Role And Mechanism Of Dax1in Regulating Embryonic Stem Cell And Induced Pluripotent State

Embryonic stem cells (ESCs), derived from the inner cell mass (ICM) of blastocyst-stageembryos, can maintain self-renewal and multilineage differentiation potential in vitro. ESCscan differentiate into all cell types of the adult organism. Understanding mechanisms formaintenance of pluripotency not only promotes advances in tissue engineering, embryonicdevelopment (including immune system development), and cancer research, but also allowsgeneration of induced pluripotent stem cell (iPSC) technology.Nanog plays key roles in regulating pluripotent state of ESCs. However, Nanogexpression is not only heterogeneous but also dynamic in ESCs. NanoghighESCs possess highself-renewal efficiency, whereas NanoglowESCs exhibit increased propensity to differentiation.So far, little is known about the mechanism for stabilizing pluripotency during the transitionbetween Nanoghighand Nanoglowstates.Gata6is the key regulator in the formation of extra-embryonic endoderm(ExEn).However, Nanoglowor even NanognullESCs retain the capacity of self-renewal andpluripotency with up-regulation of Gata6, suggesting that the increased Gata6is insufficientto induce ExEn differentiation. Therefore, possibly other ESC-specific transcription factors,which should be functionally independent of Nanog, may be involved in inhibiting fullactivation of Gata6and ExEn differentiation in Nanoglow/nullESCs.Dax1(dosage-sensitive sex reversal-AHC-critical region on the X-chromosome gene1,also known as Nr0b1) has been identified as a major component in both Oct4-centered andNanog-centered protein interaction networks. Dax1also co-occupies promoters in associationwith the pluripotency regulators Oct4and Nanog, and integrates into the core regulatorycircuitry of ES cells. Moreover, Dax1is highly expressed in ESCs and quicklydown-regulated during differentiation. Transient knockdown Dax1with siRNA inducesdifferentiation of ESCs into ExEn like cells, which indicates the role of Dax1in inhibition ofExEn differentiation. However, it is still unclear that the function relationship of Dax1with Nanog and Oct4, as well as the molecular mechanism of Dax1action for self-renewal andpluripotency.Results:1). Dax1stable knockdown(KD) ESCs generated more differentiation-like cellsand up-regulated expression of ExEn markers. However Dax1KD ESCs maintainedexpression of pluripotency-associated transcripts and could differentiate into cell types fromall three germ layers.2). Without Lif and feeders, Dax1stable overexpression(OE) ESCscould form undifferentiated alkaline phosphatase(AP)-positive colonies with a typical ESCmorphology, maintained expression of Oct4/Nanog and repressed all differentiation markers.3). Dax1repressed Gata6promotor activity through direct binding to its DNA element. OE ofGata6in Dax1OE ESCs resulted in completely differentiation.4). Dax1plus Nanog KD cellswere fully differentiated indicating that a simultaneous depletion of Dax1and Nanog inducedadditive effects.5). Dax1could not completely rescue increased expression of ExEn markersinduced by Nanog KD,and vice versa.6). Nanog KD could induce Gata6expression in Dax1+cells, whereas Dax1KD induced Gata6derepression mainly in Nanog-cells and in only aminority of Nanog+cells.7). Dax1KD-Nanog:GFPlowpopulations had more dramaticallyupregulated ExEn transcripts and were hardly expanded. Dax1KD-Nanog:GFPhighcellsregenerated GFPlowcells, but Dax1KD-Nanog:GFPlowcells could not give rise to GFPhighcells.8). During reprogramming, Dax1KD or Nanog KD Oct4:GFP MEF-derived cellsyielded few GFP+cells and reduced endogenous Sox2and Rex1expression.9). Dax1KD orOE had no significant effect on Oct4expression, whereas Oct4KD reduced Dax1and Oct4OE increased Dax1mRNA levels. ChIP-qPCR analysis showed that Oct4bound to thepromoter of Dax1, whereas the enrichment of Dax1on the Oct4gene locus was not observed.10). Dax1OE could not prevent differentiation induced by Oct4KD. Nevertheless, Dax1OEcould partially rescue Oct4KD-induced upregulation of ExEn and TE markers expression.11).Neither Dax1OE nor Dax1KD affected the phosphorylation state of Stat3and Erk1/2in thepresence or absence of LIF.Conclusions:1). Dax1stable KD mESCs are predisposed towards differentiation but donot lose pluripotency.2). Dax1OE supports Lif-independent self-renewal.3). Inhibition ofExEn differentiation by Dax1is directly mediated by Gata6.4). Dax1and Nanog co-operateto silence Gata6and prevent ExEn differentiation.5). Although partially complementary,Dax1and Nanog function independently and cannot replace one another.6). Dax1is indispensable for self-renewal of NanoglowmESCs and is required for stabilizing pluripotencyduring the transition between Nanoghighand Nanoglowstates.7). Both Dax1and Nanog arerequired for full reprogramming to induced pluripotency.8). Dax1may also mediateinhibition of trophectoderm differentiation independent or as a downstream effector of Oct4.Taken together, Dax1and Nanog offer “double-insurance” that stabilizes ESCs and inducedpluripotency. These findings provide new clues for further understanding mechanisms ofESCs fate determination, dynamic fluctuation of pluripotency and somatic cellreprogramming.