Dax1-knockdown ESCs can maintain self-renewal

The Dax1 gene was expressed highly in ESCs and was rapidly downregulated during differentiation20,21, suggesting a functional role for Dax1 in maintaining pluripotency of ESCs. To investigate this, we used short hairpin RNA (shRNA) lentiviral vectors to stably knockdown (KD) Dax1 in ESCs. Seven shRNAs targeting different regions of Dax1 complementary DNA were tested. Dax1 was effectively silenced by two constructs, designated Dax1 KD-2 and Dax1 KD-5, which target the CDS (coding sequence) and 3′ untranslated region (3′ UTR), respectively (Fig. 1a,b; Supplementary Fig. 1a,b). We found that the depletion of Dax1 resulted in more differentiation-like cells and slower cell expansion compared with wild-type (WT) and luciferase KD (Luc KD, serving as a negative control) cells (Fig. 1c,d). However, Dax1 KD cells could be continuously propagated (for at least 30 passages) in the presence of leukaemia inhibitory factor (LIF) and retained the capacity to form ESC colonies (Fig. 1c). Colony formation assays showed that Dax1 KD cells formed less wholly undifferentiated alkaline phosphatase (AP)-positive colonies and more mixed (partially differentiated) colonies compared with control cells (Fig. 1e; Supplementary Fig. 1c). Consistent with the increase in partially differentiated colonies, ExEn markers (Gata6, Foxa2, Sox17, Afp, Ihh, LamininB1 and Dab2) were significantly upregulated in Dax1 KD cells as shown by quantitative reverse transcriptase–PCR (qRT–PCR) analysis (Fig. 1f). Also, there was a weak induction of mesoderm (Flk1) and TE markers (Hand1, Cdx2 and Eomes), but not of the neuroectoderm (Sox1 and Nestin) and other mesodermal markers (Brachyury and Goosecoid). Nonetheless, expression of pluripotency-associated transcripts were maintained (Fig. 1g). Immunostaining revealed that the number of Gata6-positive cells increased, but Oct4 expression appeared to be comparable in Dax1 KD and Luc KD cells (Fig. 1h).

Figure 1: Dax1 knockdown predisposes ESCs to differentiation but with no loss of self-renewal capacity. (a,b) qRT–PCR (a) and immunoblot (b) analyses after Dax1 KD. (c) Morphology of colonies formed by the indicated lines. Cells were grown with LIF for three passages after zeocin selection. Scale bar, 100 μm. (d) ESCs (200 cells per cm2 in 12-well plates) were cultured for 5 days with LIF and cell numbers were counted. (e) Quantitative analysis of colony formation assay in the indicated lines. Cells were plated at clonal density and cultured for 6 days with LIF. Colonies were fixed and stained for AP and scored as undifferentiated (undiff.), mixed or differentiated (diff.). (f,g) qRT–PCR analyses of germ layer (f) and pluripotency (g) marker expression levels in the indicated lines cultured with LIF. All data are normalized to Gapdh and shown relative to WT ESCs (set at 1.0). (h) Immunofluorescence analysis of Oct4 (green) and Gata6 (red) in the indicated lines. Cells were cultured with LIF for 5 days and counterstained with DAPI (blue). Scale bar, 100 μm. Data in a,d–g are represented as mean±s.d.; n=3. *P<0.05; **P<0.01. All P values were calculated using Student’s t-test. Full size image

To rule out shRNA off-target effects, we transfected an expression vector containing Dax1 cDNA (Dax1 OE) into Dax1 KD-5 ESCs. Because cDNA does not contain the 3′ UTR of Dax1, the Dax1 KD-5 construct does not affect this exogenous transcript (Supplementary Fig. 1a,d). Re-expression of Dax1 could completely rescue the upregulation of ExEn markers in Dax1 KD cells (Supplementary Fig. 1e), indicating that the observed phenotypes are specifically due to loss of Dax1 function.

Furthermore, stable Dax1 KD were also generated in an Oct4:GFP reporter iPSC line. We found that Oct4:GFP was expressed in Dax1 KD iPSCs (Supplementary Fig. 1f), whereas Gata6-positive cells increased and were mainly Nanoglow cells (Supplementary Fig. 1g). A colony-forming assay and qRT–PCR analysis for pluripotency and differentiation markers also produced consistent results with that of Dax1 KD ESCs (data not shown). Collectively, these results suggest that a large number of, if not all, Dax1 KD ESCs/iPSCs can maintain undifferentiated states, despite an increased differentiation propensity and reduced self-renewal efficiency.

Dax1 KD ESCs retain multilineage differentiation potential

To assess whether Dax1 KD affects the multilineage differentiation potential of ESCs, we measured marker gene expression in day 9 embryoid bodies (EB) derived from Dax1 KD ESCs. As shown in Fig. 2a, pluripotency genes were downregulated in Dax1 KD cells on EB differentiation similar to Luc KD cells. No consistent significant variation was detected for the transcripts of neuroectoderm, mesoderm and TE, but ExEn markers were upregulated more when compared with control cells (Fig. 2a). Next, we used lineage-specific differentiation models to clarify the effect of Dax1 KD. In the presence of low-dose retinoic acid (RA), which promotes ExEn differentiation22, ~85% of Dax1 KD cells were Gata6 positive, compared with ~60% of Luc KD cells (Fig. 2b). Under conditions for neuroectoderm23, TE24 or mesoderm25 differentiation, however, expression of lineage-specific markers did not significantly differ from those in control cells (Fig. 2c,d). Notably, Dax1 KD ESCs also had the capacity to generate teratomas consisting of all three germ-derived tissues, suggesting that these cells still maintain pluripotency in vivo (Fig. 2e). These data indicate that Dax1 KD ESCs retain a multilineage differentiation potential, but have an enhanced propensity for differentiating into ExEn lineages.

Figure 2: Dax1-knockdown ESCs retain multilineage differentiation potential. (a) qRT–PCR analysis of gene expression in the indicated lines after 9 days of EB differentiation. All data are normalized to Gapdh and shown relative to WT ESCs (set at 1.0). Data are represented as mean±s.d.; n=3. (b) Enhanced ExEn differentiation is observed in Dax1 KD ESCs. Monolayer cultures were treated with RA (0.1 μM) for 4 days and costained with Gata6 and DAPI. Scale bar, 100 μm. Proportions of Gata6+ cells are shown in the bar graph next to the images. Data are represented as mean±s.e.m. (n=3). *P<0.05. P values were calculated using Student’s t-test. (c) Flow cytometric analysis of mesoderm induction, as measured by the Flk1+ population, from Luc KD (control) and Dax1 KD ESCs. Numbers in quadrants indicate the percentage of each population. (d) Immunofluorescent analyses of neuroectoderm (Nestin, red) and TE (Cdx2, red) induction from Luc KD (control) and Dax1 KD ESCs. Scale bar, 100 μm. Proportions of Nestin+ cells and Cdx2+ cells are shown in the bar graph next to the images. Data are represented as mean±s.e.m. (n=3). (e) Neuroectoderm, mesoderm and ExEn-derived structures are present in teratomas from Dax1 KD ESCs. Hematoxylin and eosin-stained tissue sections are shown. Scale bar, 50 μm. Full size image

Dax1 OE confers LIF-independent self-renewal on ESCs

To investigate the effect of Dax1 gain of function on ESC self-renewal and pluripotency, full-length cDNA for Dax1 was cloned into the pPyCAGIP-based vector and stable Dax1 OE ESC lines were established. Under +LIF conditions, Dax1 OE cells formed more disorganized semi-differentiated-like colonies (Fig. 3a, left). qRT–PCR and immunostaining analyses showed that expression of some differentiation markers (Flk1, Goosecoid, Cdx2 and Hand1) were upregulated, whereas that of the pluripotency markers were normal (Fig. 3b, upper; Supplementary Fig. 2a). After two passages without LIF, the control cells (empty vector-transfected) underwent complete differentiation. In contrast, Dax1 OE cells could form undifferentiated AP-positive colonies with a typical ESC morphology (Fig. 3a, right; Fig. 3c,d). Both mRNAs and proteins examined for lineage markers’ expression showed that, in the absence of LIF, expression of Oct4 and Nanog continued, and all differentiation markers were significantly repressed in Dax1 OE cells (Fig. 3b, lower; Fig. 3e). Cell proliferation experiments showed that despite reduced growth and increased apoptosis under +LIF conditions (Fig. 3f; Supplementary Fig. 2b), Dax1 OE ESCs could be serially passaged without LIF (Fig. 3g).

Figure 3: Dax1 overexpression confers LIF-independent self-renewal on ESCs. (a) Morphology of empty vector and Dax1-overexpressing ESCs. Cells were grown for 5 days with LIF (left), or induced to form EBs for 5 days followed by culture without LIF for two passages (right). Scale bar, 100 μm. (b) qRT–PCR analysis of gene expression in control (Empty) and Dax1 OE ESCs. Cells were cultured with (upper) or without LIF (lower) for 5 days. All data are normalized to Gapdh and shown relative to WT ESCs (set at 1.0). (c) Control (Empty) or Dax1 OE ESCs were passaged every 4 days without LIF and stained for AP. Scale bar, 100 μm. (d) Quantitation of colony types formed by cell lines shown in c. (e) Immunoblot analysis of Dax1, Oct4, Nanog and Gata4 protein in the indicated lines after 5 days of culture without LIF. β-Actin was used as an internal control. (f) Control (Empty) or Dax1 OE ESCs (200 cells per cm2 in 12-well plates) were cultured for 5 days with LIF and cells were counted. (g) Total cell number of the indicated lines cultured for multiple passages without LIF. Data in b,d,f,g are represented as mean±s.d.; n=3. *P<0.05; **P<0.01; ***P<0.001. All P values were calculated using Student’s t-test. Diff., differentiated; Undiff., undifferentiated. Full size image

Overexpression of Dax1 at high levels creates the possibility of neomorphic effects, which may impair self-renewal of ESCs by sequestering other pluripotency factors, as reported previously26,27. We therefore used low-dose puromycin selection to generate an ESC line expressing Dax1 at nearly endogenous levels (Dax1-NE; Supplementary Fig. 2c). In contrast to the pro-differentiation phenotype induced by Dax1 OE, these cells exhibited normal ESC morphology and marker gene expression under +LIF conditions, with only a slight reduction in the AP-positive colony-forming and proliferation ability (Supplementary Fig. 2d–h). However, in the absence of LIF, Dax1-NE ESCs recapitulate the phenotype of Dax1 OE cells, including: (1) undifferentiated morphology (Supplementary Fig. 2d); (2) continuous expression of pluripotency genes; (3) decreased expression of differentiation markers (Supplementary Fig. 2e); and (4) capacity to form self-renewing colonies (Supplementary Fig. 2f,g) and to expand (Supplementary Fig. 2h). Taken together, these results indicate that Dax1 can prevent differentiation and confer LIF-independent self-renewal in ESCs, but it may not be a self-renewal-promoting factor.

Dax1 directly inhibits Gata6 transcription

The above observations suggested that Dax1 may play a role in inhibiting ExEn differentiation, in which Gata6 transcriptional activation is the key11. We thus performed chromatin immunoprecipitation (ChIP) to assess whether Dax1 binds to the Gata6 locus in vivo. Significantly, an enhanced enrichment of Dax1 on Gata6 proximal promoter was observed (Fig. 4a). To rule out other pluripotency factors that may mediate Dax1 binding to the Gata6 promoter, we co-transfected 3Flag-Dax1 expression vector and the Gata6 proximal promoter fragment into 293FT cells. ChIP-qPCR analysis validated that Dax1 specifically bound to this DNA segment (Supplementary Fig. 3a).

Figure 4: Inhibition of ExEn differentiation by Dax1 is mediated by Gata6. (a) ChIP-qPCR analysis of Dax1 occupancy at Gata6 locus. Numbered grey bars indicate primer locations (upper). ESCs were transfected with 3Flag-tagged Dax1 expression vector or the 3Flag-empty vector as a control. ChIP was performed using anti-Flag antibody and qPCR analysis was performed with the primers indicated. Values are expressed as percent of input DNA (lower). (b) Luciferase reporter analysis shows that Dax1 OE downregulated, whereas Dax1 KD upregulated the Gata6 promoter activity. ESCs were co-transfected with the Gata6 promoter–reporter construct and vectors as indicated. Transfected cells were cultured without LIF for 2 days and then luciferase activity was measured. Values are normalized to a Renilla luciferase control. The mean value of cells transfected with the Gata6 promoter–reporter construct was set at 1.0. (c) Immunoblot analysis of Gata6 and Dax1 proteins in control (Empty), Gata6 OE (WT ESCs transfected with Gata6 expression vector) and Dax1 OE/Gata6 OE (Dax1 OE ESCs transfected with Gata6 expression vector) cells after 5 days of culture with LIF. β-Actin was used as an internal control. (d) Morphology of colonies formed by control (Empty), Dax1 OE, Gata6 OE and Dax1 OE/Gata6 OE cells after 5 days of culture with LIF. Scale bar, 100 μm. (e) Quantitative analysis of undifferentiated colony formed by cells shown in d. Cells were plated at clonal density, cultured for 6 days with LIF and stained for AP. (f) qRT–PCR analysis of pluripotency and ExEn markers in the indicated cells cultured with or without LIF. All data are normalized to Gapdh and shown relative to the mean of control cells (set at 1.0). (g) Immunofluorescence analysis of Oct4 (green) and Gata4 (red) in the indicated lines. ESCs were transfected with each expression vector, selected for 6 days in the presence of LIF and counterstained with DAPI. Scale bar, 100 μm. Data in a,b,e,f are represented as mean±s.d.; n=3. Full size image

To determine whether the binding was associated with the regulation of Gata6 transcription, we performed a luciferase assay. Data showed that Gata6 promoter activity was repressed by Dax1 OE and increased by Dax1 KD (Fig. 4b). Likewise, Dax1 OE effectively repressed Gata6 promoter activity in RA-treated differentiated cells (Supplementary Fig. 3b), in which Gata6 expression was activated, whereas Oct4 and Nanog were undetectable22. These results suggest that Dax1 can repress Gata6 transcription in the absence of other pluripotent factors. To map the Dax1-binding region, we generated serial deletion Gata6 promoter/enhancer reporter constructs. The luciferase assay indicated that the Dax1-binding motif was located between −710 and −570 bp of the Gata6 promoter (Supplementary Fig. 3c).

To verify whether Dax1-mediated Gata6 repression contributes to the ExEn differentiation defect of Dax1 OE ESCs, we compared phenotypes of Dax1 OE, Gata6 OE and Dax1 OE/Gata6 OE cells (Fig. 4c). Under +LIF conditions, Gata6 OE and Dax1 OE/Gata6 OE cells exhibited a completely differentiated morphology (Fig. 4d). No AP-positive colonies appeared in these cell cultures (Supplementary Figs 3d and 4e). mRNA and protein analyses showed that pluripotency markers were lost and ExEn lineage markers were strongly upregulated in both Gata6 OE and Dax1 OE/Gata6 OE cells (Fig. 4f,g; Supplementary Fig. 3e). These data indicate that as a downstream target of Dax1, Gata6 can compensate for ExEn differentiation defects caused by Dax1 OE.

Dax1 and Nanog function in parallel to maintain pluripotency

Features of Dax1 strongly suggest a functional similarity to Nanog8,9,15. Analysis using published microarray data sets28,29 showed that, in contrast to significant downregulation after Oct4 or Sox2 KD, pluripotency genes only slightly changed with respect to expression after Dax1 or Nanog KD (Supplementary Fig. 4a), as validated by qRT–PCR (Supplementary Fig. 4b). After 72 h of Dax1 and Nanog depletion, 242 and 761 genes were differentially expressed, respectively, and 133 genes were common (Supplementary Fig. 4c). In contrast, 298 and 490 genes had ≥1.5-fold expression changes after induction of Dax1 or Nanog (Supplementary Fig. 4d). The statistically significant P values were corrected using the Benjamini and Hochberg false discovery rate test (FDR<0.05)30. Interestingly, only nine differentially expressed genes were found in both Dax1 OE and Nanog OE ESCs, suggesting that they may function independently.

To better understand the functional relationship between Dax1 and Nanog, we compared the phenotypic differences on Nanog KD, Dax1 KD and Dax1 plus Nanog KD (Dax1 KD/Nanog KD) in ESCs under LIF conditions (Fig. 5a). Nanog KD cells could be continuously propagated and form undifferentiated AP-positive colonies that express Oct4 and Dax1, despite an increase in differentiated-like cells and upregulation of ExEn markers (Fig. 5a–e). These phenotypes resembled those of Dax1 KD cells (Fig. 5a–e) and were consistent with the previously described phenotypes of Nanog−/− cells15. In contrast to individual KD cells, Dax1 KD/Nanog KD cells were fully differentiated and could not generate AP-positive colonies. Oct4 expression was markedly downregulated, and ExEn differentiation markers were significantly further upregulated (Fig. 5a–e), indicating that a simultaneous depletion of Dax1 and Nanog induces terminal differentiation of ESCs even in the presence of LIF.

Figure 5: Dax1 and Nanog function in parallel and independently to regulate pluripotency of ESCs. (a) Immunoblot analysis of Nanog, Dax1 and Oct4 proteins in the indicated lines. ESCs were infected with luciferase shRNA (Luc KD), Nanog shRNA (Nanog KD), Dax1 shRNA (Dax1 KD) or Dax1 plus Nanog shRNA (Dax1 KD/Nanog KD) lentivirus and selected in the presence of LIF for 5 days. β-Actin was used as an internal control. (b) Representative morphologies of colonies formed by the indicated lines shown in a. Cells were cultured for 5 days with LIF. Scale bar, 100 μm. (c) Immunofluorescence analysis of Nanog (green), Dax1 (blue) and Gata6 (red) in the indicated lines after 5 days culture with LIF. Scale bar, 100 μm. (d) AP staining of colonies formed by plating the indicated shRNA-transduced cells at clonal density and culturing for 6 days in the presence of LIF (left). Scale bar, 100 μm. Percentage of colony types formed by cells is shown (right). Data are represented as mean±s.d.; n=3. (e) qRT–PCR analysis of gene expression in the indicated lines after 5 days of culture with LIF. All data are normalized to Gapdh and shown relative to WT ESCs (set at 1.0). Data are means±s.d.; n=3. Diff., differentiated; Undiff., undifferentiated. Full size image

In addition to Nanog, Klf4 and Esrrb have also been shown to be dispensable for ESC self-renewal. Artificial expression of Klf4 or Esrrb is sufficient to maintain pluripotency in the absence of LIF31,32,33,34,35. Therefore, functional analyses of the single KD, as well as double KD of each gene with Dax1 and Nanog were performed in parallel. Two shRNA sequences, which have been previously validated35,36, were used to silence Klf4 and Esrrb, respectively (Supplementary Fig. 5a). In accordance with previous studies35,37,38, no obvious phenotypic changes were observed in Klf4 KD cells, whereas Esrrb KD cells displayed an increased differentiation propensity, with the upregulation of TE and neuroectoderm markers (Supplementary Fig. 5b–d). By comparison, however, Dax1/Klf4, Dax1/Esrrb, Nanog/Klf4 and Nanog/Esrrb double KD all showed significantly weaker additive effects than Dax1/Nanog KD (Supplementary Fig. 5b–d).

Next, to test whether Nanog and Dax1 are functionally redundant, we measured the expression of ExEn markers in Dax1 KD/Nanog OE and Nanog KD/Dax1 OE cells. Immunoblotting analysis showed that both Dax1 and Nanog were comparably overexpressed after transfection (Supplementary Fig. 6a,b). Under +LIF conditions, Dax1 could not completely rescue increased expression of ExEn markers induced by Nanog KD. Also, Nanog could not completely rescue the effect of Dax1 KD (Fig. 6a). Under RA-induced differentiation conditions, individual Dax1 OE or Nanog OE effectively inhibited the expression of Gata6 and formed undifferentiated AP-positive colonies. In contrast, both Dax1 KD/Nanog OE and Nanog KD/Dax1 OE cells could not completely suppress Gata6 upregulation, although they partially retained the capacity to form AP-positive colonies (Fig. 6b; Supplementary Fig. 6c). These results indicate that functions of Dax1 and Nanog are partially complementary but cannot replace each other, even though they are overexpressed.

Figure 6: Functions of Dax1 and Nanog are partially complementary but they cannot replace each other. (a) qRT–PCR to measure expression of pluripotency and ExEn markers in the indicated lines after 5 days culture with LIF. All data are normalized to Gapdh and shown relative to WT ESCs (set at 1.0). Data are means±s.d.; n=3. (b) Immunofluorescence analysis of Gata6 in the indicated lines. Monolayer cultures were treated with RA (0.1 μM) for 4 days and counterstained with DAPI. Scale bar, 100 μm. Full size image

Collectively, these data suggest that Nanog is not a downstream effector of Dax1, and vice versa. These two factors most likely function in a parallel and cooperative manner to maintain pluripotency.

Dax1 is indispensable for self-renewal of Nanoglow ESCs

ESCs have been reported to fluctuate between a Nanoghigh and a Nanoglow state15. In contrast, Dax1 was uniformly expressed in ESCs (Supplementary Fig. 7a). Nanog KD could induce Gata6 expression in Dax1+ cells, whereas Dax1 KD induced Gata6 derepression mainly in Nanog− cells and in only a minority of Nanog+ cells (Supplementary Fig. 7b–d). In addition, fluorescence-activated cell sorting (FACS)-sorted Nanoglow ESCs had reduced expression of Klf4 and Esrrb but relatively normal expression of Oct4 and Dax1 (Supplementary Fig. 7e). These results are consistent with the previous findings that Klf4 and Esrrb are direct downstream targets of Nanog33,34, and suggest that Dax1, but not Klf4 or Esrrb, is required for preventing Nanoglow ESCs from differentiating. To confirm this, we established stable Dax1 KD in Nanog:GFP reporter ESCs (Supplementary Fig. 7f).

In agreement with previous reports15, GFPhigh (green fluorescent protein) and GFPlow populations sorted from control (Luc KD) cells were interconvertible, although GFPlow cells showed obviously reduced ability of self-renewal and proliferation (Fig. 7). Comparatively, Dax1 KD-Nanog:GFPhigh populations exhibited a higher self-renewal efficiency than Luc KD-Nanog:GFPlow cells. However, upregulated ExEn marker expression as well as reduced colony-forming and proliferation abilities were observed in contrast to control Nanog:GFPhigh cells (Fig. 7b–g), indicating a relatively increased differentiation propensity in Dax1 KD-Nanoghigh cells. In contrast, Dax1 KD-Nanog:GFPlow populations had more dramatically upregulated ExEn transcripts and were hardly expanded (Fig. 7b–g), indicating that Dax1 KD-Nanoglow cells cannot survive and/or self-renew. Accordingly, Dax1 KD-GFPhigh cells regenerated GFPlow cells, but GFPlow cells could not give rise to GFPhigh cells (Fig. 7a,f). These data suggest that Dax1 is not only essential but also sufficient for stabilizing ESC pluripotency during the dynamic transition between Nanoghigh and Nanoglow states.

Figure 7: Dax1 is indispensable for self-renewal of Nanoglow ESC. (a) Luc KD- and Dax1 KD-Nanog:GFP cells were sorted into Nanog:GFPlow and Nanog:GFPhigh populations (day 0). Cells were cultured in the presence of LIF for 6 days and FACS analyses were repeated. Number given is the percentage of cells in each of the indicated gates. Immunoblot analyses confirmed that GFP fluorescence reflected Nanog expression and Dax1 was efficiently silenced in both sorted Dax1 KD-Nanog:GFP populations. (b) qRT–PCR to measure expression of ExEn markers in the indicated FACS-purified cells. All data are normalized to Gapdh and shown relative to the mean of Luc KD-Nanog:GFPhigh cells (set at 1.0). Data are means±s.d.; n=3. (c) The indicated FACS-sorted cells (1 × 103 cells per cm2 in 12-well plates) were cultured for 6 days with LIF and stained for AP. (d) Percentage of colony types formed by cells shown in c. Data are means±s.d.; n=3. NA, not available. (e) Total colony number counts after plating the indicated FACS-sorted cells at a density of 1 × 103 cells per cm2 in 12-well plates and culturing for 6 days with LIF. Data are means±s.d.; n=3. (f) Morphology and GFP fluorescence of the indicated FACS-sorted cells cultured with LIF. Scale bar, 100 μm. (g) Growth curves of the indicated FACS-sorted cells cultured with LIF. Diff., differentiated; Undiff., undifferentiated. Full size image

Dax1 is required for full somatic cell reprogramming

Given the importance of Dax1 in pluripotency maintenance, we then asked whether it is necessary for efficient somatic cell reprogramming. Dax1 expression was absent in mouse embryonic fibroblasts (MEFs) but upregulated during iPSC generation alongside Nanog, Sox2 and Rex1 (Fig. 8a; Supplementary Fig. 8a). It has been reported that Dax1 is not in the original reprogramming factor cocktail Oct4, Sox2, Klf4 and c-Myc (OSKM)3. Moreover, addition of Dax1 to this quartet has not been shown to increase efficiencies39. However, whether Dax1 is, like Nanog40, required for attaining pluripotency at the final stage of the reprogramming process is unclear. Thus, we introduced Dax1 RNA interference lentivirus into Oct4-GFP MEFs41 together with OSKM reprogramming factors (Fig. 8b).

Figure 8: Dax1 and Nanog are both required for full reprogramming to induce pluripotency. (a) qRT–PCR analysis of the endogenous Rex1, Nanog and Dax1 mRNA expressions during the course of reprogramming. All data are normalized to Gapdh and shown relative to the mean of MEFs (set at 1.0). Data are means±s.d.; n=3. (b) Scheme and strategy for functional studies of Dax1 and Nanog in reprogramming. (c) Oct4-GFP MEFs were infected with the TetO-4F2A encoding Oct4, Sox2, Klf4 and c-Myc (OSKM) and M2rtTA lentiviruses in combination with luciferase shRNA (Luc KD), Dax1 shRNA (Dax1 KD) or Nanog shRNA (Nanog KD) lentivirus. The number of AP+ colonies was counted 18 days after induction. Data are means±s.d.; n=3. (d) Flow cytometric analysis of Oct4-GFP reporter activity and SSEA-1 expression in the respective lentiviral-infected Oct4-GFP MEF-derived cells 18 and 24 days after induction. Numbers in quadrants indicate the percentage of each population. (e) qRT–PCR analysis of the endogenous Sox2 and Rex1 mRNA expression in the respective lentiviral-infected Oct4-GFP MEF-derived cells 24 days after induction. All data are normalized to Gapdh and shown relative to the mean of OSKM+Luc KD cells (set at 1.0). Data are means±s.d.; n=3. (f) Immunofluorescence analysis of Oct4-GFP (green) and Nanog (red) in the indicated lentiviral-infected Oct4-GFP MEF-derived cells 24 days after induction. Cells were counterstained with DAPI (blue). Scale bar, 50 μm. d, day. Full size image

In our experiment, cell colonies started to emerge at day 9 post infection. After AP staining at day 18, no differences were found between Luc KD and Dax1 KD, as well as Nanog KD MEFs (Fig. 8c). As described previously42, rarer cells were GFP positive at this stage, indicating incomplete induced reprogramming (Fig. 8d). After replating onto gelatin-coated dishes and inducing for an additional 6 days, an estimated 25.4% of control cells were GFP positive. But in Dax1 KD cells, this ratio was <3.2%, which was approximate to that of Nanog KD cells (Fig. 8d). A significant decrease in the number of Nanog/Oct4-GFP-positive colonies and a marked reduced endogenous Sox2 and Rex1 expression were observed in both Dax1 KD and Nanog KD MEF-derived cells (Fig. 8e,f). Notably, these cells were SSEA-1 positive and proliferation was not adversely affected (Fig. 8d; Supplementary Fig. 8b). A similar reprogramming block was also observed when we used a clonal line of Oct4-GFP MEF-derived pre-iPSCs. This cell line expressed SSEA-1 and was able to spontaneously give rise to fully reprogrammed cells after passaging43. As Supplementary Fig. 8c–e showed, SSEA-1 expression was unchanged in both Dax1 KD and Nanog KD pre-iPSCs, but the GFP-positive cells appeared at a significantly lower frequency after prolonged culture, in comparison with control pre-iPSCs. These data suggest that Dax1 is not required for the initiation phase but may play a role in acquiring the authentic pluripotency at the final stage of somatic cell reprogramming.

Relationship of Dax1 with other ESC regulators

Activation of STAT3, a key downstream transcription factor of the LIF/gp130 pathway, is sufficient and necessary for self-renewal in ESCs44. Dax1 OE can maintain ESC self-renewal without LIF, but STAT3 activation was affected by neither Dax1 OE nor Dax1 KD in the presence or absence of LIF (Fig. 9a). Suppression of the extracellular signal-regulated kinase (ERK) pathway, which is also activated by LIF signalling, promotes self-renewal of ESCs45. Total ERK and ERK phosphorylation were shown to be indistinguishable in WT, Dax1 OE or Dax1 KD cells (Fig. 9a). Therefore, Dax1 OE ESC self-renewal in LIF-independent cultures is an unlikely consequence of directly activating STAT3, or suppression of the ERK pathway.

Figure 9: Relationship of Dax1 with other ESC regulators. (a) Immunoblot analysis of phospho-STAT3, total STAT3, phospho-ERK and total ERK in the indicated cells after treatment with or without LIF for 12 h. (b) qRT–PCR analysis of the Dax1 mRNA expression in the Oct4 KD (left) or Oct4 OE (right) vector-transfected ESCs at 12, 24 and 36 h after transfection. All data are normalized to Gapdh and shown relative to the mean of untreated cells (0 h; set at 1.0). Data are means±s.d.; n=3. (c) ChIP-qPCR analysis of Oct4 occupancy at Dax1 locus. Numbered grey bars indicate primer locations (upper). ESCs were transfected with 3Flag-tagged Oct4 expression vector or the 3Flag-empty vector as a control. ChIP was performed using anti-Flag antibody and qPCR analysis was performed with the primers indicated. Values are expressed as percent of input DNA (lower). Data are means±s.d.; n=3. (d) ChIP-qPCR analysis of Dax1 occupancy at Oct4 locus. Data are means±s.d.; n=3. (e) Immunoblot analysis of Oct4 and Dax1 protein levels in WT, Dax1 OE, Oct4 KD and Oct4 KD/Dax1 OE (Dax1 OE ESCs transduced with Oct4 shRNA lentivirus) cells after 5 days of culture with LIF. β-Actin was used as an internal control. (f) AP staining of colonies formed by plating the indicated cells at clonal density and culturing for 6 days with LIF (upper). Scale bar, 100 μm. Percentage of colony types formed by cells is shown (lower). Data are means±s.d.; n=3. (g) qRT–PCR analysis of gene expression in Luc KD, Oct4 KD and Oct4 KD/Dax1 OE cells after 5 days of culture with LIF. All data are normalized to Gapdh and shown relative to WT ESCs (set at 1.0). Data are means±s.d.; n=3. *P<0.05; **P<0.01; ***P<0.001. All P values were calculated using Student’s t-test. Diff., differentiated; Undiff., undifferentiated. Full size image

Oct4 is a master regulator for ESC pluripotency. Dax1 KD or OE had no significant effect on Oct4 expression, whereas Oct4 KD reduced Dax1 and Oct4 OE increased Dax1 mRNA levels as early as 12 h post transfection (Fig. 9b). ChIP-qPCR analysis showed that Oct4 bound to the promoter of Dax1, whereas the enrichment of Dax1 on the Oct4 gene locus (spanning from ~5 kb upstream to 2 kb downstream) was not observed (Fig. 9c,d). These results and those of previous reports46 support that Dax1 is a direct downstream target of Oct4.

We then examined the functional relationship between Dax1 and Oct4. Although forced expression of Dax1 conferred LIF-independent self-renewal of ESCs, Dax1 OE could not prevent differentiation induced by Oct4 KD, as shown by colony-forming assays (Fig. 9e,f). These data indicate that Oct4 persistence is required for Dax1-mediated self-renewal. Nevertheless, Dax1 OE could partially rescue Oct4 KD-induced upregulation of ExEn and TE markers’ expression (Fig. 9g), suggesting that the function of Oct4 in the maintenance of pluripotency is at least partly mediated by Dax1.

It has been reported that Dax1 binds to Oct4 and inhibits its transcriptional activity in ESCs, which in turn induces TE differentiation26. Consistently, our earlier findings showed that Dax1 OE led to an increased expression of TE markers (Fig. 3b). However, we also found that Dax1 KD induced the derepression of Cdx2 and Eomes without altering Oct4 expression (Fig. 1f,g) and, moreover, Dax1 OE rescued Oct4 KD-induced upregulation of these transcription factors to a large extent (Fig. 9g). These data therefore suggest that Dax1 can inhibit TE differentiation independently or as a downstream effector of Oct4.