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PubMed1997

A role for Indian hedgehog in extraembryonic endoderm differentiation in F9 cells and the early mouse embryo.

Hedgehog genes in Drosophila and vertebrates control patterning of a number of different structures during embryogenesis. They code for secreted signaling proteins that are cleaved into an active aminopeptide and a carboxypeptide. The aminopeptide can mediate local and long range events and can act as a morphogen, inducing differentiation of distinct cell types in a concentration-dependent manner. We demonstrate here that the expression of Indian hedgehog mRNA and protein is upregulated dramatically as F9 cells differentiate in response to retinoic acid, into either parietal endoderm or embryoid bodies, containing an outer visceral endoderm layer. The ES cell line D3 forms embryoid bodies in suspension culture without addition of retinoic acid and also upregulates Indian hedgehog expression. RT-PCR analysis of blastocyst outgrowth cultures demonstrates that whereas little or no Indian hedgehog message is present in blastocysts, significant levels appear upon subsequent days of culture, coincident with the emergence of parietal endoderm cells. In situ hybridization analysis for Indian hedgehog mRNA expression demonstrates the presence of elevated levels of message in the outer visceral endoderm cells relative to the core cells in mature embryoid bodies and in the visceral endoderm of Day 6.5 embryos. Whole-mount in situ hybridization analysis of Day 7.5 and 8.5 embryos indicates that Indian hedgehog expression is highest in the visceral yolk sac at this stage. F9 cell lines expressing a full length Indian hedgehog cDNA express a number of characteristics of differentiated cells, in the absence of retinoic acid. Taken together, these data suggest that Indian hedgehog is involved in mediating differentiation of extraembryonic endoderm during early mouse embryogenesis.

Animals↗
PubMed2000

The endoderm plays an important role in patterning the segmented pharyngeal region in zebrafish (Danio rerio).

The development of the vertebrate head is a highly complex process involving tissues derived from all three germ layers. The endoderm forms pharyngeal pouches, the paraxial mesoderm gives rise to endothelia and muscles, and the neural crest cells, which originate from the embryonic midbrain and hindbrain, migrate ventrally to form cartilage, connective tissue, sensory neurons, and pigment cells. All three tissues form segmental structures: the hindbrain compartmentalizes into rhombomeres, the mesoderm into somitomeres, and the endoderm into serial gill slits. It is not known whether the different segmented tissues in the head develop by the same molecular mechanism or whether different pathways are employed. It is also possible that one tissue imposes segmentation on the others. Most recent studies have emphasized the importance of neural crest cells in patterning the head. Neural crest cells colonize the segmentally arranged arches according to their original position in the brain and convey positional information from the hindbrain into the periphery. During the screen for mutations that affect embryonic development of zebrafish, one mutant, called van gogh (vgo), in which segmentation of the pharyngeal region is absent, was isolated. In vgo, even though hindbrain segmentation is unaffected, the pharyngeal endoderm does not form reiterated pouches and surrounding mesoderm is not patterned correctly. Accordingly, migrating neural crest cells initially form distinct streams but fuse when they reach the arches. This failure to populate distinct pharyngeal arches is likely due to the lack of pharyngeal pouches. The results of our analysis suggest that the segmentation of the endoderm occurs without signaling from neural crest cells but that tissue interactions between the mesendoderm and the neural crest cells are required for the segmental appearance of the neural crest-derived cartilages in the pharyngeal arches. The lack of distinct patches of neural crest cells in the pharyngeal region is also seen in mutants of one-eyed pinhead and casanova, which are characterized by a lack of endoderm, as well as defects in mesodermal structures, providing evidence for the important role of the endoderm and mesoderm in governing head segmentation.

Animals↗
PubMed1997

Primary endodermal sinus tumor of the vulva: a case report and review of the literature.

BACKGROUND: Extragonadal endodermal sinus tumors arising in the external genitalia represent an exceedingly rare malignancy in women. Six cases of endodermal sinus tumors of the vulva have been reported to date, with three cases failing to respond to conservative surgery and vincristine-based chemotherapy. We report a seventh case of vulvar endodermal sinus tumor that was treated with radical surgery and platinum-based chemotherapy. CASE: RT is an 18-year-old female who presented with a vulvar mass that was diagnosed as endodermal sinus tumor at the time of biopsy. She was subsequently treated with modified radical vulvectomy and ipsilateral groin lymphadenectomy, followed by bleomycin, etoposide, and cisplatin chemotherapeutic regimen. She has since remained free of disease for 18 months as evidenced by serum alpha-fetoprotein and physical exam at 18 months. CONCLUSIONS: Vulvar endodermal sinus tumors represent a very small number of germ cell tumors in women. Based on the previous accounts, this disease appears to be more fatal than endodermal sinus tumor arising at other sites. These tumors also have a predilection for local metastasis. Due to the previous accounts, we chose to treat this patient with radical surgery and platinum-based chemotherapy. This treatment regimen has resulted in a disease-free state for 18 months.

Adult↗
PubMed2002

The guts of endoderm formation.

In this chapter, we will review the formation of the definitive endoderm, the population of cells that give rise to the lining of the digestive tract, its associated organs and the pharyngeal pouches. At the cellular level, we will describe the location and movement of endodermal cells from the onset of epiboly until the end of gastrulation. At the molecular level, we will discuss the genes associated with endoderm formation beginning with Nodal signaling. For convenience, we use the term involution, sometimes referred to as internalization; we also separate endoderm formation into the pre-involution (blastula) and post-involution (gastrula) periods although of course endoderm formation involves a continuous series of events. In addition, we refer to the cells that contribute to the endoderm as progenitors prior to their involution and precursors after their involution.

Animals↗
PubMed1978

Endoderm regeneration in the chick embryo studied by SEM.

Regeneration of the area pellucida endoderm of the chick embryo was studied by scanning electron microscopy (SEM). A new endoderm was formed by in situ changes in the shape and relationships of mesoderm cells. Initially the cells flattened and lost their processes except along cell boundaries. Later even these processes were lost and an epithelium was formed. The area of regenerated endoderm coincided with the area of mesoderm at the time of endoderm removal, confirming the mesodermal origin of the new layer. Remnants of the original endoderm did not contribute to the regenerated layer. Contact inhibition was observed at the boundary between original and regenerated endoderms.

Animals↗
PubMed1981

The head-process and the formation of the definitive endoderm in the mouse embryo.

The formation of the secondary or definitive endoderm was studied by light microscopy (1-mu m sections) and (scanning) electron microscopy. The results show that the primary endoderm disappears axially, and a hiatus appears in this layer. The development of this hiatus may be caused by cell degeneration, which is observed in the primary endoderm, or by some activity of the underlying head-process. The apical parts of a number of head-process cells converge towards a hiatus. These cells are organized into a conical configuration which may participate in the formation of the hiatus. The cone cells reach through the hiatus into the yolk sac cavity, and comprise the secondary endoderm. The consequence is that in mice, the definitive endoderm develops from the head-process mesoderm rather than from the primary endoderm.

Animals↗
PubMed2005

Use of a fluorescent membrane probe to identify zooxanthellae in hospite among dissociated endoderm cell culture from coral.

Preparation of homogeneous endoderm cells and culture is a prerequisite to understanding the cellular and molecular mechanism of endosymbiosis in the cnidarian-dinoflagellate association. During the cell isolation from the stony coral Euphyllia glabrescens, various amounts of symbiotic endoderm cells were found to release their symbionts (Symbiodinium spp., or zooxanthellae in generic usage) into the culture. Due to the bulky occupation by zooxanthellae inside the endoderm cell, the symbiotic endoderm cells, or zooxanthellae in hospite, are difficult to be distinguished from released zooxanthellae by microscopic examination. We now report a method for this identification using a fluorescent analogue of sphingomyelin, N-[5-(5,7-dimethyl boron dipyrromethene difluoride)-1-pentanoyl]-D-erythro-sphingosylphosphorylcholine (C(5)-DMB-SM). Incubation of symbiotic endoderm cells with C(5)-DMB-SM-defatted bovine serum albumin (DF-BSA) complex results in bright fluorescent membrane staining. Nevertheless, the membrane staining of free-living or released zooxanthellae by this complex is significantly decreased or even diminished. This method has provided a fast and reliable assay to identify symbiotic endoderm cells and will greatly accelerate the progress of endosymbiosis research.

Animals↗
PubMed1983

Tunicamycin reversibly inhibits the terminal differentiation of teratocarcinoma stem cells to endoderm.

The differentiation of aggregates of certain teratocarcinoma stem cell lines begins with the formation of an outer layer of primary endoderm cells characterized by the production of plasminogen activator and the absence of histochemically detectable alkaline phosphatase activity. After several days of culture these outer cells develop into a mixture of two types of terminally differentiated endoderm: parietal endoderm which produces a thick layer of underlying basement membrane and visceral endoderm which produces alpha-fetoprotein (AFP). We report here that in the presence of tunicamycin, a drug that inhibits glycosylation of N-asparagine linked glycoproteins, a primary endoderm-like cell is formed which is alkaline phosphatase negative and plasminogen activator positive. However, terminal differentiation of these cells is inhibited as manifested by the lack of accumulation of a thick basement membrane and the absence of immunologically detected AFP. Such inhibition is reversible following removal of the tunicamycin. Terminal differentiation of endoderm depends, therefore, upon N-asparagine linked glycoproteins.

Alkaline Phosphatase↗
PubMed1984

Migration of I-cells from ectoderm to endoderm in Hydra attenuata Pall (Cnidaria, Hydrozoa) and their subsequent differentiation.

The cellular composition of isolated ecto- and endoderm of the gastric column of Hydra attenuata Pall were recorded qualitatively and quantitatively. The endoderm contains a small population of I-cells ("basal cells") which give rise to the endodermal neurons. The recombination of live ecto- and endoderm, one of which had previously been [3H]thymidine labeled, revealed that the endodermal I-cells and their neural derivatives originate from ectodermal I-cells which migrate across the mesoglea. No other cell types were found to pass from one cell layer to the other. The experiments support the idea that the endodermal gland cells constitute an autoreproductive cell line independent of the pluripotent I-cells.

Animals↗
PubMed1987

An adhesion-defective variant of F9 embryonal carcinoma cells fails to differentiate into visceral endoderm.

Adhesion-defective EC cells were isolated from a population of mutagenized F9 cells by serial transfer of cells that did not adhere to gelatin-coated dishes. The variant cells grew in suspension as multicellular clusters of loosely aggregated cells. The cells adhered to, but did not flatten on, fibroblast monolayers and extracellular matrix produced by parietal-like endoderm. Two different mutant cell lines exhibited increased sensitivity to the lectin abrin and decreased sensitivity to wheat germ agglutinin, suggesting that changes in cell surface glycosylation are associated with the mutant phenotype. These adhesion-defective mutants were used to study the relationship between cell-cell adhesion and endodermal differentiation. Unlike wild-type cells, when cultured with low concentrations of retinoic acid (RA) in suspension culture, the mutant cells did not form embryoid bodies but remained as loosely adhering strings of cells. Electron microscopic examination revealed that most of the differentiated variant cells resembled parietal endoderm, and this was confirmed by immunofluorescent staining for TROMA-3 marker. The levels of some of the markers that characterize the differentiative pathways were examined by immunoprecipitation and by enzyme-linked immunosorbent assay (ELISA). The variant line produced higher levels of laminin and type IV collagen compared to the wild-type cells. alpha-Fetoprotein (AFP) was produced at a significantly lower level by the variant compared to wild-type F9 cells during the differentiative process. The results show that variant cells differentiated toward parietal endoderm but have a very much restricted ability to differentiate to visceral endoderm. We conclude that aggregation and/or compaction provide some essential signals during the differentiation of F9 cells into epithelial layers of visceral endoderm.

Abrin↗
PubMed2004

Inhibition of FGF signaling causes expansion of the endoderm in Xenopus.

Fibroblast growth factor (FGF) is established as an initiator of signaling events critical for neurogenesis and mesoderm formation during early Xenopus embryogenesis. However, less is known about the role FGF signaling plays in endoderm specification. Here, we show for the first time that endoderm-specific genes are induced when FGF signaling is blocked in animal cap explants. This block of FGF signaling is also responsible for a significant enhancement of endodermal gene expression in animal cap explants that are injected with a dominant-negative BMP-4 receptor (DNBR) RNA or treated with activin, however, neural and mesoderm gene expression is diminished. Consistent with these results, the injection of dominant-negative FGF receptor (DNFR) RNA expands endodermal cell fate boundaries while FGF treatment dramatically reduces endoderm in whole embryos. Taken together, these results indicate that inhibition of FGF signaling promotes endoderm formation, whereas the presence of active FGF signaling is necessary for neurogenesis/mesoderm formation.

Abdomen↗
PubMed2004

Zebrafish pou5f1/pou2, homolog of mammalian Oct4, functions in the endoderm specification cascade.

pou5f1, also known as Oct4, is required to establish the pluripotent cell population necessary for embryogenesis in mouse. Additional roles during development, including endoderm formation, have been proposed. In zebrafish, the zygotic pou5f1/pou2 mutant spiel ohne grenzen (spg) shows neural plate patterning defects and reduced endoderm at the tailbud stage. To investigate the function of maternal and early zygotic pou5f1 expression, we rescued zygotic spg(m793) mutants by injecting pou5f1 mRNA at the one-cell stage and raised them into fertile homozygous spg(m793) adults that mate to produce maternal-zygotic spg (MZspg) mutant embryos. Although neurectoderm, mesoderm, and germ cells develop in MZspg mutants, gastrulation is delayed and proceeds abnormally. Further, MZspg mutants do not maintain expression of sox32/casanova, express little or no sox17, and fail to develop endodermal tissue. Constitutively active Nodal receptor TARAM-A or sox32 overexpression induces ubiquitous sox17 expression in wild-type embryos, but not in MZspg mutants. Overexpression of a Pou5f1-VP16 activator fusion protein can rescue gastrulation and endodermal tissues in MZspg mutants. We propose that pou5f1 plays an activating role in zebrafish endodermal development, where it maintains sox32 expression during gastrulation and acts with sox32 to induce sox17 expression in endodermal precursor cells.

Animals↗
PubMed2006

An FGF response pathway that mediates hepatic gene induction in embryonic endoderm cells.

While particular combinations of mesodermal signals are known to induce distinct tissue-specific programs in the endoderm, there is little information about the response pathways within endoderm cells that control their specification. We have used signaling inhibitors on embryo tissue explants and whole-embryo cultures as well as genetic approaches to reveal part of an intracellular network by which FGF signaling helps induce hepatic genes and stabilize nascent hepatic cells within the endodermal epithelium. Specifically, we found that hepatic gene induction is elicited by an FGF/MAPK pathway. Although the PI3K pathway is activated in foregut endoderm cells, its inhibition does not block hepatic gene induction in explants; however, it does block tissue growth. We also found that at the onset of hepatogenesis, the FGF/MAPK and PI3K pathways do not crossregulate in the endoderm. The finding of separate pathways for endoderm tissue specification and growth provides insights for guiding cellular regeneration and stem cell differentiation.

Adaptor Proteins, Signal Transducing↗
PubMed2005

Endodermal expression of Nkx6 genes depends differentially on Pdx1.

Nkx family members are essential for normal development of many different tissues such as the heart, lungs, thyroid, prostate, and CNS. Here, we describe the endodermal expression pattern of three Nkx6 family genes of which two shows conserved expression in the early pancreatic epithelium. In chicken, Nkx6.1 expression is not restricted to the presumptive pancreatic area but is more broadly expressed in the endoderm. In mice, expression of Nkx6.1 is restricted to the pancreatic epithelium. In both mice and chicken, Nkx6.2 and Pdx1 are expressed in very similar domains, identifying Nkx6.2 as a novel marker of pancreas endoderm. Additionally, our results show that Nkx6.3 is expressed transiently in pancreatic endoderm in chicken but not mouse embryos. At later stages, Nkx6.3 is found in the caudal stomach and rostral duodenum in both species. Finally, we demonstrate that Pdx1 is required for Nkx6.1 but not Nkx6.2 expression in mice and that ectopic Pdx1 can induce Nkx6.1 but not Nkx6.2 or Nkx6.3 expression in anterior chicken endoderm. These results demonstrate that Nkx6.1 lies downstream of Pdx1 in a genetic pathway and that Pdx1 is required and sufficient for Nkx6.1 expression in the early foregut endoderm.

Amino Acid Sequence↗
PubMed2006

Sonic hedgehog in the pharyngeal endoderm controls arch pattern via regulation of Fgf8 in head ectoderm.

Fgf8 signalling is known to play an important role during patterning of the first pharyngeal arch, setting up the oral region of the head and then defining the rostral and proximal domains of the arch. The mechanisms that regulate the restricted expression of Fgf8 in the ectoderm of the developing first arch, however, are not well understood. It has become apparent that pharyngeal endoderm plays an important role in regulating craniofacial morphogenesis. Endoderm ablation in the developing chick embryo results in a loss of Fgf8 expression in presumptive first pharyngeal arch ectoderm. Shh is locally expressed in pharyngeal endoderm, adjacent to the Fgf8-expressing ectoderm, and is thus a candidate signal regulating ectodermal Fgf8 expression. We show that in cultured explants of presumptive first pharyngeal arch, loss of Shh signalling results in loss of Fgf8 expression, both at early stages before formation of the first arch, and during arch formation. Moreover, following removal of the endoderm, Shh protein can replace this tissue and restore Fgf8 expression. Overexpression of Shh in the non-oral ectoderm leads to an expansion of Fgf8, affecting the rostral-caudal axis of the developing first arch, and resulting in the formation of ectopic cartilage. Shh from the pharyngeal endoderm thus regulates Fgf8 in the ectoderm and the role of the endoderm in pharyngeal arch patterning may thus be indirectly mediated by the ectoderm.

Animals↗
PubMed2003

Dynamic morphogenetic events characterize the mouse visceral endoderm.

Several lines of evidence suggest that the extraembryonic endoderm of vertebrate embryos plays an important role in the development of rostral neural structures. In mice, neural inductive signals are thought to reside in an area of visceral endoderm that expresses the Hex gene. Here, we have conducted a morphological and lineage analysis of visceral endoderm cells spanning pre- and postprimitive streak stages. Our results show that Hex-expressing cells have a tall, columnar epithelial morphology, which distinguishes them from other visceral endoderm cells. This region of visceral endoderm thickening (VET) is found overlying first the distal and then one side of the epiblast at stages between 5.5 and 5.75 days post coitum (d.p.c.). In addition, we show that the epiblast has an anteroposterior-compressed appearance that is aligned with the position of the VET. Intracellular labeling of VET/Hex-expressing cells reveals an anterior and anterolateral shift from their distal epiblast position. VET/Hex-expressing cells are first localized to the anterior side of the epiblast by 5.75 d.p.c. and form a crescent on the anterior half of the embryo at the onset of gastrulation. Subsequently, VET descendants are distributed along the embryonic/extraembryonic boundary by headfold stages at 7.5 d.p.c. The morphological characteristics and position of VET/Hex-expressing cells distinguishes the future anteroposterior axis of the embryo and provide landmarks to stage mouse embryos at preprimitive streak stages. Moreover, the morphological characteristics of pregastrulation mouse embryos together with the stereotyped shift in the position of visceral endoderm cells reveal similarities among amniote embryos that suggest an evolutionary conservation of the mechanisms that pattern the rostral neurectoderm at pregastrula stages.

Animals↗
PubMed1997

Xsox17alpha and -beta mediate endoderm formation in Xenopus.

We have isolated two Xenopus relatives of murine Sox17 expressed in gastrula presumptive endoderm. Xsox17alpha and -beta expression can be induced in animal caps by activin, but not by FGF. Ectopic expression of these genes in animal caps induces the expression of endoderm markers; this induction is blocked by overexpression of a fusion of the Xsox17beta HMG domain to the Drosophila Engrailed repressor domain, as is induction of endoderm markers by activin and the expression of endodermal markers in whole embryos and isolated vegetal poles. These experiments, as well as the effects of the mRNAs on embryo phenotypes, suggest that the Xsox17 genes mediate an activin-induced endoderm differentiation pathway in animal caps and are involved in normal endoderm differentiation in embryos.

Activins↗
PubMed2002

Xhex-expressing endodermal tissues are essential for anterior patterning in Xenopus.

Two regions expressing Hex in the early gastrula contribute to organizing the anterior of the vertebrate embryo. In Xenopus, these include the anterior yolky endoderm and the suprablastoporal endoderm (SBE), which is fated to form the epithelial lining of the gut. These tissues may correspond to the anterior visceral endoderm and anterior definitive endoderm of amniotes. Genetic studies in mice have demonstrated the important roles of these tissues in producing anterior identity in the adjacent neural ectoderm. In Xenopus, both the anterior endoderm and the SBE have anterior inducing properties; furthermore, the SBE can organize a full anterior-posterior axis. Inhibition of Xhex function shows that both these Xhex-expressing endodermal tissues are required for anterior development in Xenopus.

Animals↗