In vitro production of Reichert's membrane by mouse embryo-derived parietal endoderm cell lines

We report the isolation of eight independent cell lines from preimplantation mouse embryos, which have a parietal endoderm phenotype. When grown as aggregates, these cell lines produce large amounts of a basement membrane matrix, that contains laminin, nidogen, heparan sulfate proteoglycan, collagen IV, and BM-40. The biosynthetic profiles of all eight cell lines are very similar to parietal endoderm cells in vivo which synthesize Reichert's membrane. The structure of the matrix produced by the parietal endoderm cell lines (PEC lines) resembles more closely Reichert's membrane than the Engelbreth—Holm—Swarm (EHS) tumor in susceptibility to proteolytic degradation. Since these cell lines produce large quantities of basement membrane they will be useful for structural and functional comparison of a Reichert's membrane matrix with the basement membrane produced by the EHS tumor.     

Growth and proliferation in mouse parietal yolk sac during whole embryo culture

Use of the culture techniques for postimplantation rodent embryos, modified by explanting Day 9 or Day 10 embryos with the trophoblast cells removed but the remainder of the parietal yolk sac left intact, resulted in significant expansion of Reichert's membrane, accompanied by a marked increase in the numbers of the adherent parietal endoderm cells which synthesize the membrane. The surface area of the parietal endoderm/Reichert's membrane complex roughly doubled during culture, and the combined protein content of the cells and their basement membrane was also raised after incubation. Parietal endoderm cell numbers, calculated from area and cell density measurements on flattened membranes, increased by 54-190%, depending on the age of the embryo.     

The embryonic rat parietal yolk sac. The role of the parietal endoderm in the biosynthesis of basement membrane collagen and glycoprotein in vitro.

Basement membrane biosynthesis in vitro was studied in a rapidly growing embryonic tissue, the rat parietal yolk sac. This tissue consists of a thick, nonvascular basement membrane (Reichert's membrane) separating two cellular layers (parietal endoderm and trophoblast). Morphologically, Reichert's membrane appeared similar to other basement membranes. Previous analysis of the amino acid and carbohydrate composition of acellular Reichert's membrane showed it to be typical of basement membranes isolated from other tissues and species. Analysis of [14-C]proline incorporation and hydroxy [14-C]proline synthesis during the third quarter ogestation in vitro showed that basement membrane collagen synthesis in the parietal yolk sac was maximal around the 14th day of gestation. At this time, basement membrane collagen represented nearly 10% of the newly synthesized protein. The collagen synthesized in this system was characteristic of basement membrane collagen in that about 11% of the total hydroxy [14-C]proline was present as the 3-isomer. In addition, after incubation in the presence of [14-C]lysine, 83 to 94% of the hydroxy[14-C]lysine was glycosylated, with the predominant form being glucosylgalactosylhydroxy[14-C]lysine. When the parietal endoderm and trophoblast were incubated separately with [14-C]proline, it was determined that the former was solely responsible for the synthesis of basement membrane collagen since essentially all of the 4-hydroxy[14-C]proline was associated with this cell type. Autoradiographic experiments with [3-H]glucosamine also served to localize the synthesis of noncollagen basement membrane glycoprotein components to the parietal endoderm. As with the results reported for basement membrane collagen secretion in embryonic chick lens cells, there appeared to be approximately a 60-min delay between the incorporation of [14-C]proline into protein and the secretion of collagen as measured by the appearance of 4-hydroxy[14-C]proline in the culture medium. Experiments utilizing [3H]glucosamine to monitor glycoprotein synthesis did not show a delay between the incorporation of [3H]glucosamine and the secretion of nondialyzable 3-H into the medium. The results obtained using the parietal yolk sac system to study basement membrane biosynthesis were compared to those previously obtained using the kidney glomerular and embryonic chick lens systems. It was concluded that the parietal yolk sac system is superior for a number of reasons: (a) the extracellular matrix appeared to contain only basement membrane components; there was no contamination by acid mucopolysaccharides or other types of collagen; (b) only a single cell type appeared to be responsible for the synthesis of basement membrane components; and (c) a relatively large percentage of the newly synthesized protein was basement membrane collagen.     

A scanning electron microscope study of the extraembryonic endoderm of the 8th-day mouse embryo

Abstract. Late primitive streak embryos were dissected to reveal the junction between the visceral (VE) and parietal (PE) extraembryonic endoderm. Scanning electron microscopy showed that the two cell types differ markedly in their surface morphology and intercellular organization: the VE cells have numerous apical microvilli and form part of a continuous epithelial layer, while the smoother PE cells are scattered individually over the surface of Reichert's membrane. One interpretation of the morphology of the junction between the two tissues is that visceral endoderm cells in this region are detaching from the epithelial layer, migrating on to Reichert's membrane and differentiating into parietal endoderm. Preparatory to this, the visceral endoderm cells in the junctional zone may undergo extensive reorganization of their surface membranes.     

In vitro production of Reichert's membrane by mouse embryo-derived parietal endoderm cell lines

We report the isolation of eight independent cell lines from preimplantation mouse embryos, which have a parietal endoderm phenotype. When grown as aggregates, these cell lines produce large amounts of a basement membrane matrix, that contains laminin, nidogen, heparan sulfate proteoglycan, collagen IV, and BM-40. The biosynthetic profiles of all eight cell lines are very similar to parietal endoderm cells in vivo which synthesize Reichert's membrane. The structure of the matrix produced by the parietal endoderm cell lines (PEC lines) resembles more closely Reichert's membrane than the Engelbreth-Holm-Swarm (EHS) tumor in susceptibility to proteolytic degradation. Since these cell lines produce large quantities of basement membrane they will be useful for structural and functional comparison of a Reichert's membrane matrix with the basement membrane produced by the EHS tumor.     

Localization of fibronectin, laminin-entactin, and entactin in Reichert's membrane by immunoelectron microscopy.

Immunoelectron microscopy using protein A-colloidal gold complexes of different sizes was used to study the relative distribution of extracellular matrix glycoproteins within Reichert's membrane (RM) of 13.5-day mouse embryos. Labelling for fibronectin was distributed asymmetrically; the highest concentration occurring in the outermost layer adjacent to the trophoblast cells and negligible labelling in the inner matrix, beneath the parietal endoderm cells. Within the main body of the membrane, fibronectin was concentrated in discrete electron-opaque deposits. Antibodies raised against the native complex between laminin and entactin , and against entactin alone labelled the RM more uniformly.     

Deposition of fibronectin and laminin in the basement membrane of the rat parietal yolk sac: immunohistochemical and biosynthetic studies

Rat parietal yolk sacs (PYS) at gestational ages 7.5, 9.5, 11.5, 13.5, 14.5, and 16.5 d were reacted with antibodies against laminin or plasma fibronectin. At all times studied, laminin consistently gave a positive reaction with Reichert's membrane and with the cytoplasm of PYS cells. In contrast, fibronectin gave a negative reaction with Reichert's membrane at day 7.5, was weakly positive at day 9.5, and from then on was increasingly positive with maximum reactivity at 14.5 d. By electron microscopic immunohistochemistry, antilaminin reacted strongly with 14.5-d Reichert's membrane and with the contents of the rough endoplasmic reticulum RER cisternae of the PYS cells. Antifibronectin had some spotty reactivity with Reichert's membrane, but the cytoplasm of the PYS cells was negative. The contents of the vitelline vessels and the interface between trophoblast and Reichert's membrane were strongly positive. Metabolic labeling of PYS cells in organ culture clearly demonstrated the presence of laminin, type IV procollagen, and entactin both in the medium and in tissues, but fibronectin was absent. No component in the medium bound to gelatin-Sepharose columns. These studies demonstrate that PYS cells, which actively synthesize and secrete basement membrane components, do not synthesize any detectable fibronectin. Furthermore, the anti-fibronectin staining pattern in the vitelline vessels and trophoblast-Reichert's membrane interface strongly suggests that the fibronectin present in Reichert's membrane is derived from the maternal circulation and is merely "trapped" in the membrane.     

A new differentiated cell line (Dif 5) derived by retinoic acid treatment of F9 teratocarcinoma cells capable of extracellular matrix production and growth in the absence of serum

Treatment of F9 teratocarcinoma cells with all trans retinoic acid (RA) causes them to differentiate into two or three morphologically distinct cell types. Whereas the majority of these retinoid-derived cells exhibit properties resembling parietal endoderm, a small percentage of this differentiated cell population manifests properties distinct from the parietal endoderm cell type. The isolation and partial characterization of such a non-parietal endoderm cell line (Dif 5) derived from F9 cells following prolonged (44 days) exposure to 1 μM retinoic acid are described.Unlike the retinoid-induced parietal endoderm-like cell population, which exhibits a dramatic, characteristic morphological change upon treatment with 8-bromo cAMP, Dif 5 cells do not show any morphological change with exposure to this cAMP analog. Dif 5 cells synthesize and deposit an extracellular matrix consisting of several components of Reichert's membrane (fibronectin, laminin, and type IV collagen). This new cell line does not synthesize α-fetoprotein but does secrete plasminogen activator.An interesting property of these cells is their ability to grow in the absence of serum or other hormonal supplements. Yet the Dif 5 cells do exhibit density-dependent inhibition of growth. Unlike the parent F9 cells or parietal yolk sac (PYS-2) cells, these cells do possess specific cell surface receptors for epidermal growth factor (EGF). The growth-arrested Dif 5 cells can be reinitiated to proliferate by the addition of fetal calf serum (FCS) or EGF.The properties of Dif 5 cells determined fail to fulfill all the characteristics described for either parietal or visceral endodermal cells. This raises the possibility that Dif 5 cells might represent an endodermal cell type which is intermediate in differentiation to either parietal or visceral endoderm but which lacks the biochemical signal to complete this stage of differentiation. This new Dif 5 cell line should be of considerable value in studying the modulation of growth requirements and extracellular matrix formation during early embryonic development.     

Establishment and characterization of a parietal endoderm-like cell line derived from Engelbreth-Holm-Swarm tumor (EHSPEL), a possible resource for an engineered basement membrane matrix.

Engelbreth-Holm-Swarm (EHS) tumor produces large amounts of basement membrane (BM) components, which are widely used as cell culture substrates mimicking BM functions. EHS tumor arose spontaneously in an ST/Eh strain mouse and has been propagated by transplantation. In the present study, we established a cell line, EHSPEL (EHS Parietal Endoderm-Like), which can be cultured ex vivo and preserves the capacity to form tumors in vivo. EHSPEL cells secreted large amounts of laminin-1 into the medium and deposited BM components onto dishes. To further characterize EHSPEL cells, their gene expression profile was compared to those of parietal endoderm cells from Reichert's membrane at embryonic day 13.5, differentiated F9 embryonal carcinoma cells, and PYS-2 parietal endoderm cells. These analyses outlined not only common features of parietal endoderm-like cells that underlie the efficient production of BM components, but also germline cell-like features of EHSPEL cells, at least some of which may play crucial roles in their capacity to form tumors that accumulate abundant BM components in vivo. Karyotyping of EHSPEL cells using chromosome painting probes showed a large number of interchromosomal rearrangements and partial chromosome hyperploidy. Exogenous introduction of a human laminin-alpha(4)-EGFP fusion protein into EHSPEL cells resulted in the production and deposition of human-mouse-hybrid laminin-8. This strategy should be applicable for creating efficient systems to produce chimeric laminins as well as BM-like gels with modified biological activity.     

Extraembryonic Endoderm cells as a model of endoderm development

In recent years the multipotent extraembryonic endoderm (XEN) stem cells have been the center of much attention. In vivo, XEN cells contribute to the formation of the extraembryonic endoderm, visceral and parietal endoderm and later on, the yolk sac. Recent data have shown that the distinction between embryonic and extraembryonic endoderm is not as strict as previously thought due to the integration, and not the displacement, of the visceral endoderm into the definitive embryonic endoderm. Therefore, cells from the extraembryonic endoderm also contribute to definitive endoderm. Many research groups focused on unraveling the potential and ability of XEN cells to both support differentiation and/or differentiate into endoderm‐like tissues as an alternative to embryonic stem (ES) cells. Moreover, the conversion of ES to XEN cells, shown recently without genetic manipulations, uncovers significant and novel molecular mechanisms involved in extraembryonic endoderm and definitive endoderm development. XEN cell lines provide a unique model for an early mammalian lineage that complements the established ES and trophoblast stem cell lines. Through the study of essential genes and signaling requirements for XEN cells in vitro, insights will be gained about the developmental program of the extraembryonic and embryonic endodermal lineage in vivo. This review will provide an overview on the current literature focusing on XEN cells as a model for primitive endoderm and possibly definitive endoderm as well as the potential of using these cells for therapeutic applications.     

Blastocyst implantation in the Chinese hamster (Cricetulus griseus)

Embryonic development of the Chinese hamster (Cricetulus griseus) was studied from the onset of implantation to the formation of the parietal yolk sac placenta. Implantation began on day 6 of pregnancy, when the embryo became fixed to the uterine luminal epithelium. At this time there was no zona pellucida, and microvilli of the trophoblast and uterine epithelium were closely apposed. Stromal cells immediately adjacent to the implantation chamber began to enlarge and accumulate glycogen. By day 7 the mural trophoblast penetrated the luminal epithelium in discrete area. The trophoblast appeared to phagocytize uterine epithelial cells, although epithelium adjoining the points of penetration was normal. In other areas nascent apical protrusions from the uterine epithelium indented the surface of the trophoblast. The epiblast had enlarged and both visceral and parietal endoderm cells were present. The well-developed decidual cells were epithelioid and completely surrounded the implantation chamber. On day 8 the uterine epithelium had disappeared along the mural surface of the embryo. The embryonic cell mass was elongated and filled the yolk sac cavity. Reichert's membrane was well developed. The uterine epithelial basal lamina was largely disrupted, and the trophoblast was in direct contact with decidual cells. Primary and secondary giant trophoblast cells were present and in contact with extravasated maternal blood. The mural trophoblast formed channels in which blood cells were found in close proximity to Reichert's membrane. Decidual cells were in contact with capillary epithelium and in some cases formed part of the vessel wall. Structural changes occurring in the embryo and endometrium during implantation in the Chinese hamster are described for the first time in this report and are compared to those described for some other myomorph rodents.     

Synthesis and localization of two sulphated glycoproteins associated with basement membranes and the extracellular matrix

Two sulphated glycoproteins (sgps) of apparent molecular weight (Mr) 180,000 and 150,000, are synthesized by murine PYS and PF HR9 parietal endoderm and Swiss 3T3 cells. The Mr 150,000 sgp has a similar chemical structure to the sulphated glycoprotein, C, synthesized and laid down in Reichert's membrane by mouse embryo parietal endoderm cells (Hogan, B. L.M., A. Taylor, and A.R. Cooper, 1982, Dev. Biol., 90:210-214). Both the Mr 180,000 and 150,000 sgps are deposited in the detergent- insoluble matrix of cultured cells, but they do not apparently undergo any disulphide-dependent intermolecular interactions and are not precursors or products of each other. They contain asparagine-linked oligosaccharides, but these are not the exclusive sites of sulphate labeling. Antiserum raised against the Mr 150,000 sgp C of Reichert's membranes has been used in an immunohistochemical analysis of rat skin. In early foetal and adult skin the antigen is present only in basement membranes, but transiently before and after birth it is also found throughout the upper part of the dermis. This suggests that 150,000 sgp C is at times synthesized by nonepithelial cells and contributes to the extracellular matrix of mesenchymal tissues.     

Intracellular localization of basement membrane precursors in the endodermal cells of the rat parietal yolk sac. III. Immunostaining for laminin and its precursors

Reichert's membrane and the endodermal cells of the parietal yolk sac were examined for the presence of laminin antigenicity using anti-laminin antibodies and the peroxidase-antiperoxidase sequence. Immunostaining was observed through the full width of Reichert's membrane and within endodermal cells. In these cells immunostaining was observed in rough endoplasmic reticulum (rER) cisternae and Golgi apparatus. The Golgi staining could occur in any saccule, but predominated in components interpreted as the last saccule of the stack, the GERL element, and associated prosecretory granules. The secretory granules found in the ectoplasm were also immunostained. Finally, multivesicular bodies showed some staining. The immunostaining of Reichert's membrane indicates the presence of laminin itself, while that of rER cisternae and the Golgi apparatus is attributed to laminin precursors. Presumably the biosynthesis of laminin occurs along the usual protein pathway, that is, from rER through Golgi saccules and the GERL element to secretory granules, which release their content into Reichert's membrane. The laminin immunostaining of Reichert's membrane and endodermal cells is similar to that of type IV collagen. It is, therefore, likely that the two substances are processed and secreted simultaneously.     

In vitro matrix assembly induced by critical assembly concentration (CAC).

L-2 cells are an immortalized cell line derived from yolk sac parietal endoderm cells, which are responsible for the production of Reichert's membrane, a thick basement membrane produced during rat gestation. Although the L-2 cells secrete all the major components of the basal lamina, they do not assemble a robust matrix in cell culture. We hypothesized that the reason L-2 cells fail to assemble a matrix in cell culture is because the concentrations of matrix components necessary for this matrix assembly do not reach a critical association concentration (CAC) under standard cell culture conditions. To limit the diffusion of secreted molecules while maintaining a nutrient-rich environment for the cells to thrive, we developed a technique that uses a dialysis membrane to limit protein diffusion in a 2-well plate format. This technique permits L-2 cells to assemble a robust matrix in as little as 24 hr that continues to be formed for at least 72 hr. This technique may address some of the physical limitations imposed by cell culture and could be readily applied to other cell types and medium conditions.     

Radioautographic tracing of 3H-proline in the endodermal cells of the parietal yolk sac as an indicator of the biogenesis of basement membrane components

The biogenesis of basement-membrane components was investigated in the endodermal cells of the rat parietal yolk sac in 12.5-day pregnant rats; 3H-proline was injected into conceptuses. After various time intervals, the parietal yolk sac, including endodermal cells and the associated Reichert's membrane, was removed and processed for electron-microscopic radioautography. Silver grains were counted over endodermal cell organelles and Reichert's membrane. At 2 and 5 min after 3H-proline injection, endodermal cells showed heavy labeling in rough endoplasmic reticulum (rER). Silver grain density over the rER decreased from 2 to 20 min and then remained at a plateau. Grain density was moderate over the Golgi apparatus initially but rose to a peak at 2 hr and decreased by 4 hr and later. Grain density was negligible over secretory granules at 2 and 5 min and increased moderately with time to reach a maximum at 8 hr. Thus, radioautographic peaks occurred sequentially in rER, Golgi apparatus, and secretory granules. By 4 hr and later, silver grains accumulated over Reichert's membrane. These results indicated that endodermal cells incorporated labeled proline into substances which were processed from the rER through the Golgi apparatus, transported from there to the cell surface by secretory granules, and released for export to Reichert's membrane. To clarify the nature of the exported substances, the amount of label present in proline and hydroxyproline residues after 3H-proline injection was measured in Reichert's membrane with or without the associated endodermal cells. Within the cells, 61.8% of the labeled proteins were classified as "sedentary" and 38.2% as "exportable." Of the label exported to Reichert's membrane, 66.3% consisted of type IV collagen and the rest of other basement-membrane components. The results obtained with this model suggest that basement-membrane proteins, including type IV collagen, are elaborated by the associated cells through the classical pathway: rER-Golgi apparatus-secretory granules.     

BMP signaling induces visceral endoderm differentiation of XEN cells and parietal endoderm

The extraembryonic endoderm of mammals is essential for nutritive support of the fetus and patterning of the early embryo. Visceral and parietal endoderm are major subtypes of this lineage with the former exhibiting most, if not all, of the embryonic patterning properties. Extraembryonic endoderm (XEN) cell lines derived from the primitive endoderm of mouse blastocysts represent a cell culture model of this lineage, but are biased towards parietal endoderm in culture and in chimeras. In an effort to promote XEN cells to adopt visceral endoderm character we have mimicked different aspects of the in vivo environment. We found that BMP signaling promoted a mesenchymal-to-epithelial transition of XEN cells with up-regulation of E-cadherin and down-regulation of vimentin. Gene expression analysis showed the differentiated XEN cells most resembled extraembryonic visceral endoderm (exVE), a subtype of VE covering the extraembryonic ectoderm in the early embryo, and during gastrulation it combines with extraembryonic mesoderm to form the definitive yolk sac. We found that laminin, a major component of the extracellular matrix in the early embryo, synergised with BMP to promote highly efficient conversion of XEN cells to exVE. Inhibition of BMP signaling with the chemical inhibitor, Dorsomorphin, prevented this conversion suggesting that Smad1/5/8 activity is critical for exVE induction of XEN cells. Finally, we show that applying our new culture conditions to freshly isolated parietal endoderm (PE) from Reichert's membrane promoted VE differentiation showing that the PE is developmentally plastic and can be reprogrammed to a VE state in response to BMP. Generation of visceral endoderm from XEN cells uncovers the true potential of these blastocyst-derived cells and is a significant step towards modelling early developmental events ex vivo.     

Structure and affinity for antithrombin of heparan sulfate chains derived from basement membrane proteoglycans

Metabolically 35S- or 3H-labeled heparan sulfate was isolated from murine Reichert's membrane, an extraembryonic basement membrane produced by parietal endoderm cells, and from the basement membrane-producing Engelbreth-Holm-Swarm mouse tumor. The polysaccharides were subjected to structural analysis involving identification of products formed on deamination of the polysaccharides with nitrous acid. The polysaccharide from Reichert's membrane contained N- and O-sulfate groups in approximately equal proportions. It bound almost quantitatively and with high affinity to antithrombin. A high proportion of antithrombin-binding sequence was also indicated by the finding that 3-O-sulfated glucosamine residues accounted for about 10% of the total O-sulfate groups. In contrast, at least 80% of the sulfate residues in the heparan sulfate isolated from the mouse tumor were N-substituents. Only a minor proportion of this polysaccharide bound with high affinity to antithrombin, and no 3-O-sulfated glucosamine residues were detected. These results are discussed in relation to the possible functional role of heparan sulfate in basement membranes.     

Organ cultures of the embryonic rat parietal yolk sac. II. Synthesis, accumulation, and turnover of collagen and noncollagen basement membrane glycoproteins.

Synthesis, accumulation, and turnover of basement membrane components have been studied in organ cultures of 13.5- and 14.5-day embryonic rat parietal yolk sac tissues on a nutrient agar substrate. The biochemical studies described in this report were correlated with morphologic and autoradiographic studies described in the companion paper (Minor et al., Develop. Biol.48, 1976). These studies showed that basement membrane is the only extracellular matrix synthesized, it is only synthesized by the parietal endodermal cells, and its synthesis is maintained for at least 6 days. In these cultures, synthesis and degradation of collagen and noncollagen proteins varied independently in response to environmental changes, such as the frequency of feeding or presence of trophoblast. The turnover of basement membrane collagen was much slower than that of the noncollagen proteins and this difference in the rate of turnover of the components had a major role in determining the composition of the newly synthesized basement membrane.     

Compositional and structural requirements for laminin and basement membranes during mouse embryo implantation and gastrulation

Laminins are components of all basement membranes and have well demonstrated roles in diverse developmental processes, from the peri-implantation period onwards. Laminin 1 (alpha1beta1gamma1) is a major laminin found at early stages of embryogenesis in both embryonic and extraembryonic basement membranes. The laminin gamma1 chain has been shown by targeted mutation to be required for endodermal differentiation and formation of basement membranes; Lamc1(-/-) embryos die within a day of implantation. We report the generation of mice lacking laminin alpha1 and laminin beta1, the remaining two laminin 1 chains. Mutagenic insertions in both Lama1 and Lamb1 were obtained in a secretory gene trap screen. Lamb1(-/-) embryos are similar to Lamc1(-/-) embryos in that they lack basement membranes and do not survive beyond embryonic day (E) 5.5. However, in Lama1(-/-) embryos, the embryonic basement membrane forms, the embryonic ectoderm cavitates and the parietal endoderm differentiates, apparently because laminin 10 (alpha5beta1gamma1) partially compensates for the absent laminin 1. However, such compensation did not occur for Reichert's membrane, which was absent, and the embryos died by E7. Overexpression of laminin alpha5 from a transgene improved the phenotype of Lama1(-/-) embryos to the point that they initiated gastrulation, but this overexpression did not rescue Reichert's membrane, and trophoblast cells did not form blood sinuses. These data suggest that both the molecular composition and the integrity of basement membranes are crucial for early developmental events.