ABSTRACT
The lack of a widely accessible method for expressing genes of interest in wild-type embryos is a fundamental obstacle to understanding genetic regulation during embryonic development. In particular, only a few methods are available for introducing gene expression vectors into cells prior to neural tube closure, which is a period of drastic development for many tissues. In this study, we present a simple technique for injecting vectors into the amniotic cavity and allowing them to reach the ectodermal cells and the epithelia of endodermal organs of mouse embryos at E8.0 via in utero injection, using only a widely used optical fiber with an illuminator. Using this technique, retroviruses can be introduced to facilitate the labeling of cells in various tissues, including the brain, spinal cord, epidermis, and digestive and respiratory organs. We also demonstrated in utero electroporation of plasmid DNA into E7.0 and E8.0 embryos. Taking advantage of this method, we reveal the association between Ldb1 and the activity of the Neurog2 transcription factor in the mouse neocortex. This technique can aid in analyzing the roles of genes of interest during endo- and ectodermal development prior to neural tube closure.
ABSTRACT
Recent studies have demonstrated that epigenetic modifications are deeply involved in neurogenesis; however, the precise mechanisms remain largely unknown. To determine the role of UTX (also known as KDM6A), a demethylase of histone H3K27, in neural development, we generated Utx-deficient mice in neural stem/progenitor cells (NSPCs). Since Utx is an X chromosome-specific gene, the genotypes are sex-dependent; female mice lose both Utx alleles (UtxΔ/Δ ), and male mice lose one Utx allele yet retain one Uty allele, the counterpart of Utx on the Y chromosome (UtxΔ/Uty ). We found that UtxΔ/Δ mice exhibited fetal ventriculomegaly and died soon after birth. Immunofluorescence staining and EdU labeling revealed a significant increase in NSPCs and a significant decrease in intermediate-progenitor and differentiated neural cells. Molecular analyses revealed the downregulation of pathways related to DNA replication and increased H3K27me3 levels around the transcription start sites in UtxΔ/Δ NSPCs. These results indicate that UTX globally regulates the expression of genes required for proper neural development in NSPCs, and UTX deficiency leads to impaired cell cycle exit, reduced differentiation, and neonatal death. Interestingly, although UtxΔ/Uty mice survived the postnatal period, most died of hydrocephalus, a clinical feature of Kabuki syndrome, a congenital anomaly involving UTX mutations. Our findings provide novel insights into the role of histone modifiers in neural development and suggest that UtxΔ/Uty mice are a potential disease model for Kabuki syndrome.
Subject(s)
Histones , Hydrocephalus , Animals , Female , Male , Mice , Fetal Development , Histone Demethylases/genetics , Hydrocephalus/genetics , Neurogenesis , Stem Cells , Neural Stem CellsABSTRACT
Dysregulation of inflammatory cytokines in keratinocytes promote the pathogenesis of the skin inflammation, such as allergic contact dermatitis (ACD). High-mobility group box 1 protein (HMGB1) has been implicated in the promotion of skin inflammation upon its extracellular release as a damage-associated molecular pattern molecule. However, whether and how HMGB1 in keratinocytes contributes to ACD and other skin disorders remain elusive. In this study, we generated conditional knockout mice in which the Hmgb1 gene is specifically deleted in keratinocytes, and examined its role in ACD models. Interestingly, the mutant mice showed exacerbated skin inflammation, accompanied by increased ear thickening in 2,4-dinitrofluorobenezene-induced ACDs. The mRNA expression of interleukin-24 (IL-24), a cytokine known to critically contribute to ACD pathogenesis, was elevated in skin lesions of the mutant mice. As with constitutively expressed, IL-4-induced Il24 mRNA, expression was also augmented in the Hmgb1-deficient keratinocytes, which would account for the exacerbation of ACD in the mutant mice. Mechanistically, we observed an increased binding of trimethyl histone H3 (lys4) (H3K4me3), a hallmark of transcriptionally active genes, to the promoter region of the Il24 gene in the hmgb1-deficient cells. Thus, the nuclear HMGB1 is a critical "gate keeper" in that the dermal homeostasis is contingent to its function in chromatin remodeling. Our study revealed a facet of nuclear HMGB1, namely its antiinflammatory function in keratinocytes for the skin homeostasis.
Subject(s)
Chromatin Assembly and Disassembly , Dermatitis, Allergic Contact/metabolism , HMGB1 Protein/metabolism , Histones/metabolism , Interleukins/metabolism , Keratinocytes/metabolism , Animals , Dermatitis, Allergic Contact/genetics , Dermatitis, Allergic Contact/prevention & control , Dinitrofluorobenzene/toxicity , Disease Models, Animal , Ear/pathology , Gene Deletion , Gene Expression Regulation/genetics , HMGB1 Protein/deficiency , HMGB1 Protein/genetics , Inflammation/genetics , Inflammation/metabolism , Interleukin-4/pharmacology , Interleukins/genetics , Mice , Mice, Knockout , Promoter Regions, Genetic , Skin/immunology , Skin/metabolism , Skin/pathology , Transplantation ChimeraABSTRACT
Neuropathic pain is the most difficult type of pain to control, and patients lose their motivation for the purposive pursuit with a decrease in their quality of life. Using a functional magnetic resonance imaging analysis, we demonstrated that blood oxygenation level-dependent signal intensity was increased in the ipsilateral nucleus accumbens (N.Acc.) following peripheral nerve injury. microRNAs are small, noncoding RNA molecules that direct the post-transcriptional suppression of gene expression, and play an important role in regulating synaptic plasticity. In this study, we found that sciatic nerve ligation induced a drastic decrease in the expression of miR200b and miR429 in N.Acc. neurons. The expression of DNA methyltransferase 3a (DNMT3a), which is the one of the predicted targets of miR200b/429, was significantly increased in the limbic forebrain including N.Acc. at 7 d after sciatic nerve ligation. Double-immunolabeling with antibodies specific to DNMT3a and NR1 showed that DNMT3a-immunoreactivity in the N.Acc. was located in NR1-labeled neurons, indicating that increased DNMT3a proteins were dominantly expressed in postsynaptic neurons in the N.Acc. area under a neuropathic pain-like state. The results of these analyses provide new insight into an epigenetic modification that is accompanied by a dramatic decrease in miR200b and miR429 along with the dysfunction of "mesolimbic motivation/valuation circuitry" under a neuropathic pain-like state. These phenomena may result in an increase in DNMT3a in neurons of the N.Acc. under neuropathic pain, which leads to the long-term transcription-silencing of several genes.
Subject(s)
Adaptation, Physiological/physiology , MicroRNAs/metabolism , Neurons/physiology , Nucleus Accumbens/pathology , Nucleus Accumbens/physiopathology , Sciatica/pathology , Adaptor Proteins, Signal Transducing/metabolism , Analysis of Variance , Animals , Cytoskeletal Proteins/metabolism , DNA (Cytosine-5-)-Methyltransferases/metabolism , DNA Methyltransferase 3A , Disease Models, Animal , Gene Expression Regulation , Homeodomain Proteins/metabolism , Hyperalgesia/pathology , Image Processing, Computer-Assisted , In Vitro Techniques , Magnetic Resonance Imaging/methods , Male , Membrane Glycoproteins/metabolism , Methyl-CpG-Binding Protein 2/metabolism , Mice , Mice, Inbred C57BL , Nerve Tissue Proteins/metabolism , Nucleus Accumbens/blood supply , Oxygen/blood , Repressor Proteins/metabolism , Synaptosomal-Associated Protein 25/metabolism , Zinc Finger E-box Binding Homeobox 2ABSTRACT
We previously demonstrated that the discriminative stimulus effects of the κ-opioid receptor agonist U-50,488H were associated with its aversive effects in rats. However, its molecular signaling mechanisms are not fully understood. The aim of this study was to investigate the intracellular signaling that plays a role in mediating the discriminative stimulus effect induced by U-50,488H. To better understand the involvement of molecular signaling mechanisms in the discriminative stimulus effects of U-50,488H, rats were subjected to a drug discrimination paradigm, and levels of immunoreactivity and mRNA expression were determined in these rats. Although U-50,488H-trained rats did not show changes in the mRNA expression of typical dopamine (DA) receptors, NMDA receptor subunits, or transcriptional activators, there were remarkable changes in the levels of immunoreactivity of several phosphorylated protein kinases. The levels of immunoreactivity of phosphorylated p38 MAPK and phosphorylated calcium/calmodulin-dependent protein kinase II (CaMKII) were significantly increased in the nucleus accumbens and amygdala in lever-press, yoked and discrimination groups compared to a naive group. Furthermore, the level of phosphorylated cAMP response element-binding protein was also increased in both the discrimination and yoked groups. In contrast, the immunoreactivity of phosphorylated extracellular signaling-regulated kinase (ERK 1/2) was specifically increased in the discrimination group. These results suggest that the ERK signaling pathway in the nucleus accumbens and amygdala may be critical for the expression of the discriminative stimulus effects of U-50,488H.
Subject(s)
3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer/pharmacology , Amygdala/drug effects , Analgesics, Non-Narcotic/pharmacology , Discrimination Learning/drug effects , Extracellular Signal-Regulated MAP Kinases/metabolism , Mitogen-Activated Protein Kinase 1/metabolism , Nucleus Accumbens/drug effects , Amygdala/enzymology , Animals , Cyclic AMP Response Element-Binding Protein/genetics , Cyclic AMP Response Element-Binding Protein/metabolism , Discrimination Learning/physiology , Enzyme Activation/drug effects , Enzyme Activation/physiology , Extracellular Signal-Regulated MAP Kinases/physiology , Male , Mitogen-Activated Protein Kinase 1/physiology , Nucleus Accumbens/enzymology , Phosphorylation/drug effects , Rats , Rats, Inbred F344ABSTRACT
Insomnia is a common problem for people with chronic pain. Cortical GABAergic neurons are part of the neurobiological substrate that underlies homeostatic sleep regulation. In the present study, we confirmed that sciatic nerve ligation caused thermal hyperalgesia and tactile allodynia in mice. In this experimental model for neuropathic pain, we found an increase in wakefulness and a decrease in non-rapid eye movement sleep under a neuropathic pain-like state. Under these conditions, membrane-bound GABA (γ-aminobutyric acid) transporters (GATs) on activated glial fibrillary acidic protein-positive astrocytes were significantly increased in the cingulate cortex, and extracellular GABA levels in this area after depolarization were rapidly decreased by nerve injury. Furthermore, sleep disturbance induced by sciatic nerve ligation was improved by the intracingulate cortex injection of a GAT-3 inhibitor. These findings provide novel evidence that sciatic nerve ligation decreases extracellular-released GABA in the cingulate cortex of mice. These phenomena may, at least in part, explain the insomnia in patients with neuropathic pain. Neuropathic pain-like stimuli suppress the GABAergic transmission with increased GABA (γ-aminobutyric acid) transporters located on activated astrocytes in the cingulate cortex related to sleep disturbance.
Subject(s)
Gyrus Cinguli/metabolism , Sciatic Neuropathy/complications , Sciatic Neuropathy/pathology , Sleep Wake Disorders/etiology , gamma-Aminobutyric Acid/metabolism , Analysis of Variance , Animals , Anisoles/pharmacology , Disease Models, Animal , Electroencephalography , Electromyography , GABA Plasma Membrane Transport Proteins/genetics , GABA Plasma Membrane Transport Proteins/metabolism , Gene Expression Regulation/drug effects , Gene Expression Regulation/physiology , Glial Fibrillary Acidic Protein/metabolism , Gyrus Cinguli/drug effects , Hyperalgesia/drug therapy , Hyperalgesia/physiopathology , Hypnotics and Sedatives/therapeutic use , Mice , Mice, Inbred C57BL , Mice, Inbred ICR , Microdialysis , Midazolam/pharmacology , Midazolam/therapeutic use , Nipecotic Acids/pharmacology , Pain Measurement , Pain Threshold/physiology , Physical Stimulation/adverse effects , Pyridines/therapeutic use , RNA, Messenger/metabolism , Reflex/drug effects , Reflex/physiology , Sciatic Neuropathy/drug therapy , ZolpidemABSTRACT
Despite the importance of prefrontal cortical dopamine in modulating reward, little is known about the implication of the specific subregion of prefrontal cortex in opioid reward. We investigated the role of neurons projecting from the ventral tegmental area (VTA) to the anterior cingulate cortex (ACG) in opioid reward. Microinjection of the retrograde tracer fluorogold (FG) into the ACG revealed several retrogradely labelled cells in the VTA. The FG-positive reactions were noted in both tyrosine hydroxylase (TH)-positive and -negative VTA neurons. The released levels of dopamine and its major metabolites in the ACG were increased by either the electrical stimulation of VTA neurons or microinjection of a selective µ-opioid receptor (MOR) agonist, (D-Ala²,N-MePhe4,Gly-ol5) enkephalin (DAMGO), into the VTA. MOR-like immunoreactivity was seen in both TH-positive and -negative VTA neurons projecting to the ACG. The conditioned place preference induced by intra-VTA injection of DAMGO was significantly attenuated by chemical lesion of dopaminergic terminals in the ACG. The depletion of dopamine in the ACG induced early extinction of µ-opioid-induced place preference. The levels of phosphorylated DARPP32 (Thr34) and phosphorylated CREB (Ser133) were increased in the ACG of rats that had maintained the morphine-induced place preference, whereas the increases of these levels induced by morphine were blocked by pre-treatment of a selective dopamine D1 receptor antagonist SCH23390. These findings suggest that VTA-ACG transmission may play a crucial role in the acquisition and maintenance of µ-opioid-induced place preference. The activation of DARPP32 and CREB through dopamine D1 receptors in the ACG could be implicated in the maintenance of µ-opioid-induced place preference.