Early Embryoid Bodies Research Articles

GCN5 Regulates FGF Signaling and Activates Selective MYC Target Genes during Early Embryoid Body Differentiation.

SummaryPrecise control of gene expression during development is orchestrated by transcription factors and co-regulators including chromatin modifiers. How particular chromatin-modifying enzymes affect specific developmental processes is not well defined. Here, we report that GCN5, a histone acetyltransferase essential for embryonic development, is required for proper expression of multiple genes encoding components of the fibroblast growth factor (FGF) signaling pathway in early embryoid bodies (EBs). Gcn5−/− EBs display deficient activation of ERK and p38, mislocalization of cytoskeletal components, and compromised capacity to differentiate toward mesodermal lineage. Genomic analyses identified seven genes as putative direct targets of GCN5 during early differentiation, four of which are cMYC targets. These findings established a link between GCN5 and the FGF signaling pathway and highlighted specific GCN5-MYC partnerships in gene regulation during early differentiation.

Functions of retinoic acid on the biological characteristics of human embryonic stem cells

To analyze the influence of retinoic acid (RA) on the undifferentiated state and EB formation abilities of human embryonic stem cells. The biological characteristics of H9 ESCs after RA treatment were characterized by real-time PCR, MTS proliferation assay and immunofluorescence staining. The expression of three germ layers markers, osteogenic differentiation markers and adipogenic differentiation markers in H9-differentiated embryoid bodies (EBs) with RA treatment were quantified by real time PCR. The proliferation of H9 ESCs in the early logarithmic growth phase was accelerated by RA treatment. In addition, RA induced differentiation of H9 ESC coupled with morphology changes, decreased expression of undifferentiated markers Oct4, Nanog, Sox2 and OCT4 mRNA binding protein Lin28 at mRNA level, and reduced expression of Oct4 at protein level. RA induced formation of cavities in EBs. Real time PCR results showed that the expressions of ectodermal markers: NeuroD1, Noggin; mesodermal markers: Brachyury, Twist and endodermal markers: AFP, GATA-4 were significantly increased (P < 0.05), especially for AFP (P < 0.01), by RA treatment in a dose-dependent manner. In addition, the expression of adipogenic differentiation marker C/EBPalpha was increased while the osteogenic differentiation marker OPN was decreased in EBs after RA treatment for 5 days. High concentrations of RA induced the loss of stemness in H9 ESCs and excessive differentiation in EBs, and damaged the balance between osteogenic and adipogenic differentiation during early EB differentiation, which may be relevant to the congenital malformations.

Modeling Congenital Dyserythropoietic Anemia Type I Through Patient-Derived Induced Pluripotent Stem Cells

AbstractAbstract 3196Congenital dyserythropoietic anemias (CDAs) are rare inherited disorders characterized by impaired red blood cell formation (dyserythropoiesis) and signature cytopathologies. CDA type I is an autosomal recessive disease with macrocytic anemia and occasional bone abnormalities. Erythroid precursors exhibit pathognomonic abnormalities including internuclear chromatin bridges and spongy (“Swiss cheese”) heterochromatin. The disease is caused by biallelic mutations in the gene CDANI (Dgany et al., 2002), which encodes codanin-1, a ubiquitously expressed protein that is believed to have fundamental roles in cell cycle control and chromatin structure (Noy-Lotan et. al, 2009). Animal models for the study of CDA I are suboptimal and clinical samples are scarce. Thus, we have developed an experimental model for the study of CDA I by generating induced pluripotent stem cells (iPSCs) from affected patients. We reprogrammed fibroblasts from CDA I patients and normal subjects using a single lentiviral vector encoding OCT4, KLF4, SOX2, and MYC. The resultant iPSCs exhibited standard criteria for pluripotency and the integrated reprogramming vector was excised using Cre-lox technology.We differentiated CDA I and control iPSCs into erythroid progenitors by inducing the formation of embryoid bodies (EBs) with stepwise additions of supportive cytokines. Beginning at about day 8, hematopoietic progenitors with erythroid potential were detected within EBs and as free-floating cells in the medium. Our differentiation protocol showed two waves of erythroid precursor production. Early EBs (days 12 to 23) produced erythroid cells that expressed mainly epsilon globin, resembling early yolk sac type “primitive” erythropoiesis. In contrast, erythroblasts produced from later EBs (days 27 to 50) expressed mainly gamma globins, resembling “definitive” erythroid cells produced by late stage yolk sac and fetal liver. Our preliminary studies, indicate that CDA I iPSCs produce normal numbers of primitive and definitive erythrocytes. No defects in survival or maturation were detected by flow cytometry assessing the expression of annexin V and the developmental stage markers CD235/CD71/forward scatter. However, definitive type (but not primitive) erythroblasts derived from CDA I iPSCs exhibit some characteristic pathological features including occasional internuclear chromatin bridging visible by light microscopy and spongy “Swiss cheese” heterochromatin revealed by transmission electron microscopy. Thus, patient-derived iPSCs can model at least some aspects of CDA I and provide the basis for future studies to define the actions of codanin-1 and the pathophysiology of this disorder. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

JAK/STAT Signal Transduction Pathway Activation During Hematopoietic Differentiation From Human Embryonic Stem Cells (hESC)

Abstract 2346The ability to produce hematopoietic cells from human embryonic stem cells (hESC) has been demonstrated, using different multistage culture systems with multiple growth factor combinations. However, very little is understood about the molecular mechanisms that regulate the differentiation from hESC to hematopoietic stem and progenitor cells and further to specific lineages of differentiated hematopoietic cells. Among many signaling pathways involved in stem and progenitor cell differentiation, the JAK/STAT pathways are known to play critical roles in hematopoietic stem cell maintenance and hematopoietic differentiation. STAT3 activation is known to be essential for maintenance of murine ESC, but not human ESC, but it appears not to play a major role in myeloid cell differentiation. Although different levels of JAK2 and STAT5 signaling are important for erythroid and megakaryocytic differentiation, JAK/STAT signaling is not thought to play a role in hESC maintenance or differentiation and is not known to be essential for early stages of differentiation to hematopoietic stem and progenitor cells (HSC/HPC). We have established a serum-free, feeder cell-free system for maintaining hESC (H1 and H9 cells) and for differentiating the hESC to embryoid bodies (EB), from which end-stage hematopoietic cells, notably megakaryocytes and platelets, are produced. We used a multi-stage culture system to produce megakaryocytes and platelets from EBs, including 2 days with vascular endothelial growth factor (VEGF) and bone morphogenic protein (BMP4), 2 more days with VEGF, BMP4, stem cell factor (SCF), Flt3 ligand (FL), and thrombopoietin (TPO), 10 days with VEGF, BMP4, TPO, SCF, FL, IL3, IL6, and IL11, and 2–6 weeks with TPO, SCF, FL, IL3, IL6, and IL11. We used serial western blots, immunofluorescence with confocal microscopy and systematically observed changes of JAK/STAT signal transduction molecule activation. We found a consistent, dynamic change of STAT5 protein phosphorylation during the hematopoietic differentiation process. Interestingly, although JAK2, STAT3 and STAT5 protein were present, and JAK2 and STAT3 were phosphorylated in hESC, there was no evidence of STAT5 phosphorylation/activation in the undifferentiated hESC. During the early phases of differentiation of hESC-derived EBs toward hematopoietic progenitors in the presence of hematopoiesis-related cytokines, STAT5 was phosphorylated and activated in CD34+ HSCs and in CD61+/CD235a (glycophorin A)+ or CD41+/CD235a+ early megakaryocytic/erythroid progenitor cells (MEP). Although there was no detectable change in total STAT5 protein expression levels through hematopoietic differentiation, there was a slowly progressive decrease in phosphorylated/activated STAT5 with further maturation to megakaryocytes that express CD42b+, platelet factor 4, and von Willebrand factor and form proplatelets and platelets. Thus, in spite of hESC containing abundant phosphorylated JAK2, which is a known activator of STAT5, there was no phosphorylation/activation of STAT5 in undifferentiated hESC or early EBs. However, STAT5 became phosphorylated/activated early in hematopoiesis and declined over the course of progressive differentiation along the megakaryocytic lineage. These findings imply that activated JAK2 does not drive the activation of STAT5 that is an early event in differentiation from EBs and mesoderm to HSC and HPC in vitro. Disclosures:No relevant conflicts of interest to declare.

An organogenesis network-based comparative transcriptome analysis for understanding early human development in vivo and in vitro

BackgroundIntegrated networks hold great promise in a variety of contexts. In a recent study, we have combined expression and interaction data to identify a putative network underlying early human organogenesis that contains two modules, the stemness-relevant module (hStemModule) and the differentiation-relevant module (hDiffModule). However, owing to its hypothetical nature, it remains unclear whether this network allows for comparative transcriptome analysis to advance our understanding of early human development, both in vivo and in vitro.ResultsBased on this integrated network, we here report comparisons with the context-dependent transcriptome data from a variety of sources. By viewing the network and its two modules as gene sets and conducting gene set enrichment analysis, we demonstrate the network's utility as a quantitative monitor of the stem potential versus the differentiation potential. During early human organogenesis, the hStemModule reflects the generality of a gradual loss of the stem potential. The hDiffModule indicates the stage-specific differentiation potential and is therefore not suitable for depicting an extended developmental window. Processing of cultured cells of different types further revealed that the hStemModule is a general indicator that distinguishes different cell types in terms of their stem potential. In contrast, the hDiffModule cannot distinguish between differentiated cells of different types but is able to predict differences in the differentiation potential of pluripotent cells of different origins. We also observed a significant positive correlation between each of these two modules and early embryoid bodies (EBs), which are used as in vitro differentiation models. Despite this, the network-oriented comparisons showed considerable differences between the developing embryos and the EBs that were cultured in vitro over time to try to mimic in vivo processes.ConclusionsWe strongly recommend the use of these two modules either when pluripotent cell types of different origins are involved or when the comparisons made are constrained to the in vivo embryos during early human organogenesis (and an equivalent in vitro differentiation models). Network-based comparative transcriptome analysis will contribute to an increase in knowledge about human embryogenesis, particularly when only transcriptome data are currently available. These advances will add an extra dimension to network applications.

Wnt-Catenin Signaling System Functions in Embryoid Bodies Aggregated from Human Embryonic Stem Cell

As an essential molecule in Wnt/β-catenin signaling, β-Catenin plays a crucial role in the decision making for tissue differentiation in embryogenesis and pathogenesis. The signaling was implicated in the development of skin. In mouse skin, the deferential fate of the skin stem cell depends on β-catenin, which organizes stem cells into follicular or epidermal lineages. These analyses indicate that Wnt/β-catenin signaling should also function in the development and differentiation of human embryonic stem cell. To study the function of β-catenin in early differentiation of human stem cells, we cultured H9 stem cell and aggregated them into embryoid bodies (EB). In this study, we revealed that in early EBs some guarding cells were first differentiated from EB stem cell aggregates. These early differentiated cells for guarding epithelial cells have strongest expression of β-catenin within EB. These cells were flattened on the surface of EB, covering the surface by connections formed through protein interactions. At certain confocal sections of EB, instead of a round boundary, a polygonal boundary was observed even though the EB appeared round under conventional microscope. In these polygonal boundaries, β-catenin positive guarding epithelial cells were positioned on every corner of the polygon. In the inner portion of the EB, undifferentiated β-catenin positive cells express β-catenin in the nucleus. As the initially simple shape of EB becomes more and more intricate during development, we revealed that more β-catenin positive cells were also observed in this complex structure. Based on these results, we predicted β-catenin to play different roles while guarding epithelial cells or undifferentiated stem cells in the inner portion of EB in in vitro culture system. Phosphorylation of β-catenin may be a critical factor for fate determination of the human stem cell.

A Three Dimensional Anchorage Independent In Vitro System for the Prolonged Growth of Embryoid Bodies to Study Cancer Cell Behaviour and Anticancer Agents

We describe a three dimensional (3D) anchorage independent in vitro protocol for the prolonged growth of human embryoid bodies (EBs) up to 90 days. We grew hESCs (46XX) in methylcellulose (MC) in motion culture in the presence of EB medium (EB), EB medium with Matrigel (EB + MAT), bulk culture medium (BCM), and BCM medium with Matrigel (BCM + MAT). All four experimental groups produced embryoid bodies (EBs) which with prolonged growth to 90 days acquired blood vessels and tissues from all three germ layers. Based on histology, microarray gene expression profiles and the definition for experimental teratomas, we could classify the EBs into early EBs, mature EBs and teratomas. The EB + MAT group produced the highest number of teratomas and their microarray data suggested the presence of inductive microenvironment niches and activation of pathways for self-organization, morphogenesis and growth. When we microinjected hepatocarcinoma-Green Fluorescent Protein cells (HepG2-GFP) (46XY) into the teratomas, after 10 days the HepG2-GFP cells had grown inside the teratoma as confirmed by confocal microscopy and SRY gene analysis. This 3D-MC-(EB + MAT) in vitro system requires few cells to produce many teratomas, can be used to test pluripotency of potential human embryonic and induced pluripotent stem cell lines (hESC, hiPSC), and is an experimental humanized platform to study cancer cell behavior.

Use of Long-term Cultured Embryoid Bodies May Enhance Cardiomyocyte Differentiation by BMP2

PurposeHuman embryonic stem cells (hESCs) can proliferate for a prolonged period and differentiate into cardiomyocytes in vitro. Recent studies used bone morphogenetic protein 2 (BMP2) to generate cardiomyocytes from hESCs, however, all those studies used early embryoid bodies (EBs) and did not retrieve cardiomyocytes with a high yield. In this study, we treated long-term cultured EBs with BMP2 in order to promote differentiation into cardiomyocytes from hESCs.Materials and MethodshESC lines, including SNUhES3 and SNUhES4, were used in this study. Undifferentiated hESC colonies were detached to form EBs and cultured for up to 30 days. These long-term cultured EBs were differentiated into cardiomyocytes in serum-containing media. In our protocol, BMP2 was applied for 5 days after attachment of EBs. Cardiac specific markers, beating of differentiated cells and electron microscopic (EM) ultrastructures were evaluated and analyzed.ResultsCompared to 10-day or 20-day EBs, 30-day EBs showed a higher expression level of cardiac specific markers, Nkx2.5 and α-myosin heavy chain (αMHC). Treatment of BMP2 increased expression of cardiac troponin (cTn) I and α-actinin when evaluated at 20 days after attachment of 30-day EBs. Beating of differentiated cells was observed from 7 to 20 days after attachment. Moreover, EM findings demonstrated fine structures such as Z bands in these differentiated cardiomyocytes. These long-term cultured EBs yielded cardiomyocytes with an efficiency of as high as 73.6% when assessed by FACS.ConclusionWe demonstrated that the use of long-term cultured EBs may enhance differentiation into cardiomyocytes from hESCs when treated with BMP2.

Differential expression of glucose transporter isoforms during embryonic stem cell differentiation

In mouse blastocysts six facilitative glucose transporter isoforms (GLUT)1–4, 8 and 9 are expressed. We have used the mouse embryonic stem (ES) cell line D3 and spontaneously differentiating embryoid bodies (EB) to investigate GLUT expression and the influence of glucose during differentiation of early embryonic cells. Both ES cells and EBs (2d–20d) expressed GLUT1, 3, and 8, whereas the isoforms 2 and 4 were detectable exclusively in EBs. Differentiation-associated expression of GLUT was analyzed by double staining with stage-specific embryonic antigen (SSEA-1), cytokeratins (CK18, 19), nestin, and desmin. Similar to trophoblast cells in mouse blastocysts the outer cell layer of endoderm-like cells showed a high GLUT3 expression in early EBs. In 20-day-old EBs no GLUT3 protein and only minor GLUT3 mRNA amounts could be detected. A minimal glucose concentration of 5 mM applied during 2 and 8 days of EB culture resulted in up-regulated GLUT4, Oct-4 and SSEA-1 levels and a delay in EB differentiation. We conclude that GLUT expression depends on cellular differentiation and that the expression is modulated by glucose concentration. The developmental and glucose-dependent regulation of GLUT strongly suggests a functional role of glucose and glucose transporters in ES cell differentiation and embryonic development.

Lymphoid, Long-Term Repopulating, and Endothelial Potential of the Embryonic Hemangioblast.

Hematopoietic and endothelial cells are thought to arise from a common progenitor termed the hemangioblast. Direct evidence for the hemangioblast was first obtained from embryonic stem cells differentiated in vitro as embryoid bodies (EBs). Flk-1+ cells from early EBs generate colonies in response to VEGF and SCF (the BL-CFC assay) which can be replated to give secondary hematopoietic or endothelial cells. Bipotent BL-CFCs have also recently been derived from the posterior primitive streak of neural plate stage mouse embryos. However, a direct relationship between the early embryonic hemangioblast defined by the BL-CFC assay and the hematopoietic stem cell remains unproven. Hemangioblast-derived hematopoiesis in vitro is transient and restricted to myelo-erythroid differentiation. Lymphoid potential and long-term repopulation, two hallmarks of the definitive hematopoietic stem cell, have eluded detection to date. Previous work has shown that the homeodomain transcription factor, HoxB4, by enhancing self-renewal in vitro, can reveal latent definitive HSC activity of transient embryonic hematopoietic progenitors. Using an ES cell line with doxycycline-inducible HoxB4 expression, we have investigated the definitive hematopoietic and endothelial potential of individual hemangioblast colonies. BL-CFC numbers were unaffected by HoxB4 expression during EB differentiation, however they were increased threefold by induction during the BL-CFC assay. By replating one half of the cells from an individual blast colony in endothelial medium and the other half on an OP9 monolayer with hematopoietic cytokines, we show that the majority (60%) of HoxB4-induced BL-CFCs are bipotent. HoxB4 expression was compatible with endothelial differentiation and allowed exponential expansion of hematopoietic progenitors on OP9 cocultures. When switched to OP9-DL1 with lymphoid cytokines, T-lymphopoiesis was observed characterized by CD25 expression followed by CD4, CD8, and CD3epsilon expression. To assay long-term repopulation, individual blast colonies were picked and divided into endothelial medium and OP9 monolayers. The hematopoietic arms of colonies defined retrospectively to have been bipotent (endothelial differentiation was observed in vitro) were transplanted into sublethally irradiated Rag2; gamma-c; CD45.1 immunodeficient mice. Mice with long-term hematopoietic engraftment were identified by the presence of CD45.2 cells in peripheral blood 3 months post-transplant. Lymphoid and myeloid contribution was evaluated by costaining with Gr-1, B220, CD19, CD4, and CD8. The donor-derived component of these hematopoietic chimeras, including their entire lymphoid arm (approximately 1/3 of engrafted mice showed lymphoid differentiation) is by definition clonally derived from a single hemangioblast. These results clearly show that the embryonic hemangioblast is not intrinsically limited in its hematopoietic potential. Under conditions that favor self-renewal, lymphoid differentiation and long-term repopulation become evident, revealing the link between endothelial development and definitive hematopoiesis at the clonal level.

Expression of the liver‐specific gene Cyp7a1 reveals hepatic differentiation in embryoid bodies derived from mouse embryonic stem cells

Hepatic differentiation from mouse embryonic stem (ES) cells via the formation of embryoid bodies (EBs) has been revealed by the expression of hepatocyte-related genes such as alpha-fetoprotein and albumin. It is known, however, that the visceral endoderm differentiates in early EBs and expresses these hepatocyte-related genes. Thus, it remains unclear whether ES cells are capable of differentiating into hepatocytes derived from definitive endoderm in vitro. In the present study, yolk sac tissues isolated from the foetal mouse were found to express many hepatocyte-related genes. Among the hepatocyte-related genes examined, cytochrome P450 7A1 (Cyp7a1) was identified as a liver-specific gene that was not expressed in the yolk sac. Cyp7a1 was induced in developing EBs, and hepatic differentiation was preferentially observed in the developing EBs in attached culture as compared to those in suspension culture. Leukaemia inhibitory factor permitted the differentiation of visceral endoderm, but inhibited the expression of gastrulation-related genes and the hepatic differentiation in cultured EBs. ES cells expressing green fluorescent protein (GFP) under the control of the Cyp7a1 enhancer/promoter showed that cultured EBs contained GFP-positive epithelial-like cells. These results demonstrate that ES cells can differentiate in vitro into hepatocytes derived from definitive endoderm.

Embryoid-body cells derived from a mouse embryonic stem cell line show differentiation into functional hepatocytes

Embryonic stem (ES) cells have a potential to differentiate into various progenitor cells. Here we investigated the differentiation capacity of mouse ES cells into hepatocytes both in vitro and in vivo. During the culture of embryoid bodies (EBs) derived from ES cells, albumin (ALB) messenger RNA (mRNA) was expressed within 12 days after removal of leukemia inhibitory factor, and α-fetoprotein (AFP) mRNA was observed within 9 days without additional exogenous growth factors. In ES cells and early EBs, by contrast, neither ALB mRNA nor AFP mRNA was observed. ALB protein was first detected at day 15 and the level increased with the culture period. The differentiation of EBs facilitated the synthesis of urea with the culture period, whereas early EBs and ES cells produced no urea. These results suggest that cultured EBs contain hepatocytes capable of producing ALB and urea. ES cells and the isolated cells from EBs were transplanted through portal vein to the liver after 30% partial hepatectomy of female mice pretreated with 2-acetylaminofluorene. Four weeks after transplantation with isolated cells from day-9 EBs, ES-derived cells containing Y-chromosome in the liver were positive for ALB (0.2% of total liver cells), whereas teratoma was found in mice transplanted with ES cells or EBs up to day 6. The incidence of teratoma was decreased with the culture duration and no teratoma was observed in the liver transplanted with isolated cells from day-9 EBs. In conclusion, our in vitro and in vivo experiments revealed that cultured EBs contain functional hepatocytes or hepatocyte-like cells. (H EPATOLOGY 2002;36:22-29.)

Forced Expression of the Homeobox-Containing Gene Pem Blocks Differentiation of Embryonic Stem Cells

Similarities in the differentiation of mouse embryos and ES cell embryoid bodies suggest that aspects of early mammalian embryogenesis can be studied in ES cell embryoid bodies. In an effort to understand the regulation of cellular differentiation during early mouse embryogenesis, we altered the expression of the Pem homeobox-containing gene in ES cells. Pem is normally expressed in the preimplantation embryo and expressed in a lineage-restricted fashion following implantation, suggesting a role for Pem in regulating cellular differentiation in the early embryo. Here, we show that the forced expression of Pem from the mouse Pgk-1 promoter in ES cells blocks the in vitro and in vivo differentiation of the cells. In particular, embryoid bodies produced from these Pgk-Pem ES cells do not differentiate into primitive endoderm or embryonic ectoderm, which are prominent features of early embryoid bodies from normal ES cells. This Pgk-Pem phenotype is also different from the null phenotype, as embryoid bodies derived from ES cells in which endogenous Pem gene expression has been blocked show a pattern of differentiation similar to that of normal ES cells. When the Pgk-Pem ES cells were introduced into subcutaneous sites of nude mice, only undifferentiated EC-like cells were found in the teratomas derived from the injected cells. The Pem-dependent block of ES cell differentiation appears to be cell autonomous; Pgk-Pem ES cells did not differentiate when mixed with normal, differentiating ES cells. A block to ES cell differentiation, resulting from the forced expression of Pem, can also be produced by the forced expression of the nonhomeodomain region of Pem. These studies are consistent with a role for Pem in regulating the transition between undifferentiated and differentiated cells of the early mouse embryo.

Inhibition of Phosphatidylinositol-3-kinase Blocks Development of Functional Embryonic Cardiomyocytes

Culturing murine embryonic stem (ES) cells within embryoid bodies (EBs) has been reported to reproduce cardiomyocyte development from primitive precursor cells to highly specialized phenotypes of cardiac tissue. We show here that the specific inhibitor of phosphatidylinositol-3-kinase (PI-3-kinase), LY294002, blocks the growth and induces apoptosis as well as necrosis of D3 ES cells within early EBs. Treatment of EBs from day 3 to day 7 with 50 μM LY294002 resulted in a massive loss of α-actinin-stained cardiomyocytes after plating the EBs for additional 7 days. In parallel we observed a strong decrease in the number of EBs containing area(s) with beating cardiomyocytes. The specific action of the PI-3-kinase inhibitor on development of cardiomyocytes was demonstrated by the observation that formation of endothelial cells was not affected in the same EBs. Our results provide the first evidence that signal transduction via the PI-3-kinase pathway is essential for mammalian early cardiomyocyte development.

Homeobox-Containing Genes in Teratocarcinoma Embryoid Bodies: A Possible Role for Hox-D12 (Hox-4.7) in Establishing the Extraembryonic Endoderm Lineage in the Mouse

We wish to identify genes involved in mediating early lineage decisions in the mouse embryo. F9 teratocarcinoma cells treated with retinoic acid (RA) in suspension culture develop into embryoid bodies (EBs) with an outer layer of visceral endoderm. In order to identify genes that are involved in establishing this extraembryonic endoderm lineage we have employed a PCR-based approach using cDNAs from early EBs as templates. PCR reactions were performed with degenerate oligonucleotide primers coding for the highly conserved regions of the homeodomains of the Drosophila Antennapedia, bicoid, and zerknüllt proteins. Among the PCR products were representatives of previously identified mouse genes, including Hox-A5 (1.3), A1 (1.6), A9 (1.7), B8 (2.4), B2 (2.8), C8 (3.1), and D12 (4.7). Whole mount in situ hybridization analysis, performed to examine the temporal and spatial distribution of transcripts, suggests a possible role for the Hox-D12 gene during endoderm differentiation in F9 EBs. Whereas the expression patterns of several other homeobox genes are essentially uniform throughout the aggregates, Hox-D12 expression is restricted to the outer surface of early EBs at a time when lineage decisions may be occurring. In order to establish the relationship between the Hox-D12 expression pattern and the role of RA in inducing F9 EB differentiation, we examined PSA-1 EBs that differentiate in the absence of added RA. PSA-1 EBs show similar temporal and spatial localization of Hox-D12 when compared to F9 EBs. These data suggest that the pattern of Hox-D12 expression correlates with endoderm differentiation and not with RA treatment and point to a possible role for homeobox-containing genes during the early stages of mouse embryogenesis.