Biomedical subjects

Terry Magnuson

Publications and source records attributed to Terry Magnuson.

At least 19 recordsLinked to original sources

PubMed2007

A simple enzymatic method for parietal yolk sac removal in early postimplantation mouse embryos.

Crucial aspects of axial development in mice occur at early postimplantation stages from the time of implantation to the appearance of the primitive streak. However, this period of development is notoriously refractory to experimental approaches due to the small size of the conceptus and to the presence of the parietal yolk sac, a protective tripartite membrane that surrounds the developing egg cylinder. Here, we describe a method that combines enzymatic digestion and mechanical manipulation to remove the parietal yolk sac of conceptuses at stages between 5.5 and 6.5 days post coitum. This method, which is compatible with whole-mount in situ hybridization and immunostaining techniques, offers a significant improvement over conventional dissection techniques, and it will greatly facilitate research in early mammalian development.

Animals↗

PubMed2006

Juxtaposed Polycomb complexes co-regulate vertebral identity.

Best known as epigenetic repressors of developmental Hox gene transcription, Polycomb complexes alter chromatin structure by means of post-translational modification of histone tails. Depending on the cellular context, Polycomb complexes of diverse composition and function exhibit cooperative interaction or hierarchical interdependency at target loci. The present study interrogated the genetic, biochemical and molecular interaction of BMI1 and EED, pivotal constituents of heterologous Polycomb complexes, in the regulation of vertebral identity during mouse development. Despite a significant overlap in dosage-sensitive homeotic phenotypes and co-repression of a similar set of Hox genes, genetic analysis implicated eed and Bmi1 in parallel pathways, which converge at the level of Hox gene regulation. Whereas EED and BMI1 formed separate biochemical entities with EzH2 and Ring1B, respectively, in mid-gestation embryos, YY1 engaged in both Polycomb complexes. Strikingly, methylated lysine 27 of histone H3 (H3-K27), a mediator of Polycomb complex recruitment to target genes, stably associated with the EED complex during the maintenance phase of Hox gene repression. Juxtaposed EED and BMI1 complexes, along with YY1 and methylated H3-K27, were detected in upstream regulatory regions of Hoxc8 and Hoxa5. The combined data suggest a model wherein epigenetic and genetic elements cooperatively recruit and retain juxtaposed Polycomb complexes in mammalian Hox gene clusters toward co-regulation of vertebral identity.

Animals↗

PubMed2006

Maternal BRG1 regulates zygotic genome activation in the mouse.

Zygotic genome activation (ZGA) is a nuclear reprogramming event that transforms the genome from transcriptional quiescence at fertilization to robust transcriptional activity shortly thereafter. The ensuing gene expression profile in the cleavage-stage embryo establishes totipotency and is required for further development. Although little is known about the molecular basis of ZGA, oocyte-derived mRNAs and proteins that alter chromatin structure are likely crucial. To test this hypothesis, we generated a maternal-effect mutation of Brg1, which encodes a catalytic subunit of SWI/SNF-related complexes, utilizing Cre-loxP gene targeting. In conditional-mutant females, BRG1-depleted oocytes completed meiosis and were fertilized. However, embryos conceived from BRG1-depleted eggs exhibited a ZGA phenotype including two-cell arrest and reduced transcription for approximately 30% of expressed genes. Genes involved in transcription, RNA processing, and cell cycle regulation were particularly affected. The early embryonic arrest is not a consequence of a defective oocyte because depleting maternal BRG1 after oocyte development is complete by RNA interference (RNAi) also resulted in two-cell arrest. To our knowledge, Brg1 is the first gene required for ZGA in mammals. Depletion of maternal BRG1 did not affect global levels of histone acetylation, whereas dimethyl-H3K4 levels were reduced. These data provide a framework for understanding the mechanism of ZGA.

Acetylation↗

PubMed2006

A DNA insulator prevents repression of a targeted X-linked transgene but not its random or imprinted X inactivation.

Some genes on the inactive X chromosome escape silencing. One possible escape mechanism is that heterochromatization during X inactivation can be blocked by boundary elements. DNA insulators are candidates for blocking because they shield genes from influences of their chromosomal environment. To test whether DNA insulators can act as boundaries on the X chromosome, we inserted into the mouse X-linked Hprt locus a GFP transgene flanked with zero, one, or two copies of a prototypic vertebrate insulator from the chicken beta-globin locus, chicken hypersensitive site 4, which contains CCCTC binding factor binding sites. On the active X chromosome the insulators blocked repression of the transgene, which commences during early development and persists in adults, in a copy number-dependent manner. CpG methylation of the transgene correlated inversely with expression, but the insulators on the active X chromosome were not methylated. On the inactive X chromosome, insulators did not block random or imprinted X inactivation of the transgene, and both the insulator and transgene were almost completely methylated. Thus, the chicken hypersensitive site 4 DNA insulator is sufficient to protect an X-linked gene from repression during development but not from X inactivation.

Animals↗

PubMed2006

The Polycomb group protein EED is dispensable for the initiation of random X-chromosome inactivation.

The Polycomb group (PcG) proteins are thought to silence gene expression by modifying chromatin. The Polycomb repressive complex 2 (PRC2) plays an essential role in mammalian X-chromosome inactivation (XCI), a model system to investigate heritable gene silencing. In the mouse, two different forms of XCI occur. In the preimplantation embryo, all cells undergo imprinted inactivation of the paternal X-chromosome (Xp). During the peri-implantation period, cells destined to give rise to the embryo proper erase the imprint and randomly inactivate either the maternal X-chromosome or the Xp; extraembryonic cells, on the other hand, maintain imprinted XCI of the Xp. PRC2 proteins are enriched on the inactive-X during early stages of both imprinted and random XCI. It is therefore thought that PRC2 contributes to the initiation of XCI. Mouse embryos lacking the essential PRC2 component EED harbor defects in the maintenance of imprinted XCI in differentiating trophoblast cells. Assessment of PRC2 requirement in the initiation of XCI, however, has been hindered by the presence of maternally derived proteins in the early embryo. Here we show that Eed-/- embryos initiate and maintain random XCI despite lacking any functional EED protein prior to the initiation of random XCI. Thus, despite being enriched on the inactive X-chromosome, PcGs appear to be dispensable for the initiation and maintenance of random XCI. These results highlight the lineage- and differentiation state-specific requirements for PcGs in XCI and argue against PcG function in the formation of the facultative heterochromatin of the inactive X-chromosome.

Alleles↗

PubMed2006

A novel mouse Smad4 mutation reduces protein stability and wild-type protein levels.

Smad4 is a key signal transducer of the transforming growth factor-beta (TGF-beta) superfamily of growth factors that are critical regulators of embryonic patterning and adult tissue homeostasis. The biological activity of the TGF-beta signaling is tightly controlled at multiple levels, including the abundance of SMAD4 proteins. We previously recovered a novel allele of Smad4 in a gene-based screen in N-ethyl-N-nitrosourea (ENU)-mutagenized mouse embryonic stem cells. The mutation resulted in an unstable truncated protein that is degraded through proteasomal pathways. In the heterozygous state, this allele acts in a dominant negative fashion to reduce the wild-type protein level as well as signaling output. Biochemical characterization indicated that the truncated protein is able to form a complex with the wild-type protein, thus targeting it for proteasomal degradation as well. Phenotypic analyses of the heterozygous animals provided insight into the threshold requirement of Smad4-dependent signaling in vivo.

Alleles↗

PubMed2006

The Polycomb group protein Eed protects the inactive X-chromosome from differentiation-induced reactivation.

The Polycomb group (PcG) encodes an evolutionarily conserved set of chromatin-modifying proteins that are thought to maintain cellular transcriptional memory by stably silencing gene expression. In mouse embryos that are mutated for the PcG protein Eed, X-chromosome inactivation (XCI) is not stably maintained in extra-embryonic tissues. Eed is a component of a histone-methyltransferase complex that is thought to contribute to stable silencing in undifferentiated cells due to its enrichment on the inactive X-chromosome in cells of the early mouse embryo and in stem cells of the extra-embryonic trophectoderm lineage. Here, we demonstrate that the inactive X-chromosome in Eed(-/-) trophoblast stem cells and in cells of the trophectoderm-derived extra-embryonic ectoderm in Eed(-/-) embryos remain transcriptionally silent, despite lacking the PcG-mediated histone modifications that normally characterize the facultative heterochromatin of the inactive X-chromosome. Whereas undifferentiated Eed(-/-) trophoblast stem cells maintained XCI, reactivation of the inactive X-chromosome occurred when these cells were differentiated. These results indicate that PcG complexes are not necessary to maintain transcriptional silencing of the inactive X-chromosome in undifferentiated stem cells. Instead, PcG proteins seem to propagate cellular memory by preventing transcriptional activation of facultative heterochromatin during differentiation.

Animals↗

PubMed2006

An essential role for SNX1 in lysosomal sorting of protease-activated receptor-1: evidence for retromer-, Hrs-, and Tsg101-independent functions of sorting nexins.

Sorting nexin 1 (SNX1) and SNX2 are the mammalian homologues of the yeast Vps5p retromer component that functions in endosome-to-Golgi trafficking. SNX1 is also implicated in endosome-to-lysosome sorting of cell surface receptors, although its requirement in this process remains to be determined. To assess SNX1 function in endocytic sorting of protease-activated receptor-1 (PAR1), we used siRNA to deplete HeLa cells of endogenous SNX1 protein. PAR1, a G-protein-coupled receptor, is proteolytically activated by thrombin, internalized, sorted predominantly to lysosomes, and efficiently degraded. Strikingly, depletion of endogenous SNX1 by siRNA markedly inhibited agonist-induced PAR1 degradation, whereas expression of a SNX1 siRNA-resistant mutant protein restored agonist-promoted PAR1 degradation in cells lacking endogenous SNX1, indicating that SNX1 is necessary for lysosomal degradation of PAR1. SNX1 is known to interact with components of the mammalian retromer complex and Hrs, an early endosomal membrane-associated protein. However, activated PAR1 degradation was not affected in cells depleted of retromer Vps26/Vps35 subunits, Hrs or Tsg101, an Hrs-interacting protein. We further show that SNX2, which dimerizes with SNX1, is not essential for lysosomal sorting of PAR1, but rather can regulate PAR1 degradation by disrupting endosomal localization of endogenous SNX1 when ectopically expressed. Together, our findings establish an essential role for endogenous SNX1 in sorting activated PAR1 to a distinct lysosomal degradative pathway that is independent of retromer, Hrs, and Tsg101.

Carrier Proteins↗

PubMed2005

A Brg1 mutation that uncouples ATPase activity from chromatin remodeling reveals an essential role for SWI/SNF-related complexes in beta-globin expression and erythroid development.

The Brg1 catalytic subunit of SWI/SNF-related complexes has been implicated in many developmental and physiological processes, but null homozygotes die as blastocysts prior to implantation. To circumvent this early embryonic lethality, we performed an ENU mutagenesis screen and generated a Brg1 hypomorph mutation in the ATPase domain. The mutant Brg1 protein is stable, assembles into SWI/SNF-related complexes, and exhibits normal ATPase activity but is unable to establish DNase I hypersensitivity sites characteristic of open chromatin. Mutant embryos develop normally until midgestation but then exhibit a distinct block in the development of the erythroid lineage, leading to anemia and death. The mutant Brg1 protein is recruited to the beta-globin locus, but chromatin remodeling and transcription are perturbed. Histone acetylation and DNA methylation are also affected. To our knowledge, Brg1 is the first chromatin-modifying factor shown to be required for beta-globin regulation and erythropoiesis in vivo. Not only does this mutation establish a role for Brg1 during organogenesis, it also demonstrates that ATPase activity can be uncoupled from chromatin remodeling.

Acetylation↗

PubMed2005

Primitive streak formation in mice is preceded by localized activation of Brachyury and Wnt3.

The prevalent model for the generation of axial polarity in mouse embryos proposes that a radial to a linear transition in the expression of primitive streak markers precedes the formation of the primitive streak on one side of the epiblast. This model contrasts with the models of mesoderm formation in other vertebrates as it suggests that the primitive streak is initially established in a radial pattern rather than a localized region of the epiblast. Here, we examine the proposed correlation between the expression of Brachyury and Wnt3, two genes reported as expressed radially in the proximal epiblast, with the movements of proximal anterior epiblast cells at stages leading to the formation of the primitive streak. Our results reveal that neither Brachyury nor Wnt3 forms a ring of expression in the proximal epiblast as previously thought. In embryos dissected between 5.5 and 6.5 dpc, Brachyury is first expressed in the distal extra-embryonic ectoderm and subsequently on one side of the epiblast. Wnt3 expression is evident first in the posterior visceral endoderm of 5.5 dpc embryos and later in the posterior epiblast. Lineage analysis shows that the movements of the proximal epiblast do not restrict Brachyury expression to the posterior epiblast. Our data suggest a model whereby the localized expression of these genes in the posterior epiblast, and hence the formation of the primitive streak, is the result of local cell-cell interactions in the future posterior portion of the egg cylinder rather than regionalization of a radial pattern of expression in proximal epiblast cells.

Animals↗

PubMed2005

Genetic evidence for a mammalian retromer complex containing sorting nexins 1 and 2.

We have previously shown that the putative mammalian retromer components sorting nexins 1 and 2 (Snx1 and Snx2) result in embryonic lethality when simultaneously targeted for deletion in mice, whereas others have shown that Hbeta58 (also known as mVps26), another retromer component, results in similar lethality when targeted for deletion. In the current study, we address the genetic interaction of these mammalian retromer components in mice. Our findings reveal a functional interaction between Hbeta58, SNX1, and SNX2 and strongly suggest that SNX2 plays a more critical role than SNX1 in retromer activity during embryonic development. This genetic evidence supports the existence of mammalian retromer complexes containing SNX1 and SNX2 and identifies SNX2 as an important mediator of retromer biology. Moreover, we find that mammalian retromer complexes containing SNX1 and SNX2 have an essential role in embryonic development that is independent of cation-independent mannose 6-phosphate receptor trafficking.

Animals↗

PubMed2005

The murine polycomb group protein Eed is required for global histone H3 lysine-27 methylation.

PcG proteins mediate heritable transcriptional silencing by generating and recognizing covalent histone modifications. One conserved PcG complex, PRC2, is composed of several proteins including the histone methyltransferase (HMTase) Ezh2, the WD-repeat protein Eed, and the Zn-finger protein Suz12. Ezh2 methylates histone H3 on lysine 27 (H3K27), which serves as an epigenetic mark mediating silencing. H3K27 can be mono-, di-, or trimethylated (1mH3K27, 2mH3K27, and 3mH3K27, respectively). Hence, either PRC2 must be regulated so as to add one methyl group to certain nucleosomes but two or three to others, or distinct complexes must be responsible for 1m-, 2m-, and 3mH3K27. Consistent with the latter possibility, 2mH3K27 and 3mH3K27, but not 1mH3K27, are absent in Suz12-/- embryos, which lack both Suz12 and Ezh2 protein. Mammalian proteins required for 1mH3K27 have not been identified. Here, we demonstrate that unlike Suz12 and Ezh2, Eed is required not only for 2m- and 3mH3K27 but also global 1mH3K27. These results provide a functionally important distinction between PRC2 complex components and implicate Eed in PRC2-independent histone methylation.

Animals↗

PubMed2005

Ablation of MEKK4 kinase activity causes neurulation and skeletal patterning defects in the mouse embryo.

Skeletal disorders and neural tube closure defects represent clinically significant human malformations. The signaling networks regulating normal skeletal patterning and neurulation are largely unknown. Targeted mutation of the active site lysine of MEK kinase 4 (MEKK4) produces a kinase-inactive MEKK4 protein (MEKK4(K1361R)). Embryos homozygous for this mutation die at birth as a result of skeletal malformations and neural tube defects. Hindbrains of exencephalic MEKK4(K1361R) embryos show a striking increase in neuroepithelial cell apoptosis and a dramatic loss of phosphorylation of MKK3 and -6, mitogen-activated protein kinase kinases (MKKs) regulated by MEKK4 in the p38 pathway. Phosphorylation of MAPK-activated protein kinase 2, a p38 substrate, is also inhibited, demonstrating a loss of p38 activity in MEKK4(K1361R) embryos. In contrast, the MEK1/2-extracellular signal-regulated kinase 1 (ERK1)/ERK2 and MKK4-Jun N-terminal protein kinase pathways were unaffected. The p38 pathway has been shown to regulate the phosphorylation and expression of the small heat shock protein HSP27. Compared to the wild type, MEKK4(K1361R) fibroblasts showed significantly reduced phosphorylation of p38 and HSP27, with a corresponding heat shock-induced instability of the actin cytoskeleton. Together, these data demonstrate MEKK4 regulation of p38 and that substrates downstream of p38 control cellular homeostasis. The findings are the first demonstration that MEKK4-regulated p38 activity is critical for neurulation.

Animals↗

PubMed2004

The knockout mouse project.

Mouse knockout technology provides a powerful means of elucidating gene function in vivo, and a publicly available genome-wide collection of mouse knockouts would be significantly enabling for biomedical discovery. To date, published knockouts exist for only about 10% of mouse genes. Furthermore, many of these are limited in utility because they have not been made or phenotyped in standardized ways, and many are not freely available to researchers. It is time to harness new technologies and efficiencies of production to mount a high-throughput international effort to produce and phenotype knockouts for all mouse genes, and place these resources into the public domain.

Alleles↗

PubMed2004

Genetic and haplotype diversity among wild-derived mouse inbred strains.

With the completion of the mouse genome sequence, it is possible to define the amount, type, and organization of the genetic variation in this species. Recent reports have provided an overview of the structure of genetic variation among classical laboratory mice. On the other hand, little is known about the structure of genetic variation among wild-derived strains with the exception of the presence of higher levels of diversity. We have estimated the sequence diversity due to substitutions and insertions/deletions among 20 inbred strains of Mus musculus, chosen to enable interpretation of the molecular variation within a clear evolutionary framework. Here, we show that the level of sequence diversity present among these strains is one to two orders of magnitude higher than the level of sequence diversity observed in the human population, and only a minor fraction of the sequence differences observed is found among classical laboratory strains. Our analyses also demonstrate that deletions are significantly more frequent than insertions. We estimate that 50% of the total variation identified in M. musculus may be recovered in intrasubspecific crosses. Alleles at variants positions can be classified into 164 strain distribution patterns, a number exceeding those reported and predicted in panels of classical inbred strains. The number of strains, the analysis of multiple loci scattered across the genome, and the mosaic nature of the genome in hybrid and classical strains contribute to the observed diversity of strain distribution patterns. However, phylogenetic analyses demonstrate that ancient polymorphisms that segregate across species and subspecies play a major role in the generation of strain distribution patterns.

Animals↗

PubMed2003

The role of Brg1, a catalytic subunit of mammalian chromatin-remodeling complexes, in T cell development.

Mammalian SWI-SNF-related complexes use brahma-related gene 1 (Brg1) as a catalytic subunit to remodel nucleosomes and regulate transcription. Recent biochemical data has linked Brg1 function to genes important for T lymphocyte differentiation. To investigate the role of SWI-SNF-related complexes in this lineage, we ablated Brg1 function in T lymphocytes. T cell-specific Brg1-deficient mice showed profound thymic abnormalities, CD4 derepression at the double negative (DN; CD4- CD8-) stage, and a developmental block at the DN to double positive (CD4+ CD8+) transition. 5'-bromo-2'-deoxyuridine incorporation and annexin V staining establish a role for Brg1 complexes in the regulation of thymocyte cell proliferation and survival. This Brg1-dependent cell survival is specific for developing thymocytes as indicated by the presence of Brg1-deficient mature T lymphocytes that have escaped the developmental block in the thymus. However, reductions in peripheral T cell populations lead to immunodeficiency and compromised health of mutant mice. These results highlight the importance of chromatin-remodeling complexes at different stages in the development of a mammalian cell lineage.

Animals↗

PubMed2003

Dynamic morphogenetic events characterize the mouse visceral endoderm.

Several lines of evidence suggest that the extraembryonic endoderm of vertebrate embryos plays an important role in the development of rostral neural structures. In mice, neural inductive signals are thought to reside in an area of visceral endoderm that expresses the Hex gene. Here, we have conducted a morphological and lineage analysis of visceral endoderm cells spanning pre- and postprimitive streak stages. Our results show that Hex-expressing cells have a tall, columnar epithelial morphology, which distinguishes them from other visceral endoderm cells. This region of visceral endoderm thickening (VET) is found overlying first the distal and then one side of the epiblast at stages between 5.5 and 5.75 days post coitum (d.p.c.). In addition, we show that the epiblast has an anteroposterior-compressed appearance that is aligned with the position of the VET. Intracellular labeling of VET/Hex-expressing cells reveals an anterior and anterolateral shift from their distal epiblast position. VET/Hex-expressing cells are first localized to the anterior side of the epiblast by 5.75 d.p.c. and form a crescent on the anterior half of the embryo at the onset of gastrulation. Subsequently, VET descendants are distributed along the embryonic/extraembryonic boundary by headfold stages at 7.5 d.p.c. The morphological characteristics and position of VET/Hex-expressing cells distinguishes the future anteroposterior axis of the embryo and provide landmarks to stage mouse embryos at preprimitive streak stages. Moreover, the morphological characteristics of pregastrulation mouse embryos together with the stereotyped shift in the position of visceral endoderm cells reveal similarities among amniote embryos that suggest an evolutionary conservation of the mechanisms that pattern the rostral neurectoderm at pregastrula stages.

Animals↗

PubMed2003

Genome imprinting regulated by the mouse Polycomb group protein Eed.

Epigenetic regulation is essential for temporal, tissue-specific and parent-of-origin-dependent gene expression. It has recently been found that the mouse Polycomb group (PcG) gene Eed (embryonic ectoderm development) acts to maintain repression of the imprinted X chromosome. Here, we investigated whether Eed is also required for regulation of autosomal imprinted loci. Expression analyses showed that transcripts from the silent alleles of a subset of paternally repressed genes were present in Eed(-/-) embryos. Parent-of-origin methylation was preserved in these embryos, but we observed changes in the methylation status of specific CpGs in differentially methylated regions (DMRs) at affected but not at unaffected loci. These data identify Eed as a member of a new class of trans-acting factors that regulate parent-of-origin expression at imprinted loci.

Alleles↗