Gene-based chemical mutagenesis in mouse embryonic stem cells.
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Biomedical subjects
Terry Magnuson
Publications and source records attributed to Terry Magnuson.
An allelic series of mutations in Smad2 and Smad4 identified in a genotype-based screen of N-ethyl-N- nitrosourea-mutagenized mouse embryonic stem cells.
Using selectable genes as proof of principle, a new high-throughput genotype-based mutation screen in mouse embryonic stem (ES) cells was developed [Chen et al. (2002) Nat. Genet. 24, 314-317]. If expanded to nonselectable genes, this approach would allow one to proceed quickly from sequence to whole-animal phenotypes. Here data are presented showing that a screen of a cryopreserved library of clonal, germ line competent, N-ethyl-N-nitrosurea (ENU) mutagenized ES cells can identify a large series of allelic mutations in Smad2 and Smad4, two nonselectable genes of the transforming growth factor beta superfamily of signaling molecules. Whole animal phenotypic analyses of some of these alleles provided evidence for novel developmental processes mediated by these components of transforming growth factor beta signaling, demonstrating the utility of non-null alleles created by chemical mutagens. The accurately assessed mutation load of the ES cell library indicates that it is a valuable resource for developing mouse lines for genetic and functional studies. This methodology can conceptually be applied for the generation of an allelic series of subtle mutations at any locus of interest in the mouse.
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The Polycomb-group gene eed regulates thymocyte differentiation and suppresses the development of carcinogen-induced T-cell lymphomas.
The mouse Polycomb-group gene, embryonic ectoderm development (eed), appears to regulate cellular growth and differentiation in a developmental and tissue specific manner. During embryogenesis, eed regulates axial patterning, whereas in the adult eed represses proliferation of myeloid and B cell precursors. The present report demonstrates two novel functional activities of eed: alteration of thymocyte maturation and suppression of thymic lymphoma development. Mice that inherit the viable hypomorphic 17Rn5(1989SB) eed allele sustain a partial developmental block at or before the CD4(-)CD8(-)CD44(-)CD25(+) stage of thymocyte differentiation. Furthermore, mice that are homozygous or heterozygous for the hypomorphic eed allele have an increased incidence and decreased latency of N-methyl-N-nitrosourea-induced thymic lymphoma compared to wild-type littermates. These findings support the notion that Polycomb-group genes exert pleiotropic effects dictated by developmental stage and cellular context.
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The mouse PcG gene eed is required for Hox gene repression and extraembryonic development.
The Polycomb group (PcG) of genes was first identified in Drosophila as maintenance factors for long-term transcriptional repression of homeotic genes. In mice, the PcG protein Eed (Embryonic ectoderm development) is present in a distinct complex that interacts with histone deacetylase (HDAC) and the PcG member Ezh2 (Enhancer of zeste homolog 2), but not in the larger Polycomb repressive complex 1 (PRC1) formed by several other PcG proteins. eednull mutants manifest a distinct early gastrulation defect that occurs prior to homeotic gene expression. To determine whether Eed is also required for regulating homeotic genes, a later acting eedhypomorph mutation was analyzed. The anterior expression boundaries of several Hox genes were shifted rostrally by one segment, indicating that Eed is required for stable repression of homeotic genes. Furthermore, although the eednull/hypomorph compound heterozygotes die during mid-gestation stage, they did not show a more severe derepression of Hox genes than the eedhypomorph/hypomorph homozygotes. A detailed analysis of the mid-gestation lethality associated with the eednull/hypomorph compound heterozygotes revealed a novel function for eed in the development of secondary trophoblast giant cells during murine placenta formation. Tetraploid rescue experiments demonstrated that the defect is cell autonomous in the extraembryonic lineage. Mash2, a paternally imprinted gene important for trophoblast development, was ectopically expressed in the eed mutants. However, genetic crosses with a Mash2 null allele suggested that Eed was not required to maintain Mash2 imprinting, but could be required in a lineage specific fashion to suppress Mash2 expression.
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Genetic analysis of sorting nexins 1 and 2 reveals a redundant and essential function in mice.
Sorting nexins 1 (Snx1) and 2 (Snx2) are homologues of the yeast gene VPS5 that is required for proper endosome-to-Golgi trafficking. The prevailing thought is that Vps5p is a component of a retrograde trafficking complex called the retromer. Genetic and biochemical evidence suggest mammals may have similar complexes, but their biological role is unknown. Furthermore, if SNX1 and SNX2 belong to such complexes, it is not known whether they act together or separately. Herein, we show that mice lacking SNX1 or SNX2 are viable and fertile, whereas embryos deficient in both proteins arrest at midgestation. These results demonstrate that SNX1 and SNX2 have a highly redundant and necessary function in the mouse. The phenotype of Snx1(-/-);Snx2(-/-) embryos is very similar to that of embryos lacking another retromer homologue, Hbeta58. This finding suggests that SNX1/SNX2 and Hbeta58 function in the same genetic pathway, providing additional evidence for the existence of mammalian complexes that are structurally similar to the yeast retromer. Furthermore, the viability of Snx1(-/-) and Snx2(-/-) mice demonstrates that it is not necessary for SNX1 and SNX2 to act together. Electron microscopy indicates morphological alterations of apical intracellular compartments in the Snx1(-/-);Snx2(-/-) yolk-sac visceral endoderm, suggesting SNX1 and SNX2 may be required for proper cellular trafficking. However, tetraploid aggregation experiments suggest that yolk sac defects cannot fully account for Snx1(-/-); Snx2(-/-) embryonic lethality. Furthermore, endocytosis of transferrin and low-density lipoprotein is unaffected in mutant primary embryonic fibroblasts, indicating that SNX1 and SNX2 are not essential for endocytosis in all cells. Although the two proteins demonstrate functional redundancy, Snx1(+/-);Snx2(-/-) mice display abnormalities not observed in Snx1(-/-);Snx2(+/-) mice, revealing that SNX1 and SNX2, or their genetic regulation, are not equivalent. Significantly, these studies represent the first mutations in the mammalian sorting nexin gene family and indicate that sorting nexins perform essential functions in mammals.
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