Published online before print April 6, 2006, doi: 10.1161/01.RES.0000220650.26555.1d
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© 2006 American Heart Association, Inc.
Integrative Physiology |
Gene Expression Profile Signatures Indicate a Role for Wnt Signaling in Endothelial Commitment From Embryonic Stem Cells
From the Carolina Cardiovascular Biology Center (H.W., P.C.C., Y.W., R.R., X.P., M.M., V.B., C. Patterson) and Departments of Medicine (C. Patterson), Biology (V.B.), and Genetics (C. Perou), University of North Carolina, Chapel Hill; and Laboratory of Cellular and Molecular Biology, National Cancer Institute (M.B.-K., J.S.R.), Bethesda, Md.
Correspondence to Cam Patterson, MD, Director, Division of Cardiology and Carolina Cardiovascular Biology Center, 8200 Medical Biomolecular Research Building, Chapel Hill, NC 27599-7126. E-mail cpatters{at}med.unc.edu
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Abstract |
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We have used global gene expression analysis to establish a comprehensive list of candidate genes in the developing vasculature during embryonic (ES) cell differentiation in vitro. A large set of genes, including growth factors, cell surface molecules, transcriptional factors, and members of several signal transduction pathways that are known to be involved in vasculogenesis or angiogenesis, were found to have expression patterns as expected. Some unknown or functionally uncharacterized genes were differentially regulated in flk1+ cells compared with flk1– cells, suggesting possible roles for these genes in vascular commitment. Particularly, multiple components of the Wnt signaling pathway were differentially regulated in flk1+ cells, including Wnt proteins, their receptors, downstream transcriptional factors, and other components belonging to this pathway. Activation of the Wnt signal was able to expand vascular progenitor populations whereas suppression of Wnt activity reduced flk1+ populations. Suppression of Wnt signaling also inhibited the formation of matured vascular capillary-like structures during late stages of embryoid body differentiation. These data indicate a requisite and ongoing role for Wnt activity during vascular development, and the gene expression profiles identify candidate components of this pathway that participate in vascular cell differentiation.
Key Words: hemangioblast • angiogenesis • microarray • signaling
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Introduction |
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The vascular system is a complex network of vessels that perfuses all organs and tissues and that is universally required for their normal development and function. Vascular anomalies are found in many congenital and acquired diseases such as cardiovascular disorders, hypertension, diabetes, and neoplasms. Therefore, much effort has focused on understanding the mechanisms of vascular development for possible therapeutic applications. Unfortunately, our knowledge of endothelial progenitors and particularly of the transcriptional programs that regulate their development is still underdeveloped, and many gaps exist in our understanding of the molecular players responsible for the initiation of stem cell differentiation and subsequent commitment to a blood vessel phenotype.
Studies of vascular development have been hampered by difficulties in accessing the embryo before establishment of blood islands and by the limited number of cells available at this stage. The in vitro embryonic stem (ES) cell differentiation system provides an alternate approach.1 This system is a powerful model system to determine the cellular and molecular mechanisms of vascular development.2,3 ES cells can differentiate spontaneously, resulting in the formation of embryo-like structures called embryoid bodies (EBs) that have the potential to generate a variety of embryonic cell lineages. Blood cells and endothelial cells develop within EBs in a manner that faithfully follows developmental progression in vivo.4,5 Many aspects of normal endothelial growth and development, up to and including the formation of vascular channels, have been reported in this system.1,6 In addition, a number of intermediate cell populations, such as precursors of endothelial cells, smooth muscle cells (SMC), and blood cells, have been identified.7–9 In vitro ES cell differentiation experiments have shown that flk1-expressing cells have developmental potential uniquely restricted to hematopoietic and endothelial lineages,5,8,9 which makes flk1 a useful marker for understanding early steps in vascular development. In this study, we used the in vitro ES cell-derived EB system to establish global gene expression profiles of endothelial differentiation. Among other observations, we have used this dataset to define a critical role for Wnt signaling in endothelial cell differentiation, suggesting that modification of Wnt activity may be a potential tool to regulate vascular patterning in vivo.
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Materials and Methods |
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ES Cell Culture
R1 mouse ES cells were maintained on gelatin-coated plates with conditioned medium from 5637 cells as a source of leukemia inhibitory factor (LIF). In vitro differentiation was induced as previously described.10 Differentiation cultures were fed every other day, and in some cases the medium was supplemented with LiCl (10 mmol/L), sFRP-1 (10 µg/mL), or conditioned media as indicated from the initiation of differentiation of EBs.
Fluorescence-Activated Cell Sorting and RT-PCR
For fluorescence-activated cell sorting (FACS) analysis of flk1 expression, EBs were dissociated with trypsin and stained with phycoerythrin-conjugated anti-flk1 antibody before analysis on a FACScan (Becton Dickinson). Cells were sorted using a MoFlo (Cytomation) and reanalyzed on a FACScan. Specific primers used for RT-PCR are indicated in Table I of the online data supplement available at http://circres.ahajournals.org.
Microarray Hybridization and Data Analysis
Total RNA was isolated from undifferentiated ES cells and from differentiated EBs at 72 hours, 84 hours, and 95 hours and 8 days and from sorted flk1+ and flk1– cells at 84 hours, 95 hours, and 8 days. Microarray hybridization and data analysis are described in detail in the text of the online data supplement.
Production of Conditioned Media and Detection of Activity of Wnt Protein in Conditioned Media
The Wnt3a-producing L cell line and control L cell line were from the American Type Culture Collection. We also used a Drosophila S2 cell line expressing Wnt2 and a control S2 cell line, which were generous gifts from Roel Nusse (Stanford University). Wnt2 S2 cells were cultured in Schneider’s media supplemented with 10% FBS under selection with hygromycin (125 µg/mL) for 2 to 3 passages before collection of media.
Assays for Proliferation and Apoptosis
EBs treated with Wnt2-CM or S2-CM were fixed and triple-labeled with rabbit anti-phosphohistone H3, rat anti-PECAM, and the DNA-binding dye DAPI. Triple-labeled images were analyzed as previously described.11 Endothelial mitotic indices were calculated by dividing the number of PECAM+/phosphohistone H3+ cells by the total number of PECAM+ cells. Nonendothelial mitotic indices were also calculated on a per field basis by dividing the number of PECAM+/phosphohistone H3+ cells by the total number of PECAM– cells.
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Results |
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Identifying Genes Within the Developmental Vascular Niche by Gene Expression Profiling
We used an unbiased analysis to identify patterns of gene expression during the earliest stages of endothelial cell differentiation using flk1 as a marker, as we have previously described.10 We hypothesized that a global understanding of vascular cell gene expression profiles can identify unanticipated molecular and cellular events in vascular development. In initial experiments, we found that differentiated ES cells did not express detectable flk1 by flow cytometry until approximately 84 hours after differentiation (Figure 1). The expression of flk1 peaked at 95 hours and was maintained in 30% of the culture until at least 8 days, when a mature vascular phenotype with capillary-like structures is fully developed.12 Based on this temporal pattern, EBs were sampled into flk1+ and flk1– populations after the onset of flk1 expression at the key times during differentiation (84 hours, 95 hours, and 8 days). RNAs were hybridized after amplification to oligonucleotide arrays that contain more than 20 000 mouse genes related to development. Figure 1 summarizes the experimental design and indicates the outflow of millions of total data points. Validation of this data set is described in text of the online data supplement and supplemental Table I and supplemental Figures I and II.
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Differential Gene Expression Within the flk1+ Lineage
Overall, in comparing flk1+ with flk1– cells, there were 802 differentially regulated mRNAs at 84 hours, 486 at 95 hours, and 270 at day 8 days (supplemental Figure III). Seventy mRNAs were consistently differentially expressed at all time points, including flk1 itself, as expected (Table
). Among these, more genes were downregulated than were upregulated in flk1+ cells, which likely indicates that flk1– cells are a more heterogeneous population. In addition, there were 1134 probe sets that were differentially regulated in flk1+ cells in comparison with flk1– cells at any of the 3 time points. One thousand forty-three of these have Unigene identifiers and actually correspond to 993 unique genes. Among this broader group, a number of genes associated with the endothelial lineage—coding for proteins such as GATA2, CXCR4, neuropilin-1, and endoglin as well as components of the angiopoietin, ephrin, and notch pathways—were preferentially expressed in flk1+ cells at 1 or more time points. It is also remarkable that genes associated with the cardiomyocyte lineage—including GATA4, myosin light chain 2a, and Mesp1—appear in the early flk1+ populations.
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Using hierarchical clustering and TreeView analysis, we compared the expression pattern of those genes differentially expressed in flk1+ cells compared with flk1– cells during stem cell differentiation (Figure 2). Unsupervised hierarchical clustering of the gene expression pattern from individual samples produced groupings consistent with their development stage and flk1 expression level. Later stage samples (8 days) were grouped together, apart from early stage samples (84 and 95 hours), and flk1+ samples clustered separately from flk1– samples. Three clusters were selected for further analysis. Cluster A includes flk1 and represents genes mostly upregulated in flk1+ cells independent of time, such as Wnt2, Wnt5a, Nkd1 (Naked Cuticle 1, a Wnt signaling antagonist) and ccnd2 (cyclin D2, a Wnt target gene), cardiomyocyte lineage-associated genes (Hand1, GATA6, GATA4) and transforming growth factor family genes (Tgfb2 and Bmp4). Cluster B contains genes upregulated in early (84 and 95 hours) flk1+ cells but not during the later time points. Again, Wnt signaling associated genes such as Frzb, Cdh2 (N-cadherin), Msx2, Ccnd1 (Cyclin D1), and Dkk1 (Dickkopf 1) are present in this cluster, as are Notch1, GATA2, GATA3, Tgfb1, Bmp7, and Smad1. Cluster C contains genes downregulated in flk1+ cells relative to flk1– cells. Representative genes in this cluster include E-cadherin and transcriptional factors Sox2, Foxo1, and Foxa2, which collective are associated with endoderm- and ectoderm-derived tissues.13,14 Notably, E-cadherin and Sox2 are negative regulators of Wnt signaling.15 The presence of multiple clusters of genes showing temporally coordinated patterns of expression during a critical development stage suggests a common mechanism of transcriptional regulation (see online data supplement).
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Differential Expression of Genes Within the Wnt Signaling Pathway
The Wnt signaling pathway has a well-defined role in development. Although not investigated systematically, a few recent studies have suggested a role for Wnt signaling in angiogenesis, but the exact Wnts involved and their roles have not been well delineated. Canonical Wnt signaling is initiated when Wnts bind to a coreceptor complex containing a Frizzled receptor and lipoprotein receptor-related proteins 5 or 6 (LRP-5/6) (Figure 3). ß-Catenin is the key effector of the canonical Wnt signaling pathway. In the absence of Wnt, cytosolic ß-catenin is phosphorylated by a protein complex containing glycogen synthase kinase-3ß, axin, and adenomatous polyposis coli and is degraded rapidly by the ubiquitin–proteasome pathway. Activation of Wnt signaling inhibits ß-catenin phosphorylation via disheveled (Dsh). This results in accumulation of cytosolic ß-catenin, which then translocates to the nucleus and binds the LEF-1/TCF family of transcriptional factors and induces transcriptional activation of Wnt target genes. There are also several negative regulators of Wnt signaling, including Frizzled-related protein, Dickkopf 1, Naked Cuticle, and the Sox family transcription factors. Cadherins may also act as negative regulators by binding to ß-catenin and reducing the availability of cytosolic ß-catenin.15 In our array data, multiple components of the Wnt signaling pathway were differentially regulated in flk1+ cells during differentiation, including Wnt2, Wnt5a, Fzd7, Lef1, Frzb (FRP-3), Dickkopf 1, Nkd1, Sox17, Sox2, and N-cadherin (each of which are marked with stars in Figure 3). Known Wnt target genes including Msx1, Msx2, fibronectin, cyclin D1, cyclin D2, and Myc16,17 were also specifically upregulated in flk1+ cells, indicating enhanced Wnt activity in these cells. This pattern suggested to us a significant role for Wnt signaling in vascular development. Activation of both positive and negative regulators indicates the importance of tight control of Wnt signaling during this process.
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To confirm the microarray data, we performed RT-PCR for selected genes in the Wnt signaling pathway, including Wnt2, Wnt5a, frizzled-receptor 7 (Fzd7), frizzled-receptor 5 (Fzd5), Lef1, Frzb, Nkd1, Cdh2 (N-cadherin), Msx2, and ß-catenin. Fzd5 was not printed on the Agilent mouse array, bur we included it in the RT-PCR analysis because Fzd5–/– mice have defects in yolk sac angiogenesis.18 Again, RT-PCR results correlated closely with our array data (Figure 4). Wnt2 transcripts were first detected by 84 hours of differentiation with peak levels at day 8. Wnt2, which signals via the canonical Wnt pathway, was upregulated in flk1+ cells at all 3 time points and was not detected in flk1– cells at 84 and 95 hours. It is notable that mice lacking Wnt2 have placental angiogenic defects, although a thorough evaluation of vascular development in these mice has not been reported.19 Expression of Wnt5a, which typically activates the noncanonical Wnt pathway, was detected in undifferentiated ES cells with a gradual elevation following differentiation until day 8 and was preferentially expressed in flk1+ cells at 84 and 95 hours. Transcripts of other Wnt-associated genes were also upregulated in early flk1+ cells, including receptor Fzd7, nuclear effector Lef1, negative regulators Frzb and Nkd1, N-cadherin, and Wnt target gene Msx2. Fzd5 was expressed at higher levels in flk1– cells than in flk1+ cells. Remarkably, it has been suggested that both Wnt2 and Wnt5a are possible ligands of Fzd5.18,20
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Role of the Wnt Signaling Pathway in Endothelial Differentiation
Based on the consistent and highly specific expression pattern of components of the Wnt signaling pathway within the flk1+ population, we tested the hypothesis that Wnt activation regulates endothelial cell maturation. We focused on canonical Wnt signaling based on the impressive upregulation of Wnt2, which operates through this pathway. The absence of specific antibodies and recombinant proteins as reagents makes analysis of Wnt signaling cascades notoriously difficult. We therefore used lithium (to selectively inhibit GSK-3ß activity, which in turn mimics activation of the Wnt pathway21) and sFRP-1 (to bind secreted Wnts and suppress Wnt receptor activation22) as tools to examine the effect of Wnt signaling on vascular progenitors using the in vitro ES cell differentiation system. Addition of lithium (10 mmol/L) to the differentiation medium moderately but significantly expanded the flk1+ cell population as detected by flow cytometry after 96 hours of differentiation (Figure 5A and 5B). On the contrary, treatment of EBs with sFRP-1 (10 µg/mL) almost completely depleted the flk1+ cell population following 96 hours of differentiation. In both cases, overall morphologies of EB cultures were grossly normal and no evident increase in cell death was noted.
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To extend these observations, we also examined the direct effects of Wnt proteins on vascular progenitors in the same model using conditioned media to circumvent the challenges associated with purifying Wnts that retain biological activity. In preliminary experiments, both Wnt3a-CM and Wnt2-CM led to stabilization of ß-catenin in L cells (data not shown), indicating that the secreted Wnts retained activity in conditioned medium. Similar to the effects of lithium, both Wnt3a and Wnt2 (which activate the same canonical pathway) were able to expand the flk1+ cell population (Figure 5C and 5D). The absolute percentage of flk1+ cells in the Wnt2 treatment group was lower than after Wnt3a treatment, but this is attributable to the different cell types producing the conditioned medium. Even so, in comparison with S2-CM, Wnt2-CM was still able to significantly increase the percentage of flk1+ cells. Both sFRP-1 and Wnt3a are modulating canonical Wnt signaling in these assays, as indicated by the ability of sFRP1 in increase, and Wnt3a to decrease, the ratio of phosphorylated to total GSK-3ß (see supplemental Figure IV).
There were several possible explanations for the role of Wnt signaling on expansion of vascular progenitors, including the following: transcriptional activation of flk1, induction of differentiation to vascular progenitors, and/or increase of proliferation of vascular progenitors. Treatment of EBs with sFRP-1 was able to reduce the flk1 transcriptional level as examined by RT-PCR at 96 hour (Figure 6A). However, flk1 transcripts were still detectable at low levels, which is not entirely consistent with complete depletion of flk1+ cells with sFRP-1 treatment at this time point (Figure 5). We also did not find that Wnt activation altered the kinetics of endothelial differentiation in EBs (see supplemental Figure V). To further elucidate the mechanism of the effects of Wnt activity, we treated EBs with sFRP-1 until day 8 and then stained them with the endothelial marker PECAM. With or without sFRP-1, cultures were healthy and EBs differentiated appropriately and appeared grossly normal, as indicated by phase contrast microscopy (Figure 6B). Without sFRP-1, PECAM+ capillary-like structures readily formed, as we have previously described.10,12 However, vascular networks never formed after sFRP-1 treatment, although rare, isolated PECAM+ endothelial cells were seen.
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We next analyzed the possible contribution of several cellular parameters by Wnt signaling to vascular network formation in the same model. First, we labeled day 8 EBs treated with or without sFRP-1 with antibody to PECAM and to M30 CytoDEATH antibody, which recognizes caspase-cleaved cytokeratin 18 and detects early apoptotic cells. However, no change in positive apoptotic signals were noted (data not shown), which suggests that inhibition of vascular network formation by sFRP-1 is not caused by induction of apoptosis. We did find that Wnt3a enhanced migratory responses of mouse embryonic endothelial cells in Boyden chamber assays (see supplemental Figure VI). To address the contributions of proliferation, EBs were labeled with antibodies to the endothelial marker PECAM and to the mitotic marker phosphohistone H3 (marking cells in the G2–M phase) and then stained with DAPI (Figure 7). (Experiments with sFRP-1 were not informative for proliferation assays because so few cells are PECAM positive after sFRP-1 treatment.) Visual observation indicated that EBs treated with Wnt2-CM had more robust vascular structures and had more PECAM+ cells that were colabeled with the anti-phosphohistone H3 antibody compared with EBs treated with S2-CM. Digital images from multiple wells in 3 separate experiments were processed and used to calculate the endothelial mitotic indices. There was no difference in nonendothelial cell mitotic indices between Wnt2-CM and S2-CM treatment (data not shown). However, day 8 EBs treated with Wnt2-CM had endothelial mitotic indices (normalized to corresponding nonendothelial cell mitotic indices) that were more than 2-fold higher than EBs treated with S2-CM. These results indicate that the canonical Wnt pathway enhances proliferation of endothelial precursors with specificity and without large effects on viability and that these effects account at least in part for the expansion of the endothelial compartment by Wnt activation in our studies.
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Discussion |
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Differentiation of embryonic stem cells in vitro has proven to be a reliable and reproducible tool for understanding cellular events and signaling responses that occur during embryonic development and is particularly well adapted for characterization of hematovascular lineages,5,12,23,24 which are otherwise difficult to characterize in whole embryos. Our focus in the studies presented here is on the transcriptome of cells bearing the surface marker flk1 at different times during embryoid body differentiation. This receptor for VEGF was first considered a specific marker of the vascular endothelium in adults,25,26 and further analyses—including studies using differentiating embryonic stem cells—have indicated that flk1 marks a common progenitor for hematopoietic and endothelial lineages called the hemangioblast.5,24 Our studies are consistent with the general dogma that, at their first appearance, cells expressing flk1 have primitive characteristics and that over time they develop molecular signatures of mature endothelium that are concordant with the adoption of vascular features at the cellular level.
The patterns of expression of a number of classical endothelium-restricted genes (eg, PECAM, ICAM2, VE-cadherin, flk1 itself) within our microarray dataset are as predicted. However, it is striking that many of the mRNA species that are enriched within flk1+ cell populations are incompletely characterized and/or have not been associated with hematovascular development. The hierarchical clustering analysis suggests that these genes may participate in regulatory networks involving multiple signaling pathways that are required for appropriate maturation of flk1+ cells during the developmental plan, and thus additional analysis of the genes identified within these transcriptional clusters may provide new insights into the molecular events in blood vessel development. Remarkably, we found that a number of genes within the canonical Wnt signaling pathway were preferentially expressed in ES cell-derived flk1+ cells (Figure 3). Both positive and negative regulators of Wnt signaling were identified, suggesting that this signaling pathway requires tight control during the vascular developmental program, and the upregulation of known Wnt target genes (Msx1, Msx2, and Ccnd1) is consistent with activation of canonical Wnt signaling within the flk1+ cellular niche.
Several lines of evidence point to a role for signaling via Wnt family members in vascular development. A transgenic Lef/tcf:lacZ reporter mouse strain experiment has demonstrated activation of canonical Wnt signaling in endothelial cells during intraembryonic angiogenesis.27 Loss of Fzd5, a Wnt receptor, during mouse development severely impairs yolk sac vascularization and causes intrauterine demise soon after the onset of vascular development,18 and Wnt2–/– mice have defects in placental angiogenesis.19 Endothelial cell-specific inactivation of ß-catenin disrupts intercellular junctions and enhances vascular fragility,28 although it is not possible to determine whether this phenotype results from dysregulation of Wnt signaling or from effects of ß-catenin on cell–cell adhesions via interactions with members of the cadherin family. We therefore used a systematic approach of activation and inhibition of Wnt signaling to better define a role for the canonical Wnt signaling pathway in regulating endothelial differentiation in ES cell cultures. We find that Wnt activation is both necessary and sufficient for endothelial cell differentiation and assembly into vascular-like structures in embryoid bodies, in part through regulation of differentiation and proliferation of endothelial progenitors. Wnt proteins have been linked to enhanced proliferation in numerous settings,29 including stem cell systems.30,31
The studies presented here provide further support for a role of Wnt signaling and identify for the first time a specific requirement for the canonical Wnt signaling pathway in endothelial cell differentiation and maturation. It is notable that recent evidence points toward a critical role for Wnt activity in hematopoietic stem cell renewal,31 and the studies presented here suggest that a larger role may exist for Wnts during hematovascular differentiation. The observations here also raise the broader possibility that Wnts may have a general role in the regulation of angiogenesis under physiological and pathological conditions in adulthood. However, many new questions are also raised by our studies, chief among these being the specific players within the canonical Wnt signaling pathway that are operative during vascular development. Additional studies of the candidates identified through our gene expression profiles are likely to develop a clearer picture of the role of Wnts and their downstream targets in the development of the vascular system and in adult angiogenesis in general.
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Acknowledgments |
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This work was supported by NIH grants HL 61656, HL 03658, and HL 072347 (to C. Patterson). C. Patterson is an Established Investigator of the American Heart Association and a Burroughs Wellcome Fund Clinician Scientist in Translational Research. We thank Rebecca Rapaport for technical advice with ES cell differentiation.
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Footnotes |
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Original received December 29, 2005; revision received March 14, 2006; accepted March 27, 2006.
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References |
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H. Wang, J. B. Gilner, V. L. Bautch, D.-Z. Wang, B. J. Wainwright, S. L. Kirby, and C. Patterson Wnt2 Coordinates the Commitment of Mesoderm to Hematopoietic, Endothelial, and Cardiac Lineages in Embryoid Bodies J. Biol. Chem., January 5, 2007; 282(1): 782 - 791. [Abstract] [Full Text] [PDF]
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