Murine embryonic stem cells (ES cells) are derived from the inner cell mass (ICM) of blastocyst stage embryos and can be maintained indefinitely in vitro
while retaining the ability to subsequently differentiate into all the cells of the adult animal. Understanding properties of ES cells and how self-renewal and pluripotency are regulated will have a large impact on developmental biology studies and regenerative medicine. Several transcription factors are required for ES cell self-renewal and pluripotency, including Oct4, Sox2 and Nanog, and inactivation of these genes leads to loss of pluripotent stem cells and aberrant differentiation into extraembryonic trophoblast in the case of Oct4 and Sox2, or primitive endoderm in the case of Nanog1-5
. Recently, overexpression of a cocktail of transcription factors (Oct4, Sox2, c-Myc and Klf4 or Oct4, Sox2, Lin-28 and Nanog) has resulted in the induction of pluripotency in somatic cells 6-10
. These “induced pluripotent stem cells” (iPSCs) have all the properties of ES cells, but the mechanism of this induction is still unclear. Identification of factors immediately downstream of these transcription factors will be crucial.
Foxd3 is a forkhead transcription factor required for maintenance of progenitor cells in the ICM, trophoblast and neural crest lineages11-13
embryos die shortly after implantation and cells in the mutant ICM and epiblast undergo extensive programmed cell death11
. ES cells express Foxd3
and expression is dramatically downregulated when cells are induced to differentiate 14
, suggesting that Foxd3 expression in pluripotent stem cells is functionally significant. Together, this work illustrates the important role Foxd3 plays to maintain multipotent progenitor cells from divergent embryonic lineages, but the early lethality of Foxd3−/−
embryos and therefore inability to establish Foxd3−/−
ES cell lines, hampered efforts to study the role Foxd3 plays in ES cell maintenance.
To circumvent this problem, we derived ES cell lines in which Cre-mediated inactivation of Foxd3 function can be temporally regulated. These Foxd3fl/fl;CAGG Cre-ERTM (Foxd3fl/fl;Cre-ER) ES cells are indistinguishable from normal ES cells in culture, and the Foxd3 coding region is deleted when cells are cultured in the presence of 4-hydroxytamoxifen (TM). Using this inducible system, we demonstrate that Foxd3 is not required for cell proliferation, but that mutant ES cells undergo increased apoptosis indicating Foxd3 is required for ES cell survival. Mutant ES cells were defective in their ability to form colonies from single cells, illustrating a requirement for Foxd3 in stem cell self-renewal. At the same time, while maintained under differentiation inhibiting conditions, Foxd3 mutant ES cells do not respond to these cues and undergo extensive differentiation despite the maintenance of expression of multiple stem cell genes. Together, our results shape a deeper understanding of the biological roles of this transcription factor in murine ES cells and allow us to propose a model that will further our comprehension of mechanisms regulating maintenance of self-renewal and multipotency, the defining characteristics of all stem cells.