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Nuclear beta-catenin-dependent Wnt8 signaling in vegetal cells of the early sea urchin embryo regulates gastrulation and differentiation of endoderm and mesodermal cell lineages.

The entry of beta-catenin into vegetal cell nuclei beginning at the 16-cell stage is one of the earliest known molecular asymmetries seen along the animal-vegetal axis in the sea urchin embryo. Nuclear beta-catenin activates a vegetal signaling cascade that mediates micromere specification and specification of the endomesoderm in the remaining cells of the vegetal half of the embryo. Only a few potential target genes of nuclear beta-catenin have been functionally analyzed in the sea urchin embryo. Here, we show that SpWnt8, a Wnt8 homolog from Strongylocentrotus purpuratus, is zygotically activated specifically in 16-cell-stage micromeres in a nuclear beta-catenin-dependent manner, and its expression remains restricted to the micromeres until the 60-cell stage. At the late 60-cell stage nuclear beta-catenin-dependent SpWnt8 expression expands to the veg2 cell tier. SpWnt8 is the only signaling molecule thus far identified with expression localized to the 16-60-cell stage micromeres and the veg2 tier. Overexpression of SpWnt8 by mRNA microinjection produced embryos with multiple invagination sites and showed that, consistent with its localization, SpWnt8 is a strong inducer of endoderm. Blocking SpWnt8 function using SpWnt8 morpholino antisense oligonucleotides produced embryos that formed micromeres that could transmit the early endomesoderm-inducing signal, but these cells failed to differentiate as primary mesenchyme cells. SpWnt8-morpholino embryos also did not form endoderm, or secondary mesenchyme-derived pigment and muscle cells, indicating a role for SpWnt8 in gastrulation and in the differentiation of endomesodermal lineages. These results establish SpWnt8 as a critical component of the endomesoderm regulatory network in the sea urchin embryo.

Animals↗

The relationship between the presence of cysteine lyase in the yolk sac endoderm and the disposition of the area vasculosa in the chicken blastoderm.

Two series of experiments were carried out to test the possible relationship between the presence of cysteine lyase and area vasculosa in the chicken blastoderm. In the first series, the localization of cysteine lyase was compared with the disposition of the area vasculosa at the successive stages of gastrulation and neurulation. In definitive streak stage chicken blastoderms, the enzyme first appeared laterally at the area pellucida-area opaca border. At older stages, this positivity extended rostrally and only finally did it appear in the caudal area. Based on these in toto observations, the relationship with area vasculosa, the distribution of which has been studied by Settle ('54), seems only incidental. In the second series, intermediate streak stage blastoderms were transversely dissected so that the rostral part did not contain any mesodermal component. The presence of cysteine lyase in this rostral part after 15 hr of culture suggests that the appearance of cysteine lyase in the yolk sac endoderm is not dependent upon the presence of area vasculosa. Moreover, the results suggest that the yolk sac endoderm of the chicken blastoderm is determined to produce cysteine lyase from the intermediate streak stage on.

Animals↗

Non-midline endodermal sinus tumor in the head and neck region: a case report.

Germ cell tumors, in particular teratomas, are some of the most commonly found tumors in childhood. Six percent of all germ cell tumors are located in the head and neck region. Endodermal sinus tumors (yolk sac tumors) of the head and neck, exclusive of the central nervous system, are rare. This study reports a 20-month-old girl with a mass of 5 x 5 cm on the left temporal area and with bone destruction on CT. The histopathological examination of the excised mass revealed an endodermal sinus tumor. The serum AFP and the LDH levels were elevated at diagnosis. The patient was treated by the BEP protocol (bleomycin, etoposide, and cisplatin), six cycles every three weeks. Partial response was attained after the first and complete response after the fourth cycle. The patient, who is being followed up, has been in remission for five months.

Endodermal Sinus Tumor↗

Endodermal sinus tumor of the vagina.

Malignant germ cell tumors of the vagina represent a critical site for local treatment. We report on our experience with two vaginal endodermal sinus tumors. Treatment with PEB regimen induced both tumor regression and alpha fetoprotein normalization. One patient had a residual lesion that was biopsied twice and no viable tumor cells were ever found. Both patients remained alive and disease-free, without having had surgical procedures except for biopsies. Platinum-based chemotherapy is able to achieve complete remissions and should be considered for vaginal endodermal sinus tumors.

Antineoplastic Combined Chemotherapy Protocols↗

A new tinman-related gene, nkx2.7, anticipates the expression of nkx2.5 and nkx2.3 in zebrafish heart and pharyngeal endoderm.

The Drosophila homeobox gene tinman and its vertebrate homologs Nkx-2.5 and Nkx-2.3 are critical determinants of cardiac development. We report here the identification of a new tinman-related gene, nkx2.7, as well as orthologs of Nkx-2.5 and Nkx-2.3 in the zebrafish. Analysis of their expression in the developing zebrafish embryo reveals that nkx2.7 transcripts are the first to appear in cardiac mesodermal and pharyngeal endodermal precursors of the anterior hypoblast, anticipating both temporally and spatially the later expression of nkx2.5 and nkx2.3 in these lineages. The preeminence of nkx2.7 in these embryonic lineages is consistent with a key role in cell fate determination, perhaps in part through the induction of nkx2.5 and nkx2.3. The findings provide the first molecular clues as to the spatial organization of endodermal and cardiac mesodermal precursors in the zebrafish hypoblast immediately following gastrulation. They suggest a coordinate role for these three tinman-related genes in the development of the heart and pharyngeal arches, and reinforce the paradigm of gene duplication and subspecialization between Drosophila and vertebrate species. The results provide a framework in which to analyze potential changes in tinman-related gene expression during abnormal zebrafish development.

Amino Acid Sequence↗

Chicken Nkx-2.8: a novel homeobox gene expressed in early heart progenitor cells and pharyngeal pouch-2 and -3 endoderm.

Members of the NK family of homeobox transcription factors regulate critical steps of organogenesis during vertebrate development. In the studies described in this report, we have isolated and functionally characterized the chicken Nkx-2.8 (cNkx-2.8) cDNA and protein and defined the temporal and spatial pattern of cNkx-2.8 gene expression during chicken development. cNkx-2.8 transcripts are first detectable at HH stage 7 in the splanchnopleura. At stage 10(+), the cNkx-2.8 gene is expressed in the linear heart tube and the dorsal half of the vitelline vein. However, after looping, HH stage 13, cNkx-2.8 is no longer expressed in the looped heart tube, but is expressed in the ventral pharyngeal endoderm. At stage 15, in addition to the pharyngeal expression pattern, cNkx-2.8 is expressed in the ectoderm of the pharyngeal arches and the aortic sac. By HH Stage 17, cNkx-2.8 expression is detectable in lateral endoderm of the second and third pharyngeal pouches, the posterior portion of the aortic sac, and the sinus venosus. cNkx-2.8 binds to previously characterized Nkx2-1 and Nkx2-5 DNA-binding sites and overexpression of cNkx-2.8 transactivates a minimal promoter which contains multimerized Nkx-2 DNA-binding sites. In addition, cNkx-2.8 and serum response factor can coactivate a minimal cardiac alpha-actin promoter. These data are consistent with a model in which cNkx-2.8 performs a unique temporally and spatially restricted function in the developing embryonic heart and pharyngeal region. Moreover, the coexpression of cNkx-2.5 and -2.8 raises the possibility that cNkx-2. 8 may have a redundant role with cNkx-2.5 in the coalescing heart tube and may play an important role in the transcriptional program(s) that underlies thymus formation. The existence of multiple NK-2 family members and their partially overlapping patterns of expression are discussed within the framework of a "Nkx code."

Amino Acid Sequence↗

Developmental competence of the gut endoderm: genetic potentiation by GATA and HNF3/fork head proteins.

A long-standing problem in developmental biology has been to understand how the embryonic germ layers gain the competence to differentiate into distinct cell types. Genetic studies have shown that members of the GATA and HNF3/fork head transcription factor families are essential for the formation and differentiation of gut endoderm tissues in worms, flies, and mammals. Recent in vivo footprinting studies have shown that GATA and HNF3 binding sites in chromatin are occupied on a silent gene in endoderm that has the potential to be activated solely in that germ layer. These and other data indicate that these evolutionarily conserved factors help impart the competence of a gene to be activated in development, a phenomenon called genetic potentiation. The mechanistic implications of genetic potentiation and its general significance are discussed.

Animals↗

Cooperation of endoderm-derived BMP2 and extraembryonic ectoderm-derived BMP4 in primordial germ cell generation in the mouse.

The primordial germ cells (PGCs) of the mouse are derived from proximal epiblast cells that are adjacent to the extraembryonic ectoderm during gastrulation. Previous studies have demonstrated that extraembryonic ectoderm-derived BMP4 and BMP8B are both required for PGC generation. Here we show that Bmp2, a member of the Dpp class of the Bmp superfamily, also plays a role in PGC generation. PGC number is significantly reduced in Bmp2 heterozygous and homozygous embryos at the N2 generation onto C57BL/6 background. Bmp2 homozygous embryos also have a short allantois and about 50% of them do not undergo normal chorioallantoic fusion. Using whole-mount in situ hybridization, we show that Bmp2 is primarily expressed in the endoderm of mouse pregastrula and gastrula embryos. Using a genetic approach, we further show that Bmp2 and Bmp4, but not Bmp2 and Bmp8b, have an additive effect on PGC generation. These results suggest that PGC generation in the mouse embryo is regulated not only by extraembryonic ectoderm-derived BMP4 and BMP8B, but also by endoderm-derived BMP2.

Allantois↗

Function and differential regulation of the alpha 6 integrin isoforms during parietal endoderm differentiation.

F9 embryonal carcinoma cells treated with retinoic acid differentiate in monolayer into parietal endoderm (PE) or in suspension into embryoid bodies with an outer layer of visceral endoderm (VE) surrounding a core of largely undifferentiated cells. Previous reports have shown that cell-extracellular matrix interactions mediated by the beta 1 integrins play a critical role in the differentiation and migration of PE. In the present study we investigated the pattern of expression and function of the integrin alpha 6 beta 1 during the differentiation of F9 cells into VE and PE. F9 cells express integrin subunits alpha 3, alpha 5, alpha 6, and beta 1. Cell adhesion and migration assays demonstrate that alpha 6 beta 1 is the major laminin receptor in undifferentiated F9 cells as well as F9-derived PE cells. However, the amount of alpha 6 protein decreases significantly upon F9 cell differentiation into either VE or PE, as revealed by immunofluorescent staining and immunoprecipitation analysis. In contrast, the amount of steady-state alpha 6 message stays constant before and after F9 cell differentiation, suggesting that the down-regulation of alpha 6 beta 1 occurs post-transcriptionally. In view of previous reports of two alpha 6 isoforms generated by alternative RNA processing, we carried out reverse transcription-PCR analysis and show that, while alpha 6B is the major mRNA isoform before and after F9 cell differentiation, alpha 6A mRNA is weakly expressed in undifferentiated F9 cells and is substantially increased following F9 differentiation into PE. Immunoprecipitations using the isoform-specific antibodies show an increase in alpha 6A and a dramatic decrease in alpha 6B protein following PE differentiation. Pulse-chase experiments indicate that, whereas the stability of alpha 6B protein is unaltered, synthesis of alpha 6B protein is decreased at least threefold following PE differentiation. Further experiments demonstrate that alpha 6A localizes to focal contacts in PE cells. The switch from alpha 6B to alpha 6A and the localization of alpha 6A at focal contacts correlate with the acquisition of PE cell motility, which suggests distinct functions for the two alpha 6 isoforms.

Animals↗

The involvement of tissue-type plasminogen activator in parietal endoderm outgrowth.

When F9 stem cells are treated in suspension with retinoic acid, they differentiate into embryoid bodies (EBs) consisting of an inner core of undifferentiated stem cells surrounded by an outer layer of visceral endoderm (VE). When these EBs are plated onto a fibronectin (FN)-coated substrate, VE-derived parietal endoderm (PE) cells migrate onto the substrate. It has been suggested that increased levels of tPA associated with the emerging PE cells may help mediate PE outgrowth. We now show that goat anti-human tPA, an anticatalytic antibody that crossreacts with mouse tPA, and a panel of serine protease inhibitors partially inhibit PE outgrowth. Extracellular matrix (ECM) degradation analysis demonstrates that PE cell-mediated degradation of [3H]proline-labeled ECM is time- and cell concentration-dependent. A serine protease inhibitor reduced the extent of degradation, suggesting that tPA might play a role in PE outgrowth by cleaving the ECM. In support of this contention, we demonstrate that incubation of purified FN with conditioned medium plus plasminogen results in FN proteolysis. The degradation of FN is blocked by either serine protease inhibitors or goat anti-human tPA. Our data suggest that enhanced production of tPA during PE outgrowth may facilitate the migratory behavior of PE cells by mediating the degradation of ECM components such as FN.

Animals↗

Modulation of alpha 6/beta 1 integrin expression during differentiation of F9 murine embryonal carcinoma cells to parietal endoderm.

In vitro differentiation of the murine embryonal carcinoma (EC) cell line F9 parallels that of the early blastocyst, where visceral (VE) and parietal endoderm (PE) diverge from a common precursor, the primitive endoderm. This differentiation pathway is induced by retinoic acid (RA) and dibutyryl cyclicAMP (dcAMP) and is accompanied by progressive and dramatic changes in cell morphology and functions. Within 7 days of treatment the cells organize their cytoskeleton and synthesize large amounts of extracellular matrix proteins, becoming fully differentiated migratory cells; all these changes are likely to involve integrins expression and organization. We have investigated the changes in beta 1 integrin expression, its maturation, and organization on the cell surface in association with alpha 6, during the transition from undifferentiated F9 stem cells to migrating PE cells. By Western blotting and immunoprecipitation we showed a gradual decrease in the amount of the beta 1 subunit on the cell surface and a parallel progressive accumulation of immature protein, indicating that the control of beta 1 expression during F9 cells differentiation occurs first at post-translational level and then at the level of transcription. Moreover, the induction of differentiation produces a marked decrease of alpha 6B and its association to a high molecular weight protein, while alpha 6A level increases. By immunofluorescence we found that upon differentiation there is a relocation of the beta 1 and alpha 6B integrin subunits from cell-cell contacts to focal contacts where they colocalize with vinculin. On the contrary alpha 6A, weakly present in F9 stem cells, is present in the focal contacts of PE cells and along the stress fibers. We suggest different roles for the two alpha 6 isoforms.

Animals↗

Primary endodermal sinus tumor of the endometrium presenting as "recurrent" endometrial adenocarcinoma.

BACKGROUND: Primary endodermal sinus tumor of the endometrium is an extremely rare malignancy with few reports in the world literature. CASE: A case of primary endodermal sinus tumor of the endometrium is presented. The case is unusual in several aspects: it occurred in a patient with a history of breast cancer and long-standing tamoxifen use, and was diagnosed only after presenting as an apparent unexpected recurrence of endometrial adenocarcinoma. The tumor recurred despite initial cytoreductive surgery and combination chemotherapy. CONCLUSION: Rare types of endometrial cancers may present as unexpected recurrences of previously resected endometrial adenocarcinomas. Appropriate therapy depends on obtaining sufficient tissue to establish an accurate diagnosis to ensure selection of proper chemotherapeutic agents.

Adenocarcinoma↗

Ultrastructural localization of calcium in the chick yolk sac membrane endodermal cells as revealed by cytochemistry and X-ray microanalysis.

The yolk sac membrane (YSM) of the chick embryo transports calcium from the yolk into the embryonic circulation during the first half of development, but the intracellular pathway of calcium transport is poorly understood. In the present study, the ultrastructural localization of calcium was investigated in cells of the YSM of 9-day chick embryos. X-ray microanalysis as well as cytochemical techniques performed on yolk sac membrane cells treated with potassium oxalate, potassium ferricyanide and potassium antimonate demonstrated accumulation of calcium in yolk granules, digested yolk products, electron-dense bodies (EDBs; 100-400 nm diameter) and electron-dense granules (EDGs; 30-50 nm diameter). When strontium ions were injected into the yolk, they were incorporated into the endodermal cells and sequestered specifically in EDGs. From these results, we propose that calcium enters the endodermal cells by endocytosis of calcium-containing yolk granules, as well as through calcium channels in the apical cell membrane. In the cytoplasm, digested yolk products, EDBs, and EDGs act as sites of sequestration and accumulation of calcium. Extrusion of intracellular calcium into the extracellular space and embryonic circulation is accomplished by exocytosis of calcium-containing material and via an ion pump in the basal cell membrane.

Animals↗

Three-dimensional architecture of the tubular endocytic apparatus and paramembranous networks of the endoplasmic reticulum in the rat visceral yolk-sac endoderm.

The three-dimensional architecture of the tubular endocytic apparatus and the endoplasmic reticulum in the rat yolk-sac endoderm was investigated after loading with horseradish peroxidase-conjugated concanavalin A by intrauterine administration. After 30 min, small vesicles (50-150 nm in diameter), small tubules (80-100 nm in diameter) and large vacuoles (0.2-1.0 microns in diameter) in the apical cytoplasm were labeled with the tracer, but lysosomes (1.0-3.5 microns in diameter) in the supranuclear cytoplasm were not labeled until 60 min after loading. Stereo-viewing of the labeled small tubules in thick sections revealed that they were not isolated structures but formed three-dimensional anastomosing networks, which were also confirmed by scanning electron microscopy after maceration with diluted osmium tetroxide. Their earlier labeling with the endocytic tracer, localization in the apical cytoplasm and three-dimensional network formation indicated that the labeled small tubules represented tubular endosomes (tubular endocytic apparatus). These well-developed membranous networks provided by the tubular endosomes are suggested to facilitate the receptor-mediated endocytosis and transcytosis of the maternal immunoglobulin in the rat yolk-sac endoderm. Scanning electron microscopy further revealed lace-like networks of the smooth endoplasmic reticulum near the lateral plasma membrane. Their possible involvement in transport of small molecules or electrolytes is discussed.

Animals↗

Cytochemical demonstration of phosphatases in membrane-recycling structures of endodermal cells.

We applied cytochemical procedures to demonstrate the presence of acid and alkaline phosphatase in the visceral yolk-sac endoderm of rats using frozen, aldehyde-fixed tissue with cerium as the capture agent. This procedure allowed more detailed topochemical localization than was possible using unfrozen tissue or with lead as the capture agent. Acid phosphatase was found to be present in lysosomes as well as in a small number of apical canaliculi, which are thought to be recycling structures of the cell membranes in endodermal cells. Reaction products of alkaline phosphatase were observed on the outer surface of apical, lateral, and basal cell membranes. In addition, some apical vacuoles contained alkaline phosphatase, and more apical canaliculi were positive for alkaline phosphatase than for acid phosphatase. However, most of the apical canaliculi were negative for both enzymes. It is suggested that acid and alkaline phosphatase are taken up by different numbers of apical canaliculi during the detachment of apical canaliculi from lysosomes and resorption vacuoles.

Acid Phosphatase↗

Emergence of mature teratoma following treatment of sacrococcygeal endodermal sinus tumor: CT and MR imaging with pathological correlation.

We report a case of pseudoretroconversion in which, clinically and pathologically, it appeared that an endodermal sinus tumor retroconverted to mature teratoma. An 8-month-old white female infant with a sacrococcygeal tumor, diagnosed by biopsy as endodermal sinus tumor (yolk sac tumor), was treated with chemotherapy. The residual tumor after treatment was mature teratoma confirmed by imaging and by pathological examination. We present the computerized tomography (CT) and magnetic resonance imaging (MRI) findings with pathological correlation.

Endodermal Sinus Tumor↗

Comparison of c-Fos immunoreactivity in pancreatic beta cells and cells with neural crest, endoderm and mesoderm origin in rats.

AIMS: Pancreatic beta cells are known to share many similarities with neuronal cells, but their origin remains controversial. It has been hypothesized that pancreatic beta cells are derived from neural crest cells. The aim of this study was to investigate the similarities between pancreatic beta cells and cells with neural crest, endoderm and mesoderm origin with respect to c-Fos immunoreactivity (c-Fos-ir), which has a role in important cellular processes including cellular proliferation, growth, differentiation and apoptosis. METHODS: c-Fos-ir was analyzed by immunohistochemical methods in formaline-fixed, paraffin-embedded sections of rat pancreatic beta cells (BCs), in adrenal medullary chromaffin cells (CCs) that are derived from neural crest, in exocrine pancreatic acinar cells (ACs) that are derived from endoderm, and in adrenal cortex zona reticularis cells (RCs) that are derived from mesoderm. RESULTS: The statistical comparisons revealed no significant differences between BCs and CCs with respect to c-Fos-ir (p>0.05). However, a highly significant difference (p<0.001) with respect to c-Fos-ir both between ACs and RCs, and between these two cell types and each of the two other cell types was noted. CONCLUSIONS: As opposed to findings in cells without neural crest origin, the observed similarity between BCs and CCs with respect to c-Fos-ir, provides additional evidence for the similarity of these cells with cells derived from neural crest.

Adrenal Cortex↗