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1.
Cell ; 186(6): 1162-1178.e20, 2023 03 16.
Article in English | MEDLINE | ID: mdl-36931244

ABSTRACT

Germline histone H3.3 amino acid substitutions, including H3.3G34R/V, cause severe neurodevelopmental syndromes. To understand how these mutations impact brain development, we generated H3.3G34R/V/W knock-in mice and identified strikingly distinct developmental defects for each mutation. H3.3G34R-mutants exhibited progressive microcephaly and neurodegeneration, with abnormal accumulation of disease-associated microglia and concurrent neuronal depletion. G34R severely decreased H3K36me2 on the mutant H3.3 tail, impairing recruitment of DNA methyltransferase DNMT3A and its redistribution on chromatin. These changes were concurrent with sustained expression of complement and other innate immune genes possibly through loss of non-CG (CH) methylation and silencing of neuronal gene promoters through aberrant CG methylation. Complement expression in G34R brains may lead to neuroinflammation possibly accounting for progressive neurodegeneration. Our study reveals that H3.3G34-substitutions have differential impact on the epigenome, which underlie the diverse phenotypes observed, and uncovers potential roles for H3K36me2 and DNMT3A-dependent CH-methylation in modulating synaptic pruning and neuroinflammation in post-natal brains.


Subject(s)
DNA Methyltransferase 3A , Histones , Animals , Mice , DNA (Cytosine-5-)-Methyltransferases/genetics , DNA Methylation/genetics , DNA Modification Methylases/genetics , Histones/metabolism , Neuroinflammatory Diseases
2.
Methods Mol Biol ; 2429: 515-531, 2022.
Article in English | MEDLINE | ID: mdl-35507186

ABSTRACT

Genetically engineered mouse models (GEMMs) are very powerful tools to study lineage hierarchy and cellular dynamics of stem cells in vivo. Stem cell behavior in various contexts such as development, normal homeostasis and diseases have been investigated using GEMMs. The strategies to generate GEMMs have drastically changed in the last decade with the development of the CRISPR/Cas9 system for manipulation of the mammalian genome. The advantages of the CRISPR/Cas9 are its simplicity and efficiency. The bioinformatics tools available now allow us to quickly identify appropriate guide RNAs and design experimental conditions to generate the targeted mutation. In addition, the genome can be manipulated directly in the zygote which reduces the time to modify target genes compared to other technologies such as Embryonic Stem (ES) cells. Equally important is that we can manipulate the genome of any mouse background with the CRISPR/Cas9 system which omits time-consuming backcrossing processes, accelerates research and increases flexibility. Here, we will summarize basic allelic types and our standard strategies of how to generate them.


Subject(s)
CRISPR-Cas Systems , Gene Editing , Animals , CRISPR-Cas Systems/genetics , Genome/genetics , Mammals/genetics , Mice , RNA, Guide, Kinetoplastida/genetics , Zygote
3.
Sci Adv ; 7(51): eabl4391, 2021 Dec 17.
Article in English | MEDLINE | ID: mdl-34910520

ABSTRACT

Follicle-stimulating hormone (FSH), a key regulator of ovarian function, is often used in infertility treatment. Gonadal inhibins suppress FSH synthesis by pituitary gonadotrope cells. The TGFß type III receptor, betaglycan, is required for inhibin A suppression of FSH. The inhibin B co-receptor was previously unknown. Here, we report that the gonadotrope-restricted transmembrane protein, TGFBR3L, is the elusive inhibin B co-receptor. TGFBR3L binds inhibin B but not other TGFß family ligands. TGFBR3L knockdown or overexpression abrogates or confers inhibin B activity in cells. Female Tgfbr3l knockout mice exhibit increased FSH levels, ovarian follicle development, and litter sizes. In contrast, female mice lacking both TGFBR3L and betaglycan are infertile. TGFBR3L's function and cell-specific expression make it an attractive new target for the regulation of FSH and fertility.

4.
PLoS One ; 16(8): e0249439, 2021.
Article in English | MEDLINE | ID: mdl-34437552

ABSTRACT

We demonstrate that simple, non-invasive environmental DNA (eDNA) methods can detect transgenes of genetically modified (GM) animals from terrestrial and aquatic sources in invertebrate and vertebrate systems. We detected transgenic fragments between 82-234 bp through targeted PCR amplification of environmental DNA extracted from food media of GM fruit flies (Drosophila melanogaster), feces, urine, and saliva of GM laboratory mice (Mus musculus), and aquarium water of GM tetra fish (Gymnocorymbus ternetzi). With rapidly growing accessibility of genome-editing technologies such as CRISPR, the prevalence and diversity of GM animals will increase dramatically. GM animals have already been released into the wild with more releases planned in the future. eDNA methods have the potential to address the critical need for sensitive, accurate, and cost-effective detection and monitoring of GM animals and their transgenes in nature.


Subject(s)
Animals, Genetically Modified/genetics , DNA, Environmental/genetics , Transgenes/genetics , Animals , Characidae/genetics , Drosophila melanogaster/genetics , Environmental Monitoring/methods , Mice/genetics
5.
Development ; 143(9): 1534-46, 2016 05 01.
Article in English | MEDLINE | ID: mdl-27143755

ABSTRACT

Cellular interactions are key for the differentiation of distinct cell types within developing epithelia, yet the molecular mechanisms engaged in these interactions remain poorly understood. In the developing olfactory epithelium (OE), neural stem/progenitor cells give rise to odorant-detecting olfactory receptor neurons (ORNs) and glial-like sustentacular (SUS) cells. Here, we show in mice that the transmembrane receptor neogenin (NEO1) and its membrane-bound ligand RGMB control the balance of neurons and glial cells produced in the OE. In this layered epithelium, neogenin is expressed in progenitor cells, while RGMB is restricted to adjacent newly born ORNs. Ablation of Rgmb via gene-targeting increases the number of dividing progenitor cells in the OE and leads to supernumerary SUS cells. Neogenin loss-of-function phenocopies these effects observed in Rgmb(-/-) mice, supporting the proposal that RGMB-neogenin signaling regulates progenitor cell numbers and SUS cell production. Interestingly, Neo1(-/-) mice also exhibit increased apoptosis of ORNs, implicating additional ligands in the neogenin-dependent survival of ORNs. Thus, our results indicate that RGMB-neogenin-mediated cell-cell interactions between newly born neurons and progenitor cells control the ratio of glia and neurons produced in the OE.


Subject(s)
Gene Expression Regulation, Developmental , Membrane Proteins/genetics , Nerve Tissue Proteins/genetics , Neural Stem Cells/cytology , Neurogenesis/genetics , Olfactory Mucosa/embryology , Olfactory Receptor Neurons/cytology , Animals , Apoptosis/genetics , Cell Adhesion Molecules, Neuronal , Cell Proliferation/genetics , GPI-Linked Proteins , Membrane Proteins/biosynthesis , Mice , Mice, Inbred C57BL , Mice, Knockout , Nerve Tissue Proteins/biosynthesis , Neurogenesis/physiology , Neuroglia/cytology , Olfactory Mucosa/cytology , Olfactory Receptor Neurons/metabolism , Signal Transduction/physiology
6.
J Biol Chem ; 289(43): 30133-43, 2014 Oct 24.
Article in English | MEDLINE | ID: mdl-25225289

ABSTRACT

Coordinated control of the growth cone cytoskeleton underlies axon extension and guidance. Members of the collapsin response mediator protein (CRMP) family of cytosolic phosphoproteins regulate the microtubule and actin cytoskeleton, but their roles in regulating growth cone dynamics remain largely unexplored. Here, we examine how CRMP4 regulates the growth cone cytoskeleton. Hippocampal neurons from CRMP4-/- mice exhibited a selective decrease in axon extension and reduced growth cone area, whereas overexpression of CRMP4 enhanced the formation and length of growth cone filopodia. Biochemically, CRMP4 can impact both microtubule assembly and F-actin bundling in vitro. Through a structure function analysis of CRMP4, we found that the effects of CRMP4 on axon growth and growth cone morphology were dependent on microtubule assembly, whereas filopodial extension relied on actin bundling. Intriguingly, anterograde movement of EB3 comets, which track microtubule protrusion, slowed significantly in neurons derived from CRMP4-/- mice, and rescue of microtubule dynamics required CRMP4 activity toward both the actin and microtubule cytoskeleton. Together, this study identified a dual role for CRMP4 in regulating the actin and microtubule growth cone cytoskeleton.


Subject(s)
Actin Cytoskeleton/metabolism , Growth Cones/metabolism , Microtubules/metabolism , Nerve Tissue Proteins/metabolism , Animals , Axons/metabolism , Cell Size , Female , Hippocampus/cytology , Mice, Inbred BALB C , Mice, Inbred C57BL , Microtubule-Associated Proteins/metabolism , Nerve Tissue Proteins/chemistry , Nerve Tissue Proteins/deficiency , Protein Structure, Tertiary , Tubulin/metabolism
7.
Lab Anim (NY) ; 40(10): 305-12, 2011 Sep 21.
Article in English | MEDLINE | ID: mdl-22358207

ABSTRACT

The authors implemented a PCR protocol to rapidly screen for Pasteurella pneumotropica and to accurately identify contaminated laboratory mice in a clinical setting. This protocol was implemented in response to a severe outbreak of P. pneumotropica in their animal facility. Although a sentinel program was in place to routinely screen for P. pneumotropica, it was inadequate for the identification of contaminated animals. As a result, several additional strains of mice were contaminated and developed clinical signs of infection. The authors implemented a screening method using PCR with reported primer pairs previously developed to identify the biotype isolates of P. pneumotropica in laboratory mice. Throat culture swabs were collected from live mice and placed in a bacterial culture. The DNA from these cultures was isolated and screened by PCR. This procedure enabled the authors to eliminate P. pneumotropica from several animal housing rooms. The assay can be easily applied in most animal facilities.


Subject(s)
Mice , Pasteurella Infections/veterinary , Pasteurella pneumotropica/genetics , Polymerase Chain Reaction/methods , Rodent Diseases/microbiology , Animals , Housing, Animal , Mice, Inbred C57BL , Pasteurella Infections/microbiology , Pasteurella pneumotropica/isolation & purification , Polymerase Chain Reaction/veterinary
8.
Proc Natl Acad Sci U S A ; 101(17): 6605-10, 2004 Apr 27.
Article in English | MEDLINE | ID: mdl-15096582

ABSTRACT

Vascular permeability plays a key role in a wide array of life-threatening and sight-threatening diseases. Vascular endothelial growth factor can increase vascular permeability. Using a model system for nonproliferative diabetic retinopathy, we found that pigment epithelium-derived factor (PEDF) effectively abated vascular endothelial growth factor-induced vascular permeability. A 44-amino acid region of PEDF was sufficient to confer the antivasopermeability activity. Additionally, we identified four amino acids (glutamate-101, isoleucine-103, leucine-112, and serine-115) critical for this activity. PEDF, or a derivative, could potentially abate or restore vision loss from diabetic macular edema. Furthermore, PEDF may represent a superior therapeutic approach to sepsis-associated hypotension, nephrotic syndrome, and other sight-threatening and life-threatening diseases resulting from excessive vascular permeability.


Subject(s)
Capillary Permeability/physiology , Eye Proteins , Nerve Growth Factors , Proteins/physiology , Serpins/physiology , Amino Acid Sequence , Animals , Binding Sites , Cell Line , Fluorescein Angiography , Humans , Mice , Mice, Inbred C57BL , Molecular Sequence Data , Proteins/chemistry , Proteins/metabolism , Recombinant Proteins/metabolism , Retinal Vessels/physiology , Sequence Homology, Amino Acid , Serpins/chemistry , Serpins/metabolism , Vascular Endothelial Growth Factor A/physiology
9.
Science ; 303(5655): 197-202, 2004 Jan 09.
Article in English | MEDLINE | ID: mdl-14716005

ABSTRACT

The lasting effects of neuronal activity on brain development involve calcium-dependent gene expression. Using a strategy called transactivator trap, we cloned a calcium-responsive transactivator called CREST (for calcium-responsive transactivator). CREST is a SYT-related nuclear protein that interacts with adenosine 3',5'-monophosphate (cAMP) response element-binding protein (CREB)-binding protein (CBP) and is expressed in the developing brain. Mice that have a targeted disruption of the crest gene are viable but display defects in cortical and hippocampal dendrite development. Cortical neurons from crest mutant mice are compromised in calcium-dependent dendritic growth. Thus, calcium activation of CREST-mediated transcription helps regulate neuronal morphogenesis.


Subject(s)
Calcium/metabolism , Dendrites/physiology , Neurons/physiology , Trans-Activators/metabolism , Transcription, Genetic , Transcriptional Activation , Amino Acid Sequence , Animals , Blotting, Northern , Brain/cytology , Brain/embryology , Brain/growth & development , Brain/metabolism , CREB-Binding Protein , Calcium Channels/metabolism , Cell Line , Cells, Cultured , Cerebral Cortex/cytology , Cerebral Cortex/embryology , Cerebral Cortex/metabolism , Cloning, Molecular , Dendrites/ultrastructure , Gene Expression Profiling , Gene Expression Regulation, Developmental , Gene Library , Gene Targeting , Humans , In Situ Hybridization , Mice , Mice, Knockout , Molecular Sequence Data , Mutation , Nervous System/embryology , Nervous System/growth & development , Nervous System/metabolism , Neurons/ultrastructure , Nuclear Proteins/metabolism , Protein Structure, Tertiary , Rats , Recombinant Fusion Proteins/metabolism , Trans-Activators/chemistry , Trans-Activators/genetics , Transfection
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