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1.
Development ; 140(23): 4672-82, 2013 Dec.
Article in English | MEDLINE | ID: mdl-24173802

ABSTRACT

Mammalian neuroepithelial stem cells divide using a polarized form of cytokinesis, which is not well understood. The cytokinetic furrow cleaves the cell by ingressing from basal to apical, forming the midbody at the apical membrane. The midbody mediates abscission by recruiting many factors, including the Kinesin-6 family member Kif20b. In developing embryos, Kif20b mRNA is most highly expressed in neural stem/progenitor cells. A loss-of-function mutant in Kif20b, magoo, was found in a forward genetic screen. magoo has a small cerebral cortex, with reduced production of progenitors and neurons, but preserved layering. In contrast to other microcephalic mouse mutants, mitosis and cleavage furrows of cortical stem cells appear normal in magoo. However, apical midbodies show changes in number, shape and positioning relative to the apical membrane. Interestingly, the disruption of abscission does not appear to result in binucleate cells, but in apoptosis. Thus, Kif20b is required for proper midbody organization and abscission in polarized cortical stem cells and has a crucial role in the regulation of cerebral cortex growth.


Subject(s)
Cerebral Cortex/metabolism , Cytokinesis/physiology , Kinesins/metabolism , Neural Stem Cells/metabolism , Animals , Cell Polarity/genetics , Gene Expression , Kinesins/genetics , Mice , Mice, Inbred C57BL , Microtubules/metabolism , RNA, Messenger/biosynthesis
2.
Neural Dev ; 6: 3, 2011 Jan 07.
Article in English | MEDLINE | ID: mdl-21214893

ABSTRACT

BACKGROUND: The dorsal thalamus acts as a gateway and modulator for information going to and from the cerebral cortex. This activity requires the formation of reciprocal topographic axon connections between thalamus and cortex. The axons grow along a complex multistep pathway, making sharp turns, crossing expression boundaries, and encountering intermediate targets. However, the cellular and molecular components mediating these steps remain poorly understood. RESULTS: To further elucidate the development of the thalamocortical system, we first created a thalamocortical axon reporter line to use as a genetic tool for sensitive analysis of mutant mouse phenotypes. The TCA-tau-lacZ reporter mouse shows specific, robust, and reproducible labeling of thalamocortical axons (TCAs), but not the overlapping corticothalamic axons, during development. Moreover, it readily reveals TCA pathfinding abnormalities in known cortical mutants such as reeler. Next, we performed an unbiased screen for genes involved in thalamocortical development using random mutagenesis with the TCA reporter. Six independent mutant lines show aberrant TCA phenotypes at different steps of the pathway. These include ventral misrouting, overfasciculation, stalling at the corticostriatal boundary, and invasion of ectopic cortical cell clusters. An outcross breeding strategy coupled with a genomic panel of single nucleotide polymorphisms facilitated genetic mapping with small numbers of mutant mice. We mapped a ventral misrouting mutant to the Emx2 gene, and discovered that some TCAs extend to the olfactory bulbs in this mutant. Mapping data suggest that other lines carry mutations in genes not previously known for roles in thalamocortical development. CONCLUSIONS: These data demonstrate the feasibility of a forward genetic approach to understanding mammalian brain morphogenesis and wiring. A robust axonal reporter enabled sensitive analysis of a specific axon tract inside the mouse brain, identifying mutant phenotypes at multiple steps of the pathway, and revealing a new aspect of the Emx2 mutant. The phenotypes highlight vulnerable choice points and latent tendencies of TCAs, and will lead to a refined understanding of the elements and interactions required to form the thalamocortical system.


Subject(s)
Axons/physiology , Cerebral Cortex , Gene Expression Regulation, Developmental/genetics , Homeodomain Proteins/genetics , Mutation/genetics , Phenotype , Thalamus , Transcription Factors/genetics , Alkylating Agents/pharmacology , Animals , Body Patterning/drug effects , Body Patterning/genetics , Cell Adhesion Molecules, Neuronal/genetics , Cell Adhesion Molecules, Neuronal/metabolism , Cerebral Cortex/embryology , Cerebral Cortex/growth & development , Cerebral Cortex/metabolism , Embryo, Mammalian , Ethylnitrosourea/pharmacology , Extracellular Matrix Proteins/genetics , Extracellular Matrix Proteins/metabolism , Female , Gene Expression Regulation, Developmental/drug effects , Genetic Testing/methods , Lac Operon/genetics , Male , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Transgenic , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Neural Pathways/abnormalities , Neural Pathways/embryology , Neural Pathways/growth & development , Reelin Protein , Serine Endopeptidases/genetics , Serine Endopeptidases/metabolism , Thalamus/embryology , Thalamus/growth & development , Thalamus/metabolism , beta-Galactosidase/metabolism
3.
J Neurosci ; 28(12): 3123-30, 2008 Mar 19.
Article in English | MEDLINE | ID: mdl-18354015

ABSTRACT

Recent evidence challenges the prevalent view that neural factors induce the formation of a de novo postsynaptic apparatus during development of the vertebrate neuromuscular junction. The latest experiments suggest an alternative model in which the muscle fiber induces a nascent postsynaptic apparatus and sets the location of the future synapse. On axonal contact, these sites, laid out in a prepattern in the central area of developing muscle fibers, mature into synapses by the combined action of neural factors such as agrin and ACh. We sought to test in mammals these two models of neuromuscular synaptogenesis. Previously, we showed that continuous prenatal muscle expression of constitutively active ErbB2 (CAErbB2) led to synaptic loss, exuberant axonal sprouting, and lethality at birth. Here, we transiently induced CAErbB2 during midgestation and examined synapse restoration after inducer withdrawal. Centrally enriched ACh receptor (AChR) transcription and clustering were abolished after transient CAErbB2 induction. After inducer withdrawal, synapses were restored but were distributed widely over the entire diaphragm muscle. Under the nerve-dependent model, this distribution is explained by the wide pattern of axonal sprouting triggered by CAErbB2. Yet, in the absence of the nerve, introduced in our animals by mating to Hb9(+/-) mice, a very similar, wide distribution of aneural AChR clusters resulted. Thus, transient expression of CAErbB2 in skeletal muscles leads to reprogramming of the endogenous muscle AChR prepattern. This, and not the nerve, seems primarily responsible for the widely distributed pattern of synapses in our experimental animals.


Subject(s)
Muscle Fibers, Skeletal/physiology , Muscle, Skeletal/physiology , Neuromuscular Junction/physiology , Animals , Animals, Newborn , Bungarotoxins/pharmacokinetics , Diaphragm/cytology , Doxycycline/pharmacology , Embryo, Mammalian , Female , Gene Expression Regulation, Developmental/drug effects , Gene Expression Regulation, Developmental/physiology , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Homeodomain Proteins , Luciferases/metabolism , Male , Mice , Mice, Transgenic , Microscopy, Confocal , Receptor Protein-Tyrosine Kinases/metabolism , Receptor, ErbB-2/genetics , Receptors, Cholinergic/genetics , Receptors, Cholinergic/metabolism , Transcription Factors/deficiency
4.
Mol Cell Neurosci ; 31(2): 334-45, 2006 Feb.
Article in English | MEDLINE | ID: mdl-16278083

ABSTRACT

We overexpressed a constitutively active form of the neuregulin receptor ErbB2 (CAErbB2) in skeletal muscle fibers in vivo and in vitro by tetracycline-inducible expression. Surprisingly, CAErbB2 expression during embryonic development was lethal and impaired synaptogenesis yielding a phenotype with loss of synaptic contacts, extensive axonal sprouting, and diffuse distribution of acetylcholine receptor (AChR) transcripts, reminiscent of agrin-deficient mice. CAErbB2 expression in cultured myotubes inhibited the formation and maintenance of agrin-induced AChR clusters, suggesting a muscle- and not a nerve-origin for the defect in CAErbB2-expressing mice. Levels of tyrosine phosphorylated MuSK, the signaling component of the agrin receptor, were similar, while tyrosine phosphorylation of AChRbeta subunits was dramatically reduced in CAErbB2-expressing embryos relative to controls. Thus, a gain-of-function manipulation of ErbB2 signaling pathways renders an agrin-deficient-like phenotype that uncouples MuSK and AChR tyrosine phosphorylation.


Subject(s)
Muscle, Skeletal/cytology , Muscle, Skeletal/embryology , Neuromuscular Junction/embryology , Receptor, ErbB-2/metabolism , Synapses/physiology , Agrin/genetics , Agrin/metabolism , Animals , Mice , Mice, Transgenic , Muscle, Skeletal/metabolism , Neuromuscular Junction/metabolism , Protein Isoforms/genetics , Protein Isoforms/metabolism , Receptor Protein-Tyrosine Kinases/genetics , Receptor Protein-Tyrosine Kinases/metabolism , Receptor, ErbB-2/genetics , Receptors, Cholinergic/genetics , Receptors, Cholinergic/metabolism , Signal Transduction/physiology , Tyrosine/metabolism
5.
Neuroreport ; 16(3): 271-5, 2005 Feb 28.
Article in English | MEDLINE | ID: mdl-15706234

ABSTRACT

Genetic and behavioral studies in humans and mouse mutants have implicated the gene encoding neuregulin-1 (Nrg-1) as a candidate susceptibility gene for schizophrenia. We examined the behavior of mice heterozygous for a mutation in neuregulin-1's immunoglobulin (Ig)-like domain (Ig-nrg-1 mice). We found that these animals displayed behaviors related to a schizophrenia-like phenotype, such as clozapine suppression of open-field and running wheel activity and impaired latent inhibition. Contrary to findings with other nrg-1 mutants, Ig-nrg-1 mice did not exhibit significantly elevated locomotion relative to littermate controls. These results suggest that Ig-Nrg-1's contribute to some - but not all - aspects of the schizophrenia-like phenotype of nrg-1 mutants, and further support nrg-1 as a candidate gene for schizophrenia.


Subject(s)
Antipsychotic Agents/therapeutic use , Clozapine/therapeutic use , Neural Inhibition/drug effects , Neuregulin-1/chemistry , Neuregulin-1/genetics , Schizophrenia/drug therapy , Animals , Behavior, Animal/physiology , Disease Models, Animal , Exploratory Behavior/drug effects , Immunoglobulins , Mice , Mice, Mutant Strains , Motor Activity/drug effects , Phenotype , Protein Structure, Tertiary/physiology , Running
6.
J Biol Chem ; 277(21): 18489-93, 2002 May 24.
Article in English | MEDLINE | ID: mdl-11889124

ABSTRACT

Nonsense-mediated decay (NMD) is an RNA surveillance pathway that degrades mRNAs containing premature termination codons (PTC). T-cell receptor (TCR) and immunoglobulin (Ig) transcripts, which are encoded by genes that very frequently acquire PTCs during lymphoid ontogeny, are down-regulated much more dramatically in response to PTCs than are other known transcripts. Another feature unique to TCR, Ig, and a subset of other mRNAs is that they are down-regulated in response to nonsense codons in the nuclear fraction of cells. This is paradoxical, as the only well recognized entity that recognizes nonsense codons is the cytoplasmic translation apparatus. Therefore, we investigated whether translation is responsible for this nuclear-associated mechanism. We found that the down-regulation of TCR-beta transcripts in response to nonsense codons requires several features of translation, including an initiator ATG and the ability to scan. We also found that optimal down-regulation depends on a Kozak consensus sequence surrounding the initiator ATG and that it can be initiated by an internal ribosome entry site, neither of which has been demonstrated before for any other PTC-bearing mRNA. At least a portion of this down-regulatory response is mediated by the NMD pathway as antisense hUPF2 transcripts increased the levels of PTC-bearing TCR-beta transcripts in the nuclear fraction of cells. We conclude that a hUPF2-dependent RNA surveillance pathway with translation-like features operating in the nuclear fraction of cells prevents the expression of potentially deleterious truncated proteins encoded by non-productively rearranged TCR genes.


Subject(s)
Down-Regulation , Fungal Proteins/physiology , Protein Biosynthesis , RNA, Messenger/genetics , Receptors, Antigen, T-Cell, alpha-beta/genetics , Saccharomyces cerevisiae Proteins , Trans-Activators/physiology , Adaptor Proteins, Signal Transducing , Base Sequence , Codon, Nonsense , DNA Primers , Fungal Proteins/genetics , Mutagenesis, Site-Directed , Plasmids , Trans-Activators/genetics
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