Studies described here advance our understanding of the molecular properties of mouse endoderm, leading to practical innovations for marking, generating, purifying, and assessing both native and in vitro-
derived DE. While previous studies had reported gene expression profiles of mouse endoderm partially purified by microdissection or FACS [2
], separation of definitive from VE was not previously achieved, thereby precluding discovery of a specific gene expression profile of DE. Thus, our use of classical FACS- and microarray-based methods to purify and analyze native DE from other primary germ layers and extraembryonic mouse cells generated a unique “molecular signature” of this tissue that will aid studies of the development of this germ layer and its derivatives both in vivo and in vitro. These signatures proved heuristic in suggesting signaling pathway modifications that fine-tuned the development of Sox17-eGFP+
ES cell progeny toward DE, while reducing its similarity to E7.5 DE. Thus, our study provides a paradigm for using the rigor of genomic-scale expression profiling of native endoderm to guide ES cell differentiation toward endodermal fates, a strategy not used in previous studies.
One impediment to investigating mouse endoderm biology has been a relative paucity of molecular probes for fundamental aspects of endoderm development, such as axial patterning, tissue specification, and differentiation. In this study, we generated highly specific gene expression profiles of DE and VE, which identified several endodermal markers we characterized and authenticated using flow cytometry and in situ hybridization. To generate endoderm purification strategies independent of transgene marking or mouse genotype, we focused attention on markers predicted by microarray analysis to be cell surface proteins expressed in endoderm, such as CD24, CD55, and CD38. Thus, our flow cytometry studies showing that native DE at E7.5 and E8.25 is EpCAM+
provide a unique strategy for purifying endoderm from mice in a variety of genetic conditions. Combined with other methods [38
], these findings should facilitate studies of endoderm formation.
DE is patterned along all 3 major embryonic axes, most strikingly the anterior-posterior axis, and DE isolated by FACS based on CD24 or CD55 was enriched for a variety of markers expressed throughout the endoderm axis, such as Gfpt2
in foregut, and Krt19
in hindgut. Thus, results reported here should facilitate region specific DE cell labeling and purification, allowing further developmental studies of mechanisms underlying foregut, midgut, and hindgut endoderm regionalization, a highly dynamic process [10
]. Our studies also reveal methods to isolate and investigate embryonic VE, a vital source of extraembryonic tissues such as the yolk sac, where embryonic hematopoiesis initiates.
Previous studies have reported differentiation of endoderm-like cells from mouse and human embryonic stem cells after treatment with growth factors or small molecules [4
]. These differentiated cell populations were found to be heterogeneous, containing undifferentiated cells, ectoderm and mesodermal derivatives in addition to endoderm-like cells. To address this, transgenic mouse ES cell lines expressing fluorescent protein markers from loci such as Gsc, FoxA2
, and Sox17
were used to isolate endoderm from these cell mixtures by FACS, but only 1 previous report described attempts to purify DE from genetically unmodified mouse ES cells [7
]. In that study, antibodies to E-cadherin and Cxcr4 permitted FACS isolation of mesendoderm-like progeny of mES cells, but did not permit FACS purification of native mouse DE, largely because E-caderin and Cxcr4 are expressed in ectoderm and mesoderm, respectively [42
]. Here, we identified new combinations of antibodies recognizing cell surface markers for FACS-based purification of native definitive and VE, and of endoderm-like progeny from ES cell lines. Thus, our studies of endodermal development produced unique methods to purify relevant cells from heterogeneous in vitro cultures or embryonic tissues, which should accelerate understanding of mechanisms regulating endodermal development and organogenesis.
Our findings correlate with a subset of findings by Hoodless and colleagues [2
] that coupled mouse embryonic microdissection with Serial Analysis of Gene Expression to identify markers of mouse endoderm (Supplementary Fig. S2
). For example, similar to an earlier study, we also found enrichment of Nepn,
, and Trh
DE and of Apoc2
, and Cubilin
VE. This correlation supports the use of CD24 as a marker that can distinguish between DE and VE in the mouse embryo. We also identified additional DE markers not described in Hou et al. (2007), including Eppk1
, Gfpt2, Sorcs2, Nedd9
, and Krt19.
Our gene expression profiling also corroborates a subset of data reported by Sherwood et al. (2007), who used an FACS-based strategy to isolate endoderm from embryonic mice harboring a transgene encoding Sox17 cis-
regulatory elements that drove expression of a modified yellow fluorescent protein in all 3 primary embryonic germ layers (Supplementary Fig. S2
). However, unlike that previous study, our gene expression profiling clearly distinguishes DE and VE, allowing for several innovations, including separation and purification of genetically unmodified DE and VE.
Our understanding of signaling pathways that regulate endoderm development has grown in recent years [10
], but much remains to be learned about the cell interactions and signaling pathways that govern endodermal differentiation, which occurs in the context of complex morphogenetic movement of the primary germ layers. Experiments in frogs, fish, and mice provide strong evidence that Wnt and Nodal signaling regulate early endoderm development [10
], and addition of purified Wnt and Activin A to mouse and human ES cell cultures is a “standard” strategy for inducing differentiation of endoderm-like progeny [46
]. However, results described here demonstrate substantial differences between the gene expression profile of purified native Sox17+
DE and that of Sox17+
endoderm-like progeny derived from Wnt and Activin A treated ES cells, including pathways regulated by FGFs, BMP, and RA. For the FGF pathway, these data support conclusions from recent studies on FGF regulation of mouse DE development [9
]. Collectively, our findings may prove useful for verifying the quality of endoderm-like cells produced from multipotent sources such as ES or induced pluripotent stem (iPS) cells. Moreover, our gene expression findings motivated us to modify FGF, Noggin, and RA signaling in mouse ES cell cultures, resulting in an increased correlation of the gene expression patterns between ES-derived endoderm-like cells and native DE. Thus, our studies have proved heuristic for optimizing derivation and purification of ES cell progeny with features of DE. BMP and FGF signaling are also thought to direct axial specialization of native endoderm; thus, endodermal development from ES progeny with modulators of these signaling pathways may reflect both refined formation of DE-like cells and recapitulation of axial patterning in our cultures. We postulate that the remaining gap between native and ES cell-derived DE reflects the absence of additional signaling interactions in ES cell cultures, including the complex, dynamic reciprocal signaling known to occur between DE and adjacent mesoderm. We further speculate that our results should facilitate small molecule screens [4
] to identify index compounds that promote endoderm differentiation or maturation.
Based on progress from studies with our mouse Sox17-eGFP
transgenic ES cell line, we used a homologous recombination to build a similar human ES cell line to investigate the development of human endoderm-like cells [16
]. Here, we showed that human SOX17-eGFP+
cells derived from cultures exposed to Wnt and Activin A can be FACS purified based on eGFP expression to assess endoderm differentiation. For example, similar to our mouse studies, FACS purification of human SOX17-eGFP+
cells allowed microarray assessment of cells cultured with specific conditions to develop endoderm-like cells. Fine-tuning of an endoderm-like gene expression signature in hSOX17-eGFP+
cells derived from cultures exposed to unique combinations of growth factors including Wnt, Activin A, FGF, Noggin, and RA provides evidence that this new human ES cell line will be a useful tool for studies of human endoderm differentiation. Moreover, our work suggests that studies of native mouse DE can guide hES cell differentiation in vitro.