By targeting GFP to the brachyury locus, we have established a model system with which to study the development of mesoderm and its specification to the hematopoietic and vascular lineages after ES cell differentiation in culture. Using this approach, we have demonstrated that it is possible to separate cells with mesoderm potential from other cell types, including those of the neuroectoderm lineage. Our data highlight the power of such a strategy for isolating the primary germ cell lineages as they develop in this model system. By combining GFP and Flk1 expression, we were able to isolate and characterize three distinct populations that define a premesoderm stage of development as GFP–Flk1–, a prehemangioblast mesoderm stage as GFP+Flk1– and the hemangioblast stage as GFP+Flk1+.
One of the original goals of this targeting approach was to further characterize the developmental relationship between the BL-CFC and mesoderm by determining whether or not this hemangioblast-like progenitor expressed brachyury. The outcome of this study clearly demonstrates that the entire Flk1+ population at the BL-CFC stage of development expresses GFP. Although all BL-CFC are GFP+, the RT-PCR analyses in Fig. 5 indicate that the levels of brachyury are reduced compared with the GFP+Flk1– population suggesting that these progenitors are downregulating brachyury as they differentiate to the hematopoietic and endothelial lineages. Previous findings demonstrating that progeny of the BLCFC no longer express brachyury (Kennedy et al., 1997
; Robertson et al., 2000) are consistent with this interpretation. Collectively, these observations would position the BL-CFC at a stage of development that represents mesodermal cells committed to the hematopoietic and vascular lineages.
In the early mouse embryo, the first mesodermal cells generated within the primitive streak migrate to the extra-embryonic region where they differentiate and form the hematopoietic and endothelial lineage of the blood islands (Moore and Metcalf, 1970; Kinder et al., 1999). Although most cells within the primitive streak are brachyury positive, expression is rapidly downregulated as they exit the streak and begin migrating (Wilkinson et al., 1990). Flk1 is expressed in these migrating cells and subsequently in the blood islands and the developing vasculature of the yolk sac (Shalaby et al., 1995). Given that brachyury is widely expressed in the primitive streak, it is assumed that the Flk1+ cells and ultimately the hematopoietic and endothelial lineages derive from brachyury expressing mesoderm. The findings in this report formally demonstrate that brachyury+ mesodermal progenitors do indeed give rise to the BL-CFC and to cells of the hematopoietic lineage within the ES/EB model system.
In addition to providing further characterization of the BLCFC, the GFP-Bry ES cell line has enabled us to segregate the Flk1– fraction of day 3.0-3.5 EBs into GFP– and GFP+ populations, representing cells with pre-mesoderm and prehemangioblast mesoderm potential, respectively. The strongest evidence in support of this interpretation is provided by the experiment using reaggregation cultures in which each isolated population was found to differentiate rapidly to the subsequent stage of development. One of the most striking developmental changes was observed with the GFP+Flk1– population that contained little, if any, BL-CFC potential prior to culture. After culture, a substantial number of the cells upregulated Flk1 and with this change in expression the population acquired the capacity to generate blast cell colonies. This is an important observation as it enables one, for the first time, to access the immediate progenitors of the BL-CFC.
If the GFP+Flk1+ population is indicative of cells emerging from the primitive streak, then the GFP+Flk1– population should represent cells within the primitive streak, whereas the GFP–Flk1– population would be equivalent to the pregastrulation, pre-streak stage of development. The gene expression profiles of the three populations are consistent with this interpretation. Two of the most restricted expression patterns were observed with the Wnt genes that are upregulated with the onset of brachyury expression and downregulated with the acquisition of Flk1. In the early mouse embryo, expression of Wnt3a and Wnt8a are overlapping with that of brachyury, both are expressed in the primitive streak and then downregulated with migration and patterning associated with the formation of extra-embryonic mesoderm of the yolk sac (Bouillet et al., 1996; Takada et al., 1994; Yamaguchi et al., 1999). Studies in the chick embryo (Marvin et al., 2001) and in Xenopus (Schneider and Mercola, 2001) have implicated Wnt3a and Wnt8 as important molecules in the specification of mesoderm to a hematopoietic fate. When expressed in cells of the cardiac crescent, Wnt3a displayed the potential to respecify these cells to a hematopoietic fate. Conversely, when the function of both was blocked in cells fated to the hematopoietic lineage, these cells acquired cardiac potential. Expression of both Wnt3a and Wnt8a in the GFP+Flk1– population that contains cells undergoing commitment to the BL-CFC suggests that these factors could have a similar role in the specification of hematopoietic mesoderm in the mouse. The expression pattern of Fgf5 and Nodal is also consistent with the assigned developmental potential of the three populations. In the mouse embryo, Fgf5 is initially expressed in cells of the epiblast and following gastrulation is found in cells that form the primitive streak and subsequently in the paraxial subpopulation of mesoderm (Haub and Goldfarb, 1991; Hebert et al., 1991). Nodal is expressed in the epiblast and primitive endoderm, the primitive streak and ultimately in a subset of cells found in the node (Varlet et al., 1997). Expression of both genes in the GFP–Flk1– and GFP+Flk1– populations but not in the GFP+Flk1+ cells would be consistent with a progression from epiblast-like cells to cells representing the primitive streak and finally specification to the hemangioblast.
The isolation of cell populations based on brachyury expression described here together with our previous studies and findings from others provide the basis for a model of mesoderm induction and specification as outlined in Fig. 7. Based on surface marker analysis, the undifferentiated ES cells can be defined as CD31+ Kit+ brachury– Flk1–. Over a 2.5-3.0-day period of differentiation, the ES cells differentiate and give rise to three different populations, the most immature of which is considered as pre-mesoderm and represented as GFP–Flk1–. The majority of these cells has downregulated CD31 (Fig. 4), but retain some expression of Kit. This population also expressed Fgf5, Nodal and low levels of Wnt8a. The next stage of development, the pre-hemangioblast mesoderm has upregulated brachyury (GFP+) and continues to express Kit. Fgf5, Nodal, Wnt3a, Wnt8a and Bmp2 are expressed at readily detectable levels at this stage. The most mature population, the hemangioblast, continues to express some brachyury, has upregulated Flk1 and downregulated Kit. These cells no longer express Fgf5 or the Wnt genes, but do express Bmp2 and Bmp4 as well as Runx1 and Scl.
Access to populations that represent the pre-hemangioblast stage of development as outlined in Fig. 7 provides a unique approach for studying the induction of mesoderm and its specification to the hematopoietic and vascular lineages. Although previous studies have implicated a role for factors such as BMP4, FGF and activin in the induction of mesoderm in EBs, they were unable to define the precise stage at which these molecules exerted their effect (Faloon et al., 2000; Johansson and Wiles, 1995). With the GFP-Bry ES cells, we will be able to assess the role of these and other factors on distinct developmental steps, the first being the induction of mesoderm as defined by the development of the GFP+Flk1– population and the second being the specification of mesoderm to the hemangioblast lineages as characterized by the progression to GFP+Flk1+ cells. In addition to defining the role of known factors on mesoderm induction and specification, the large numbers of mesodermal cells accessible with the GFPBry model will provide a novel approach for defining new factors and genes that regulate these developmental steps.
ACKNOWLEDGMENTS
We thank members of the Keller laboratory for critical reading of the manuscript. This work was supported by National Institutes of Health grants RO1 HL48834 and RO1 HL65169 and Human Frontiers in Science grant RG0345/1999-M 103. H.J.F. is supported by Sonderforschungsbereich (SFB) 497-Projekt A7. Part of this work has been carried out at the former Basel Institute for Immunology supported by F. Hoffmann-La Roche.
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