Ever since the implication of Tet proteins in DNA demethylation and maintenance of pluripotency, research has focused on establishing the biological relevance of these proteins (Ito et al., 2010
; Koh et al., 2011b
; Tahiliani et al., 2009
). Targeted deletion of Tet1 in ES cells and generating Tet1 knockout mice has provided a clean and stable system to study Tet1 function in pluripotency and development. Here, we have provided three lines of evidence to establish that Tet1 is dispensable for ES cell maintenance and its loss compatible with embryonic development and postnatal survival. First, Tet1−/−
mESCs remain undifferentiated in vitro
and express pluripotency markers Oct4, Sox2 and Nanog and can contribute to tissues of the three embryonic germ layers in teratomas. Second, Tet1 null mESCs can support normal development of the embryo proper in a tetraploid complementation assay suggesting that they are pluripotent and can support post-implantation embryonic development. Third, Tet1 homozygous mutant mice are viable and fertile and born at normal Mendelian ratio, albeit with slight reduction in body size, establishing that loss of Tet1 does not interfere with postnatal survival.
Our findings expand upon the previously published in vitro
studies on Tet1 and bring new insights into defining the actual physiological requirements of the gene in pluripotency and development. Previously reported shRNA-based in vitro
knockdown studies (Ito et al., 2010
; Koh et al., 2011a
) together with recent findings on the dual roles of Tet1 in transcriptional regulation and repression of polycomb controlled developmental genes (Williams et al., 2011
; Wu et al., 2011
), suggested that Tet1 is likely a key player in ES cell maintenance, lineage specification and embryogenesis. One report showed that Tet1 knockdown in mESCs diminished Nanog expression and impaired self-renewal (Ito et al., 2010
). We did not observe such a phenotype in Tet1−/−
cells and it is possible that ES cell background differences and/or off target effects of shRNAs may explain these differences. Consistent with our findings, another study using a different set of shRNAs to knockdown Tet1 and Tet2 reported that depletion of these proteins does not affect Nanog expression, ES cell self-renewal and pluripotency (Koh et al., 2011b
). However, this study found that Tet1 deficiency skews lineage specification toward mesendoderm and trophectoderm at the expense of neuroectoderm in a teratoma assay leading to formation of large hemorrhagic tumors. Although we observed more hemorrhage and an increase in trophoblast-like cells in Tet1 knockout teratomas, we did not observe any overt reduction in formation of neuroectoderm or contribution to placenta upon injection of mutant ESCs into wild-type blastocysts. EBs generated from Tet1 knockout ES cells were capable to form neural progenitors in culture, even though the overall yield of EB formation from knockout cells were substantially lower due to their increased tendency to attach to the plastic surface and differentiate.
The generation of viable mutant mice argues against profound defects in lineage specification due to loss of Tet1. It is possible, therefore, that the teratoma assay or differentiation to EBs uncovered abnormalities that are only detectable in vitro and do not overtly affect embryonic development. We consider two possibilities to explain the seemingly normal in vivo development of mutant embryos and the in vitro differentiation abnormalities of mutant ES cells. (1) Tet1 knockout ES cells exhibit skewed differentiation in non-physiological conditions such as a teratoma or in in vitro assays. (2) Tet1 knockout ES cells have lineage specification defects but any such defects are less pronounced in the context of an embryo compared to a teratoma or EB and are compatible with embryogenesis consistent with the failure of mutant ES cells to contribute to the trophectoderm lineage in vivo.
The generation of viable and fertile Tet1 mutant mice unequivocally indicated that Tet1 deficiency does not prevent embryonic and postnatal development. However, the slight reduction in postnatal body size and weight of Tet1 knockout mice is consistent with Tet1 playing a role during development. Given the evidence that Tet1 regulates gene expression, particularly by repressing polycomb regulated developmental genes (Williams et al., 2011
; Wu et al., 2011
), it is possible that subtle deregulation of these developmental regulators interfere with timely progression of embryogenesis and trigger a mild developmental delay. Because normal sized knockout mice were generated by tetraploid complementation it is possible that the role of Tet1 is more pronounced in placental function than in embryo development. However, further molecular and histological analysis of embryos at various stages during development is needed to establish if Tet1 loss leads to placental defects.
The three Tet enzymes are differentially regulated during early development. Tet3 is the only Tet enzyme expressed in the oocyte and zygote and is believed to have a role in paternal pronucleus demethylation (Iqbal et al., 2011
; Wossidlo et al., 2011
). As the zygote develops Tet3 levels diminish at two cell stage and by the blastocyst stage Tet1 and Tet2 are mainly expressed in the inner cell mass (ICM). Both Tet1 and Tet2 are expressed in ES cells with Tet2 expression levels being five fold less than Tet1 (Koh et al., 2011a
). We found that loss of Tet1 did not lead to induction of Tet2 and Tet3 mRNA levels in Tet1−/−
mESCs. Given that Tet1 knockout ES cells have only a 35% reduction in 5hmC levels, it is likely that expression of Tet2 can compensate for Tet1 loss consistent with the observation that a 60% knockdown of Tet2 in Tet1−/−
ES cells further reduced 5hmC levels. Generation of double and triple knockout ES cells and mice for Tet genes will be important for dissecting the developmental roles of the different Tet proteins and 5hmC in vivo
Embryonic development is a multi-step and complex process. Although we have generated viable Tet1 knockout mice and shown that loss of Tet1 is compatible with pluripotency and development, we cannot exclude subtle defects during development whose effects would manifest later in life, such as neurological and behavioral phenotypes, particularly given the high levels of 5hmC and Tet1 in the adult brain and their potential role in active DNA demethylation (Guo et al., 2011
). Moreover, despite the presence of germ cells in knockout gonads, it is important to further study if Tet1 plays any role in gametogenesis, particularly methylation erasure and germ cell maintenance, which requires detailed investigation. Further characterization of Tet1 knockout mice will also allow studying the physiological significance of Tet1 during adult development.