Disruption of the Mouse mTOR Gene Leads to Early Postimplantation Lethality and Prohibits Embryonic Stem Cell Development

Abstract

The mammalian target of rapamycin (mTOR) is a key component of a signaling pathway which integrates inputs from nutrients and growth factors to regulate cell growth. Recent studies demonstrated that mice harboring an ethylnitrosourea-induced mutation in the gene encoding mTOR die at embryonic day 12.5 (E12.5). However, others have shown that the treatment of E4.5 blastocysts with rapamycin blocks trophoblast outgrowth, suggesting that the absence of mTOR should lead to embryonic lethality at an earlier stage. To resolve this discrepancy, we set out to disrupt the mTOR gene and analyze the outcome in both heterozygous and homozygous settings. Heterozygous mTOR (mTOR ؉/؊ ) mice do not display any overt phenotype, although mouse embryonic fibroblasts derived from these mice show a 50% reduction in mTOR protein levels and phosphorylation of S6 kinase 1 T389, a site whose phosphorylation is directly mediated by mTOR. However, S6 phosphorylation, raptor levels, cell size, and cell cycle transit times are not diminished in these cells. In contrast to the situation in mTOR ؉/؊ mice, embryonic development of homozygous mTOR ؊/؊ mice appears to be arrested at E5.5; such embryos are severely runted and display an aberrant developmental phenotype. The ability of these embryos to implant corresponds to a limited level of trophoblast outgrowth in vitro, reflecting a maternal mRNA contribution, which has been shown to persist during preimplantation development. Moreover, mTOR ؊/؊ embryos display a lesion in inner cell mass proliferation, consistent with the inability to establish embryonic stem cells from mTOR ؊/؊ embryos.

The role of oxygen availability in embryonic development and stem cell function

“…Germline disruption of mTOR in mice causes embryonic lethality during blastocyst implantation [99][100][101] . Explanted mTOR-null blastocysts appear normal, but the ICM and trophoblast giant cells fail to expand during culture.…”

Rapamycin and mTOR kinase inhibitors

“…Mouse embryos lacking mTOR die at E5.5-6.5 days [10,11]. Ablation of Raptor to disrupt mTORC1 is equally lethal at approximately E6.5 days [12].…”

Stress and mTORture signaling

“…mTOR disruption in mice is incompatible with passage through embryogenesis, and mutant animals die in early postimplantation stages (Gangloff et al, 2004). Along these lines, yolk protein synthesis in Aedes aegypti stimulated after a bloodmeal is critically dependent on TOR/S6K, as interference with their function lowers Vitellogenin levels and severely disrupts egg development (Hansen et al, 2005).…”