Yolk Sac Endoderm Research Articles

Trinucleotide Repeat Containing 6a (Tnrc6a)-mediated MicroRNA Function Is Required for Development of Yolk Sac Endoderm

Tnrc6 family members (Tnrc6a/b/c) are key components of the RNA-induced silencing complex in microRNA (miRNA)-mediated gene suppression. Here, we show that Tnrc6a, also known as GW182, is selectively expressed in the yolk sac endoderm and that gene trap disruption of GW182 leads to growth arrest and apoptosis. We found that targets of miRNAs highly expressed in the yolk sac are significantly derepressed in GW182(gt/gt) mutant mice, although levels of miRNAs are not altered. Specifically, growth arrest and apoptosis phenotype are associated with significant derepression of Cdkn1a (p21), Cdkn1c (P27), Lats1, Lats2, Rb1, Rbl, Bim, and Pten, known targets of miRNAs from miR-17/20/93/106 clusters highly expressed in yolk sac endoderm. Together, these data strongly suggest that GW182 is an essential functional component in the RNA-induced silencing complex for miRNA-mediated gene silencing in vivo, and selectively regulation of miRNA activity plays an important role in the proper development of yolk sac.

A light- and electron microscopic study of primordial germ cells in the zebra fish (Danio rerio)

In sexually reproducing organisms, primordial germ cells (PGCs) give rise to the cells of the germ line, the gametes. In many animals, PGCs are set apart from somatic cells early during embryogenesis. This study explores the origin of primordial germ cells (PGCs) of the zebra fish and examines their morphology during early development (1st day-15th day). PGCs were selectively stained by the alkaline phosphatase histochemical reaction and viewed by light and electron microscopy from the time they are first detectable in the yolk sac endoderm. PGCs occurred in the subendodermal space on the syncytial periblast; differing from the surrounding endodermal cells. Later the PGCs moved to between the blastoderm and yolk sac and transferred to the dorsal mesentery where they formed gonadal anlage with mesoderm cells. PGCs were easily distinguished from somatic cells by their morphology and low electron density of their nuclei. Under light microscopy, PCGs were rounded with a distinct cytoplasmic membrane.

The secondary human yolk sac has an immunophenotype indicative of both hepatic and intestinal differentiation

Although the microscopy of the secondary human yolk sac (SHYS) is well known, few studies have addressed its immunohistochemical profile. The SHYS is involved in the synthesis, absorption and transfer of various proteins and behaves as a temporary liver and intestine. The objective of this study was to evaluate the presence of immunohistochemical markers of hepatic and intestinal function in the SHYS. We performed a retrospective histological and immunohistochemical study of 26 SHYS from spontaneous abortions and tubal pregnancies, 15 of which were from the 7th to 8th week. The antibodies used were against alpha-foetoprotein (AFP), glypican 3 (GLP3), hepatocyte-paraffin-1 (HepPar-1), villin, CDX2, SALL4 and podoplanin (D2-40). Early SHYS from the 5th to the 8th week revealed a network of intracellular vesicles communicating with the lumen of endodermal tubules that were highlighted by intense membrane AFP expression. Endodermal cells consistently expressed AFP, GLP3, SALL4, hep-par-1, villin and CDX2, while mesothelial cells only expressed D2-40. The endodermal layer of the SHYS from the 5th to the 8th week revealed a transient canalicular network which was highlighted by strong membranous AFP expression; this may represent the substrate of a SHYS transport system during its period of maximal activity. The synthetic and transfer functions of the yolk sac endoderm were reflected in a hybrid immunophenotype in which proteins characteristic of hepatic function such as AFP, GLP3, SALL4 and hep-par-1 were coexpressed simultaneously with others such as villin and CDX2, indicative of an intestinal role.

BMP4 signaling directs primitive endoderm-derived XEN cells to an extraembryonic visceral endoderm identity

The visceral endoderm (VE) is an epithelial tissue in the early postimplantation mouse embryo that encapsulates the pluripotent epiblast distally and the extraembryonic ectoderm proximally. In addition to facilitating nutrient exchange before the establishment of a circulation, the VE is critical for patterning the epiblast. Since VE is derived from the primitive endoderm (PrE) of the blastocyst, and PrE-derived eXtraembryonic ENdoderm (XEN) cells can be propagated in vitro, XEN cells should provide an important tool for identifying factors that direct VE differentiation. In this study, we demonstrated that BMP4 signaling induces the formation of a polarized epithelium in XEN cells. This morphological transition was reversible, and was associated with the acquisition of a molecular signature comparable to extraembryonic (ex) VE. Resembling exVE which will form the endoderm of the visceral yolk sac, BMP4-treated XEN cells regulated hematopoiesis by stimulating the expansion of primitive erythroid progenitors. We also observed that LIF exerted an antagonistic effect on BMP4-induced XEN cell differentiation, thereby impacting the extrinsic conditions used for the isolation and maintenance of XEN cells in an undifferentiated state. Taken together, our data suggest that XEN cells can be differentiated towards an exVE identity upon BMP4 stimulation and therefore represent a valuable tool for investigating PrE lineage differentiation.

Yolk sac endoderm is the major source of serum proteins and lipids and is involved in the regulation of vascular integrity in early chick development

An important function of the vascular system is nutrient delivery. In adult animals, this is mediated through a close contact of the mesoderm-derived vasculature with the endoderm-derived enterocytes and hepatocytes. During embryonic development, the yolk sac (YS) endoderm has been suggested to play a similar role. Physiological and molecular nature of the contact between the YS endoderm and the vasculature is not well-understood. To understand roles of the YS endoderm in early development, we used the avian model and carried out a gene expression profiling analysis of isolated area vasculosa YS endoderm tissues from embryonic day 2-4 chick embryos, covering the first 48 hr of postcirculation development. Genes involved in lipid metabolism are highly enriched, indicating an active modification of lipid components during their transfer from the yolk to the circulatory system. We also uncovered genes encoding major serum proteins and key regulators of vascular integrity. In particular, PTGDS, an enzyme controlling the last step of prostaglandin D2 production, shows high expression in the YS endoderm. Experimental introduction of prostaglandin D2 into embryonic circulation led to intraembryonic vessel rupture. These data suggest that the YS endoderm is the major, if not exclusive, source of lipid and protein constituents of the early embryonic serum and plays an important role in the regulation of vascular integrity in developing embryo.

The visceral yolk sac endoderm provides for absorption of nutrients to the embryo during neurulation

Neural tube defects (NTDs) represent some of the most common congenital malformations in humans. The causes of NTDs are complex with both genetic and environmental contributing factors. Periconception nutrition is an important environmental factor influencing the penetrance of NTDs. NTDs arise from failure to close the neural tube completely during development, an event that occurs before establishment of the chorioallantoic placenta. During neurulation, nutrients are absorbed by histotrophic mechanisms and absorbed by endocytosis in the endoderm-derived cell layer of the visceral yolk sac (VYS). Here we review the histotrophic mechanisms by which nutrients are delivered to the human embryo during this critical time period. Because more detailed studies on the molecular mechanisms regulating uptake of nutrients have been performed using rodent models, most importantly mouse and rat models, we will also review nutrient uptake in these model organisms to set the stage for presentation of experimental data that have provided valuable information about how nutrients are delivered to the neurulating embryo.

IgG is transported across the mouse yolk sac independently of FcγRIIb

While generally accepted that FcRn of the human syncytiotrophoblast and the mouse yolk sac endoderm is the major IgG transporter, the finding of a different Fc receptor FcgammaRIIb (RIIb) in the human placental endothelium has suggested the existence of an additional IgG transporter. Testing our hypothesis in mouse, we found that while RIIb is expressed in the yolk sac vasculature, IgG concentrations in fetuses of wild-type mice (RIIb(+/+)) and mice with a null mutation in the gene encoding RIIb (RIIb(-/-) mice) are not different, and we thus reject our hypothesis that yolk sac RIIb transports IgG in utero in the mouse. However, the capillary bed in the mouse yolk sac is structurally more complex than in human placenta, consisting of three types of cells: an RIIb-negative endothelium, a unique RIIb-bearing cell that also expresses 2 out of 4 macrophage markers but not endothelial cell or pericyte markers, and pericytes. As in the human placenta the b2 isoform of RIIb predominates in the mouse yolk sac. Remarkably only a single capillary channel rather than 2 channels with a loop is found in each yolk sac villus, which, along with intracapillary erythrocytes, suggests that blood flow is peristaltic, mediated by pericytes. It is not clear whether RIIb in the human placental villus might mediate an IgG transport function in light of the mouse yolk sac equivalent failing to do so.

Significant contributions of the extraembryonic membranes and maternal genotype to the placental pathology in heterozygous Nsdhl deficient female embryos

Mutations in the gene encoding the cholesterol biosynthetic enzyme NSDHL are associated with the X-linked male-lethal bare patches (Bpa) mouse. Mutant male embryos for several Nsdhl alleles die in midgestation with placental insufficiency. We examined here a possible role of the maternal genotype in such placental pathology. Pre-pregnancy plasma cholesterol levels were similar between wild-type (WT) and Bpa(1H)/+ dams fed a standard, cholesterol-free diet. However, there was a marked decrease in cholesterol levels between embryonic day (E)8.5 and E10.5 for both genotypes. Further, there was a significant lag between E11.5 and E13.5 (P = 0.0011) in the recovery of levels in Bpa(1H)/+ dams to their pre-pregnancy values. To investigate possible effects of the maternal genotype on fetal placentation, we generated transgenic mice that expressed human NSDHL and rescued the male lethality of the Bpa(1H) null allele. We then compared placenta area at E10.5 in WT and Bpa(1H)/+ female embryos where the mutant X chromosome was transmitted from a heterozygous mother or a rescued mutant father. In mutant conceptuses, placental areas were approximately 50% less than WT. Surprisingly, expression of Nsdhl in trophoblast lineages of the placenta and yolk sac endoderm, which occurs only from the maternally inherited allele in a female embryo, had the largest effect on placental area (-0.681 mm(2); P < 0.0001). The maternal genotype had a smaller effect, independent of the fetal genotype (-0.283 mm(2); P = 0.024). These data demonstrate significant effects of the mother and fetal membranes on pregnancy outcome, with possible implications for cholesterol homeostasis during human pregnancy.

Local Guidance of Emerging Vessel Sprouts Requires Soluble Flt-1

Blood vessel networks form via sprouting of endothelial cells from parent vessels. Extrinsic cues guide sprouts after they leave the initiation site, but these cues are likely insufficient to regulate initial outward movement, and many embryonic vessel networks form in the absence of a strong extrinsic gradient. We hypothesized that nascent sprouts are guided by spatial cues produced along their own vessels, and that soluble Flt-1 (sFlt-1) participates in this guidance. Analysis of developing vessels with perturbed flt-1 function revealed misguided emerging sprouts, and transgenic sFlt-1 rescued sprout guidance parameters. sflt-1 activity in endothelial cells immediately adjacent to the emerging sprout significantly improved local sprout guidance. Thus, we propose that a vessel-intrinsic system initially guides emerging sprouts away from the parent vessel, utilizing spatially regulated expression of sFlt-1 in conjunction with exogenous VEGF-A. Local sprout guidance defects are predicted to contribute to vessel dysmorphogenesis during perturbed development and disease.

Evidence for crucial role of hindgut expansion in directing proper migration of primordial germ cells in mouse early embryogenesis

During mouse gastrulation, primordial germ cells (PGCs) become clustered at the base of the allantois and move caudally into the hindgut endoderm before entering the genital ridges. The precise roles of endoderm tissues in PGC migration, however, remain unclear. By using Sox17 mutants with a specific endoderm deficiency, we provide direct evidence for the crucial role of hindgut expansion in directing proper PGC migration. In Sox17-null embryos, PGCs normally colonize in the allantois and then a small front-row population of PGCs moves properly into the most posterior gut endoderm. Defective hindgut expansion, however, causes the failure of further lateral PGC movement, resulting in the immobilization of PGCs in the hindgut entrance at the later stages. In contrast, the majority of the remaining PGCs moves into the visceral endoderm layer, but relocate outside of the embryonic gut domain. This leads to a scattering of PGCs in the extraembryonic yolk sac endoderm. This aberrant migration of Sox17-null PGCs can be rescued by the supply of wildtype hindgut cells in chimeric embryos. Therefore, these data indicate that hindgut morphogenic movement is crucial for directing PGC movement toward the embryonic gut side, but not for their relocation from the mesoderm into the endoderm.

FcRn in the Yolk Sac Endoderm of Mouse Is Required for IgG Transport to Fetus

In adults, the nonclassical MHC class I molecule, FcRn, binds both IgG and albumin and rescues both from a degradative fate, endowing both proteins with high plasma concentrations. FcRn also transports IgG from mother to young during gestation. Anticipating that a detailed understanding of gestational IgG transport in the mouse may give us a useful model to understand FcRn function in the human placenta, we have studied FcRn in the mouse yolk sac placenta in detail. Analyzing day 19-20 fetuses of the three FcRn genotypes resulting from matings of FcRn(+/-) parents, we found that FcRn(-/-) fetuses showed negligible IgG concentrations (1.5 microg/ml), whereas IgG concentrations in FcRn(+/-) fetuses were about a half (176 microg/ml) that of FcRn(+/+) fetuses (336 microg/ml), indicating that FcRn is responsible for virtually all IgG transport from mother to fetus. Immunofluorescence and immunoblotting studies indicated that FcRn is expressed in the endoderm of the yolk sac placenta but not in other cells of the yolk sac placenta or in the chorioallantoic placenta. IgG was found in the endoderm of both FcRn(+/+) and FcRn(-/-) yolk sac placentas and in the mesenchyme of FcRn(+/+) but was missing from the mesenchyme of FcRn(-/-) yolk sac placentas, indicating that IgG enters the endoderm constitutively but is moved out of the endoderm by FcRn. The similarities of these results to human placental FcRn expression and function are striking.

Development of the Inverted Visceral Yolk Sac in Three Species of Caviids (Rodentia, Caviomorpha, Caviidae)

Guinea pig related rodents possess numerous derived placental characters. We attempt to identify diversity within the visceral yolk sac and its association with the chorioallantoic placenta in three species of caviids, two of them possessing a capsule formed by the decidua that covers the chorioallantoic placenta. The results verify that in early pregnancy all three species have an inverted yolk sac placenta. In advanced pregnancy the species differ: Galea spixii, as representative without a capsule, bear a yolk sac in apposition to the chorioallantoic placenta with signs of exchange activity until term. Galea is similar to other caviomorphs in this respect. In Dasyprocta leporina and Cuniculus paca, the representatives possessing a capsule, the yolk sac endoderm lacks signs of substance exchange. Evidently, the presence of a capsule prevents such an interaction. The variations established here must be considered if animal models for human placentation are required which have restricted access to the chorioallantoic placenta from the outside.

Significance of aquaporins and sodium potassium ATPase subunits for expansion of the early equine conceptus

Expansion of the equine conceptus can be divided into blastocoel and yolk sac phases. The endodermal layer transforming the blastocoel into the yolk sac is completed around day 8 of pregnancy. From that time, the size of the spherical conceptus increases tremendously due mainly to the accumulation of fluid rather than cell multiplication. In this study, we have investigated the abundance and localisation of Na(+)/K(+)-ATPases and aquaporins (AQP) in the equine conceptus on days 8, 10, 12, 14 and 16 by multiplex reverse transcriptase PCR, Western blot and immunohistochemistry. During conceptus expansion, the ectoderm of the yolk sac exhibited basolateral abundance of alpha1ATPase, apical localisation of AQP5, and membrane and cytoplasmic expression of AQP3. With increasing conceptus size its cells showed an extensive enlargement of the apical membrane surface by microvilli. From day 14 onwards, the yolk sac endoderm forms arc-like structures with attaching sites to the ectodermal layer and shows intensive staining for alpha1ATPase, AQP5 and AQP3 in the membrane as well as in the cytoplasm. In the yolk sac ectoderm, the arrangement of these proteins is comparable with the collecting ducts of kidney with AQP2 being replaced by the closely related AQP5. The detection of phosphorylation sites for protein kinase A suggests a similar AQP5 traffic and regulation as known for AQP2 in the collecting ducts of the kidney. The arrangement of these proteins in equine embryos indicates at least partially the mechanism of conceptus expansion.

Adaptive Responses by Mouse Early Embryos to Maternal Diet Protect Fetal Growth but Predispose to Adult Onset Disease1

Poor maternal nutrition during pregnancy can alter postnatal phenotype and increase susceptibility to adult cardiovascular and metabolic diseases. However, underlying mechanisms are largely unknown. Here, we show that maternal low protein diet (LPD), fed exclusively during mouse preimplantation development, leads to offspring with increased weight from birth, sustained hypertension, and abnormal anxiety-related behavior, especially in females. These adverse outcomes were interrelated with increased perinatal weight being predictive of later adult overweight and hypertension. Embryo transfer experiments revealed that the increase in perinatal weight was induced within blastocysts responding to preimplantation LPD, independent of subsequent maternal environment during later pregnancy. We further identified the embryo-derived visceral yolk sac endoderm (VYSE) as one mediator of this response. VYSE contributes to fetal growth through endocytosis of maternal proteins, mainly via the multiligand megalin (LRP2) receptor and supply of liberated amino acids. Thus, LPD maintained throughout gestation stimulated VYSE nutrient transport capacity and megalin expression in late pregnancy, with enhanced megalin expression evident even when LPD was limited to the preimplantation period. Our results demonstrate that in a nutrient-restricted environment, the preimplantation embryo activates physiological mechanisms of developmental plasticity to stablize conceptus growth and enhance postnatal fitness. However, activation of such responses may also lead to adult excess growth and cardiovascular and behavioral diseases.

Primordial germ cell formation in birds.

At the early somite stage, chicken embryo primordial germ cells comprise a population of about 200 dispersed, morphologically distinct cells, located in the extraembryonic germinal crescent. Tracing the origin of primordial germ cells at earlier stages is at present not possible by either cytological or immunohistochemical techniques. To resolve this difficulty we have microdissected embryos at early embryonic stages, isolated fragments from various positions and incubated them to the somite stage, when the primordial germ cells can be visualized accurately. From these studies a distinct developmental pattern has emerged. (1) At the uterine stage, when the area pellucida starts to thin out, there is already a defined area, confined to the most central part of the blastodisc, which constitutes the sole source of the future primordial germ cells. (2) As development proceeds, the area that gives rise to the primordial germ cells expands peripherally. (3) Primordial germ cells thereafter migrate as isolated cells, descending to the lower layer, the hypoblast. (4) This gradual process starts at the onset of blastulation and terminates towards the end of gastrulation. (5) The hypoblast, destined to become the yolk sac endoderm, is pushed anteriorly during gastrulation by morphogenetic movements to be replaced by the endoderm proper; the primordial germ cells are carried forwards with it and occupy an extraembryonic location.

Loss of the Atp2c1 Secretory Pathway Ca2+-ATPase (SPCA1) in Mice Causes Golgi Stress, Apoptosis, and Midgestational Death in Homozygous Embryos and Squamous Cell Tumors in Adult Heterozygotes

Loss of one copy of the human ATP2C1 gene, encoding SPCA1 (secretory pathway Ca(2+)-ATPase isoform 1), causes Hailey-Hailey disease, a skin disorder. We performed targeted mutagenesis of the Atp2c1 gene in mice to analyze the functions of this Golgi membrane Ca(2+) pump. Breeding of heterozygous mutants yielded a normal Mendelian ratio among embryos on gestation day 9.5; however, null mutant (Spca1(-/-)) embryos exhibited growth retardation and did not survive beyond gestation day 10.5. Spca1(-/-) embryos had an open rostral neural tube, but hematopoiesis and cardiovascular development were ostensibly normal. Golgi membranes of Spca1(-/-) embryos were dilated, had fewer stacked leaflets, and were expanded in amount, consistent with increased Golgi biogenesis. The number of Golgi-associated vesicles was also increased, and rough endoplasmic reticulum had fewer ribosomes. Coated pits, junctional complexes, desmosomes, and basement membranes appeared normal in mutant embryos, indicating that processing and trafficking of proteins in the secretory pathway was not massively impaired. However, apoptosis was increased, possibly the result of secretory pathway stress, and a large increase in cytoplasmic lipid was observed in mutant embryos, consistent with impaired handling of lipid by the Golgi. Adult heterozygous mice appeared normal and exhibited no evidence of Hailey-Hailey disease; however, aged heterozygotes had an increased incidence of squamous cell tumors of keratinized epithelial cells of the skin and esophagus. These data show that loss of the Golgi Ca(2+) pump causes Golgi stress, expansion of the Golgi, increased apoptosis, and embryonic lethality and demonstrates that SPCA1 haploinsufficiency causes a genetic predisposition to cancer.

Insulin is imprinted in the placenta of the marsupial, Macropus eugenii

Therian mammals (marsupials and eutherians) rely on a placenta for embryo survival. All mammals have a yolk sac, but while both chorio-allantoic and chorio-vitelline (yolk sac) placentation can occur, most marsupials only develop a yolk sac placenta. Insulin (INS) is unusual in that it is the only gene that is imprinted exclusively in the yolk sac placenta. Marsupials, therefore, provide a unique opportunity to examine the conservation of INS imprinting in mammalian yolk sac placentation. Marsupial INS was cloned and its imprint status in the yolk sac placenta of the tammar wallaby, Macropus eugenii, examined. In two informative individuals of the eight that showed imprinting, INS was paternally expressed. INS protein was restricted to the yolk sac endoderm, while insulin receptor, IR, protein was additionally expressed in the trophoblast. INS protein increased during late gestation up to 2 days before birth, but was low the day before and on the day of birth. The conservation of imprinted expression of insulin in the yolk sac placenta of divergent mammalian species suggests that it is of critical importance in the yolk sac placenta. The restriction of imprinting to the yolk sac suggests that imprinting of INS evolved in the chorio-vitelline placenta independently of other tissues in the therian ancestor of marsupials and eutherians.

RAT EMBRYONIC STEM-LIKE (ES-LIKE) CELLS CAN CONTRIBUTE TO MULTIPLE EARLY EMBRYO LINEAGES IN VIVO

The rat is an important disease-based model used extensively in biomedical research. For many human diseases, including cardiovascular diseases and diabetes, the rat is a more appropriate animal model than the mouse for deciphering mechanisms underlying both normal and pathological function, not only due to the similarity of its physiology to that of humans, but also simply due to its larger size, facilitating experimental manipulation. Unfortunately, at present, no genuine rat embryonic stem (ES) cells are available, as judged by the capacity to contribute to all tissues as well as to the germline of chimeras. Therefore, in sharp contrast to the mouse, the use of gene-targeting technology in the rat via chimera generation is not yet possible. Indeed, no in vivo contribution of rat ES-like cell lines to any developing or adult tissues has been reported following blastocyst injection. Recently, for the first time, we have shown that one of our rat ES-like cell lines, ICM17, can contribute to the extraembryonic tissues of developing fetuses by microsatellite analysis. However, this PCR-based approach does not allow us to identify which specific cell types are actual descendants of the ESlike cells. The reasons underlying the apparent incapacity of rat ES-like cells to maintain the ability to contribute to developing tissues of the embryo proper are unclear. We hypothesized that these rat ES-like cells, previously derived and characterized in our lab, possess an intrinsic propensity to contribute exclusively to extraembryonic tissues, and that this in vivo bias was a reflection of the phenotype observed in vitro. Using a transgenic rat ES-like cell line expressing fluorescent proteins of the GFP family, we sought to determine in finer detail the extent to which our cells were able to contribute to the different lineages of the early embryo. Analysis of late midgestation fetuses by PCR on genomic DNA for detection of the transgene shows that the transgenic rat ES-like cells contribute exclusively to extraembryonic tissues of fetuses, confirming the results obtained by microsatellite analysis. In addition, the results obtained by immunohistochemistry on chimeric extraembryonic tissues reveal that the rat ES-like cells injected into blastocysts are able to contribute specifically to tissues of the visceral yolk sac endoderm, parietal yolk sac endoderm, as well as trophoblast giant cell lineages, clearly demonstrating the multipotency of the rat ES-like cells. Furthermore, RT-PCR and immunofluorescence analysis of the rat ES-like cultures reveals that in addition to expressing molecular markers of the undifferentiated state (Oct4), markers of primitive endoderm (Gata4/6), visceral endoderm (Foxa2), and parietal endoderm (Sparc) are also expressed in vitro. Taken together, these results suggest that although the rat ES-like cells do not contribute to tissues of the embryo proper, they are sufficiently multipotent to contribute to multiple lineages of the late midgestation rat embryo. Also, it appears that contribution to these lineages can be foreshadowed by the detection of particular lineage markers expressed in the original cultures. This research therefore represents a step forward in the search for bona fide rat ES cells. Supported by a Natural Sciences and Engineering Research Council of Canada Industrial Postgraduate Scholarship to S.-P. Demers and by Clonagen inc. (platform)

Isolation and characterization of porcine visceral endoderm cell lines derived from in vivo 11-day blastocysts

Two porcine cell lines of yolk-sac visceral endoderm, designated as PE-1 and PE-2, were derived from in vivo 11-d porcine blastocysts that were either ovoid (PE-1) or at the early tubular stage of elongation (PE-2). Primary and secondary culture of the cell lines was done on STO feeder cells. The PE-1 and PE-2 cells morphologically resembled visceral endoderm previously cultured from in vivo-derived ovine and equine blastocysts and from in vitro-derived bovine blastocysts. Analysis of the PE-1- and PE-2-conditioned medium by 2D-gel electrophoresis and matrix-assisted laser desorption/ionization-time-of-flight-mass spectrometry demonstrated that they produced serum proteins. Reverse transcriptase polymerase chain reaction analysis showed that the cells expressed several genes typical for yolk-sac endoderm differentiation and function including GATA-6, DAB-2, REX-1, HNF-1, transthyretin, alpha-fetoprotein, and albumin. Unlike a porcine liver cell line, the PE-1 and PE-2 cell lines had relatively low inducible P-450 content and EROD activity, and, while they cleared ammonia from the cell culture medium, they did not produce urea. Transmission electron microscopy revealed that the cells were a polarized epithelium connected by complex junctions resembling tight junctions and by lateral desmosomes. Rough endoplasmic reticulum was prominent within the cells. Immunocytochemistry indicated that the PE-1 cells expressed cytokeratin 18 and had robust microtubule networks similar to those observed in in vivo porcine yolk-sac endoderm. Metaphase spreads prepared at passage 26 of the PE-1 cell line indicated a diploid porcine karyotype of 38 chromosomes. The cells have been grown for over 1 yr for multiple passages at 1:10 or 1:20 split ratios on STO feeder cells. The cell lines will be of interest as an in vitro model of the porcine preimplantation yolk-sac tissue.

Essential role of RGS-PX1/sorting nexin 13 in mouse development and regulation of endocytosis dynamics.

RGS-PX1 (also known as sorting nexin 13) is a member of both the regulator of G protein signaling (RGS) and sorting nexin (SNX) protein families. Biochemical and cell culture studies have shown that RGS-PX1/SNX13 attenuates Galphas-mediated signaling through its RGS domain and regulates endocytic trafficking and degradation of the epidermal growth factor receptor. To understand the functions of RGS-PX1/SNX13 in vivo, we generated mice carrying targeted mutations of Snx13 and found that systemic Snx13-null mice were embryonic lethal around midgestation. Snx13-null embryos had significant overall growth retardation and defects in neural tube closure, blood vessel formation, and the formation of the placental labyrinthine layer. Moreover, the Snx13-null visceral yolk sac endoderm cells showed dramatic changes in the organization of endocytic compartments, abundant autophagic vacuoles, and abnormal localization of several endocytic markers, including megalin, a receptor for nutrients and proteins; ARH, a coat protein that binds megalin; LAMP2; and LC3. These changes suggest that Snx13-null embryos are defective in nutrient uptake and transport, which may contribute to the other developmental abnormalities observed. Taken together, our findings demonstrate an essential role for RGS-PX1/SNX13 in mouse development and provide previously undescribed insights into its cellular function in the regulation of endocytosis dynamics.