neurogranin-like immunoreactivity in the zebrafish brain during development

Neurogranin (Nrgn) is a neural protein that is enriched in the cerebral cortex and is involved in synaptic plasticity via its interaction with calmodulin. Recently we reported its expression in the brain of the adult zebrafish (Alba-González et al. J Comp Neurol 530:1569–1587, 2022). In this study we analyze the development of Nrgn-like immunoreactivity (Nrgn-like-ir) in the brain and sensory structures of zebrafish embryos and larvae, using whole mounts and sections. First Nrgn-like positive neurons appeared by 2 day post-fertilization (dpf) in restricted areas of the brain, mostly in the pallium, epiphysis and hindbrain. Nrgn-like populations increased noticeably by 3 dpf, reaching an adult-like pattern in 6 dpf. Most Nrgn-like positive neurons were observed in the olfactory organ, retina (most ganglion cells, some amacrine and bipolar cells), pallium, lateral hypothalamus, thalamus, optic tectum, torus semicircularis, octavolateralis area, and viscerosensory column. Immunoreactivity was also observed in axonal tracts originating in Nrgn-like neuronal populations, namely, the projection of Nrgn-like immunopositive primary olfactory fibers to olfactory glomeruli, that of Nrgn-like positive pallial cells to the hypothalamus, the Nrgn-like-ir optic nerve to the pretectum and optic tectum, the Nrgn-like immunolabeled lateral hypothalamus to the contralateral region via the horizontal commissure, the octavolateralis area to the midbrain via the lateral lemniscus, and the viscerosensory column to the dorsal isthmus via the secondary gustatory tract. The late expression of Nrgn in zebrafish neurons is probably related to functional maturation of higher brain centers, as reported in the mammalian telencephalon. The analysis of Nrgn expression in the zebrafish brain suggests that it may be a useful marker for specific neuronal circuitries.

Hesperidin Improves Memory Function by Enhancing Neurogenesis in a Mouse Model of Alzheimer's Disease.

Alzheimer’s disease (AD) is an irreversible neurodegenerative disease characterized by memory and cognitive impairments. Neurogenesis, which is related to memory and cognitive function, is reduced in the brains of patients with AD. Therefore, enhancing neurogenesis is a potential therapeutic strategy for neurodegenerative diseases, including AD. Hesperidin (HSP), a bioflavonoid found primarily in citrus plants, has anti-inflammatory, antioxidant, and neuroprotective effects. The objective of this study was to determine the effects of HSP on neurogenesis in neural stem cells (NSCs) isolated from the brain of mouse embryos and five familial AD (5xFAD) mice. In NSCs, HSP significantly increased the proliferation of NSCs by activating adenosine monophosphate (AMP)-activated protein kinase (AMPK)/cAMP-response element-binding protein (CREB) signaling, but did not affect NSC differentiation into neurons and astrocytes. HSP administration restored neurogenesis in the hippocampus of 5xFAD mice via AMPK/brain-derived neurotrophic factor/tropomyosin receptor kinase B/CREB signaling, thereby decreasing amyloid-beta accumulation and ameliorating memory dysfunction. Collectively, these preclinical findings suggest that HSP is a promising candidate for the prevention and treatment of AD.

Sustained release of sulforaphane by bioactive extracellular vesicles for neuroprotective effect on chick model.

Novel studies have shown neurological treatment possibilities with extracellular vesicles (EVs) as natural particles with a special composition that are produced by different cell types. Their stability, natural structure, composition, and bioavailability make them good candidates as drug vehicles. Here, EVs were isolated from amniotic fluid (AF) through differential centrifugation, and characterized for size (<200 nm), structure, and composition, their effectiveness on the human PC12 cell line, and brain of chick embryos exposed to sodium valproate (animal autistic model). Sulforaphane (SFN) was employed as a bioactive compound and then encapsulated into Evs using three methods including passive (incubation), active (sonication), and active-passive (sonication-incubation). Further, the loading and in vitro releases of SFN fitted the Korsmeyer-Peppas (R2 = 0.99) kinetic model by non-Fickian diffusion case II (n=0.44, passive loading) and Fickian diffusion case I (n=0.41, active and active-passive loading). SFN-loaded EVs (SFN@EVs; 11 μM: 103 nM) stimulated hPC-12 cell proliferation. The gene expression analysis revealed that SFN@EVs could upregulate Nrf2 and reduce IL-6 expression. Eventually, histopathological results of the coronal cross-section of the chick embryos brain showed treatment with SFN@EVs. This treatment illustrated normality in the gray and white matter and the orientation of the bipolar neurons. Our findings showed EVs' potentially acting as a gene expression regulator in autism spectrum disorder.

De novo variants in the PABP domain of PABPC1 lead to developmental delay

PurposeThe study aimed to investigate the role of PABPC1 in developmental delay (DD). MethodsChildren were examined by geneticists and pediatricians. Variants were identified using exome sequencing and standard downstream bioinformatics pipelines. We performed in silico molecular modeling and coimmunoprecipitation to test if the variants affect the interaction between PABPC1 and PAIP2. We performed in utero electroporation of mouse embryo brains to enlighten the function of PABPC1. ResultsWe describe 4 probands with an overlapping phenotype of DD, expressive speech delay, and autistic features and heterozygous de novo variants that cluster in the PABP domain of PABPC1. Further symptoms were seizures and behavioral disorders. Molecular modeling predicted that the variants are pathogenic and would lead to decreased binding affinity to messenger RNA metabolism-related proteins, such as PAIP2. Coimmunoprecipitation confirmed this because it showed a significant weakening of the interaction between mutant PABPC1 and PAIP2. Electroporation of mouse embryo brains showed that Pabpc1 knockdown decreases the proliferation of neural progenitor cells. Wild-type Pabpc1 could rescue this disturbance, whereas 3 of the 4 variants did not. ConclusionPathogenic variants in the PABP domain lead to DD, possibly because of interference with the translation initiation and subsequently an impaired neurogenesis in cortical development.

Nutrient sensitive protein O-GlcNAcylation modulates the transcriptome through epigenetic mechanisms during embryonic neurogenesis.

Protein O-GlcNAcylation is a dynamic, nutrient-sensitive mono-glycosylation deposited on numerous nucleo-cytoplasmic and mitochondrial proteins, including transcription factors, epigenetic regulators, and histones. However, the role of protein O-GlcNAcylation on epigenome regulation in response to nutrient perturbations during development is not well understood. Herein we recapitulated early human embryonic neurogenesis in cell culture and found that pharmacological up-regulation of O-GlcNAc levels during human embryonic stem cells' neuronal differentiation leads to up-regulation of key neurogenic transcription factor genes. This transcriptional de-repression is associated with reduced H3K27me3 and increased H3K4me3 levels on the promoters of these genes, perturbing promoter bivalency possibly through increased EZH2-Thr311 phosphorylation. Elevated O-GlcNAc levels also lead to increased Pol II-Ser5 phosphorylation and affect H2BS112O-GlcNAc and H2BK120Ub1 on promoters. Using an in vivo rat model of maternal hyperglycemia, we show similarly elevated O-GlcNAc levels and epigenetic dysregulations in the developing embryo brains because of hyperglycemia, whereas pharmacological inhibition of O-GlcNAc transferase (OGT) restored these molecular changes. Together, our results demonstrate O-GlcNAc mediated sensitivity of chromatin to nutrient status, and indicate how metabolic perturbations could affect gene expression during neurodevelopment.

An integrated systems-level model of ochratoxin A toxicity in the zebrafish (Danio rerio) embryo based on NMR metabolic profiling

Ochratoxin A (OTA) is one of the most widespread mycotoxin contaminants of agricultural crops. Despite being associated with a range of adverse health effects, a comprehensive systems-level mechanistic understanding of the toxicity of OTA remains elusive. In the present study, metabolic profiling by high-resolution magic angle spinning (HRMAS) NMR, coupled to intact zebrafish embryos, was employed to identify metabolic pathways in relation to a systems-level model of OTA toxicity. Embryotoxicity was observed at sub-micromolar exposure concentrations of OTA. Localization of OTA, based on intrinsic fluorescence, as well as a co-localization of increased reactive oxygen species production, was observed in the liver kidney, brain and intestine of embryos. Moreover, HRMAS NMR showed significant alteration of metabolites related to targeting of the liver (i.e., hepatotoxicity), and pathways associated with detoxification and oxidative stress, and mitochondrial energy metabolism. Based on metabolic profiles, and complementary assays, an integrated model of OTA toxicity is, thus, proposed. Our model suggests that OTA hepatotoxicity compromises detoxification and antioxidant pathways, leading to mitochondrial membrane dysfunction manifested by crosstalk between pathways of energy metabolism. Interestingly, our data additionally aligns with a possible role of mitochondrial fusion as a “passive mechanism” to rescue mitochondrial integrity during OTA toxicity.

Influence of temperature during incubation on the mRNA levels of temperature sensitive ion channels in the brain of broiler chicken embryos

Thermosensation is crucial for the survival of any organism. In animals, changes in brain temperature are detected via sensory neurons, their cell bodies are located in the trigeminal ganglia. Transient receptor potential (TRP) ion channels are the largest temperature sensing family. In mammals, 11 thermoTRPs are known, as in poultry, there are only three. This research further elucidates TRP mRNA expression in the brain of broiler embryo's. Three incubation treatments were conducted on 400 eggs each: the control (C) at 37.6 °C; T1 deviating from C by providing a + 1 °C heat stimuli during embryonic day (ED) 15–20 for 8 h a day; and T2, imposing a + 2 °C heat stimuli. After each heat stimuli, 12 eggs per treatment were taken for blood sampling from the chorioallantoic membrane and brain harvesting. Incubation parameters such has residual yolk (free embryonic) weight, chick quality and hatch percentage were collected. After primer optimization, 22 target genes (13 TRPs and 9 non-TRPs) were measured on mRNA of the brain using a nanofluidic biochip (Fluidigm Corporation). Four target genes (ANO2, TRPV1, SCN5A, TRAAK) have a significant treatment effect - independent of ED. Another four (TRPM8, TRPA1, TRPM2, TRPC3) have a significant treatment effect visible on one or more ED. Heat sensitive channels were increased in T2 and to a lesser degree in T1, which could be part of an acclimatisation process resulting in later life heat tolerance by increased heat sensitivity. T2, however, resulted in a lower hatch weight, quality and hatchability. No hormonal differences were detected.

Differential effects of early or late exposure to prenatal maternal immune activation on mouse embryonic neurodevelopment

Exposure to maternal immune activation (MIA) in utero is a risk factor for neurodevelopmental and psychiatric disorders. MIA-induced deficits in adolescent and adult offspring have been well characterized; however, less is known about the effects of MIA exposure on embryo development. To address this gap, we performed high-resolution ex vivo MRI to investigate the effects of early (gestational day [GD]9) and late (GD17) MIA exposure on embryo (GD18) brain structure. We identify striking neuroanatomical changes in the embryo brain, particularly in the late-exposed offspring. We further examined the putative neuroanatomical underpinnings of MIA timing in the hippocampus using electron microscopy and identified differential effects due to MIA timing. An increase in apoptotic cell density was observed in the GD9-exposed offspring, while an increase in the density of neurons and glia with ultrastructural features reflective of increased neuroinflammation and oxidative stress was observed in GD17-exposed offspring, particularly in females. Overall, our findings integrate imaging techniques across different scales to identify differential impact of MIA timing on the earliest stages of neurodevelopment.

3D reconstructed brain images reveal the possibility of the ogg1 gene to suppress the irradiation-induced apoptosis in embryonic brain in medaka (Oryzias latipes)

The accumulation of oxidative DNA lesions in neurons is associated with neurodegenerative disorders and diseases. Ogg1 (8-oxoG DNA glycosylase-1) is a primary repair enzyme to excise 7,8-dihydro-8-oxoguanine (8-oxoG), the most frequent mutagenic base lesion produced by oxidative DNA damage. We have developed ogg1-deficient medaka by screening with a high resolution melting (HRM) assay in Targeting-Induced Local Lesions In Genomes (TILLING) library. In this study, we identified that ogg1-deficient embryos have smaller brains than wild-type during the period of embryogenesis and larvae under normal conditions. To reveal the function of ogg1 when brain injury occurs during embryogenesis, we examined the induction of apoptosis in brains after exposure to gamma-rays with 10 Gy (137Cs, 7.3 Gy/min.) at 24 h post-irradiation both in wild-type and ogg1-deficient embryos. By acridine orange (AO) assay, clustered apoptosis in irradiated ogg1-deficient embryonic brains were distributed in a similar manner to those of irradiated wild-type embryos. To evaluate possible differences of gamma-ray induced apoptosis in both types of embryonic brains, we constructed 3D images of the whole brain based on serial histological sections. This analysis identified that the clustered apoptotic volume was about 3 times higher in brain of irradiated ogg1-deficient embryos (n = 3) compared to wild-type embryos (n = 3) (P = 0.04), suggesting that irradiation-induced apoptosis in medaka embryonic brain can be suppressed in the presence of functional ogg1. Collectively, reconstruction of 3D images can be a powerful approach to reveal slight differences in apoptosis induction post-irradiation.

Moderate Folic Acid Supplementation in Pregnant Mice Results in Altered Sex-Specific Gene Expression in Brain of Young Mice and Embryos.

Food fortification and increased vitamin intake have led to higher folic acid (FA) consumption by many pregnant women. We showed that FA-supplemented diet in pregnant mice (fivefold higher FA than the recommended level (5xFASD)) led to hyperactivity-like behavior and memory impairment in pups. Disturbed choline/methyl metabolism and altered placental gene expression were identified. The aim of this study was to examine the impact of 5xFASD on the brain at two developmental stages, postnatal day (P) 30 and embryonic day (E) 17.5. Female C57BL/6 mice were fed a control diet or 5xFASD for 1 month before mating. Diets were maintained throughout the pregnancy and lactation until P30 or during pregnancy until E17.5. The 5xFASD led to sex-specific transcription changes in P30 cerebral cortex and E17.5 cerebrum, with microarrays showing a total of 1003 and 623 changes, respectively. Enhanced mRNA degradation was observed in E17.5 cerebrum. Expression changes of genes involved in neurotransmission, neuronal growth and development, and angiogenesis were verified by qRT-PCR; 12 and 15 genes were verified at P30 and E17.5, respectively. Hippocampal collagen staining suggested decreased vessel density in FASD male embryos. This study provides insight into the mechanisms of neurobehavioral alterations and highlights potential deleterious consequences of moderate folate oversupplementation during pregnancy.

Time-Dependent Monitoring of Dopamine in the Brain of Live Embryonic Zebrafish Using Electrochemically Pretreated Carbon Fiber Microelectrodes.

Neurotransmitters are involved in functions related to signaling, stress response, and pathological disorder development, and thus, their real-time monitoring at the site of production is important for observing the changes related to these disorders. Here, we demonstrate the first time-dependent quantification of dopamine in the brains of live zebrafish embryos using electrochemically pretreated carbon fiber microelectrodes (CFMEs) utilizing differential pulse voltammetry as the measurement technique. The pretreatment of the CFMEs in 0.1 M NaOH held at a potential of +1.0 V for 600 s improves the sensitivity toward dopamine and allows for reliable measurements in low ionic strength media. We demonstrate the measurement of extracellular dopamine concentrations in the zebrafish brain during late embryogenesis. The extracellular dopamine concentration in the tectum of zebrafish varies between 200 and 400 nM. The conventional pharmacological manipulation of neurotransmitter levels in the brain demonstrates the selective detection of dopamine at the implantation site. Exposure to the dopamine transporter inhibitor nomifensine induces an increase in extracellular dopamine from 201.9 (±34.9) nM to 352.2 (±20.0) nM, while exposure to the norepinephrine transporter inhibitor desipramine does not lead to a significant modulation of the measured signal. Furthermore, we report the quantitative assessment of the catecholamine stress response of embryos to tricaine, an anesthetic frequently used in zebrafish assays. Exposure to tricaine induces a short-lived increase in brain dopamine from 198.6 (±15.7) nM to a maximum of 278.8 (±14.0) nM. Thus, in vivo electrochemistry can detect real-time changes in zebrafish neurochemical physiology resulting from drug exposure.

Targeted disruption of Rab1a causes early embryonic lethality.

Guanosine nucleotide diphosphate (GDP) dissociation inhibitor 2 (GDI2) regulates the GDP/guanosine triphosphate (GTP) exchange reaction of Rab proteins by inhibiting the dissociation of GDP and the subsequent binding of GTP. The present study aimed to determine the function of Rab1a in vivo, and thus generated mice with a trapped Rab1a gene. It was demonstrated that Rab1a is essential for embryonic development. It was also found that one functional Rab1a allele was sufficient for development in a heterozygous murine embryo, whereas a double mutant led to embryonic lethality. The dissection of uteri on embryonic day (E)10.5-14.5 yielded no homozygous embryos, indicating that homozygotes die between E10.5 to E11.5. The gene trap construct contains a β-galactosidase/neomycin reporter gene, allowing for heterozygotes to be stained for β-galactosidase to determine the tissue-specific expression of Rab1a. Rab1a was found to be highly expressed in the small intestine of both adult mice and embryos, although its expression levels were low in the brains of embryos. Moreover, there was no significant change in cytokine production and survival in wild-type and heterozygous Rab1a+/− mice following a challenge with lipopolysaccharide. On the whole, the present study demonstrates that the disruption of the Rab1a gene causes embryonic lethality and homozygotes die between E10.5 and E11.5, suggesting that Rab1a is essential for the early development of mouse embryos.