The Role of Mga in the Survival of Pluripotent Cells During Peri-implantation Development

The dual specificity transcription factor Mga contains both a T-box binding domain and a basic helix-loop-helix zipper (bHLHZip) domain. Loss of Mga leads to embryonic lethality by E5.5. In vitro blastocyst culture and embryonic stem (ES) cell culture identify a lack of pluripotent inner cell mass (ICM) derived cells as the cause of embryonic lethality. Loss of Mga leads to increased apoptosis in E4.5 embryos, though there is no decrease in the amount of cell proliferation. Embryos with mutant Mga have fewer pluripotent ICM cells during delayed implantation, though the number of differentiated primitive endoderm cells remained initially stable. Despite the loss of pluripotent cells, there is no change in the pattern of expression of Nanog or Oct4, pluripotent cell markers, or Gata4, a primitive endoderm marker. Expression of Ornithine Decarboxylase (ODC), the rate-limiting enzyme in the synthesis of cellular polyamines, was identified as a possible cause of embryonic lethality based on a similar mutant phenotype as well as the presence of E-box sequences in genetic regulation loci. ODC is expressed at lower levels in the ICM of Mga mutants. Blastocyst and ES cell culture defects were rescued when cultured in the presence of exogenous putrescine, the metabolic product of ODC. These results suggest a mechanism for Mga to influence pluripotent cell survival through interactions with other bHLHZip domain proteins in the regulation of the polyamine pool in pluripotent cells of the embryo

The Role of Mga in the Survival of Pluripotent Cells During Peri-implantation Development

The dual specificity transcription factor Mga contains both a T-box binding domain and a basic helix-loop-helix zipper (bHLHZip) domain. Loss of Mga leads to embryonic lethality by E5.5. In vitro blastocyst culture and embryonic stem (ES) cell culture identify a lack of pluripotent inner cell mass (ICM) derived cells as the cause of embryonic lethality. Loss of Mga leads to increased apoptosis in E4.5 embryos, though there is no decrease in the amount of cell proliferation. Embryos with mutant Mga have fewer pluripotent ICM cells during delayed implantation, though the number of differentiated primitive endoderm cells remained initially stable. Despite the loss of pluripotent cells, there is no change in the pattern of expression of Nanog or Oct4, pluripotent cell markers, or Gata4, a primitive endoderm marker. Expression of Ornithine Decarboxylase (ODC), the rate-limiting enzyme in the synthesis of cellular polyamines, was identified as a possible cause of embryonic lethality based on a similar mutant phenotype as well as the presence of E-box sequences in genetic regulation loci. ODC is expressed at lower levels in the ICM of Mga mutants. Blastocyst and ES cell culture defects were rescued when cultured in the presence of exogenous putrescine, the metabolic product of ODC. These results suggest a mechanism for Mga to influence pluripotent cell survival through interactions with other bHLHZip domain proteins in the regulation of the polyamine pool in pluripotent cells of the embryo

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The ring finger protein PCGF6 (polycomb group ring finger 6) interacts with RING1A/B and E2F6 associated factors to form a non-canonical PRC1 (polycomb repressive complex 1) known as PCGF6-PRC1. Here, we demonstrate that PCGF6-PRC1 plays a role in repressing a subset of PRC1 target genes by recruiting RING1B and mediating downstream mono-ubiquitination of histone H2A. PCGF6-PRC1 bound loci are highly enriched for promoters of germ cell-related genes in mouse embryonic stem cells (ESCs). Conditional ablation of Pcgf6 in ESCs leads to robust de-repression of such germ cell-related genes, in turn affecting cell growth and viability. We also find a role for PCGF6 in pre- and peri-implantation mouse embryonic development. We further show that a heterodimer of the transcription factors MAX and MGA recruits PCGF6 to target loci. PCGF6 thus links sequence specific target recognition by the MAX/MGA complex to PRC1-dependent transcriptional silencing of germ cell-specific genes in pluripotent stem cells

<em>Mga</em> is essential for the survival of pluripotent cells during peri-implantation development.

The maintenance and control of pluripotency is of great interest in stem cell biology. The dual specificity T-box/basic-helix-loop-helix-zipper transcription factor Mga is expressed in the pluripotent cells of the inner cell mass (ICM) and epiblast of the peri-implantation mouse embryo, but its function has not been investigated previously. Here, we use a loss-of-function allele and RNA knockdown to demonstrate that Mga depletion leads to the death of proliferating pluripotent ICM cells in vivo and in vitro, and the death of embryonic stem cells (ESCs) in vitro. Additionally, quiescent pluripotent cells lacking Mga are lost during embryonic diapause. Expression of Odc1, the rate-limiting enzyme in the conversion of ornithine into putrescine in the synthesis of polyamines, is reduced in Mga mutant cells, and the survival of mutant ICM cells as well as ESCs is rescued in culture by the addition of exogenous putrescine. These results suggest a mechanism whereby Mga influences pluripotent cell survival through regulation of the polyamine pool in pluripotent cells of the embryo, whether they are in a proliferative or quiescent state

Cell lineage of timed cohorts of Tbx6-expressing cells in wild-type and Tbx6 mutant embryos

Tbx6 is a T-box transcription factor with multiple roles in embryonic development as evidenced by dramatic effects on mesoderm cell fate determination, left/right axis determination, and somite segmentation in mutant mice. The expression of Tbx6 is restricted to the primitive streak and presomitic mesoderm, but some of the phenotypic features of mutants are not easily explained by this expression pattern. We have used genetically-inducible fate mapping to trace the fate of Tbx6-expressing cells in wild-type and mutant embryos to explain some of the puzzling features of the mutant phenotype. We created an inducible Tbx6-creERT2 transgenic mouse in which cre expression closely recapitulates endogenous Tbx6 expression both temporally and spatially. Using a lacZ-based Cre reporter and timed tamoxifen injections, we followed temporally overlapping cohorts of cells that had expressed Tbx6 and found contributions to virtually all mesodermally-derived embryonic structures as well as the extraembryonic allantois. Contribution to the endothelium of major blood vessels may account for the embryonic death of homozygous mutant embryos. In mutant embryos, Tbx6-creERT2-traced cells contributed to the abnormally segmented anterior somites and formed the characteristic ectopic neural tubes. Retention of cells in the mutant tail bud indicates a deficiency in migratory behavior of the mutant cells and the presence of Tbx6-creERT2-traced cells in the notochord, a node derivative provides a possible explanation for the heterotaxia seen in mutant embryos