Requirement for Foxd3 in maintaining pluripotent cells of the early mouse embryo

Abstract

Critical to our understanding of the developmental potential of stem cells and subsequent control of their differentiation in vitro and in vivo is a thorough understanding of the genes that control stem cell fate. Here, we report that Foxd3, a member of the forkhead family of transcriptional regulators, is required for maintenance of embryonic cells of the early mouse embryo. Foxd3−/− embryos die after implantation at approximately 6.5 days postcoitum with a loss of epiblast cells, expansion of proximal extraembryonic tissues, and a distal, mislocalized anterior organizing center. Moreover, it has not been possible to establish Foxd3−/− ES cell lines or to generate Foxd3−/− teratocarcinomas. Chimera analysis reveals that Foxd3 function is required in the epiblast and that Foxd3−/− embryos can be rescued by a small number of wild-type cells. Foxd3−/− mutant blastocysts appear morphologically normal and express Oct4, Sox2, and Fgf4, but when placed in vitro the inner cell mass initially proliferates and then fails to expand even when Fgf4 is added. These results establish Foxd3 as a factor required for the maintenance of progenitor cells in the mammalian embryo.[Keywords: mouse embryogenesis; stem cells; Foxd3; winged helix gene] Multipotent progenitor cells exist transiently in the mammalian embryo in several tissues including the preimplantation stage blastocyst, the gastrulating epiblast, and the neural crest. Each of these cell types can be cultured in vitro to generate multipotent stem cell lines, and when grafted ectopically, blastocysts and epiblast tissue will produce teratocarcinomas containing multipotent stem cells. Understanding the common molecular regulatory mechanisms of different stem cell types is critical to an understanding of how multipotency is maintained in vivo and how differentiation is controlled.Mammalian embryonic development initiates with a series of cleavage divisions, and at 2.5 days postcoitum (dpc) the free-floating mouse embryo undergoes compaction: cell boundaries become tightly apposed to one another and cells are no longer equivalent. Inner cells contribute to the inner cell mass (ICM) and embryo proper, whereas outer cells contribute to the trophectoderm, a tissue essential for implantation. After implantation, the ICM proliferates and extends distally to form the cuplike egg cylinder (at ∼ 5.5 dpc) consisting of an inner layer of epiblast cells and an outer layer of extraembryonic visceral endoderm (for review, see Hogan et al. 1994).Few genes have been identified that are required for the maintenance of the epiblast cell population and the establishment of ICM-derived embryonic stem (ES) cells in vitro. Oct4−/− embryos, mutant for this POU family transcription factor, die around 5.0 dpc, shortly after implantation but before the egg cylinder is formed. Oct4−/− ES cells cannot be established, as the ICM cells fail to survive (Nichols et al. 1998). Survival of multipotent cells of the ICM is highly sensitive to Oct4 expression levels; low Oct4 levels result in the differentiat...

High-throughput screen for genes predominantly expressed in the ICM of mouse blastocysts by whole mount in situ hybridization

“…All-stage WISH confirmed that the seven genes that we selected indeed showed the ICM-predominant expression. The expression patterns of Pou5f1 (Hanna et al, 2002;Pelton et al, 2002) and Nanog (Chambers et al, 2003;Mitsui et al, 2003) confirmed previously published results. Two new genes identified here were Mybl2 and Gtf2e2.…”

“…While a pore size of 20 μm is necessary for achieving efficient drainage without special instruments, much smaller pores are preferable to maintain the best morphology of small samples. As a result, transwell with pore size 12 μm which are originally designed for cell culture were introduced into WISH (Hanna et al, 2002) to retain embryos. Although, solution changes were achieved by manually transferring the transwell from one well to another, it is difficult to have good buffer exchange through smaller pores without the assistance of a special device.…”

Somatic stem cells and the origin of cancer

“…According to this theory, authors define tumor-progenitor genes as those that suffer epigenetic modification in stem cells thus promoting malignant transformation 137 . Examples of these tumor progenitor genes are, in addition to IGF2, the POU transcription factor OCT4 or the forkhead transcription factor FOXD3, whose inappropriate expression could augment proliferation and block differentiation in stem cells 145,146 . One additional example may be represented by two DNA repair genes, ataxia teleangiectasia mutated (ATM) and checkpoint kinase 2 (CHK2), frequently inactivated in mantle cell lymphoma.…”

Requirement for Foxd3 in the maintenance of neural crest progenitors

“…Foxd3 tm2.Lby is a null mutation of Foxd3 generated previously (Hanna et al, 2002) and is referred to as Foxd3 -throughout. Wnt1-Cre mice were a generous gift of Drs Andrew McMahon and David Rowitch (Harvard University, Cambridge, MA).…”

“…Foxd3 was first characterized by its expression in embryonic stem (ES) cells and multipotent cells of the NC (Labosky and Kaestner, 1998). Our previous work demonstrated that Foxd3 is required early in mouse embryogenesis; Foxd3 -/-embryos fail around the time of implantation, cells of the inner cell mass cannot be maintained in vitro, and ES cell and trophoblast stem cell lines cannot be established (Hanna et al, 2002;Tompers et al, 2005). In vivo, the pool of both embryonic and trophoblast progenitors is not maintained without Foxd3; embryonic progenitor cells die, while trophoblast progenitors give rise to an excess of trophoblast giant cells at the expense of the remaining trophoblast lineage.…”