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Kristina Vintersten

Publications and source records attributed to Kristina Vintersten.

14 recordsLinked to original sources
PubMed2006

Transgenic mouse proteomics identifies new 14-3-3-associated proteins involved in cytoskeletal rearrangements and cell signaling.

Identification of protein-protein interactions is crucial for unraveling cellular processes and biochemical mechanisms of signal transduction. Here we describe, for the first time, the application of the tandem affinity purification (TAP) and LC-MS method to the characterization of protein complexes from transgenic mice. The TAP strategy developed in transgenic mice allows the emplacement of complexes in their physiological environment in contact with proteins that might only be specifically expressed in certain tissues while simultaneously ensuring the right stoichiometry of the TAP protein versus their binding partners and represents a novelty in proteomics approaches used so far. Mouse lines expressing TAP-tagged 14-3-3zeta protein were generated, and protein interactions were determined. 14-3-3 proteins are general regulators of cell signaling and represent up to 1% of the total brain protein. This study allowed the identification of almost 40 novel 14-3-3zeta-binding proteins. Biochemical and functional characterization of some of these interactions revealed new mechanisms of action of 14-3-3zeta in several signaling pathways, such as glutamate receptor signaling via binding to homer homolog 3 (Homer 3) and in cytoskeletal rearrangements and spine morphogenesis by binding and regulating the activity of the signaling complex formed by G protein-coupled receptor kinase-interactor 1 (GIT1) and p21-activated kinase-interacting exchange factor beta (betaPIX).

14-3-3 Proteins↗
PubMed2006

Multiple epigenetic maintenance factors implicated by the loss of Mll2 in mouse development.

Epigenesis is the process whereby the daughters of a dividing cell retain a chromatin state determined before cell division. The best-studied cases involve the inheritance of heterochromatic chromosomal domains, and little is known about specific gene regulation by epigenetic mechanisms. Recent evidence shows that epigenesis pivots on methylation of nucleosomes at histone 3 lysines 4, 9 or 27. Bioinformatics indicates that mammals have several enzymes for each of these methylations, including at least six histone 3 lysine 4 methyltransferases. To look for evidence of gene-specific epigenetic regulation in mammalian development, we examined one of these six, Mll2, using a multipurpose allele in the mouse to ascertain the loss-of-function phenotype. Loss of Mll2 slowed growth, increased apoptosis and retarded development, leading to embryonic failure before E11.5. Using chimera experiments, we demonstrated that Mll2 is cell-autonomously required. Evidence for gene-specific regulation was also observed. Although Mox1 and Hoxb1 expression patterns were correctly established, they were not maintained in the absence of Mll2, whereas Wnt1 and Otx2 were. The Mll2 loss-of-function phenotype is different from that of its sister gene Mll, and they regulate different Hox complex genes during ES cell differentiation. Therefore, these two closely related epigenetic factors play different roles in development and maintain distinct gene expression patterns. This suggests that other epigenetic factors also regulate particular patterns and that development entails networks of epigenetic specificities.

Alleles↗
PubMed2006

Murine embryonic stem cells.

Embryonic stem (ES) cells are derived from preimplantation stage mouse embryos at the time when they have reached the blastocyst stage. It is at this point that the first steps of differentiation take place during mammalian embryonic development. The individual blastomeres now start to organize themselves into three distinct locations, each encompassing a different cell type: outside epithelial cells, trophectoderm; cells at the blastocele surface of the inner cell mass (ICM), the primitive endoderm; and inside cells of the ICM, the primitive ectoderm. ES cells originate from the third population, the primitive ectoderm, which is a transiently existing group of cells in the embryo. Primitive ectoderm cells diminish within a day as the embryo is entering into the next steps of differentiation. ES cells, however, while retaining the property of their origin in terms of developmental potential, also have the ability to self-renew. It is hence important to realize that ES cells do not exist in vivo; they should be regarded simply as tissue culture artifact. Nevertheless, these powerful cells have the potential to differentiate into all the cells of the embryo proper and postnatal animal. Furthermore, they retain the limitation of their origin through their inability to contribute to the trophectoderm lineage (the trophoblast of the placenta) and the lineages of the primitive endoderm, the visceral and parietal endoderm. Due to these unique features, we must admit that even if we regard ES cells as products of in vitro culture and should not compare them to true somatic stem cells found in the adult organism, they certainly offer us a fantastic tool for genetic, developmental, and disease studies.

Animals↗
PubMed2004

Gremlin-mediated BMP antagonism induces the epithelial-mesenchymal feedback signaling controlling metanephric kidney and limb organogenesis.

Epithelial-mesenchymal feedback signaling is the key to diverse organogenetic processes such as limb bud development and branching morphogenesis in kidney and lung rudiments. This study establishes that the BMP antagonist gremlin (Grem1) is essential to initiate these epithelial-mesenchymal signaling interactions during limb and metanephric kidney organogenesis. A Grem1 null mutation in the mouse generated by gene targeting causes neonatal lethality because of the lack of kidneys and lung septation defects. In early limb buds, mesenchymal Grem1 is required to establish a functional apical ectodermal ridge and the epithelial-mesenchymal feedback signaling that propagates the sonic hedgehog morphogen. Furthermore, Grem1-mediated BMP antagonism is essential to induce metanephric kidney development as initiation of ureter growth, branching and establishment of RET/GDNF feedback signaling are disrupted in Grem1-deficient embryos. As a consequence, the metanephric mesenchyme is eliminated by apoptosis, in the same way as the core mesenchymal cells of the limb bud.

Alleles↗
PubMed2004

Mouse limb deformity mutations disrupt a global control region within the large regulatory landscape required for Gremlin expression.

The mouse limb deformity (ld) mutations cause limb malformations by disrupting epithelial-mesenchymal signaling between the polarizing region and the apical ectodermal ridge. Formin was proposed as the relevant gene because three of the five ld alleles disrupt its C-terminal domain. In contrast, our studies establish that the two other ld alleles directly disrupt the neighboring Gremlin gene, corroborating the requirement of this BMP antagonist for limb morphogenesis. Further doubts concerning an involvement of Formin in the ld limb phenotype are cast, as a targeted mutation removing the C-terminal Formin domain by frame shift does not affect embryogenesis. In contrast, the deletion of the corresponding genomic region reproduces the ld limb phenotype and is allelic to mutations in Gremlin. We resolve these conflicting results by identifying a cis-regulatory region within the deletion that is required for Gremlin activation in the limb bud mesenchyme. This distant cis-regulatory region within Formin is also altered by three of the ld mutations. Therefore, the ld limb bud patterning defects are not caused by disruption of Formin, but by alteration of a global control region (GCR) required for Gremlin transcription. Our studies reveal the large genomic landscape harboring this GCR, which is required for tissue-specific coexpression of two structurally and functionally unrelated genes.

Amino Acid Sequence↗
PubMed2004

Elk-1 knock-out mice engineered by Flp recombinase-mediated cassette exchange.

Elk-1 is a member of the TCF subfamily of Ets proteins. TCFs interact with SRF at serum response elements (SREs) of immediate early genes (IEGs), such as c-fos and Egr-1, thereby mediating IEG induction upon extracellular stimulation. We previously generated an Elk-1 null allele (Elk1-137) in murine embryonic stem (ES) cells by homologous recombination. In Elk1-137, the Elk-1 gene was replaced by a Hygromycin B phosphotransferase - Thymidine Kinase (HygTk) fusion gene, flanked by two nonidentical Flp recombinase recognition (FRT) sites (Cesari et al., [2004] Mol Cell Biol, in press) to allow for the subsequent generation of alternative alleles of interest by recombinase-mediated cassette exchange (RMCE). Elk1-deficient mice derived from Elk-1((137/0)) ES cells are viable and do not reveal strong phenotypical abnormalities, apart from male sterility. However, the Elk-1 locus contains the Tk cassette, which has previously been related to this defect. Therefore, in our first experiment involving the technique of Flp RMCE we chose to remove the HygTk cassette in Elk-1((137/0)) ES cells and to generate Elk-1((RMCE16/0)) and Elk-1((RMCE16/RMCE16)) mice. In so doing, we provide evidence that the sterility of Elk1((137/0)) mice was not due to the absence of Elk-1 but rather the presence of HygTk. This is the first report of mice derived from ES cells which were subjected to Flp RMCE and thus proves that RMCE is a powerful tool for the genetic engineering of previously tagged loci in the mouse genome.

Animals↗
PubMed2004

Mouse in red: red fluorescent protein expression in mouse ES cells, embryos, and adult animals.

Spectral variants of green fluorescent protein are widely used in live samples for a broad range of applications: from visualization of protein interactions, through following gene expression, to marking particular cells in complex tissues. Higher wavelength emissions (such as red) are preferred due to the lower background-autofluorescence in tissues (Miyawaka et al., Nat Cell Biol Suppl S1-7, 2003). Until now, however, red fluorescent proteins (RFP) have displayed toxicity in murine embryos, which has hampered its application in this model. Here we report strong expression of a recently developed RFP variant, DsRed.T3, in mouse ES cells, embryos, and adult mice. Our results show that the red fluorescent wavelength has a superior tissue penetrance compared with spectral variants of lower wavelength. Furthermore, we have generated an ES cell line and a corresponding transgenic mouse line in which red fluorescence is activated upon Cre excision. Finally, we introduced cell type-specifically expressed Cre transgenes into this Cre recombinase reporter cell line, and by using the tetraploid embryo complementation assay, we could directly verify the Cre recombinase specificity on ES cell-derived embryos/animals.

Animals↗
PubMed2004

Mice deficient for the ets transcription factor elk-1 show normal immune responses and mildly impaired neuronal gene activation.

The transcription factor Elk-1 belongs to the ternary complex factor (TCF) subfamily of Ets proteins. TCFs interact with serum response factor to bind jointly to serum response elements in the promoters of immediate-early genes (IEGs). TCFs mediate the rapid transcriptional response of IEGs to various extracellular stimuli which activate mitogen-activated protein kinase signaling. To investigate physiological functions of Elk-1 in vivo, we generated Elk-1-deficient mice by homologous recombination in embryonic stem cells. These animals were found to be phenotypically indistinguishable from their wild-type littermates. Histological analysis of various tissues failed to reveal any differences between Elk-1 mutant and wild-type mice. Elk-1 deficiency caused no changes in the proteomic displays of brain or spleen extracts. Also, no immunological defects could be detected in mice lacking Elk-1, even upon infection with coxsackievirus B3. In mouse embryonic fibroblasts, Elk-1 was dispensable for c-fos and Egr-1 transcriptional activation upon stimulation with serum, lysophosphatidic acid, or tetradecanoyl phorbol acetate. However, in brains of Elk-1-deficient mice, cortical and hippocampal CA1 expression of c-fos, but not Egr-1 or c-Jun, was markedly reduced 4 h following kainate-induced seizures. This was not accompanied by altered patterns of neuronal apoptosis. Collectively, our data indicate that Elk-1 is essential neither for mouse development nor for adult life, suggesting compensatory activities by other TCFs.

Animals↗
PubMed2003

Engineering the mouse genome with bacterial artificial chromosomes to create multipurpose alleles.

The mouse is the leading vertebrate model because its genome can be altered by both random transgenesis and homologous recombination with targeting constructs. Both approaches have been hindered by the size and site limitations implicit in conventional Escherichia coli DNA-engineering methods. Homologous recombination in E. coli, or 'recombineering', has overcome these limitations for bacterial artificial chromosome (BAC) transgenesis. Here we applied Red/ET recombineering (using the lambda Redalpha/Redbeta recombinase pair) to generate a 64 kilobase targeting construct that carried two selectable cassettes permitting the simultaneous mutation of the target gene, Mll, at sites 43 kb apart in one round of mouse embryonic stem (ES) cell targeting. The targeting frequency after dual selection was 6%. Because the two selectable cassettes were flanked by FRT or loxP sites, three more alleles can be generated by site-specific recombination. Our approach represents a simple way to introduce changes at two or more sites in a genetic locus, and thereafter generate allele combinations. The size of BAC templates offers new freedom for the design of targeting constructs. Combined with the use of two selectable cassettes placed far apart, BAC-based targeting constructs may be applicable to tasks such as regional exchanges, deletions, and insertions.

Alleles↗
PubMed2003

Negative feedback regulation of MKK6 mRNA stability by p38alpha mitogen-activated protein kinase.

p38 mitogen-activated protein (MAP) kinases play an important role in the regulation of cellular responses to all kinds of stresses. The most abundant and broadly expressed p38 MAP kinase is p38alpha, which can also control the proliferation, differentiation, and survival of several cell types. Here we show that the absence of p38alpha correlates with the up-regulation of one of its upstream activators, the MAP kinase kinase MKK6, in p38alpha(-/-) knockout mice and in cultured cells derived from them. In contrast, the expression levels of the p38 activators MKK3 and MKK4 are not affected in p38alpha-deficient cells. The increase in MKK6 protein concentration correlates with increased amounts of MKK6 mRNA in the p38alpha(-/-) cells. Pharmacological inhibition of p38alpha also up-regulates MKK6 mRNA levels in HEK293 cells. Conversely, reintroduction of p38alpha into p38alpha(-/-) cells reduces the levels of MKK6 protein and mRNA to the normal levels found in wild-type cells. Moreover, we show that the MKK6 mRNA is more stable in p38alpha(-/-) cells and that the 3'untranslated region of this mRNA can differentially regulate the stability of the lacZ reporter gene in a p38alpha-dependent manner. Our data indicate that p38alpha can negatively regulate the stability of the MKK6 mRNA and thus control the steady-state concentration of one of its upstream activators.

3' Untranslated Regions↗