Sequence interpretation. Functional annotation of mouse genome sequences.
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Animals↗
Biomedical subjects
T Magnuson
Publications and source records attributed to T Magnuson.
Cell and tissue requirements for the gene eed during mouse gastrulation and organogenesis.
Mouse embryos homozygous for the allele eed(l7Rn5-3354SB) of the Polycomb Group gene embryonic ectoderm development (eed) display a gastrulation defect in which epiblast cells move through the streak and form extraembryonic mesoderm derivatives at the expense of development of the embryo proper. Here we demonstrate that homozygous mutant ES cells have the capacity to differentiate embryonic cell types both in vitro as embryoid bodies and in vivo as chimeric embryos. In chimeric embryos, eed mutant cells can respond to wild-type signals and participate in normal gastrulation movements. These results indicate a non-cell-autonomous function for eed. Evidence of mutant cell exclusion from the forebrain and segregation within somites, however, suggests that eed has cell-autonomous roles in aspects of organogenesis. A requirement for eed in the epiblast during embryonic development is supported by the fact that high-contribution chimeras could not be rescued by a wild-type extraembryonic environment.
Alleles↗
Imprinted X inactivation maintained by a mouse Polycomb group gene.
In mammals, dosage compensation of X-linked genes is achieved by the transcriptional silencing of one X chromosome in the female (reviewed in ref. 1). This process, called X inactivation, is usually random in the embryo proper. In marsupials and the extra-embryonic region of the mouse, however, X inactivation is imprinted: the paternal X chromosome is preferentially inactivated whereas the maternal X is always active. Having more than one active X chromosome is deleterious to extra-embryonic development in the mouse. Here we show that the gene eed (embryonic ectoderm development), a member of the mouse Polycomb group (Pc-G) of genes, is required for primary and secondary trophoblast giant cell development in female embryos. Results from mice carrying a paternally inherited X-linked green fluorescent protein (GFP) transgene implicate eed in the stable maintenance of imprinted X inactivation in extra-embryonic tissues. Based on the recent finding that the Eed protein interacts with histone deacetylases, we suggest that this maintenance activity involves hypoacetylation of the inactivated paternal X chromosome in the extra-embryonic tissues.
Acetylation↗
Mouse homolog of the Drosophila Pc-G gene esc exerts a dominant negative effect in Drosophila.
The Polycomb group genes are involved in maintaining long term transcriptional repression of the homeotic genes in both Drosophila and mammals. The mouse eed locus encodes the highly conserved ortholog of the Drosophila ESC protein. To test the functional conservation between the two genes, eed was introduced into the fly to determine whether it could rescue the esc mutant phenotype. eed exerted a dominant negative effect on the leg transformation phenotype associated with the esc mutation. This result is interpreted in light of in vitro protein-protein binding data and in vivo polytene chromosome staining indicating the lack of significant interaction between Eed and fly E(Z), a molecular partner of ESC. genesis 26:67-76, 2000
Animals↗
Pc-G/trx-G and the SWI/SNF connection: developmental gene regulation through chromatin remodeling.
Pc-G and trx-G genes are responsible for maintenance of transcriptional regulation and provide a cellular memory mechanism throughout development. Studies in fly, yeast, mouse, and human have implicated modulation of higher-order chromatin structure as an important component in this process. Specifically, connections between SWI/SNF complexes and trx-G genes have provided a mechanistic link between chromatin remodeling and transcriptional regulation. Here we discuss recent genetic and biochemical data that has shed light on the molecular mechanisms and pathways associated with Pc-G and trx-G function in developmental processes such as cell cycle control and hematopoiesis. genesis 26:189-197, 2000.
Animals↗
Genetic analysis of the exed region in mouse.
The extraembryonic ectoderm development (exed) mutant phenotype was described in mice homozygous for the c(6H) deletion, a radiation-induced deletion in the tyrosinase region of mouse Chromosome 7. These mutants fail to gastrulate and die around embryonic day 8.0. Several genes including, for example, embryonic ectoderm development (eed), are deleted in the c(6H) mutants; however, the portion of the chromosome responsible for the more severe exed phenotype is localized to a 20-kb region called the "exed-critical region." To understand the genetics behind the exed phenotype, we analyzed this region in two ways. First, to determine whether the 20-kb exed-critical region alone causes the mutant phenotype, we removed it from a wild-type chromosome. The resulting mice homozygous for this deletion were viable and fertile, indicating that the 20-kb exed-critical region by itself is not sufficient to cause the phenotype when deleted. We then sequenced the 20-kb exed-critical region and no expressed exons were found. Several short matches to GenBank Expressed Sequence Tag (EST) databases were identified; however, none of these ESTs mapped to the region. Taken together, these results indicate that the exed phenotype may either be a position effect on a distal gene caused by the c(6H) breakpoint or the result of composite effects of nullizygosity of multiple genes in the deletion homozygotes.
Animals↗
Molecular and genetic analysis of the mouse homolog of the Drosophila suppressor of position-effect variegation 3-9 gene.
The Drosophila melanogaster gene suppressor of position-effect variegation 3-9 [Su(var)3-9] encodes a component of heterochromatin with a chromodomain and a SET domain. Here, we describe the cloning of a mouse homolog called Suv39hl and describe the genomic organization, pattern of expression, and genetic map position. The genomic locus is approximately 10 kb and consists of five exons. The first two exons, 1a and 1b, are alternative first exons and are followed by three common exons. Two mRNAs, encompassing exon 1a or 1b, encode protein isoforms with distinct amino termini, but which are otherwise identical, including the chromodomain and SET domain. Interestingly, only one of the isoforms contains a putative nuclear localization signal. Consistent with other genes encoding proteins associated with chromatin structure, Suv39hl is expressed in a widespread manner. Interspecific backcross mapping localized Suv39hl near tattered (Td) and scurfy (sf) on the proximal X Chromosome (Chr). However, analysis of Td/Y and sf/Y mutant stocks indicated that Suv39hl is not responsible for either mutant phenotype.
Animals↗
Toward the yeastification of mouse genetics: chemical mutagenesis of embryonic stem cells.
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Animals↗
A Brg1 null mutation in the mouse reveals functional differences among mammalian SWI/SNF complexes.
Mammalian SWI/SNF complexes utilize either brahma (Brm) or brahma-related gene 1 (Brg1) catalytic subunits to remodel nucleosomes in an ATP-dependent manner. Brm was previously shown to be dispensable, suggesting that Brm and Brg1 are functionally redundant. To test this hypothesis, we have generated a Brg1 null mutation by gene targeting, and, surprisingly, homozygotes die during the periimplantation stage. Furthermore, blastocyst outgrowth studies indicate that neither the inner cell mass nor trophectoderm survives. However, experiments with other cell types demonstrate that Brg1 is not a general cell survival factor. In addition, Brg1 heterozygotes are predisposed to exencephaly and tumors. These results provide evidence that biochemically similar chromatin-remodeling complexes have dramatically different functions during mammalian development.
Animals↗
Cross-talk between epidermal growth factor receptor and protein kinase C during calcium-induced differentiation of keratinocytes.
The induction of epidermal differentiation by extracellular Ca2+ involves activation of both tyrosine kinase and protein kinase C (PKC) signaling cascades. To determine if the differentiation-dependent activation of tyrosine kinase signaling can influence the PKC pathway, we examined the tyrosine phosphorylation status of PKC isoforms in primary mouse keratinocytes stimulated to terminally differentiate with Ca2+. Elevation of extracellular Ca2+ induced tyrosine phosphorylation of PKC-delta, but not the other keratinocyte PKC isoforms (alpha, epsilon, eta, zeta). We have previously demonstrated that activation of the epidermal growth factor receptor (EGFR) pathway induces PKC-delta tyrosine phosphorylation in basal keratinocytes (Denning M F, Dlugosz A A, Threadgill D W, Magnuson T, Yuspa S H (1996) J Biol Chem 271: 5325-5331). When basal keratinocytes were stimulated to differentiate by Ca2+, the level of cell-associated transforming growth factor-alpha (TGF-alpha) increased 30-fold, while no increase in secreted TGF-alpha was detected. Furthermore, Ca2+-induced tyrosine phosphorylation of PKC-delta and phosphotyrosine-association of the receptor adapter protein Shc was diminished in EGFR -/- keratinocytes, suggesting that EGFR activation may occur during keratinocyte differentiation. Tyrosine phosphorylated PKC-delta was also detected in mouse epidermis, suggesting that this differentiation-associated signaling pathway is physiological. These results establish a requirement for the EGFR in Ca2+-induced tyrosine phosphorylation of PKC-delta, and document the production of cell-associated TGF-alpha in differentiated keratinocytes which may function independent of its usual mitogenic effects.
Animals↗
Mice mutant for Egfr and Shp2 have defective cardiac semilunar valvulogenesis.
Atrioventricular and semilunar valve abnormalities are common birth defects, but how cardiac valvulogenesis is directed remains largely unknown. During studies of genetic interaction between Egfr, encoding the epidermal growth factor receptor, and Ptpn11, encoding the protein-tyrosine-phosphatase Shp2, we discovered that Egfr is required for semilunar, but not atrioventricular, valve development. Although unnoticed in earlier studies, mice homozygous for the hypomorphic Egfr allele waved-2 (Egfrwa2/wa2) exhibit semilunar valve enlargement resulting from over-abundant mesenchymal cells. Egfr-/- mice (CD1 background) have similar defects. The penetrance and severity of the defects in Egfrwa2/wa2 mice are enhanced by heterozygosity for a targeted mutation of exon 2 of Ptpn11 (ref. 3). Compound (Egfrwa2/wa2:Ptpn11+/-) mutant mice also show premature lethality. Electrocardiography, echocardiography and haemodynamic analyses showed that affected mice develop aortic stenosis and regurgitation. Our results identify the Egfr and Shp2 as components of a growth-factor signalling pathway required specifically for semilunar valvulogenesis, support the hypothesis that Shp2 is required for Egfr signalling in vivo, and provide an animal model for aortic valve disease.
Abnormalities, Multiple↗
Genotype-based screen for ENU-induced mutations in mouse embryonic stem cells.
The ability to generate mutations is a prerequisite to functional genetic analysis. Despite a long history of using mice as a model system for genetic analysis, the scientific community has not generated a comprehensive collection of multiple alleles for most mouse genes. The chemical mutagen of choice for mouse has been N-ethyl-N-nitrosourea (ENU), an alkylating agent that mainly causes base substitutions in DNA, and therefore allows for recovery of complete and partial loss-, as well as gain-, of-function alleles . Specific locus tests designed to detect recessive mutations showed that ENU is the most efficient mutagen in mouse with an approximate mutation rate of 1 in 1,000 gametes. In fact, several genome-wide and region-specific screens based on phenotypes have been carried out. The anticipation of the completion of the human and mouse genome projects, however, now emphasizes genotype-driven genetics--from sequence to mutants. To take advantage of the mutagenicity of ENU and its ability to create allelic series of mutations, we have developed a complementary approach to generating mutations using mouse embryonic stem (ES) cells. We show that a high mutation frequency can be achieved and that modulating DNA-repair activities can enhance this frequency. The treated cells retain germline competency, thereby rendering this approach applicable for efficient generation of an allelic series of mutations pivotal to a fine-tuned dissection of biological pathways.
Amino Acid Substitution↗
Linguistic evaluation of Profet II: a pilot project.
Profet, a word prediction program, was designed to accelerate the writing process and to minimize the writing effort of persons with motor dysfunction. It has also proved to be beneficial in text construction for persons with linguistic impairment such as dyslexia. With increasing linguistic demands on support for individuals with severe reading and writing difficulties/dyslexia, the need for an improved version of Profet arose. Thus, Profet II was designed. In this study, a procedure for evaluating Profet II has been developed. Results from a single-case evaluation study with a person with dyslexia are presented. The possible implications for support and aspects such as spelling, morphology and subjective judgements of and attitudes towards texts are discussed.
Adult↗
Functional antagonism of the Polycomb-Group genes eed and Bmi1 in hemopoietic cell proliferation.
The murine Polycomb-Group (PcG) proteins Eed and Bmi1 govern axial patterning during embryonic development by segment-specific repression of Hox gene expression. The two proteins engage in distinct multimeric complexes that are thought to use a common molecular mechanism to render the regulatory regions of Hox and other downstream target genes inaccessible to transcriptional activators. Beyond axial patterning, Bmi1 is also involved in hemopoiesis because a loss-of-function allele causes a profound decrease in bone marrow progenitor cells. Here, evidence is presented that is consistent with an antagonistic function of eed and Bmi1 in hemopoietic cell proliferation. Heterozygosity for an eed null allele causes marked myelo- and lymphoproliferative defects, indicating that eed is involved in the negative regulation of the pool size of lymphoid and myeloid progenitor cells. This antiproliferative function of eed does not appear to be mediated by Hox genes or the tumor suppressor locus p16(INK4a)/p19(ARF) because expression of these genes was not altered in eed mutants. Intercross experiments between eed and Bmi1 mutant mice revealed that Bmi1 is epistatic to eed in the control of primitive bone marrow cell proliferation. However, the genetic interaction between the two genes is cell-type specific as the presence of one or two mutant alleles of eed trans-complements the Bmi1-deficiency in pre-B bone marrow cells. These studies thus suggest that hemopoietic cell proliferation is regulated by the relative contribution of repressive (Eed-containing) and enhancing (Bmi1-containing) PcG gene complexes.
Aging↗
The murine Polycomb-group gene eed and its human orthologue: functional implications of evolutionary conservation.
Similar to Drosophila, murine Polycomb-group (PcG) genes regulate anterior-posterior patterning of segmented axial structures by transcriptional repression of homeotic gene expression. The murine PcG gene eed (embryonic ectoderm development) encodes a 441-amino-acid protein with five WD motifs which, except for the amino terminus, is highly homologous to Drosophila ESC (Extra Sex Combs). Here, sequence and expression analysis as well as chromosomal mapping of the human orthologue of eed is described. Absolute conservation of the human eed protein along with significant divergence at the nucleotide level reveals functional constraints operating on all residues. The human orthologue appears to be ubiquitously expressed and maps to chromsome 11q14.2-q22.3. Using the first WD motif of the beta-subunit of the bovine G protein as a structural reference, the predicted locations of two previously identified eed point mutations (A. Schumacher et al., 1996, Nature 383: 250-253) are also reported herein. The proline substitution (L196P) in the second WD motif of the l7Rn5(3354SB) null allele maps to the internal core of the inner end of the beta-propeller blade and is likely to disrupt protein folding. In contrast, the asparagine substitution (I193N) in the second WD motif of the hypomorphic l7Rn5(1989SB) allele maps onto the surface of the beta-propeller blade near the central cavity and may affect surface interactions without compromising propeller packing. These results illustrate the critical importance of all residues for eed function in mammals and support a model whereby the amino terminus might implement function(s) related to embryonic development in higher organisms.
5' Untranslated Regions↗
Lumican regulates collagen fibril assembly: skin fragility and corneal opacity in the absence of lumican.
Lumican, a prototypic leucine-rich proteoglycan with keratan sulfate side chains, is a major component of the cornea, dermal, and muscle connective tissues. Mice homozygous for a null mutation in lumican display skin laxity and fragility resembling certain types of Ehlers-Danlos syndrome. In addition, the mutant mice develop bilateral corneal opacification. The underlying connective tissue defect in the homozygous mutants is deregulated growth of collagen fibrils with a significant proportion of abnormally thick collagen fibrils in the skin and cornea as indicated by transmission electron microscopy. A highly organized and regularly spaced collagen fibril matrix typical of the normal cornea is also missing in these mutant mice. This study establishes a crucial role for lumican in the regulation of collagen assembly into fibrils in various connective tissues. Most importantly, these results provide a definitive link between a necessity for lumican in the development of a highly organized collagenous matrix and corneal transparency.
Animals↗
NCAM-180 knockout mice display increased lateral ventricle size and reduced prepulse inhibition of startle.
NCAM-180 knockout mice, which have documented deficits in neural migration, were used to determine whether developmental abnormalities could lead to morphological changes and alterations in sensory motor gating mechanisms. Measurement of the lateral ventricle showed that NCAM-180-/- mice had marked increases in both the left and right anterior horns of the lateral ventricle. Furthermore, these mice also displayed a reduction of prepulse inhibition that was differentially affected by the dopamine agonist apomorphine. These results are discussed in light of the known increase in lateral ventricle size and reduction in prepulse inhibition that are seen in schizophrenia.
Acoustic Stimulation↗