The mesodermal marker brachyury is expressed in Flk1 positive cells
As a first step in defining the relationship between mesoderm and the BL-CFC, we analyzed the expression pattern of the brachyury gene in Flk1+ cells isolated from developing EBs. The majority of BL-CFC was found in the Flk1+ fraction of day 2.75 EBs, confirming the earlier findings of Faloon et al. (Faloon et al., 2000) (Fig. 1A,B). Expression analysis revealed that brachyury was present in both the Flk1+ and Flk1– populations, indicating that the BL-CFC may represent a subset of mesoderm (Fig. 1C). The helix-loop-helix transcription factor Scl (Tal1 – Mouse Genome Informatics) (Begley et al., 1989) was expressed only in the Flk1+ fraction, consistent with the presence of BL-CFC and cells undergoing commitment to the hematopoietic lineages.
To define more accurately the co-expression pattern of these genes, 100 individual Flk1+ cells were analyzed. As shown in Fig. 1D, which represents 40 of these single cells, different patterns were observed, the most common being co-expression of Flk1 (Kdr – Mouse Genome Informatics) and brachyury. Cells expressing either Flk1 or brachyury represented a smaller subset of the population. Scl-expressing cells were the least abundant. Of the 100 cells analyzed, 91 showed expression of the ribosomal gene L32, indicating the presence of amplified material in the sample. Out of these 91 cells, 43 expressed both Flk1 and brachyury, and of these, three also expressed Scl. Although a number of cells expressed either Flk1 or brachyury, we interpret these patterns with caution, as the lack of a signal on single cells analysis can reflect differences in cell cycle status. The high frequency of cells co-expressing Flk1 and brachyury adds support to the notion that the BL-CFC is closely related to mesoderm. To directly address the question of whether or not the BL-CFC expresses brachyury, we targeted the GFP cDNA to the brachyury locus to enable us to isolate cells that express this gene.
Development and characterization of an ES cell line carrying GFP targeted to the brachyury locus
Two different vectors were designed for targeting GFP to the brachyury locus. The vector that gave the highest level of expression contained a mini gene locus, which consisted of the GFP cDNA, followed by a splice donor site, an artificial intron and an exon encoding the SV40 polyadenylation signal sequence to prevent transcription of regions downstream of the brachyury gene (Fig. 2A). A translational stop codon was positioned downstream of the artificial intron, as it has been reported that primary transcripts with stop codons preceding intronic sequences can be recognized as aberrant messages, thereby rendering them subject to rapid degradation (Maquat, 2002). The vector was designed to replace approximately two-thirds of the first exon of the brachyury gene with the GFP expression cassette, resulting in the disruption of the targeted brachyury allele. The targeting construct was electroporated into E14.1 ES cells and several positive clones were identified. The Neo selection marker was subsequently removed from the targeted ES cells by Cre/loxP-mediated recombination (Gu et al., 1993) to minimize the impact of foreign DNA sequences and ensure that expression of the inserted GFP cassette was under the control of native brachyury regulatory elements. Two GFP targeted, Neo-deleted ES clones, referred as GFP-Bry cells, were differentiated in vitro and analyzed for GFP expression. EBs generated from these two clones expressed readily detectable levels of GFP when observed under a fluorescence microscope (Fig. 2C) and were used in the subsequent analyses. The second vector, which did not result in significant GFP expression after targeting, consisted of an insertion of a simple GFP cDNA instead of the minigene into the same segment of the brachyury locus (not shown). The comparison of these vectors highlight the importance of vector design as relatively small differences in targeting constructs resulted in significant differences in levels of GFP expression.
For the targeted GFP-Bry ES cells to be a reliable model for studying mesoderm induction and subsequent specification to the hemangioblast and to the hematopoietic and endothelial lineages, the inactivation of one allele should not be detrimental to these developmental processes. Although the viability and near normal development of the T heterozygous mouse clearly indicates that there are no significant hemizygous effects on mesoderm development (Herrmann, 1991), it is possible that this targeting could impact the in vitro differentiation potential. To address this question, we assayed the BL-CFC potential of the GFP-Bry cells and compared it with that of wild-type cells. EBs generated from the GFP-Bry cells were indistinguishable from those that developed from wild-type ES cells with respect to morphology and size (not shown). As indicated in Fig. 2B, no significant differences were observed in the kinetics and numbers of blast colonies generated by the GFP-Bry ES cells when compared with wild-type cells. Analysis of the blast colonies indicated that their hematopoietic and endothelial potential was similar to those of wild-type colonies (not shown). Taken together, these findings demonstrate that heterozygosity at the brachyury locus does not alter the specification and development of the hematopoietic program during EB differentiation.
Correlation between expression of the targeted GFP and transcription of the endogenous brachyury gene
As a marker of mesoderm formation, GFP expression must reflect the expression pattern of the endogenous gene. To determine if this is the case, EBs derived from GFP-Bry ES cells were harvested at daily intervals over a 6-day differentiation period and analyzed for brachyury transcription by RT-PCR and for GFP expression by flow cytometry (Fig. 2D,E). As shown in Fig. 2D, brachyury expression was detected between day 2 and 4 of differentiation, with the highest levels present at day 3, consistent with the previously described pattern for this gene (Robertson et al., 2000). FACS analysis revealed the presence of low numbers of GFP+ cells as early as day 2 of differentiation. The number of GFP+ cells increased dramatically over the next 48 hours, representing 65% and 85% of the total day 3 and 4 EB populations, respectively. Following this peak, the number of GFP+ cells dropped sharply to undetectable levels in day 6 EBs. The high levels of GFP detected by FACS analysis demonstrate that a large proportion of the day 3-4 EB cells express brachyury indicative of extensive mesoderm development in our differentiation conditions. The findings from this comparative analysis strongly suggest that GFP expression faithfully recapitulates brachyury expression in differentiating EBs and as such, provides a unique marker for the identification and isolation of cells expressing this gene.
Segregation of mesoderm and neuroectoderm lineages by GFP expression
In early development, brachyury expression is restricted to the primitive streak and nascent mesoderm in the region that will define the posterior part of the embryo. The epiblast cells in the anterior region of the embryo acquire a neuroectoderm fate and do not express brachyury (Herrmann, 1991). To determine if brachyury expression could also distinguish these primary germ cell populations within the ES differentiation cultures, EBs were fractionated into GFP+ and GFP– populations by cell sorting and assayed for mesoderm and neuroectoderm potential (Fig. 3). GFP+ and GFP– populations were isolated from day 3.5 EBs and subjected to gene expression and BL-CFC analysis (Fig. 3A). As shown in Fig. 3B, cells expressing brachyury segregated to the GFP+ fraction indicating that it is possible to isolate mesodermal cells based on GFP expression. Genes associated with the earliest stages of hematopoietic and endothelial commitment, including Flk1 and Runx1 (Okuda et al., 1996; Wang et al., 1996), co-segregated with brachyury to the GFP+ fraction. By contrast, markers of primitive ectoderm (Rex1; Zfp42 – Mouse Genome Informatics) (Rogers et al., 1991) and neuroectoderm (Pax6) (Walther and Gruss, 1991) were detected only in the GFP– population. BL-CFC analysis indicated that most progenitors were found in the GFP+ fraction. Segregation of the BL-CFC potential to GFP+ fraction demonstrates that it contains mesodermal derivatives and that the BL-CFC itself may retain some level of brachyury expression (Fig. 3C).
To evaluate the neuroectoderm potential of the GFP populations, day 2.5 EBs were fractionated, the cells from each population allowed to reaggregate to form EB-like structures and further cultured as described in the Materials and Methods. Day 2.5 EBs were used for this analysis as they contain significantly more neuroectoderm potential than later stage EBs in culture conditions selected to promote hematopoietic development. The reaggregated cells were cultured for an additional 3.5 days in suspension and then replated to assess their potential to generate neurites. At the end of the 6-day culture, EBs generated from the GFP– population expressed genes indicative of neural development, including Nkx2.2 (Briscoe et al., 1999), Pax6 (Walther and Gruss, 1991) and Neurod1 (previously NeuroD) (Bang and Goulding, 1996) (Fig. 3D). By contrast, none of these genes was expressed in the EBs derived from GFP+ cells. Almost all of the reaggregated EBs generated from the GFP– population developed neurite outgrowths that expressed the neuronal class III ß-tubulin (TuJ1) (Fig. 3E,F). Consistent with the gene expression profile, none of the EBs from the GFP+ fraction formed neurites. The findings from these analyses clearly demonstrate that it is possible to separate mesodermal cells from those that display neuroectoderm potential based on GFP expression under the control of the brachyury regulatory elements. Taken together with the findings from the kinetic analysis, these data demonstrate that the strategy of targeting a selectable marker to the brachyury locus was successful and that in this context, GFP provides a unique marker with which to track and isolate cells with mesoderm potential.
Temporal expression of brachyury relative to markers indicative of ES cell differentiation and BLCFC development
As shown in Fig. 2, GFP is expressed in a dynamic temporal pattern that reflects expression of the endogenous brachyury gene. To further define the stages of ES differentiation to mesoderm and subsequent specification to the hemangioblast lineages, we compared the kinetic of GFP expression with that of CD31, Kit and Flk1. Although Kit and CD31 are best known for their expression on hematopoietic (Ogawa et al., 1991) and endothelial (Vecchi et al., 1994) populations from fetal and adult tissues, they are also expressed on undifferentiated ES cells (Robson et al., 2001; Vittet et al., 1996). As both are downregulated after the onset of ES cell differentiation, their expression patterns can be used to track the early commitment steps in the formation of EBs. Undifferentiated GFP-Bry ES cells did not express significant levels of GFP or Flk1, but did have high levels of CD31 (Fig. 4A) and intermediate level of Kit (Fig. 4B). Day 2 EBs contained a small population of GFP+ cells, a fraction of which also expressed Flk1. The overall levels of CD31 on day 2 EBs were somewhat reduced compared with that found on the undifferentiated ES population. The expression patterns of all markers changed dramatically over the next 24 hours. At day 3 of differentiation, more than 40% of the EB population expressed GFP with no Flk1, while 18% of the cells expressed both GFP and Flk1. CD31 levels were significantly reduced on the entire EB population and none was expressed on the GFP+ cells. Three colors analysis revealed that the double positive GFP+Flk1+ cells had low level of Kit expression (Fig. 4B). Both the GFP+Flk1– and the GFP–Flk1– populations had intermediate levels of Kit. By day 4 of differentiation, the EB cells appear to have down regulated the levels of GFP and Flk1 expression, although a significant portion of the populations still expressed both markers. Relatively few cells expressed CD31 (Fig. 4A) or Kit (not shown) at this stage of development. These analyses clearly demonstrate that using the GFP-Bry ES line together with cell-surface markers, it is possible to track the differentiation of ES cells to brachyury-positive mesoderm and subsequently to cell populations that express Flk1 together with brachyury.
GFP and Flk1 delineate specific subpopulations of mesoderm
Expression of GFP and Flk1 on day 3 EB cells delineates three distinct populations designated as GFP–Flk1–, GFP+Flk1– and GFP+Flk1+ (Fig. 5A). In some experiments, we observed a small GFP–Flk1+ population at early stages of differentiation. When analyzed, this population appeared to represent dead and/or dying cells that had non-specifically bound the secondary streptavidin reagent. Analysis of BL-CFC potential of the three populations revealed the expected pattern of segregation. The majority of these progenitors were present in the GFP+Flk1+ population, whereas the secondary EBs that develop from residual ES cells segregated to the GFP–Flk1– fraction (Fig. 5B). The GFP+Flk1– cells did not generate significant numbers of blast colonies or secondary EBs. Gene expression analysis revealed striking differences between the populations, consistent with the observed difference in biological potential. As expected, Flk1 was restricted to the GFP+Flk1+ fraction, whereas brachyury was present in both GFP+ fractions, although at lower levels in the population that co-expressed Flk1. Nodal and Fgf5, genes found in the epiblast and early gastrulating embryo (Haub and Goldfarb, 1991; Hebert et al., 1991; Varlet et al., 1997), were expressed in the GFP–Flk1– and GFP+Flk1– fractions, but downregulated in GFP+Flk1+ cells. Two members of the Wnt family, Wnt3a and Wnt8a that are expressed in the primitive streak and early mesoderm (Bouillet et al., 1996; Takada et al., 1994; Yamaguchi et al., 1999) were restricted to the GFP+Flk1– subpopulation. Bmp2 and Bmp4, genes encoding factors that play crucial roles during mesoderm formation and specification (Hogan, 1996), displayed interesting patterns of expression. Bmp2 was detected in both GFP-positive fractions, whereas Bmp4 expression was restricted to the GFP+Flk1+ cells. Finally, genes associated with the hematopoietic lineages, Runx1 (Okuda et al., 1996; Wang et al., 1996) and Scl (Begley et al., 1989) were expressed predominantly in the GFP+Flk1+ fraction, consistent with the fact that this fraction contains the BL-CFC.
Taken together, these findings demonstrate that expression of Flk1 and GFP delineates three subpopulations within day 3.0 EBs that display distinct developmental potential as defined by gene expression profiles and progenitor cell content. The potential of these populations are consistent with the hypothesis that they represent a developmental progression from pre-mesoderm cells, defined as GFP–
cells to prehemangioblast mesoderm represented by the GFP+
fraction to the hemangioblast, defined as GFP+
Tracking the induction of mesoderm and its specification to the BL-CFC
If the three fractions defined by GFP and Flk1 represent distinct steps within a developmental program, then it should be possible to demonstrate that those representing the early stages are able to give rise to the more mature populations. To address this issue, each fraction was isolated from day 3 EBs and allowed to reaggregate at high cell density in culture for 20 hours (Fig. 6A). Although cell numbers did not change significantly during this time, the developmental potential of the populations did. In the reaggregated presort control, the GFP+Flk1+ fraction increased in size from 16% to 40% of the total population, the GFP+Flk1– fraction remained relatively constant in size whereas the GFP–Flk1– fraction decreased in size during this time. After the 20-hour culture period, the GFP–Flk1– sorted population gave rise to a significant number of GFP+Flk1– cells (27% of the total culture) and also to a small emerging population (3.5%) that expressed Flk1. In this same period, the GFP+Flk1– fraction generated a large GFP+Flk1+ population that represented 66% of the total cells recovered from the reaggregation culture. A subpopulation of cells did not acquire Flk1 and retained relatively high levels of GFP. Expression of both Flk1 and GFP was downregulated after culture of the GFP+Flk1+ fraction. Analysis of the BL-CFC and hematopoietic progenitor potential of the fractions prior to (pre-culture) and following (post-culture) the culture revealed changes that were consistent with the changes observed in Flk1 and GFP expression. Prior to culture, most of the blast colony-forming potential was found in the GFP+Flk1+ fraction (Fig. 6B). After culture, the BL-CFC potential of the two GFP+ fractions changed dramatically. The GFP+Flk1– population acquired the potential to generate blast colonies, a finding consistent with the fact that these cells upregulated Flk1 expression level during this time period. The GFP+Flk1+ population, conversely, lost BL-CFC activity but developed significant hematopoietic potential during this culture step (Fig. 6C). These changes in progenitor cell potential together with the downregulation of both Flk1 and GFP within this population are an indication of maturation beyond the hemangioblast stage to the early hematopoietic stage of development. The majority of hematopoietic progenitors that developed in the reaggregation culture of the GFP+Flk1+ fraction were of the primitive erythroid lineage, the earliest population to develop in EBs. The GFP–Flk1– fraction contained no BL-CFC prior to or following culture, consistent with the lack of significant numbers of Flk1-expressing cells in either population.
The findings from this experiment support the concept that populations defined by GFP and Flk1 expression in day 3 EBs represent distinct stages of ES cell differentiation to mesoderm and to the earliest stages of hematopoietic and endothelial development, as defined by the BL-CFC.