Extraembryonic Endoderm Formation Research Articles

Technical note: improving the efficiency of generating bovine extraembryonic endoderm cells.

The formation of extraembryonic endoderm (XEN) occurs early in embryonic development. The cell types that develop from the XEN remain poorly studied in ruminant species because of the lack of suitable cell culture model systems. The goal of this work was to establish a protocol for producing XEN cell cultures from bovine blastocysts. Previous work identified fibroblast growth factor 2 (FGF2) as a facilitator of bovine XEN development. Further refinements in culture conditions studied here included exposure to 20% fetal bovine serum and FGF2 replenishment. These modifications yielded an endoderm outgrowth formation incidence of 81.6% ± 5.5% compared with 33.3% ± 5.5% in bovine serum albumin (BSA)-supplemented controls. These cells resembled XEN when examined morphologically and contained XEN transcripts (GATA binding protein 4 [GATA4] and GATA binding protein 6 [GATA6]) as well as transcripts present in visceral (BCL2 interacting protein 1 [BNIP1] and vascular endothelial growth factor A [VEGFA]) and parietal (C-X-C motif chemokine receptor 4 [CXCR4], thrombomodulin [THBD], and hematopoietically expressed homeobox [HHEX]) XEN. Two XEN cell lines were maintained for prolonged culture. Both lines continued to proliferate for approximately 6 wk before becoming senescent. These cultures maintained an XEN-like state and continued to express GATA4 and GATA6 until senescence. An increase in the abundance of visceral and parietal XEN transcripts was observed with continued culture, suggesting that these cells either undergo spontaneous differentiation or retain the ability to form various XEN cell types. Stocks of cultured cells exposed to a freeze-thaw procedure possessed similar phenotypic and genotypic behaviors as nonfrozen cells. To conclude, a procedure for efficient production of primary bovine XEN cell cultures was developed. This new protocol may assist researchers in exploring this overlooked cell type for its roles in nutrient supply during embryogenesis.

Serum-dependent and -independent regulation of PARP2.

PARP2 belongs to a family of proteins involved in cell differentiation, DNA damage repair, cellular energy expenditure, and chromatin modeling. In addition to these overlapping functions with PARP1, PARP2 participates in spermatogenesis, T-cell maturation, extra-embryonic endoderm formation, adipogenesis, lipid metabolism, and cholesterol homeostasis. Knowledge of the functions of PARP2 is far from complete, and the mechanism(s) by which the gene and protein are regulated are unknown. In this study, we found that two different mechanisms are used in vitro to regulate PARP2 levels. In the presence of serum, PARP2 is degraded through the ubiquitin-proteasome pathway; however, when serum is removed or dialyzed with a 3.5 kDa molecular cut membrane, PARP2 rapidly becomes sodium dodecyl sulphate- and urea-insoluble. Despite the presence of a putative serum response element in the PARP2 gene, transcription is not affected by serum deprivation, and PARP2 levels are restored when serum is replaced. The loss of PARP2 affects cell differentiation and gene expression linked to cholesterol and lipid metabolism. These observations highlight the critical roles that PARP2 plays under different physiological conditions, and reveal that PARP2 is tightly regulated by distinct pathways.

A XEN-like State Bridges Somatic Cells to Pluripotency during Chemical Reprogramming

Somatic cells can be reprogrammed into pluripotent stem cells (PSCs) by using pure chemicals, providing a different paradigm to study somatic reprogramming. However, the cell fate dynamics and molecular events that occur during the chemical reprogramming process remain unclear. We now show that the chemical reprogramming process requires the early formation of extra-embryonic endoderm (XEN)-like cells and a late transition from XEN-like cells to chemically-induced (Ci)PSCs, a unique route that fundamentally differs from the pathway of transcription factor-induced reprogramming. Moreover, precise manipulation of the cell fate transition in a step-wise manner through the XEN-like state allows us to identify small-molecule boosters and establish a robust chemical reprogramming system with a yield up to 1,000-fold greater than that of the previously reported protocol. These findings demonstrate that chemical reprogramming is a promising approach to manipulate cell fates.

Redox Regulation of Canonical Wnt Signaling Affects Extraembryonic Endoderm Formation

Retinoic acid (RA) induces mouse F9 cells to form primitive endoderm (PrE) and increased levels of reactive oxygen species (ROS) accompany differentiation. ROS are obligatory for differentiation and while H2O2 alone induces PrE, antioxidants attenuate the response to RA. Evidence shows that ROS can modulate the Wnt/β-catenin pathway and in this study, we show that extraembryonic endoderm formation is dependent on the redox state of nucleoredoxin (NRX). In undifferentiated F9 cells, NRX interacted with dishevelled 2 (Dvl2) and while this association was enhanced under reduced conditions, it decreased following H2O2 treatment. Depleting NRX levels caused morphological changes like those induced by RA, while increasing protein kinase A activity further induced these PrE cells to parietal endoderm. Reduced NRX levels also correlated to an increase in T-cell-factors-lymphoid enhancer factors-mediated transcription, indicative of canonical Wnt signaling. Together these results indicate that a mechanism exists whereby NRX maintains canonical Wnt signaling in the off state in F9 cells, while increased ROS levels lift these constraints. Dvl2 no longer bound to NRX is now positioned to prime the Wnt pathway(s) required for PrE formation.

Arf tumor suppressor and miR-205 regulate cell adhesion and formation of extraembryonic endoderm from pluripotent stem cells

Induction of the Arf tumor suppressor (encoded by the alternate reading frame of the Cdkn2a locus) following oncogene activation engages a p53-dependent transcriptional program that limits the expansion of incipient cancer cells. Although the p19(Arf) protein is not detected in most tissues of fetal or young adult mice, it is physiologically expressed in the fetal yolk sac, a tissue derived from the extraembryonic endoderm (ExEn). Expression of the mouse p19(Arf) protein marks late stages of ExEn differentiation in cultured embryoid bodies (EBs) derived from either embryonic stem cells or induced pluripotent stem cells. Arf inactivation delays differentiation of the ExEn lineage within EBs, but not the formation of other germ cell lineages from pluripotent progenitors. Arf is required for the timely induction of ExEn cells in response to Ras/Erk signaling and, in turn, acts through p53 to ensure the development, but not maintenance, of the ExEn lineage. Remarkably, a significant temporal delay in ExEn differentiation detected during the maturation of Arf-null EBs is rescued by enforced expression of mouse microRNA-205 (miR-205), a microRNA up-regulated by p19(Arf) and p53 that controls ExEn cell migration and adhesion. The noncanonical and canonical roles of Arf in ExEn development and tumor suppression, respectively, may be conceptually linked through mechanisms that govern cell attachment and migration.

Ras/MAPK Pathway Confers Basement Membrane Dependence upon Endoderm Differentiation of Embryonic Carcinoma Cells

The formation of extraembryonic endoderm is one of the earliest steps in the differentiation of pluripotent cells of the inner cell mass during the early stages of embryonic development. The primitive endoderm cells and the derived parietal and visceral endoderm cells gain the capacity to produce collagen IV and laminin. The deposition of these components results in the formation of basement membrane and epithelium of the endoderm, with polarized cells covering the inner surface of the blastocoels. We used retinoic acid-induced endoderm differentiation of stem cell-like F9 embryonic carcinoma cells to study the role of the Ras pathway and its regulation in the formation of the visceral endoderm. Upon endoderm differentiation of F9 cells induced by retinoic acid, c-Fos expression, the downstream target of the Ras pathway, is suppressed by uncoupling Elk-1 phosphorylation/activation to MAPK activity. However, attachment to matrix gel greatly enhances the activation of MAPK in endoderm cells but not in undifferentiated F9 cells. Enhanced MAPK activation as a result of contact with basement membrane is able to compensate for reduced Elk-1 phosphorylation and c-Fos expression. We conclude that endoderm differentiation renders the activation of the Ras pathway basement membrane dependent, contributing to the epithelial organization of the visceral endoderm.

Identification of a retinoic acid-inducible element in the murine PTH/PTHrP (parathyroid hormone/parathyroid hormone-related peptide) receptor gene.

We have shown previously that the PTH/PTHrP (PTH-related peptide) receptor mRNA becomes expressed very early in murine embryogenesis, i.e. during the formation of extraembryonic endoderm. Retinoic Acid (RA) is a potent inducer of extraembryonic endoderm formation and PTH/PTHrP-receptor expression in embryonal carcinoma (EC) and embryonal stem (ES) cells. Using the P19 EC cell line, we have characterized promoter elements of the murine PTH/PTHrP-receptor gene that are involved in this RA-induced expression. The data show that RA-induced expression of the PTH/ PTHrP-receptor gene is mediated by the downstream P2 promoter. Analysis of promoter reporter constructs in transiently transfected P19 cells treated with RA identified an enhancer region between nucleotides -2714 and -2702 upstream of the P2 transcription start site that is involved in the RA effect. This region matches a consensus hormone response element consisting of a direct repeat with an interspacing of 1 bp (R-DR1). The R-DR1 efficiently binds retinoic acid receptor-alpha (RARalpha)-retinoid X receptor-alpha (RXRalpha) and chicken ovalbumin upstream promoter (COUP)-transcription factor I (TFI)-RXRalpha heterodimers and RXRalpha and COUP-TFI homodimers in a bandshift assay using extracts of transiently transfected COS-7 cells. RA differentiation of P19 EC cells strongly increases protein binding to the R-DR1 in a band-shift assay. This is caused by increased expression of RXR (alpha, beta, or gamma) and by the induction of expression of RARbeta and COUP TFI/TFII, which bind to the R-DR1 as shown by supershifting antibodies. The presence of RXR (alpha, beta, or gamma) in the complexes binding to the R-DR1 suggests that RXR homodimers are involved in RA-induced expression of the PTH/PTHrP-receptor gene. The importance of the R-DR1 for RA-induced expression of PTH/ PTHrP-receptor was shown by an inactivating mutation of the R-DR1, which severely impairs RA-induced expression of PTH/PTHrP-receptor promoter reporter constructs. Since this mutation does not completely abolish RA-induced expression of PTH/PTHrP-receptor promoter reporter constructs, sequences other than the R-DR1 might also be involved in the RA effect. Finally, we show that the RA-responsive promoter region is also able to induce expression of a reporter gene in extraembryonic endoderm of 7.5 day-old transgenic mouse embryos.

The Ras/Erk Pathway Induces Primitive Endoderm but Prevents Parietal Endoderm Differentiation of F9 Embryonal Carcinoma Cells

The formation of parietal endoderm (PE) is one of the first differentiation processes during mouse development and can be studied in vitro using F9 embryonal carcinoma (EC) cells. Treatment of F9 EC cells with retinoic acid (RA) induces differentiation toward primitive endoderm (PrE), while differentiation toward PE is induced by subsequent addition of parathyroid hormone (PTH) or PTH-related peptide (PTHrP). The signal transduction mechanisms involved in this two-step process are largely unclear. We show that the RA-induced differentiation toward PrE is accompanied by a sustained increase in Ras activity and that ectopic expression of oncogenic Ha-Ras is sufficient to induce PrE differentiation. Ras activity subsequently decreases upon PTH-induced differentiation toward PE. This is a necessary event, since expression of oncogenic Ha-Ras in PrE-like cells prevents PTH-induced PE differentiation. Expression of active PKA in PrE-like F9 cells mimics PTH-induced PE differentiation and is again prevented by oncogenic Ha-Ras. The effect of oncogenic Ras on both differentiation steps is abolished by the MEK inhibitor PD98059 and can be mimicked by constitutively active forms of Raf and MEK. In conclusion, our data suggest that activation of the Ras/Erk is sufficient to induce differentiation to PrE and to prevent subsequent differentiation toward PE. Activation of PKA down-regulates Ras activity, resulting in disappearance of this blockade and transmission of signal(s) triggering PE differentiation.

Signals governing extraembryonic endoderm formation in the mouse: involvement of the type 1 parathyroid hormone-related peptide (PTHrP) receptor, p21Ras and cell adhesion molecules.

The formation of parietal endoderm (PE) from primitive endoderm (PrE) immediately after implantation of the early mouse embryo can be seen as the earliest example of an epithelio-mesenchyme transition (EMT) in murine development. Since EMT and EMI (epithelium-mesenchyme interactions) are at the very heart of morphogenesis, identifying molecular mechanisms governing these processes is of utmost importance. An excellent in vitro model system to study PE formation, i.e. F9 embryonal carcinoma cells, is available to this end. In the present paper we review our own recent results and those of others using these cells, and present our current view on the molecular mechanisms involved in PE formation.

Polarized and functional epithelia can form after the targeted inactivation of both mouse keratin 8 alleles.

We have tested the requirement of keratin intermediate filaments for the formation and function of a simple epithelium. We disrupted both alleles of the mouse keratin 8 (mK8) gene in embryonic stem cells, and subsequently analyzed the phenotype in developing embryoid bodies in suspension culture. After the inactivation of the mouse keratin 8 (mK8) gene by a targeted insertion, mK8 protein synthesis was undetectable. In the absence of mK8 its complementary partners mK18 and mK19 were unable to form filaments within differentiated cells. Surprisingly, these ES cells differentiate to both simple and cystic embryoid bodies with apparently normal epithelia. Ultrastructural analysis shows an apparently normal epithelium with microvilli on the apical membrane, tight junctions and desmosomes on the lateral membrane, and an underlying basal membrane. No significant differences in the synthesis or secretion of alpha 1-fetoprotein and laminin were observed between the mK8- or wild-type embryoid bodies. Our data show that mK8 is not required for simple epithelium formation of extraembryonic endoderm.