Disabled-2 is essential for endodermal cell positioning and structure formation during mouse embryogenesis

The signal transduction adapter protein Disabled-2 (Dab2) is one of the two mammalian orthologs of the Drosophila Disabled. The brain-specific Disabled-1 (Dab1) functions in positional organization of brain cells during development. Dab2 is widely distributed and is highly expressed in many epithelial cell types. The dab2 gene was interrupted by in-frame insertion of beta-galactosidase (LacZ) in embryonic stem cells and transgenic mice were produced. Dab2 expression was first observed in the primitive endoderm at E4.5, immediately following implantation. The homozygous Dab2-deficient mutant is embryonic lethal (earlier than E6.5) due to defective cell positioning and structure formation of the visceral endoderm. In E5.5 dab2 (-/-) conceptus, visceral endoderm-like cells are present in the deformed primitive egg cylinder; however, the visceral endoderm cells are not organized, the cells of the epiblast have not expanded, and the proamniotic cavity fails to form. Disorganization of the visceral endodermal layer is evident, as cells with positive visceral endoderm markers are scattered throughout the dab2 (-/-) conceptus. Only degenerated remains were observed at E6.5 for dab2 (-/-) embryos, and by E7.5, the defective embryos were completely reabsorbed. In blastocyst in vitro culture, initially cells with characteristics of endoderm, trophectoderm, and inner cell mass were observed in the outgrowth of the hatched dab2 (-/-) blastocysts. However, the dab2 (-/-) endodermal cells are much more dispersed and disorganized than those from wild-type blastocysts, the inner cell mass fails to expand, and the outgrowth degenerates by day 7. Thus, Dab2 is required for visceral endodermal cell organization during early mouse development. The absence of an organized visceral endoderm in Dab2-deficient conceptus leads to the growth failure of the inner cell mass. We suggest that Dab2 functions in a signal pathway to regulate endodermal cell organization using endocytosis of ligands from the blastocoel cavity as a positioning cue.     

Insufficient VEGFA activity in yolk sac endoderm compromises haematopoietic and endothelial differentiation

Vascular endothelial growth factor A (VEGFA) plays a pivotal role in the first steps of endothelial and haematopoietic development in the yolk sac, as well as in the establishment of the cardiovascular system of the embryo. At the onset of gastrulation, VEGFA is primarily expressed in the yolk sac visceral endoderm and in the yolk sac mesothelium. We report the generation and analysis of a Vegf hypomorphic allele, Vegf(lo). Animals heterozygous for the targeted mutation are viable. Homozygous embryos, however, die at 9.0 dpc because of severe abnormalities in the yolk sac vasculature and deficiencies in the development of the dorsal aortae. We find that providing 'Vegf wild-type' visceral endoderm to the hypomorphic embryos restores normal blood and endothelial differentiation in the yolk sac, but does not rescue the phenotype in the embryo proper. In the opposite situation, however, when Vegf hypomorphic visceral endoderm is provided to a wild-type embryo, the 'Vegf wild-type' yolk sac mesoderm is not sufficient to support proper vessel formation and haematopoietic differentiation in this extra-embryonic membrane. These findings demonstrate that VEGFA expression in the visceral endoderm is absolutely required for the normal expansion and organisation of both the endothelial and haematopoietic lineages in the early sites of vessel and blood formation. However, normal VEGFA expression in the yolk sac mesoderm alone is not sufficient for supporting the proper development of the early vascular and haematopoietic system.     

Autotaxin/Lysophospholipase D-mediated Lysophosphatidic Acid Signaling Is Required to Form Distinctive Large Lysosomes in the Visceral Endoderm Cells of the Mouse Yolk Sac

Autotaxin, a lysophospholipase D encoded by the Enpp2 gene, is an exoenzyme that produces lysophosphatidic acid in the extracellular space. Lysophosphatidic acid acts on specific G protein-coupled receptors, thereby regulating cell growth, migration, and survival. Previous studies have revealed that Enpp2^−/− mouse embryos die at about embryonic day (E) 9.5 because of angiogenic defects in the yolk sac. However, what cellular defects occur in Enpp2^−/− embryos and what intracellular signaling pathways are involved in the phenotype manifestation remain unknown. Here, we show that Enpp2 is required to form distinctive large lysosomes in the yolk sac visceral endoderm cells. From E7.5 to E9.5, Enpp2 mRNA is abundantly expressed in the visceral endoderm cells. In Enpp2^−/− mouse embryos, lysosomes in the visceral endoderm cells are fragmented. By using a whole embryo culture system combined with specific pharmacological inhibitors for intracellular signaling molecules, we show that lysophosphatidic acid receptors and the Rho-Rho-associated coiled-coil containing protein kinase (ROCK)-LIM kinase pathway are required to form large lysosomes. In addition, electroporation of dominant negative forms of Rho, ROCK, or LIM kinase also leads to the size reduction of lysosomes in wild-type visceral endoderm cells. In Enpp2^−/− visceral endoderm cells, the steady-state levels of cofilin phosphorylation and actin polymerization are reduced. In addition, perturbations of actin turnover dynamics by actin inhibitors cytochalasin B and jasplakinolide result in the defect in lysosome formation. These results suggest that constitutive activation of the Rho-ROCK-LIM kinase pathway by extracellular production of lysophosphatidic acid by the action of autotaxin is required to maintain the large size of lysosomes in visceral endoderm cells.     

A Drosophila growth factor homolog, decapentaplegic, regulates homeotic gene expression within and across germ layers during midgut morphogenesis

The decapentaplegic (dpp) gene product, a member of the transforming growth factor-beta family, is required in Drosophila embryos for normal gastrulation and the establishment of dorsal-ventral polarity in the embryo. dpp is also expressed at specific positions in the visceral mesoderm along the developing midgut. We find that mutations that eliminate the visceral mesoderm expression of dpp lead to defects in midgut morphogenesis and alter the spatially localized expression of the homeotic genes Sex combs reduced (Scr), Ultrabithorax (Ubx), and Antennapedia (Antp) in the visceral mesoderm. The extracellular dpp protein migrates from the visceral mesoderm across the apposing endodermal cell layer in a region of the endoderm that expresses the homeotic gene labial (lab). Mesodermal expression of dpp is required for the expression of lab in these endodermal cells indicating that dpp mediates an inductive interaction between the two germ layers. We propose that extracellular dpp protein regulates gut morphogenesis, in part, by regulating homeotic gene expression in the visceral mesoderm and endoderm of the developing midgut.     

Cell position regulates endodermal differentiation in embryonal carcinoma cell aggregates

It has been suggested that cell position regulates endodermal differentiation in mouse embryo inner cell masses and in aggregates of embryonal carcinoma (EC) cells. This hypothesis states that cells at the interface between the cell mass and blastocoel fluid or culture medium differentiate into endoderm, whereas internally located cells follow alternative developmental pathways. To test the cell position hypothesis, pluripotent PSA-1 cells were aggregated with hypoxanthine phosphoribosyltransferase-deficient, parietal-like, endodermal cells. The resulting aggregates consisted of cores of PSA-1 cells surrounded by endodermal cells. Autoradiography was used to distinguish between endodermal cells that were the products of EC cell differentiation and the exogenous endoderm. Alkaline phosphatase staining was used to distinguish EC cells from endodermal cells. As predicted by the cell position hypothesis, the PSA-1 EC cells, all of which were internally located, did not differentiate into endodermal cells. Nonspecific inhibition of differentiation did not account for the lack of PSA-1-derived endoderm since the PSA-1 cells in such aggregates did differentiate into columnar ectodermal-like cells. Similar experiments were also conducted with F9 cells. In this case, aggregation cultures contained retinoic acid to induce F9 cells to differentiate into visceral endoderm. In cultures containing F9 cells surrounded by parietal-like endodermal cells, no F9-derived endoderm was detected either autoradiographically or by assaying for alpha-fetoprotein production, a visceral endoderm marker. Thus, retinoic acid-induced endodermal differentiation was also regulated by cell position. Collectively, the above results provide strong evidence for the hypothesis that cell position regulates endodermal differentiation in aggregates of EC cells.     

A Comparative Analysis of Extra-Embryonic Endoderm Cell Lines

Prior to gastrulation in the mouse, all endodermal cells arise from the primitive endoderm of the blastocyst stage embryo. Primitive endoderm and its derivatives are generally referred to as extra-embryonic endoderm (ExEn) because the majority of these cells contribute to extra-embryonic lineages encompassing the visceral endoderm (VE) and the parietal endoderm (PE). During gastrulation, the definitive endoderm (DE) forms by ingression of cells from the epiblast. The DE comprises most of the cells of the gut and its accessory organs. Despite their different origins and fates, there is a surprising amount of overlap in marker expression between the ExEn and DE, making it difficult to distinguish between these cell types by marker analysis. This is significant for two main reasons. First, because endodermal organs, such as the liver and pancreas, play important physiological roles in adult animals, much experimental effort has been directed in recent years toward the establishment of protocols for the efficient derivation of endodermal cell types in vitro. Conversely, factors secreted by the VE play pivotal roles that cannot be attributed to the DE in early axis formation, heart formation and the patterning of the anterior nervous system. Thus, efforts in both of these areas have been hampered by a lack of markers that clearly distinguish between ExEn and DE. To further understand the ExEn we have undertaken a comparative analysis of three ExEn-like cell lines (END2, PYS2 and XEN). PYS2 cells are derived from embryonal carcinomas (EC) of 129 strain mice and have been characterized as parietal endoderm-like [1], END2 cells are derived from P19 ECs and described as visceral endoderm-like, while XEN cells are derived from blastocyst stage embryos and are described as primitive endoderm-like. Our analysis suggests that none of these cell lines represent a bona fide single in vivo lineage. Both PYS2 and XEN cells represent mixed populations expressing markers for several ExEn lineages. Conversely END2 cells, which were previously characterized as VE-like, fail to express many markers that are widely expressed in the VE, but instead express markers for only a subset of the VE, the anterior visceral endoderm. In addition END2 cells also express markers for the PE. We extended these observations with microarray analysis which was used to probe and refine previously published data sets of genes proposed to distinguish between DE and VE. Finally, genome-wide pathway analysis revealed that SMAD-independent TGFbeta signaling through a TAK1/p38/JNK or TAK1/NLK pathway may represent one mode of intracellular signaling shared by all three of these lines, and suggests that factors downstream of these pathways may mediate some functions of the ExEn. These studies represent the first step in the development of XEN cells as a powerful molecular genetic tool to study the endodermal signals that mediate the important developmental functions of the extra-embryonic endoderm. Our data refine our current knowledge of markers that distinguish various subtypes of endoderm. In addition, pathway analysis suggests that the ExEn may mediate some of its functions through a non-classical MAP Kinase signaling pathway downstream of TAK1.     

Induction of embryonic hematopoietic and endothelial stem/progenitor cells by hedgehog-mediated signals

Blood and vascular endothelial cells form in all vertebrates during gastrulation, a process in which the mesoderm of the embryo is induced and then patterned by molecules whose identity is still largely unknown. Blood islands' of primitive hematopoietic cell clusters surrounded by a layer of endothelial cells form in the yolk sac, external to the developing embryo proper. These lineages arise from a layer of extraembryonic mesoderm that is closely apposed with a layer of primitive (visceral) endoderm. Despite the identification of genes such as Flk1, SCL/tal-1, Cbfa2/Runx1/AML1 and CD34 that are expressed during the induction of primitive hematopoiesis and vasculogenesis, the early molecular and cellular events involved in these processes are not well understood. Recent work has demonstrated that extracellular signals secreted by visceral endoderm surrounding the embryo are essential for the initiation of these events. A member of the Hedgehog family of signaling molecules (Indian hedgehog) is produced by visceral endoderm, can induce formation of blood and endothelial cells in explant cultures and can reprogram prospective neurectoderm along hematopoietic and endothelial cell lineages. Hedgehog proteins also stimulate proliferation of definitive hematopoietic stem/progenitor cells. These findings may have important implications for regulating hematopoiesis and vascular development for therapeutic purposes in humans and for the development of new sources of stem cells for transplantation and gene therapy.     

Origin and role of distal visceral endoderm, a group of cells that determines anterior-posterior polarity of the mouse embryo

Anterior-posterior polarity of the mouse embryo has been thought to be established when distal visceral endoderm (DVE) at embryonic day (E) 5.5 migrates toward the future anterior side to form anterior visceral endoderm (AVE). Lefty1, a marker of DVE and AVE, is asymmetrically expressed in implanting mouse embryos. We now show that Lefty1 is expressed first in a subset of epiblast progenitor cells and then in a subset of primitive endoderm progenitors. Genetic fate mapping indicated that the latter cells are destined to become DVE. In contrast to the accepted notion, however, AVE is not derived from DVE but is newly formed after E5.5 from Lefty1(-) visceral endoderm cells that move to the distal tip. Concomitant with DVE migration, all visceral endoderm cells in the embryonic region undergo global movement. In embryos subjected to genetic ablation of Lefty1-expressing DVE cells, AVE was formed de novo but the visceral endoderm including the newly formed AVE failed to migrate, indicating that DVE guides the migration of AVE by initiating the global movement of visceral endoderm cells. Future anterior-posterior polarity is thus already determined by Lefty1(+) blastomeres in the implanting blastocyst.     

Cell fate, morphogenetic movement and population kinetics of embryonic endoderm at the time of germ layer formation in the mouse

The fate of the embryonic endoderm (generally called visceral embryonic endoderm) of prestreak and early primitive streak stages of the mouse embryo was studied in vitro by microinjecting horseradish peroxidase into single axial endoderm cells of 6.7-day-old embryos and tracing the labelled descendants either through gastrulation (1 day of culture) or to early somite stages (2 days of culture). Descendants of endoderm cells from the anterior half of the axis were found at the extreme cranial end of the embryo after 1 day and in the visceral yolk sac endoderm after 2 days, i.e. they were displaced anteriorly and anterolaterally. Descendants of cells originating over and near the anterior end of the early primitive streak, i.e. posterior to the distal tip of the egg cylinder, were found after 1 day over the entire embryonic axis and after 2 days in the embryonic endoderm at the anterior intestinal portal, in the foregut, along the trunk and postnodally, as well as anteriorly and posteriorly in the visceral yolk sac. Endoderm covering the posterior half of the early primitive streak contributed to postnodal endoderm after 1 day (at the late streak stage) and mainly to posterior visceral yolk sac endoderm after 2 days. Clonal descendants of axial endoderm were located after 2 days either over the embryo or in the yolk sac; the few exceptions spanned the caudal end of the embryo and the posterior yolk sac. The clonal analysis also showed that the endoderm layer along the posterior half of the axis of prestreak- and early-streak-stage embryos is heterogeneous in its germ layer fate. Whereas the germ layer location of descendants from anterior sites did not differ after 1 day from that expected from the initial controls (approx. 90% exclusively in endoderm), only 62% of the successfully injected posterior sites resulted in labelled cells exclusively in endoderm; the remainder contributed partially or entirely to ectoderm and mesoderm. This loss from the endoderm layer was compensated by posterior-derived cells that remained in endoderm having more surviving descendants (8.4 h population doubling time) than did anterior-derived cells (10.5 h population doubling time). There was no indication of cell death at the prestreak and early streak stages; at least 93% of the cells were proliferating and more than half of the total axial population were in, or had completed, a third cell cycle after 22 h culture.(ABSTRACT TRUNCATED AT 400 WORDS)     

Regionalization of cell fates and cell movement in the endoderm of the mouse gastrula and the impact of loss of Lhx1(Lim1) function

Investigation of the developmental fates of cells in the endodermal layer of the early bud stage mouse embryo revealed a regionalized pattern of distribution of the progenitor cells of the yolk sac endoderm and the embryonic gut. By tracing the site of origin of cells that are allocated to specific regions of the embryonic gut, it was found that by late gastrulation, the respective endodermal progenitors are already spatially organized in anticipation of the prospective mediolateral and anterior-posterior destinations. The fate-mapping data further showed that the endoderm in the embryonic compartment of the early bud stage gastrula still contains cells that will colonize the anterior and lateral parts of the extraembryonic yolk sac. In the Lhx1(Lim1)-null mutant embryo, the progenitors of the embryonic gut are confined to the posterior part of the endoderm. In particular, the prospective anterior endoderm was sequestered to a much smaller distal domain, suggesting that there may be fewer progenitor cells for the anterior gut that is poorly formed in the mutant embryo. The deficiency of gut endoderm is not caused by any restriction in endodermal potency of the mutant epiblast cells but more likely the inadequate allocation of the definitive endoderm. The inefficient movement of the anterior endoderm, and the abnormal differentiation highlighted by the lack of Sox17 and Foxa2 expression, may underpin the malformation of the head of Lhx1 mutant embryos.     

The location and synthesis of transferrin in mouse embryos and teratocarcinoma cells

In early postimplantation mouse development, transferrin synthesis appears to be a marker of visceral endoderm cell types. Transferrin was identified using immunoperoxidase staining, in the proximal (visceral) endoderm of the sixth-day egg cylinder, in some tissues at later stages, and in the visceral yolk sac (VYS) at all stages examined. Since the location of a plasma protein does not necessarily indicate its site of synthesis, the incorporation of labeled amino acids into transferrin was studied. Synthesis could be detected in egg cylinders on the seventh day of gestation onwards and in the VYS at all stages. However, although endoderm was the likely tissue source, its ability to synthesize transferrin after its isolation from the embryo was either much reduced or absent. The data are suggestive of a modulating influence by mesoderm and other cell types on transferrin synthesis in visceral endoderm cells. Three types of endoderm-like cells which are produced by teratocarcinoma embryonal carcinoma (EC) cells were analyzed for transferrin synthesis to assess possible parallels with the embryo. Embryoid bodies from PSA1 EC cells contained some outer endoderm cells which stained for transferrin and others which did not. The endoderm line PSA5E but not PYS-2 synthesized transferrin. The third type of endoderm-like cell (END cells) synthesized very little (OC15S1) or no (PC13 clone 5) transferrin. The conclusion that PSA5E, OC15 END, and some differentiated PSA1 cells have visceral endoderm-like character while PYS-2 reflects parietal endoderm phenotype is in agreement with published data.     

Rac1-Dependent Collective Cell Migration Is Required for Specification of the Anterior-Posterior Body Axis of the Mouse

Cell migration and cell rearrangements are critical for establishment of the body plan of vertebrate embryos. The first step in organization of the body plan of the mouse embryo, specification of the anterior-posterior body axis, depends on migration of the anterior visceral endoderm from the distal tip of the embryo to a more proximal region overlying the future head. The anterior visceral endoderm (AVE) is a cluster of extra-embryonic cells that secretes inhibitors of the Wnt and Nodal pathways to inhibit posterior development. Because Rac proteins are crucial regulators of cell migration and mouse Rac1 mutants die early in development, we tested whether Rac1 plays a role in AVE migration. Here we show that Rac1 mutant embryos fail to specify an anterior-posterior axis and, instead, express posterior markers in a ring around the embryonic circumference. Cells that express the molecular markers of the AVE are properly specified in Rac1 mutants but remain at the distal tip of the embryo at the time when migration should take place. Using tissue specific deletions, we show that Rac1 acts autonomously within the visceral endoderm to promote cell migration. High-resolution imaging shows that the leading wild-type AVE cells extend long lamellar protrusions that span several cell diameters and are polarized in the direction of cell movement. These projections are tipped by filopodia-like structures that appear to sample the environment. Wild-type AVE cells display hallmarks of collective cell migration: they retain tight and adherens junctions as they migrate and exchange neighbors within the plane of the visceral endoderm epithelium. Analysis of mutant embryos shows that Rac1 is not required for intercellular signaling, survival, proliferation, or adhesion in the visceral endoderm but is necessary for the ability of visceral endoderm cells to extend projections, change shape, and exchange neighbors. The data show that Rac1-mediated epithelial migration of the AVE is a crucial step in the establishment of the mammalian body plan and suggest that Rac1 is essential for collective migration in mammalian tissues.     

Conditional Aurora A deficiency differentially affects early mouse embryo patterning

Aurora A is a mitotic kinase essential for cell proliferation. In mice, ablation of Aurora A results in mitotic arrest and pre-implantation lethality, preventing studies at later stages of development. Here we report the effects of Aurora A ablation on embryo patterning at early post-implantation stages. Inactivation of Aurora A in the epiblast or visceral endoderm layers of the conceptus leads to apoptosis and inhibition of embryo growth, causing lethality and resorption at approximately E9.5. The effects on embryo patterning, however, depend on the tissue affected by the mutation. Embryos with an epiblast ablation of Aurora A properly establish the anteroposterior axis but fail to progress through gastrulation. In contrast, mutation of Aurora A in the visceral endoderm, leads to posteriorization of the conceptus or failure to elongate the anteroposterior axis. Injection of ES cells into Aurora A epiblast knockout blastocysts reconstitutes embryonic development to E9.5, indicating that the extra-embryonic tissues in these mutant embryos can sustain development to organogenesis stages. Our results reveal new ways to induce apoptosis and to ablate cells in a tissue-specific manner in vivo. Moreover, they show that epiblast-ablated embryos can be used to test the potency of stem cells.     

Endoderm is required for vascular endothelial tube formation, but not for angioblast specification

Angioblasts, the precursor cells that comprise the endothelial layer of blood vessels, arise from a purely mesodermal population. Individual angioblasts coalesce to form the primary vascular plexus through a process called vasculogenesis. A number of reports in the literature suggest that signals from the adjacent endoderm are necessary to induce angioblast specification within the mesoderm. We present evidence, using both embryological and molecular techniques, indicating that endoderm is not necessary for the induction of angioblasts. Xenopus embryos that had endoderm physically removed at the onset of gastrulation still express vascular markers. Furthermore, animal caps stimulated with bFGF form angioblasts in the absence of any detectable endodermal markers. These results show that endoderm is not required for the initial formation of angioblasts. While Xenopus embryos lacking endoderm contain aggregates of angioblasts, these angioblasts fail to assemble into endothelial tubes. Endothelial tube formation can be rescued, however, by implantation of endodermal tissue from sibling embryos. Based on these studies in Xenopus, and corroborating experiments using the quail embryo, we conclude that endoderm is not required for angioblast specification, but does play an essential role in the formation of vascular tubes.     

Epiblast and primitive-streak origins of the endoderm in the gastrulating chick embryo

Gastrulation is characterized by the extensive movements of cells. Fate mapping is used to follow such cell movements as they occur over time, and prospective fate maps have been constructed for several stages of the model organisms used in modern studies in developmental biology. In chick embryos, detailed fate maps have been constructed for both prospective mesodermal and ectodermal cells. However, the origin and displacement of the prospective endodermal cells during crucial periods in gastrulation remain unclear. This study had three aims. First, we determined the primitive-streak origin of the endoderm using supravital fluorescent markers, and followed the movement of the prospective endodermal cells as they dispersed to generate the definitive endodermal layer. We show that between stages 3a/b and 4, the intraembryonic definitive endoderm receives contributions mainly from the rostral half of the primitive streak, and that endodermal movements parallel those of ingressing adjacent mesodermal subdivisions. Second, the question of the epiblast origin of the endodermal layer was addressed by precisely labeling epiblast cells in a region known to give rise to prospective somitic cells, and following their movement as they underwent ingression through the primitive streak. We show that the epiblast clearly contributes prospective endodermal cells to the primitive streak, and subsequently to definitive endoderm of the area pellucida. Finally, the relationship between the hypoblast and the definitive endoderm was defined by following labeled rostral primitive-streak cells over a short period of time as they contributed to the definitive endoderm, and combining this with in situ hybridization with a riboprobe for Crescent, a marker of the hypoblast. We show that as the definitive endodermal layer is laid down, there is cell-cell intercalation at its interface with the displaced hypoblast cells. These data were used to construct detailed prospective fate maps of the endoderm in the chick embryo, delineating the origins and migrations of endodermal cells in various rostrocaudal levels of the primitive streak during key periods in early development.     

High molecular weight extracellular proteins synthesized by endoderm cells derived from mouse teratocarcinoma cells and normal extraembryonic membranes

Rabbit antiserum raised against teratocarcinoma embryoid bodies reacts with two extracellular, collagenase-resistant glycoproteins, PYS A and B, with molecular weights of approximately 350,000 and 220,000 daltons. The 220,000-dalton protein is distinguishable from fibronectin. The two proteins are synthesized and secreted into the medium in large amounts by the teratocarcinoma-derived parietal endoderm line PYS-1, and by normal parietal endoderm cells from the 10.5-day embryo. There was no detectable synthesis of PYS A and B by normal visceral endoderm cells isolated from the 10.5-day embryo, and only trace amounts of PYS A were synthesized by the teratocarcinoma-derived visceral endoderm line PSA5E and by mesodermal cells isolated from the visceral yolk sac. The two proteins therefore seem to be good biochemical markers for distinguishing parietal from visceral endoderm cells. Synthesis and secretion of PYS A and B could not be detected in undifferentiated embryonal carcinoma cells or in endoderm cells derived from them in the presence of retinoic acid.     

The location and expression of fibroblast growth factor (FGF) in F9 visceral and parietal embryonic cells after retinoic acid-induced differentiation

It is well-established that fibroblast growth factors (FGFs) participate in mesoderm formation and patterning in the developing embryo. To identify cells in mammalian embryos that produce and/or respond to FGFs, we utilized the F9 teratocarcinoma cell system. Undifferentiated F9 cells resemble inner cell mass (ICM) cells of the mouse blastocyst by several criteria including having a characteristic high nuclear to cytoplasmic ratio and by their expression of stage-specific embryonic antigens. F9 stem cells differ from ICM cells by their low spontaneous rate of differentiation and their differentiation potential. ICM cells are heterogeneous with a proportion of the cells maintaining totipotency. In contrast, F9 stem cells appear capable of forming only endodermal derivatives. Retinoic acid (RA) treatment of F9 stem cells is required for them to differentiate, and under different culturing conditions the F9 cells will form either extraembryonic parietal or visceral endoderm. We have previously shown that FGF is synthesized by F9 parietal endoderm, but not by F9 stem cells. Our present study demonstrates that F9 aggregate cultures that contain visceral endoderm cells produce cell-associated-heparin-binding mitogens for 3T3 and endothelial cells, factors with characteristics of FGFs. Furthermore, our studies detect endothelial cell-mitogens within the extracellular matrix (ECM) of F9 parietal endoderm cells, not detected within F9 stem cell 'matrices'. Parietal endoderm cell matrix mitogens could be removed by prior treatment of the ECM with buffers containing heparin or 2 M NaCl, and could be neutralized by basic FGF antibodies.