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EMBRYONIC STEM CELLS/INDUCED PLURIPOTENT STEM CELLS Enforced Expression of Mixl1 During Mouse ES Cell Differentiation Suppresses Hematopoietic Mesoderm and Promotes Endoderm Formation SUE MEI LIM, LLOYD PEREIRA, MICHAEL S. WONG, CLAIRE E. HIRST, BENJAMIN E. VAN VRANKEN, MARJORIE PICK, ALAN TROUNSON, ANDREW G. ELEFANTY, EDOUARD G. STANLEY Monash Immunology and Stem Cell Laboratories, Monash University, Clayton, Victoria, Australia Key Words. Embryonic stem cells • Mixl1 • Endoderm • Mesoderm

ABSTRACT The Mixl1 gene encodes a homeodomain transcription factor that is required for normal mesoderm and endoderm development in the mouse. We have examined the consequences of enforced Mixl1 expression during mouse embryonic stem cell (ESC) differentiation. We show that three independently derived ESC lines constitutively expressing Mixl1 (Mixl1C ESCs) differentiate into embryoid bodies (EBs) containing a higher proportion of E-cadherin (E-Cad)ⴙ cells. Our analysis also shows that this differentiation occurs at the expense of hematopoietic mesoderm differentiation, with Mixl1C ESCs expressing only low levels of Flk1 and failing to de-

velop hemoglobinized cells. Immunohistochemistry and immunofluorescence studies revealed that Mixl1C EBs have extensive areas containing cells with an epithelial morphology that express E-Cad, FoxA2, and Sox17, consistent with enhanced endoderm formation. Luciferase reporter transfection experiments indicate that Mixl1 can transactivate the Gsc, Sox17, and E-Cad promoters, supporting the hypothesis that Mixl1 has a direct role in definitive endoderm formation. Taken together, these studies suggest that high levels of Mixl1 preferentially allocate cells to the endoderm during ESC differentiation. STEM CELLS 2009;27:363–374

Disclosure of potential conflicts of interest is found at the end of this article.

INTRODUCTION Mixl1 belongs to the Mix/Bix family of paired-like homeobox genes, which have been shown to play a role in the formation and/or specification of mesoderm and endoderm in Xenopus, zebrafish, and mice [1–12]. The founding member of this family, Mix.1, was identified in Xenopus as an immediate early response gene to activin induction in Xenopus animal pole explants [6] and subsequently as a key mediator of bone morphogenetic protein 4 signaling [13]. Experiments in Xenopus showed that Mix.1 could respecify dorsal mesoderm to a ventral fate and that overexpression of an engrailed repressor–Mix.1 fusion protein (Mix.1ENR) resulted in defective endoderm formation [5, 13, 14]. The role of Mix.1 in endoderm formation was further supported by the work of Lemaire and colleagues, who showed that overexpression of Mix.1 in the ventral marginal zone of Xenopus embryos repressed dorsal and ventral mesoderm differentiation and, in concert with the transcription factor Siamois, promoted the expansion of endoderm [5].

Mix-related genes have also been identified in zebrafish and are expressed in the marginal blastomeres and/or yolk syncytial layer, regions believed to be the functional equivalent of mouse visceral endoderm [1, 15]. In zebrafish, bonnie and clyde (bon) regulates endoderm formation and functions downstream of Nodal signaling, with bon mutants exhibiting a reduction in the number of Xsox17⫹ endodermal precursors and abnormal gut development [4]. The zebrafish gene Mixer (zMixer) has an endodermally restricted expression pattern, and promoted endodermal gene expression when ectopically overexpressed [1, 4]. In contrast with the multiple Mix/Bix-related genes found in Xenopus and zebrafish, only one Mix-related gene, Mixl1 has been identified in the mouse and human [8, 9]. In the mouse, transient Mixl1 transcripts have been detected in the proximal visceral endoderm of the pregastrula embryo. During gastrulation, expression is observed in the primitive streak and emerging mesendoderm, and becomes restricted to the posterior primitive streak by the head-fold/neurula stage [8, 9, 12]. By embryonic day 9.5 (E9.5), expression was substantially diminished, retracting caudally to the tail bud region [8, 12]. Whole-mount in situ hybridization experiments of two to three and five to six somite-stage embryos showed Mixl1 expression in the

Author contributions: S.M.L.: conception and design, collection and/or assembly of data, data analysis and interpretation, manuscript writing, final approval of manuscript; L.P.: collection and/or assembly of data, data analysis and interpretation, manuscript writing; M.W.: collection and/or assembly of data, data analysis and interpretation; C.H.: data analysis and interpretation; B.V.V.: collection and/or assembly of data; M.P.: collection and/or assembly of data, data analysis and interpretation; A.T.: financial support; A.G.E.: conception and design, financial support, collection and/or assembly of data, data analysis and interpretation, manuscript writing, final approval of manuscript; E.G.S.: Conception and design, financial support, collection and/or assembly of data, data analysis and interpretation, manuscript writing, final approval of manuscript; A.G.E. and E.G.S. contributed equally to this work. Correspondence: Ed Stanley, Ph.D., Monash Immunology and Stem Cell Laboratories, Level 3, Building 75, STRIP1, West Ring Road, Monash University, Clayton, Victoria, 3800, Australia. Telephone: 61-39905-0651; Fax: 61-39905-0680; e-mail: [email protected]. edu.au Received October 13, 2008; accepted for publication November 12, 2008; first published online in STEM CELLS EXPRESS November 26, 2008. ©AlphaMed Press 1066-5099/2009/$30.00/0 doi: 10.1634/stemcells.2008-1008

STEM CELLS 2009;27:363–374 www.StemCells.com

Mixl1 Modulates Mesoderm and Endoderm Formation

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developing hindgut, particularly the posterior intestinal portal and the midgut [12]. Gene-targeting experiments in the mouse have confirmed that Mixl1 is a crucial regulator of axial patterning and morphogenesis [10]. Mixl1-null (Mixl1⫺/⫺) embryos displayed an enlarged primitive streak, and subsequently exhibited multiple developmental abnormalities in axial morphogenesis and formation of definitive endoderm [10]. Chimera analysis showed that Mixl1⫺/⫺ embryonic stem cells (ESCs) had a cell autonomous defect in their ability to colonize gut endoderm [10]. More recent work suggests that Mixl1 is required for the movement of prospective endoderm away from the primitive streak, a function that is independent of cell movements within the mesoderm [16]. Collectively, these findings implicate Mixl1 in the formation of definitive endoderm as well as mesoderm and suggest that mouse Mixl1 functions similarly to Mix.1 during Xenopus development [10]. Indeed, injection of RNA encoding mouse Mixl1 into Xenopus embryos was sufficient to activate expression of several endodermal markers in animal cap explants [12]. ESCs represent a tractable system for analyzing gene function during the early stages of lineage specification and commitment. In serum-containing cultures, differentiating ESCs pass through a series of developmental milestones that mirror those traversed by cells within the embryo [17, 18]). Gene profiling experiments indicate that differentiating ESCs sequentially express genes marking successive stages of embryonic development, including Oct-4 and Sox2 (inner cell mass), Fgf-5 (epiblast), Brachyury, Mixl1, and Gsc (primitive streak) [17]. In this respect, transient expression of Mixl1 during the in vitro differentiation of ESCs parallels primitive streak formation in vivo [19, 20]. A number of studies have sought to use differentiating ESCs as a system to examine the function of Mixl1-related genes during early lineage commitment. Using a doxycycline-inducible Mixl1 transgene, Willey and colleagues showed that ectopic induction of Mixl1 increased the frequency of hematopoietic precursors observed in day 4 (d4) embryoid bodies (EBs) [21]. Conversely, Shiraki et al. [22] found that forced expression of the chicken Mixl1 homologue during mouse ESC (mESC) differentiation induced the expression of endodermal-associated genes such as HNF3␣, Ihh, GATA-4, GATA-6, and Sox17. In the current study, we investigated the effect of constitutive Mixl1 expression on the emergence of mesodermal and endodermal precursors during mouse ESC differentiation. We found that ESCs constitutively expressing Mixl1 (Mixl1C ESCs) failed to generate hematopoietic cells during serum-induced differentiation. Instead, differentiating Mixl1C ESCs yielded a higher frequency of FoxA2⫹ E-cadherin (E-Cad)⫹ cells, suggesting that enforced Mixl1 expression promoted endoderm formation. The appearance of endodermal cell types was preceded by the increased expression of Gsc, a marker of anterior primitive streak derivatives, including presumptive definitive endoderm [23]. Gel mobility shift and luciferase reporter assays indicated that Mixl1 could bind to and activate transcription from the Gsc promoter. Taken together, our results suggest that the level of Mixl1 expression influences the allocation of cells between mesoderm and endoderm during the differentiation of ESCs in vitro, with high levels of Mixl1 favoring endoderm formation.

vector pEFBOS-IRES-GFPNeo (Genbank EF459695). The pEFBOSMixl1-IRES-GFPNeo expression vector was constructed by inserting the XhoI-SalI cDNA fragment, encoding mouse Mixl1 (Genbank AF154573) into pEFBOS-IRES-GFPNeo. The expression vector was linearized with PvuI and electroporated into an ESC line (W9.5) [25], and G418-resistant colonies were isolated as described elsewhere [26]. Forty-eight GFP⫹ clones were differentiated as previously described [19] for 4 days to ascertain which clones maintained GFP expression. Subsequently, three independent clones that maintained the highest level of GFP expression at differentiation d4, Mix.17, Mix.33, and Mix.35, were selected for further analysis. Mixl1C ESCs were maintained in the presence of 200 ␮g/ml G418. Control ESCs were the parental line W9.5 and two cloned derivatives that expressed GFP either from a ␤-actin promoter transgene or the Pdx1 locus [27]. ESCs were maintained in serum-containing media as described elsewhere [26].

Reverse-Transcription Polymerase Chain Reaction Analysis RNA preparation and reverse-transcription polymerase chain reaction (RT-PCR) gene expression analysis of differentiating mouse ESCs was performed as described previously [19]. Oligonucleotide primer pairs additional to those previously described [9, 19] are provided in supporting information Table 1. For semiquantitative studies, the template input was standardized relative to Hprt controls as described previously [28].

Intracellular Flow Cytometry and Western Blotting Western blotting and intracellular flow cytometry using the antiMixl1 antibody 6G2 were performed as previously described [29]. Catalog numbers and dilutions used for each antibody are provided in supporting information Table 2.

Flow Cytometry Analysis Cells were prepared for flow cytometry and labeled with primary antibodies as described previously [19]. Rat anti-E-Cad antibodies (ECCD-2) (Zymed Laboratories, Inc., South San Francisco, CA, http://www.zymed.com) were detected using an allophycocyanin (APC)-conjugated goat anti-rat IgG antibody (BD Pharmingen, San Diego, http://www.bdbiosciences.com/index_us.shtml). Rat antiFlk1 antibodies (BD Pharmingen) were detected using an APCconjugated goat anti-rat IgG antibody (BD Pharmingen). This was costained with a phycoerythrin-conjugated mouse anti-c-kit (CD117) antibody (BD Pharmingen). Mouse anti-surface specific embryonic antigen 1 (SSEA1) (Chemicon, Temecula, CA, http:// www.chemicon.com) was detected with a phycoerythrin-conjugated goat anti-mouse IgG antibody (BD Pharmingen). Dilutions of antibodies are listed in supporting information Table 2.

ESC Colony-Forming Self-Renewal Assay Control and Mixl1C ESC lines were differentiated in serum-containing medium for 6 days at a density of 2 ⫻ 103 cells/ml. At d0, d4, and d6, EBs were trypsinized to form a single-cell suspension and then seeded at a density of 1 ⫻ 104 cells/well of a six-well tissue culture dish (BD Pharmingen) in ESC growth medium [26]. After 6 days of culture, colonies were fixed in 4% paraformaldehyde (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com), washed with phosphate-buffered saline, and stained for alkaline phosphatase (AP) activity using BMPurple according to the manufacturer’s instructions (Roche Diagnostics, Basel, Switzerland, //www.rocheapplied-science.com).

Endoderm Colony-Forming Assays

MATERIALS

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METHODS

Expression Vectors and ESC Lines The mammalian expression vector pEFBOS [24] was modified to include an internal ribosomal entry site (IRES) green fluorescent protein–neomycin resistance (GFPNeo) fusion gene to give the

Control and Mixl1C ESCs were differentiated in serum-containing medium for 6 days at a cell density of 2 ⫻ 103 cells/ml. At d6, mouse EBs were disaggregated into a single cell suspension and recultured at 1 ⫻ 105 cells/well of a six-well tissue culture dish (BD Pharmingen) in ␣-modified Eagle’s medium (Invitrogen, Carlsbad, CA, //www.invitrogen.com) supplemented with 10% fetal calf serum (HyClone, Logan, UT, //www.hyclone.com) and 50 ␮M 2-mercaptoethanol on type I collagen-coated dishes (Sigma). Six days

Lim, Pereira, Wong et al. later, cell morphology was analyzed following Giemsa staining or indirect immunofluorescence.

Hematopoietic Colony Assay of Differentiated mESCs Hematopoietic colony formation was assayed by combining 3 ⫻ 104 dissociated EB cells with 0.5 ml of MethoCult (Stem Cell Technologies, Vancouver, BC, Canada, //www.stemcell.com) and plating this mixture into each well of a 24-well tissue culture plate. The MethoCult medium was supplemented with the following growth factors: 5 ng/ml vascular endothelial growth factor (VEGF) (R&D Systems Inc., Minneapolis, //www.rndsystems.com), 50 ng/ml stem cell factor (SCF) (R&D systems), 1% of a supernatant from a cell line producing mouse interleukin (IL)-3 [30], and 5 U/ml of erythropoietin (Epo) (PeproTech, Rocky Hill, NJ, //www. peprotech.com). Hematopoietic colonies were scored after 10 days of methylcellulose culture.

Immunohistochemical and Immunofluorescent Cell Staining Immunohistochemical labeling of EB paraffin sections was performed as previously described [31]. Antibody specificity was verified by staining histological sections of appropriately staged mouse embryos (supporting information Fig. 8). Dilutions of FoxA2 and Sox17 antibodies are described in supporting information Table 3. Immunofluorescence labeling was performed as described in supporting information.

Luciferase Assays For luciferase assays, Balb/c 3T3 cells or C2C12 cells were cultured in 24-well plates for 24 hours prior to transfection. Transfections were performed with FuGENE six reagent as described by the manufacturer (Roche). pGL3 reporter constructs contained the luciferase gene linked to genomic fragments from Gsc (0.8 kb), Sox17 (2.0 kb), E-Cad (2.0 kb), FoxA2 (2.0 kb), or Cer-l (2.4 kb). In each case, genomic fragments represented sequences of the specified length immediately upstream from the start codon. These were cotransfected with 0, 25, 100, and 400 ng of a pEFBOS encoding FLAG-tagged mouse Mixl1. Transfection of phRTKluc (Renilla) served as a transfection control and was used to normalize luciferase activity. At 48 hours post-transfection, cells were lysed and assayed for firefly and Renilla luciferase activity as specified by the manufacturer (Promega, Madison, WI, //www.promega.com). All data shown represent an average of at least three experiments performed in triplicate.

Electrophoretic Mobility Shift Assays Electrophoretic mobility shift assays (EMSAs) were performed with 5,000 cpm of 32P-labeled double-stranded Gsc DNA probes [32]. The labeled probes were incubated with purified His-Mixl1 (supporting information) protein in a total volume of 20 ␮l containing 15 mM HEPES-KOH, pH 7.9, 60 mM KCl, 7.5% glycerol, 0.05% TX-100, 50 ␮g/ml poly [d(I-dC)], and 0.25 mg/ml bovine serum albumin for 20 minutes at room temperature. For the supershift experiments, the antibody against Mixl1 (6G2) [29] was added following DNA-protein binding and incubated for 10 minutes at room temperature. The binding reactions were resolved on a 6% nondenaturing polyacrylamide/bisacrylamide (29:1) gel buffered with 0.5⫻ Tris/borate/EDTA and run at 250V for 2–3 hours. Gels were dried and bands visualized by Phosphoimager analysis (Typhoon Trio, GE Healthcare, http://www.gelifesciences.com).

RESULTS Constitutive Expression of Mixl1 in Differentiating mESCs To examine the effect of enforced Mixl1 expression on the emergence of mesodermal and endodermal precursors during www.StemCells.com

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mESC differentiation, we generated Mixl1C ESCs using a vector in which the Mixl1 cDNA was linked by an IRES to a GFPNeo fusion gene (Fig. 1A). The vector directed transcription of a single bicistronic mRNA from the elongation factor 1␣ (EF1␣) promoter, from which both the Mixl1 and GFPNeo proteins were translated. Hence, culturing the cells in G418 simultaneously selected for cells expressing both GFPNeo and Mixl1. This vector was transfected into W9.5 ESCs, and three independent G418-resistant ESC clones that expressed high levels of GFP (Mix.17, Mix.33, and Mix.35) were selected for further analysis. In their undifferentiated state, Mixl1C ESC clones displayed uniform GFP expression and maintained a normal ESC morphology (Fig. 1B). Both control ESCs and Mixl1C ESCs expressed robust levels of the stem cell marker Oct-4 by immunofluorescence, whereas only Mixl1C ESCs expressed Mixl1 protein in undifferentiated cells (Fig. 1C, supporting information Figs. 3, 4). Western blot analysis of the nuclear and cytoplasmic fractions of undifferentiated Mixl1C ESCs confirmed the nuclear localization of exogenous Mixl1, as was previously reported for the endogenous Mixl1 protein [29] (Fig. 1D). Flow cytometric analysis of undifferentiated ESCs demonstrated a good correlation between the frequency of GFP⫹ cells (⬎90%) and the proportion of cells expressing the Mixl1 protein (⬎95%) for each of the three independent Mixl1C ESC lines (Fig. 1E). These experiments validated the use of GFP expression as a surrogate marker for exogenous Mixl1 expression in the Mixl1C ESCs. This analysis also confirmed the absence of Mixl1 expression in the parental control W9.5 ESCs. Overall, these results indicate that Mixl1C ESCs robustly express Mixl1 protein yet maintain characteristics of undifferentiated control ESCs. We then determined if transgene expression was maintained during the course of ESC differentiation. RT-PCR analysis of samples collected during differentiation time course experiments demonstrated that exogenous Mixl1 levels were sustained over the period during which endogenous Mixl1 was normally expressed (Fig. 1F, G). Furthermore, this analysis showed that a wave of endogenous Mixl1 expression still occurred in cultures of differentiating Mixl1C ESCs. However, when GFP expression was examined in differentiating Mixl1C ESCs on a per cell basis by flow cytometry, it was observed that the frequency of GFP-expressing cells remained at ⬃90% until d3 and then gradually decreased such that by d9 of differentiation, only ⬃25% of the cells were GFP⫹ (Fig. 1H). Similar expression patterns were observed for the other two Mixl1C ESC clones (Mix.35 and Mix.17) (data not shown). The reason for the reduction in the frequency of transgene-expressing cells is not clear.

Persistence of Undifferentiated Cells in Cultures of Differentiating Mixl1C ESCs Interestingly, PCR analysis suggested that the expression of genes associated with undifferentiated ESCs (Oct-4, Rex1, and Nanog) or epiblasts (Fgf-5) remained at higher levels in differentiating Mixl1C ESCs than in control cultures, even as late as d9 (Fig. 2A, B, supporting information Fig. 2A, B). Given this observation, we tested whether Mixl1C ESC-derived EBs retained a higher proportion of undifferentiated cells than control lines. In order to address this possibility, cultures of undifferentiated ESCs and d4, d6, and d9 EBs were dissociated and single cells were seeded into culture conditions routinely used to support the propagation of undifferentiated ESCs. Six days later, the plates were fixed and scored for the presence of AP⫹ colonies with ESC-like morphology. Colonies derived from undifferentiated ESCs for both control and Mixl1C ESC lines

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Figure 1. Characterization of Mixl1-overexpressing mouse embryonic stem cells (ESCs). (A): Schematic of the Mixl1 expression vector comprised of an elongation factor 1␣ (EF1␣) promoter, the Mixl1 cDNA linked by an internal ribosomal entry site (IRES) to a green fluorescent protein-neomycin resistance fusion gene (GFPNeo), and the bovine growth hormone polyadenylation signal (Poly A). (B): Bright-field, fluorescence (GFP), and overlay (Merge) images of Mixl1C ESC colonies (upper row) showing uniform GFP expression and higher power images of an individual colony (lower row) displaying typical ESC morphology. Original magnifications: upper row: ⫻50; lower row: ⫻100. (C): Immunofluorescence analysis of control (upper row) and Mix.17 ESCs (lower row) showing expression of Oct-4 (left panels) and Mixl1 (right panels). Original magnification: ⫻400. (D): Western blot analysis showing localization of Mixl1 protein to the nuclear (N) but not cytoplasmic (C) fraction of three Mixl1C ESC lines, Mix.17, Mix.33, and Mix.35. Undifferentiated parental W9.5 ESCs served as a negative control and a whole-cell lysate (W/C) of 293T cells transfected with a Mixl1 expression vector served as a positive control. The lower panel shows the level of ␤-actin in each sample (loading control). (E): Flow cytometric analysis of undifferentiated Mixl1C ESCs showing the strong correlation between GFP expression (upper panels) and intracellular Mixl1 protein (lower panels). The white peak in each panel represents GFP fluorescence and Mixl1 expression in W9.5 control ESCs. The percentage of cells falling within the region indicated by the horizontal bar is shown. (F): Semiquantitative reverse transcription-polymerase chain reaction (PCR) analysis of exogenous and endogenous Mixl1 expression in control (W9.5) and Mixl1C ESCs (Mix.35) over a 9-day differentiation time course. Hprt controls used to standardize the amount of template in each reaction are shown in supporting information Figure 1. NT, no template control. (G): Real-time quantitative PCR analysis showing sustained and elevated levels of Mixl1 transcripts in differentiating Mixl1C ESCs (black bars) and transient expression of endogenous Mixl1 in control ESCs (white bars) (n ⫽ 3; *p ⬍ .05). (H): Time course of GFP expression in differentiating W9.5 control (top row) and Mix.33 Mixl1C ESCs (bottom row) as determined by flow cytometry. The position of the flow cytometry gates were set against GFP fluorescence associated with W9.5 control ESCs, and the percentage of GFP⫹ cells is shown in the lower right of each plot.

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Figure 2. Perturbed differentiation of embryonic stem cells constitutively expressing Mixl1 (Mixl1C ESCs). (A): Semiquantitative polymerase chain reaction analysis showing persistent Oct-4 and Nanog expression in day 9 (d9) Mixl1C ESCs. (B): Graphical summary of densitometry analysis of the images shown in (A) demonstrated a trend towards persistent Oct-4 expression in Mixl1C ESCs (n ⫽ 3; d9, p ⫽ .07) and that Nanog expression remained significantly higher in Mixl1C ESCs (n ⫽ 3; d9: *p ⫽ .003). Error bars represent the standard error of the mean (SEM) from triplicate experiments. Relative gene expression was calculated as the ratio of the value obtained for the test gene relative to the value obtained for Hprt (supporting information Fig. 1). (C): Morphology of alkaline phosphatase (AP)⫹ colonies arising from d0 and d4 cultures of control (W9.5) and Mixl1C ESCs (Mix.35). (D): Histogram showing the average number of ESC-like colonies obtained from the replating of cells from d6 embryoid bodies derived from the three Mixl1C ESC lines and control ESC lines. Error bars represent the SEM from triplicate experiments (p ⫽ .051).

had well-defined boundaries and displayed strong AP expression (Fig. 2C, supporting information Fig. 5). When control cell EBs were dissociated at d4 and recultured for a further 6 days, the diffuse AP staining, flattened morphology, and lack of defined cell boundaries observed in the resultant colonies indicated that most cells in the EBs had started to differentiate and there were no longer cells present that could initiate a colony of undifferentiated ESCs (Fig. 2C). However, cells recultured from Mixl1C EBs (Fig. 2C, lower panel, supporting information Fig. 5) had an increased propensity to form colonies with a less differentiated phenotype that retained strong AP expression. This distinction was most evident when d6 EBs were dissociated and replated into ESC maintenance conditions (Fig. 2D, Mixl1C versus control ESCs; p ⫽ .051, n ⫽ 3). This trend was also observed in cultures seeded with cells derived from d4 and d9 EBs (supporting information Fig. 5). Overall, these experiments suggest that enforced Mixl1 expression perturbed ESC differentiation by maintaining stem cell-associated markers and inwww.StemCells.com

creasing the probability that undifferentiated cells persisted in later stage EBs.

Constitutive Expression of Mixl1 Suppresses Hematopoietic Differentiation The most obvious phenotype observed during the differentiation of Mixl1C EBs was their smaller size and the absence of overtly hemoglobinized cells at later differentiation time points (Fig. 3A). In order to quantify the effect of Mixl1 misexpression on hematopoiesis, differentiated Mixl1C ESCs were analyzed by flow cytometry for the expression of Flk1 and c-kit, two cell surface markers previously used to enrich for hematopoietic progenitors [21]. Analysis of differentiating control ESCs (Fig. 3B) revealed that a distinct population of Flk1⫹ cells had emerged by d5, ⬃30% of which also expressed c-kit. By d7, most of the Flk1⫹ cells were c-kit⫹, consistent with the emergence of mesoderm with hematopoietic activity [21]. Marked differences between the control and Mixl1C ESC lines were apparent by differentiation d5, with

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Figure 3. Hematopoietic differentiation is suppressed in embryonic stem cells constitutively expressing Mixl1 (Mixl1C ESCs). (A): Appearance of control and Mixl1C embryoid bodies (EBs) at differentiation day 7 (d7) showing the small size and absence of overt hemoglobinization in the latter. Original magnification: ⫻100. (B): Flow cytometric analysis of differentiating control and Mixl1C ESCs shows EBs overexpressing Mixl1 generate few Flk1⫹ cells. Flow cytometry gates were set relative to isotype controls (supporting information Fig. 6). The percentage of cells falling into each quadrant is indicated. (C): Summary of flow cytometric analysis showing the proportion of Flk1-expressing cells in control W9.5 ESCs (white bars) and Mixl1C ESCs (black bars) at d0, d5, and d7. Error bars show the standard error of the mean (SEM) for triplicate experiments (n ⫽ 3; *p ⬍ .01 for control versus Mixl1C ESCs). (D): Morphology of colonies derived following 10-day methylcellulose culture of d7 EB cells. Phase contrast images of W9.5 control EB-derived cells showing erythroid, myeloid, and mixed hematopoietic colonies arising in methylcellulose cultures containing vascular endothelial growth factor, stem cell factor, interleukin-3, and erythropoietin. Mixl1C ESC lines failed to form hematopoietic colonies, instead predominantly forming secondary EBs. Original magnification: ⫻100. (E): Histogram displaying the total number of hematopoietic colonies (black bars) and secondary EBs (white bars) derived from control and Mixl1C ESCs in methylcellulose culture. Error bars represent the SEM for three independent experiments (n ⫽ 3; *p ⬍ .01 for hematopoietic colony-forming cells (CFCs) derived from control versus Mixl1C ESCs and for secondary EBs derived from Mixl1C versus control ESCs).

a reduced frequency of Flk1-expressing cells in Mixl1C EBs (Fig. 3B). These observations were confirmed with experiments using the two additional independently derived Mixl1C ESC clones Mix.33 and Mix.17 (Fig. 3C). In order to formally establish that constitutive expression of Mixl1 suppresses the generation of hematopoietic progenitors, the frequency of hematopoietic colony-forming cells (CFCs) in Mixl1C EBs was examined using methylcellulose cultures. d7 EBs were dissociated to form a single-cell suspension, seeded into methylcellulose cultures containing VEGF, SCF, IL-3, and Epo, and hematopoietic colonies were analyzed 10 days later (Fig. 3D, 3E). Whereas control EBs generated ⬃250 hematopoietic CFCs per 3 ⫻ 104 cells (Fig. 3E, black bars), hematopoietic CFCs were virtually absent from Mixl1C EBs at a similar stage. Instead, Mixl1C EB cells gave rise to morphologically uniform cellular spheres at a high frequency in the same combination of growth factors (Fig. 3D, 3E). Microarray analysis of gene expression of these spheres indicated that they expressed

genes associated with undifferentiated ESCs (e.g., Oct-4, Rex1, Sox2) as well as genes indicative of both mesoderm (PDGFR␣) and endoderm (Afp, Sox17, FoxA2) differentiation, supporting their classification as secondary EBs (data not shown). The presence of these secondary EBs is consistent with our earlier observations suggesting the heightened retention of multipotent cells within late differentiation stage Mixl1C ESCs.

Mixl1 Promotes Endoderm Formation Overexpression of Mixl1-related proteins in Xenopus and zebrafish embryos promotes endodermal differentiation [4, 5, 12, 14, 33]. In addition, more recent experiments with mESCs indicated that enforced expression of Mix-related family members increased the transcription of endodermal-associated genes [12]. Our analysis of E-Cad expression during differentiation of Mixl1C ESCs suggested that, at late stages, EBs contained a substantial fraction of E-Cad⫹ cells (Fig. 4A). The majority of


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Figure 4. Endodermal differentiation of embryonic stem cells constitutively expressing Mixl1 (Mixl1C ESCs). (A): Histogram plot of E-cadherin (E-Cad) expression in differentiating Mixl1C ESCs (Mix.33) and control ESCs (W9.5) as determined by flow cytometry over 9 days of differentiation. The white histograms represent staining obtained with an isotype control antibody and the red histograms show the distribution of cells labeled with rat anti-E-Cad antibodies. At day 9 (d9), E-Cad was retained at higher levels in the Mix.33 ESCs (⬃15%) than in the W9.5 ESCs (⬃2%). The percentage of cells falling within the region indicated by the horizontal bar is shown. (B): Graphical summary of flow cytometric analysis of E-Cad expression in Mixl1C ESCs (black bars) and control ESCs (white bars) over 9 days of differentiation. The proportion of E-Cad⫹ cells is expressed as the mean ⫾ standard deviation from three experiments (n ⫽ 3; *p ⬍ .05). (C): Quantitative polymerase chain reaction analysis of Gsc, FoxA2, Cer-l, Afp, and Sox17 expression in control (white bars) and Mixl1C ESCs (black bars) during 9 days of differentiation (n ⫽ 3; *p ⬍ .05). The error bars represent the standard error of the mean of three experiments for control ESCs and for three experiments with each Mixl1C ESC line. (D): Immunohistochemical analysis showing Sox17 expression largely confined to peripheral visceral endoderm-like cells of control embryoid bodies (EBs) whereas Sox17⫹ cells were observed more widely throughout the Mix.35 EBs. Antibody specificity was verified by staining histological sections of appropriately staged mouse embryos (supporting information Fig. 8). (E): Immunohistochemical analysis of FoxA2 expression in d9 control and Mixl1C EBs. In contrast to control EBs, in which FoxA2 expression was restricted to cells with an epithelial morphology, robust and widespread FoxA2 expression was observed throughout Mix.35 EBs. (F): Immunofluorescence analysis of FoxA2 (red) and E-Cad (green) expression in d9 control EBs (W9.5, top panel) and Mixl1C EBs (Mix.35, bottom panel). Nuclear stain was performed using TO-PRO-3 iodide (blue). The boxed areas in the left-most images (original magnification: ⫻400) are shown at higher magnification in the remaining images for each series. Note that FoxA2 expression is not restricted to E-Cad⫹ cells in Mixl1C EBs.

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these cells at d6 and d9 were SSEA1⫺, implying that undifferentiated ESCs or epiblast-like cells represent a minor contributor to the E-Cad⫹ population at these stages (supporting information Fig. 10). The persistence of E-Cad expression was confirmed with replicate experiments using the three independently derived Mixl1C ESC clones (Fig. 4B). Between d3 and d5, the frequency of E-Cad⫹ cells in control ESCs dropped from 90% to 10%, with only ⬃5% of cells still positive at d9. In contrast, Mixl1C ESCs displayed a delayed decline in the frequency of E-Cad⫹ cells, with ⬃20% of cells positive at d9 (Fig. 4B). Gene expression analysis indicated that d9 E-Cad⫹ cells expressed both Afp and FoxA2 (supporting information Fig. 7), consistent with the presence of endodermal cells within this population. Levels of these transcripts and those representing Gsc, Cer-l, and Sox17, genes that are also associated with endoderm formation, were all elevated in differentiating Mixl1C ESCs (Fig. 4C). Immunohistochemical analysis confirmed that the proportion of Sox17- and FoxA2-expressing cells was higher in Mixl1C EBs than in those derived from control ESCs (Fig. 4D, 4E). Whereas Sox17 was largely restricted to the nucleus of visceral endoderm-like cells surrounding control EBs, expression of this factor was more widely distributed in d9 Mixl1C EBs (Fig. 4D). Likewise, FoxA2 expression in control EBs was localized to specific regions, including visceral endoderm-like cells along the periphery and in ductal epithelial-like structures (Fig. 4E). Similar to the control EBs, FoxA2 expression in Mixl1C EBs was detected around the periphery and within ductal-like structures (Fig. 4E). However, extensive FoxA2 expression was also observed in regions of the EBs that did not coincide with epithelial-like structures (Fig. 4E). In the case of control EBs, immunofluorescence analysis indicated that FoxA2 expression was always associated with E-Cad⫹ epithelial-like cells (Fig. 4F). Conversely, morphometric analysis indicated that, in control EBs, ⬎90% of E-Cad⫹ cells expressed FoxA2 (supporting information Fig. 9). A similar analysis of Mixl1C EBs showed that ⬎80% of E-Cad⫹ cells coexpressed FoxA2, supporting the conclusion that the E-Cad⫹ populations present in both d9 control and Mixl1C EBs represent endoderm (supporting information Fig. 9). However, in contrast to control EBs, FoxA2 expression was found in both E-Cad⫹ epithelial structures and in clusters of E-Cad⫺ cells within Mixl1C EBs (Fig. 4F). This separation of FoxA2 expression into the E-Cad⫹ and E-Cad⫺ fractions was also confirmed in gene-expression studies of cells sorted on the basis of E-Cad expression (supporting information Fig. 7). Furthermore, the absence of substantial Afp and Albumin expression from the E-Cad⫺ fractions suggests that the FoxA2⫹ E-Cad⫺ cells were unlikely to represent endodermal derivatives, neither visceral nor definitive (supporting information Fig. 7, data not shown). Whereas the identity of these cells is uncertain, FoxA2⫹ ECad⫺ cells are described in the notochord, a structure that originates from anterior primitive streak mesoderm [34]. The increased proportion of endodermal cells within Mixl1C EBs may have reflected the absence of posterior mesodermal derivatives and/or the presence of an endodermal precursor with enhanced proliferative capacity. To examine this latter possibility, we disaggregated d4 and d6 Mixl1C and control EBs and then plated single-cell suspensions onto type I collagen-coated plates in serum-containing medium, conditions previously reported to allow the limited proliferation of endodermal precursors [23]. Using these conditions, we observed the emergence of epithelial-like cell colonies over a period of 6 days in cultures seeded with cells from d6 Mixl1C EBs (Fig. 5A). Similar colonies were rare in parallel cultures initiated with cells from control EBs. Examination of Giemsa-stained cultures derived from Mixl1C EBs showed cell aggregates with an epithelial-like

Mixl1 Modulates Mesoderm and Endoderm Formation morphology and a clearly defined cell– cell boundary that contrasted with the flattened morphology and prominent nuclei of cells grown from control EBs (Fig. 5B). On average, epithelial colonies were approximately 120-fold more abundant in the secondary cultures derived from Mixl1C ESCs (Fig. 5C). Immunofluorescence analysis showed that the epithelial-like cells expressed surface E-Cad whereas the larger dispersed cells expressed smooth muscle actin (SMA) (Fig. 5D). Expression of these markers was always mutually exclusive, and therefore identified two discrete cell populations. Taken together, the above data suggest that d6 Mixl1C EBs contain an increased number of E-Cad⫹ endodermal progenitors and that this translates into the higher prevalence of endodermal cell types in later stage EBs.

Mixl1 Activates the Promoters of Gsc, Sox17, and E-Cad The above experiments suggest that constitutive Mixl1 expression suppresses the appearance of hematopoietic tissue, a derivative of ventral mesoderm, and increases the fraction of endodermal cells. To investigate the mechanism by which Mixl1 mediated this latter effect, we examined the potential of Mixl1 to activate the promoters of genes upregulated in Mixl1C EBs. Promoter sequences derived from the Gsc, Sox17, E-Cad, FoxA2, and Cer-l genes were cloned upstream of a luciferase reporter gene, and these constructs were transfected into Balb/c 3T3 or C2C12 cells in the presence of increasing amounts of a plasmid expressing Mixl1. These experiments demonstrated that Mixl1 could activate the promoters of the Gsc, Sox17, and E-Cad genes in a dose-dependent manner (Fig. 6A), consistent with the postulated role of Mixl1 in definitive endoderm induction [16]. Conversely, no induction of the FoxA2 or Cer-l promoters was observed, suggesting that activation of these genes by Mixl1 requires a missing cofactor [35] or is indirect, or that Mixl1 target sequences were not contained in the region of the promoter included in the reporter vector. Analysis of the promoter sequences of the above genes revealed that the Gsc promoter contained palindromic paired homeodomain binding sites [36]. These occurred in two elements located 491 (proximal element [PE]) and 573 (distal element [DE]) base pairs upstream of the Gsc translation-initiation codon (Fig. 6B). These elements both contained an inverted iteration of the canonical homeobox binding site ATTA, separated by three nucleotides. In order to determine if Mixl1 protein could directly bind to these sequences, we performed EMSAs, in which purified bacterially expressed Mixl1 protein was bound to oligonucleotides corresponding to both the DE and PE. These experiments showed that Mixl1 protein formed a complex with the DE that was supershifted in the presence of the anti-Mixl1 antibody 6G2 (Fig. 6C). Furthermore, mutations that altered the palindromic ATTA sequence within the DE abolished Mixl1 binding. An oligonucleotide corresponding to the PE also formed a complex with Mixl1 protein and this could be supershifted with anti-Mixl1 antibodies. Mutations that disrupted all three (m4) significantly affected the complex formation (Fig. 6C). Taken together, these data support the hypothesis that Mixl1 binds to and activates the Gsc promoter during ESC differentiation, a conclusion consistent with findings from experiments in the Xenopus system indicating that gsc is a direct target of Mix.1 [14].

DISCUSSION Experiments in Xenopus and zebrafish showed that members of the Mixl1 family had a role in endoderm formation, a function


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Figure 5. The frequency of endodermal colony-forming cells (CFCs) is enhanced in differentiating embryonic stem cells constitutively expressing Mixl1 (Mixl1C ESCs). (A): Time course analysis of adherent colonies formed from dissociated day 6 (d6) Mixl1C embryoid bodies (EBs) (Mix.17). Dissociated Mixl1C EBs cultured on type I collagen-coated dishes formed discrete epithelial-like colonies over a 6-day period. Original magnification: ⫻100. (B): Distinct morphologies of Giemsa-stained control versus Mixl1C EB-derived cells following 6 days of culture on collagen-coated dishes. Original magnification: ⫻200 (C): Number of epithelial colonies per 105 cells in secondary cultures of d6 control and Mixl1C ESCs. The data represent the mean ⫾ standard deviation from three experiments (n ⫽ 3, *p ⫽ .01). (D): Immunofluorescence analysis of E-cadherin (E-Cad) and smooth muscle actin (SMA) expression in d6 control and Mixl1C EB-derived cells following 6 days of culture on collagen-coated dishes. Secondary cultures of control EB-derived cells stained positive for SMA (red) but were E-Cad⫺ (green). Most colonies derived from Mixl1C EBs did not express SMA but were E-Cad⫹ (second row). A minority of Mixl1C EB-derived colonies contained two morphologically distinct cell types (third row). SMA⫹ and E-Cad⫹ staining cells sometimes abutted, but no double-positive cells were seen. Nuclei were visualized with 4⬘,6-diamidino-2-phenylindole (blue). Original magnification: ⫻400.

likely to have been conserved in Mix family proteins in mice [38]. The results presented in our study extend previous work by demonstrating that constitutive expression of Mixl1 increases the proportion of cells with characteristics of definitive endoderm. The increased prevalence of these cells coincides with the activation of two genes that have been shown to identify definitive endoderm, Gsc and Sox17 [23], and occurs in the context of diminished formation of hematopoietic mesoderm.

Mixl1 and Self-Renewal In addition to the effects on mesoderm and endoderm formation, we also observed other differences between wild-type EBs and those constitutively expressing Mixl1. These differences included a prolongation of the expression of the stem cell markers Oct-4, Rex1, and Nanog and the epiblast marker Fgf-5. The ability of Mixl1 overexpression to prolong expression of Rex1 during ESC differentiation has been observed previously [21]. This activity may reflect the nonphysiological activation of stem cell genes by ectopically expressed Mixl1 or the persistence of www.StemCells.com

undifferentiated ESCs or epiblast-like cells within Mixl1C EBs. With respect to the latter possibility, we also observed a consistent trend towards retention of ESC CFCs in d4 and d6 EBs. In addition, Mixl1C ESCs generated significantly more secondary EBs in methylcellulose cultures, indicative of the presence of multipotent cells with substantial proliferative capacity within d7 EBs. Irrespective of the mechanism, given that Mixl1 expression normally postdates the exit of cells from the undifferentiated state, these effects of Mixl1 on stem cell gene expression are likely to be nonphysiological. Such effects may reflect the ability of ectopically expressed Mixl1 to usurp the function of other homeodomain proteins that are normally involved in maintaining the expression of stem cell genes and the pluripotent state.

Mixl1 and Hematopoiesis The findings of the current study contrast with those reported by Willey and colleagues, who showed that transient overexpression of Mixl1 during ESC differentiation promoted the formation of Flk1⫹c-kit⫹ hematopoietic progenitors [21]. In our sys-

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Figure 6. The endoderm-expressed Gsc, Sox17, and E-Cad genes are activated by Mixl1. (A): Luciferase reporter assays showing the effect of Mixl1 on the activity of promoter sequences of genes upregulated in embryonic stem cells constitutively expressing Mixl1 (Mixl1C ESCs). Balb-C-3T3 (Gsc) or C2C12 (Sox17, E-Cad, FoxA2, Cer-l) cells were transiently cotransfected with pGL3 reporter constructs containing genomic fragments from the indicated genes with 0, 25, 100, and 400 ng of a pEFBOS encoding FLAG-tagged mMixl1. A Student’s t-test comparing the relative luciferase activity of samples containing 0 ng versus 400 ng of FLAG-tagged Mixl1 yielded values of p ⬍ .05 for all constructs (n ⫽ 3). (B): Schematic showing the position of conserved palindromic binding sites for prd-type homeodomain proteins within the Gsc promoter [36]. Numbers refer to the position of each element relative to the Gsc translation initiation codon. Gsc promoter electrophoretic mobility shift assay (EMSA) probe sequences representing the wild-type distal element (DE) and proximal element (PE) and mutated variants of these elements (m1–m4) are shown. Palindromic binding sites for homeodomain proteins are underlined, and mutated nucleotides are marked with an *. (C): Gsc EMSA probes containing wild-type or mutated prd binding sites (as shown in (B)) were incubated with bacterially expressed and purified His-tagged Mixl1 protein. Mixl1 protein–DNA complexes were supershifted by the addition of 6G2 anti-Mixl1 antibody (M) [30]. A rat isotype control IgG (I) was used as a control. Black arrows indicate the position of Mixl1–DNA complexes; white arrows show the complex supershifted in the presence of the anti-Mixl1 antibody.

tem, Mixl1C EBs failed to express substantial levels of Flk1, contained no hematopoietic CFCs, and did not undergo overt hemoglobinization. There may be a number of reasons for the diametrically opposed results reported by Willey and colleagues [21] and those shown here. Experiments in Xenopus showed that low-level ectopic expression of the Mix family member Bix1 induced ventral mesoderm, whereas higher levels favored endoderm formation [7]. In our experiments, Mixl1 expression was driven by the EF1␣ promoter, a sequence chosen specifically for its ability to provide high levels of expression [24]. Indeed, even though expression of the transgene waned during differentiation, quantitative PCR analysis indicated that Mixl1 expression remained at elevated levels in Mixl1C ESCs, even at later time points. In the experiments of Willey et al. [21, 38], expression was derived from a synthetic promoter configured to deliver reliable inducibility in response to doxycycline. We hypothesize that these differing results may reflect differences in the level and duration of Mixl1 expression obtained between the two systems. Thus, although Mixl1 is required for normal hematopoietic development from differentiating mouse ESCs [19], the results of the current study indicate that prolonged high-level expression of this factor favors endoderm differentiation.

Mixl1 Promotes Endoderm Formation It had previously been reported that Mixl1⫺/⫺ mutant embryos showed impaired endoderm formation, as evidenced by a reduction in the frequency of Sox17 and Cer-l-expressing cells [10].

More recent experiments showed that this phenotype resulted from the defective migration of Mixl1⫺/⫺ endoderm cells and could not be corrected by the expansion of the underlying mesoderm [16]. These results pointed to a cell-autonomous function of Mixl1 in endoderm specification and morphogenesis. Our experiments suggest that sustained expression of Mixl1 increased the number of endodermal precursor cells that formed E-Cad⫹SMA⫺ colonies with an epithelial-like morphology. This result was corroborated by the emergence of an increased proportion of endodermal cells in late-stage EBs. The consequences of enforced expression of Mixl1 mirrored the results obtained in other vertebrate systems. In Xenopus, overexpression of Mix.1 in the ventral marginal zone was shown to repress dorsal and ventral mesoderm differentiation and, in concert with the transcription factor Siamois, promoted the expansion of endoderm [5]. This synergy between Mix.1 and Siamois strongly activated the Xenopus endodermal genes endodermin, IFABP, and XlHbox8 [5]. Similarly, the zebrafish gene zMixer has an endodermally restricted expression pattern, and promoted endodermal gene expression when ectopically overexpressed [1]. The findings from our study are also consistent with the hypothesis of Izumi et al. [39], who proposed a critical role for Mixl1 in the generation of definitive endoderm [23, 39]. They also observed that Mixl1 upregulated expression of Gsc and Sox17, genes which together mark progenitors of definitive endoderm [39]. Our study supports and extends this work, by showing that Mixl1 can transactivate the promoters of these

Lim, Pereira, Wong et al. genes and promote the formation of cells with the characteristics of definitive endoderm. At least in the case of Gsc, transactivation may involve direct binding of Mixl1 to sequences in the Gsc promoter. Whether Mixl1-induced genes such as Gsc and Sox17 play a role in the suppression of hematopoietic cell fates remains to be determined. Overall, the results of this study support a role for higher levels of Mixl1 in the preferential allocation of cells to endodermal versus mesodermal compartments as they transit the primitive streak-like stage during ESC differentiation. In turn, this suggests that factors that modulate the activity of Mixl1 could form part of strategies aimed at biasing the differentiation of ESCs towards therapeutically important cell types derived from mesodermal or endodermal germ layers.

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the assistance of Mr. Stephen Firth, Dr. Judy Callaghan, Mr. Chad Johnson (Monash Micro Imaging), Mr. Andrew Fryga (Australian Stem Cell Centre Flow Cytometry Core Facility), and Mr. Ian Boundy (Department of Anatomy and Histology, Monash University). This work was supported by the National Health and Medical Research Council (NHMRC), the Juvenile Diabetes Research Foundation (JDRF), and the Australian Stem Cell Centre (ASCC). A.G.E. is an NHMRC Senior Research Fellow. M.P. is now affiliated with the Department of Genetics, Institute of Life Sciences, Hebrew University, Givat Ram Campus, Jerusalem, 91904, Israel. A.T. is now affiliated with the California Institute for Regenerative Medicine, 210 King Street, San Francisco, California, 94107, USA.

DISCLOSURE

ACKNOWLEDGMENTS We wish to thank Dr. Anna Mossman and Ms. Koula Sourris for the provision of reagents. We would also like to acknowledge

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CONFLICTS

The authors indicate no potential conflicts of interest.

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