Yolk Sac Development Research Articles

Ultrastructure of the fetal membranes of the oviparous kingsnake, Lampropeltis getula (Colubridae) as revealed by scanning electron microscopy

In reptilian sauropsids, fetal (extraembryonic) membranes that line the eggshell sustain developing embryos by providing for gas exchange and uptake of water and eggshell calcium. However, a scarcity of morphological studies hinders an understanding of functional specializations and their evolution. In kingsnakes (Lampropeltis getula), scanning electron microscopy reveals two major fetal membranes: the chorioallantois and yolk sac omphalopleure. In early development, the chorioallantois contains tall chorionic epithelial cells, avascular connective tissue, and enlarged allantoic epithelial cells. During its maturation, the chorionic and allantoic epithelia thin dramatically and become underlain by a rich network of allantoic capillaries, yielding a membrane ideally suited for respiratory gas exchange. Yolk sac development initially is like that of typical lizards and snakes, forming an avascular omphalopleure, isolated yolk mass (IYM), and yolk cleft. However, unlike the situation in most squamates studied, the omphalopleure becomes transformed into a "secondary chorioallantois" via three asynchronous events: flattening of the epithelium, regression of the IYM, and vascularization by the allantois. Progressive expansion of chorioallantois parallels growing embryonic needs for gas exchange. In early through mid-development, external surfaces of both the chorionic and omphalopleure epithelium show an abundance of irregular surface protrusions that possibly increase surface area for water absorption. We postulate that the hypertrophied allantoic epithelial cells produce allantoic fluid, a viscous substance that facilitates water uptake and storage. Our findings are consistent with a previous study on the corn snake Pantherophis guttatus, but include new observations and novel functional hypotheses relevant to a reconstruction of basal squamate patterns.

Yolk-sac larval development of the substrate-brooding cichlid Archocentrus nigrofasciatus in relation to temperature.

In order to conserve and culture the cichlid fish Archocentrus nigrofasciatus, more information about its reproductive biology and its larval behavior and morphogenesis is necessary. Currently, temperatures ranging from 21 to 27 °C are used in ornamental aquaculture hatcheries. Lower temperatures are preferred to reduce the costs of water heating, and 23 °C is usually the selected temperature. However, there is limited information on culturing protocols for ornamental species and most of the information generated on this topic remains scarce. Thus, the present study examines the morphological development of Archocentrus nigrofasciatus during the yolk-sac period up to the age of 100 h post-hatching in relation to 2 temperature regimes used in ornamental aquaculture: a temperature of 27 °C (thermal optimum) and a decreased temperature of 23 °C (thermal tolerance). The results of this study suggest that the 27 °C temperature generates intense morphological changes in yolk-sac development in a shorter period. This has advantages as it reduces the time of yolk-sac larval development, and, thus, minimizes the transition phase to exogenous feeding and maximizes the efficiency at which yolk is converted into body tissues. The present paper provides necessary information to produce freshwater ornamental fish with better practices so as to increase larval survival and capitalize on time for growth.

Loss of PiT-1 results in abnormal endocytosis in the yolk sac visceral endoderm

PiT-1 protein is a transmembrane sodium-dependent phosphate (Pi) transporter. PiT-1 knock out (KO) embryos die from largely unknown causes by embryonic day (E) 12.5. We tested the hypothesis that PiT-1 is required for endocytosis in the embryonic yolk sac (YS) visceral endoderm (VE). Here we present data supporting that PiT-1 KO results in a YS remodeling defect and decreased endocytosis in the YS VE. The remodeling defect is not due to an upstream cardiomyocyte requirement for PiT-1, as SM22αCre-specific KO of PiT-1 in the developing heart and the YS mesodermal layer (ME) does not recapitulate the PiT-1 global KO phenotype. Furthermore, we find that high levels of PiT-1 protein localize to the YS VE apical membrane. Together these data support that PiT-1 is likely required in YS VE. During normal development maternal immunoglobulin (IgG) is endocytosed into YS VE and accumulates in the apical side of the VE in a specialized lysosome termed the apical vacuole (AV). We have identified a reduction in PiT-1 KO VE cell height and a striking loss of IgG accumulation in the PiT-1 KO VE. The endocytosis genes Tfeb, Lamtor2 and Snx2 are increased at the RNA level. Lysotracker Red staining reveals a loss of distinct AVs, and yolk sacs incubated ex vivo with phRODO Green Dextran for Endocytosis demonstrate a functional loss of endocytosis. As yolk sac endocytosis is controlled in part by microautophagy, but expression of LC3 had not been examined, we investigated LC3 expression during yolk sac development and found stage-specific LC3 RNA expression that is predominantly from the YS VE layer at E9.5. Normalized LC3-II protein levels are decreased in the PiT-1 KO YS, supporting a requirement for PiT-1 in autophagy in the YS. Therefore, we propose the novel idea that PiT-1 is central to the regulation of endocytosis and autophagy in the YS VE.

Expression of microsomal triglyceride transfer protein in lipoprotein-synthesizing tissues of the developing chicken embryo

In contrast to mammals, in the chicken major sites of lipoprotein synthesis and secretion are not only the liver and intestine, but also the kidney and the embryonic yolk sac. Two key components in the assembly of triglyceride-rich lipoproteins are the microsomal triglyceride transfer protein (MTP) and apolipoprotein B (apoB). We have analyzed the expression of MTP in the embryonic liver, small intestine, and kidney, and have studied the expression of MTP in, and the secretion of apoB from, the developing yolk sac (YS). Transcript and protein levels of MTP increase during embryogenesis in YS, liver, kidney, and small intestine, and decrease in YS, embryonic liver, and kidney after hatching. In small intestine, the MTP mRNA level rises sharply during the last trimester of embryo development (after day 15), while MTP protein is detectable only after hatching (day 21). In the YS of 15- and 20-day old embryos, apoB secretion was detected by pulse-chase metabolic radiolabeling experiments and subsequent immunoprecipitation. Taken together, our data reveal the importance of coordinated production of MTP and apoB in chicken tissues capable of secreting triglyceride-rich lipoproteins even before hatching.

Cooperation between RUNX1-ETO9a and Novel Transcriptional Partner KLF6 in Upregulation of Alox5 in Acute Myeloid Leukemia

Fusion protein RUNX1-ETO (AML1-ETO, RUNX1-RUNX1T1) is expressed as the result of the 8q22;21q22 translocation [t(8;21)], which is one of the most common chromosomal abnormalities found in acute myeloid leukemia. RUNX1-ETO is thought to promote leukemia development through the aberrant regulation of RUNX1 (AML1) target genes. Repression of these genes occurs via the recruitment of the corepressors N-COR and SMRT due to their interaction with ETO. Mechanisms of RUNX1-ETO target gene upregulation remain less well understood. Here we show that RUNX1-ETO9a, the leukemogenic alternatively spliced transcript expressed from t(8;21), upregulates target gene Alox5, which is a gene critically required for the promotion of chronic myeloid leukemia development by BCR-ABL. Loss of Alox5 expression reduces activity of RUNX1-ETO9a, MLL-AF9 and PML-RARα in vitro. However, Alox5 is not essential for the induction of leukemia by RUNX1-ETO9a in vivo. Finally, we demonstrate that the upregulation of Alox5 by RUNX1-ETO9a occurs via the C2H2 zinc finger transcription factor KLF6, a protein required for early hematopoiesis and yolk sac development. Furthermore, KLF6 is specifically upregulated by RUNX1-ETO in human leukemia cells. This identifies KLF6 as a novel mediator of t(8;21) target gene regulation, providing a new mechanism for RUNX1-ETO transcriptional control.

The placental expression of Human Leukocyte Antigen HLA-F and HLA-E in early normal placentation

The placental expression of Human Leukocyte Antigen HLA-F and HLA-E in early normal placentation

Visceral endoderm expression of Yin-Yang1 (YY1) is required for VEGFA maintenance and yolk sac development.

Mouse embryos lacking the polycomb group gene member Yin-Yang1 (YY1) die during the peri-implantation stage. To assess the post-gastrulation role of YY1, a conditional knock-out (cKO) strategy was used to delete YY1 from the visceral endoderm of the yolk sac and the definitive endoderm of the embryo. cKO embryos display profound yolk sac defects at 9.5 days post coitum (dpc), including disrupted angiogenesis in mesoderm derivatives and altered epithelial characteristics in the visceral endoderm. Significant changes in both cell death and proliferation were confined to the YY1-expressing yolk sac mesoderm indicating that loss of YY1 in the visceral endoderm causes defects in the adjacent yolk sac mesoderm. Production of Vascular Endothelial Growth Factor A (VEGFA) by the visceral endoderm is essential for normal growth and development of the yolk sac vasculature. Reduced levels of VEGFA are observed in the cKO yolk sac, suggesting a cause for the angiogenesis defects. Ex vivo culture with exogenous VEGF not only rescued angiogenesis and apoptosis in the cKO yolk sac mesoderm, but also restored the epithelial defects observed in the cKO visceral endoderm. Intriguingly, blocking the activity of the mesoderm-localized VEGF receptor, FLK1, recapitulates both the mesoderm and visceral endoderm defects observed in the cKO yolk sac. Taken together, these results demonstrate that YY1 is responsible for maintaining VEGF in the developing visceral endoderm and that a VEGF-responsive paracrine signal, originating in the yolk sac mesoderm, is required to promote normal visceral endoderm development.

Does Low Gas Permeability of Rigid‐Shelled Gekkotan Eggs Affect Embryonic Development?

Parchment-shelled eggs are characteristic of most squamates, including the basal clades of gekkotan lizards. The majority of gekkotan lizards, however, produce rigid-shelled eggs that are highly impermeable to gas exchange; eggs are laid in dry sites and experience a net loss of water during incubation. We tested the hypothesis that the 1,000-fold lower rate of oxygen diffusion through the shells of rigid- compared to parchment-shelled eggs imposes a physiological cost on development. To do this, we contrasted species with rigid and with parchment shells with regards to (1) rates of embryonic metabolism and (2) rates and patterns of development of the yolk sac and chorioallantois, the vascularized extra-embryonic membranes that transport oxygen to embryonic tissues. Metabolic rates of embryos from the rigid-shelled eggs of Gehyra variegata did not differ from those of the parchment-shelled eggs of Oedura lesueurii. Moreover, maximum metabolic rates of gekkotans with rigid shells did not differ from those of gekkotan or scincid lizards with parchment shells. In contrast, the yolk sac covered more of the surface area of the egg at oviposition, and the chorioallantois reached its full extent earlier for the species with rigid shelled eggs (Chondrodactylus turneri, G. variegata) than for the species with parchment-shelled eggs (Eublepharis macularius, O. lesueurii). Differences in the temporal patterns of yolk sac and chorioallantois development would thus serve to compensate for low rates of oxygen diffusion through rigid shells of gekkotans.

Cytometry in stem cell research and therapy

recent Nobel Prize in medicine was awarded to two stemcell researchers, John Gurdon and Shinya Yamanaka, for theirachievements in stem cell research and reprogramming ofsomatic cells. Flow cytometry is by nature the ideal tool toidentify, characterize, and isolate stem and progenitor cells forresearch and potential clinical use (1). The major strength offlow cytometry is its ability to rapidly perform highlymultiplexed quantitative measurements on single cells withina heterogeneous cell population. However, when the cell typeof interest is extremely rare, as most stem and progenitor cellsare, several sources of artifact must be addressed. The impor-tance of flow cytometry as a driving force for stem cellresearch was demonstrated in a focused issue of the journalexactly three years ago (1). This current focus issue of Cytome-try A is devoted to the topic of stem cells due to numerouscurrent innovations and discoveries.Applications of stem cells include several disciplines,from embryogenesis, adult tissue maintenance, and repair,and more recently, cancer as well as for toxicity screening anddisease modeling. All of these topics are represented in thisissue, with special emphasis on the role of analytic and pre-parative flow cytometry in the elucidation of stem cell pheno-type and function, and best laboratory practices as they applyto flow cytometry. Image and flow cytometry together withcell sorting have revolutionized the study of stem cell biologyand the implications of these cells and their progeny indevelopmental biology, tissue engineering, and cellulartherapy. The number of parameters and the speed of theirsimultaneous measurements in single cells has continued toincrease with advances in hardware, reagents, and analyticalsoftware (2).

Reproductive strategy, egg characteristics and embryonic development of Greenland halibut (Reinhardtius hippoglossoides)

AbstractDomínguez-Petit, R., Ouellet, P., and Lambert, Y. 2013. Reproductive strategy, egg characteristics and embryonic development of Greenland halibut (Reinhardtius hippoglossoides). – ICES Journal of Marine Science, 70: 342–351. Despite the commercial importance of Greenland halibut (GH), important gaps exist in our knowledge of the reproductive and early life stage biology for this species. The present study examined through laboratory experiments the spawning strategy, realized fecundity, egg characteristics, biochemical composition, and embryonic development of GH. The results confirmed the hypothesis that GH is a single-batch spawner producing large eggs, resulting in low realized fecundity. Embryonic development and hatching time are highly dependent on incubation temperature; 50% hatching occurred after 46, 30, and 24 days at 2, 4, and 6°C, respectively. Few changes in the biochemical composition of the eggs are observed during embryonic development. Newly hatched larvae are not well developed, having a large yolk sac, no pigmentation and incomplete development of the jaws. Egg specific density confirmed the mesopelagic distribution of the eggs at sea. However, important buoyancy changes occurring in the last 3–4 days before hatching indicate that larvae hatch higher in the water column. These results are important for understanding advection and dispersion processes of GH eggs and larvae and the connectivity between spawning grounds and nursery areas.

Development of bovine embryos derived from reproductive techniques

Assisted reproduction techniques have improved agricultural breeding in the bovine. However, important development steps may differ from the situation in vivo and there is a high mortality rate during the first trimester of gestation. To better understand these events, we investigated the development of embryos and fetal membranes following fixed-time AI (FTAI), IVF and nuclear transfer (NT). The onset of yolk-sac development was not normal in cloned embryos. Later steps differed from conditions in vivo in all three groups; the yolk-sac was yellowish and juxtaposed with the amniotic membrane. Vascularisation of the chorioallantoic membrane was relatively late and low in NT gestations, but normal in the others. The overall development of the embryos was normal, as indicated by morphology and regression analysis of growth rate. However, NT conceptuses were significantly smaller, with the livers in some embryos occupying the abdominal cavity and others exhibiting heart abnormalities. In conclusion, the yolk-sac and the cardiovascular system seem to be vulnerable to morphogenetic alterations. Future studies will focus on gene expression and early vascularisation processes to investigate whether these changes may be responsible for the high incidence of intrauterine mortality, especially in clones.

Etv2 Is Expressed in the Yolk Sac Hematopoietic and Endothelial Progenitors and Regulates Lmo2 Gene Expression

During embryogenesis, the endothelial and the hematopoietic lineages first appear during gastrulation in the blood island of the yolk sac. We have previously reported that an Ets variant gene 2 (Etv2/ER71) mutant embryo lacks hematopoietic and endothelial lineages; however, the precise roles of Etv2 in yolk sac development remains unclear. In this study, we define the role of Etv2 in yolk sac blood island development using the Etv2 mutant and a novel Etv2-EYFP reporter transgenic line. Both the hematopoietic and the endothelial lineages are absent in the Etv2 mutant yolk sac. In the Etv2-EYFP transgenic mouse, the EYFP reporter is activated in the nascent mesoderm, expressed in the endothelial and blood progenitors, and in the Tie2(+), c-kit(+), and CD41(+) hematopoietic population. The hematopoietic activity in the E7.75 yolk sac was exclusively localized to the Etv2-EYFP(+) population. In the Etv2 mutant yolk sac, Tie2(+) cells are present but do not express hematopoietic or endothelial markers. In addition, these cells do not form hematopoietic colonies, indicating an essential role of Etv2 in the specification of the hematopoietic lineage. Forced overexpression of Etv2 during embryoid body differentiation induces the hematopoietic and the endothelial lineages, and transcriptional profiling in this context identifies Lmo2 as a downstream target. Using electrophoretic mobility shift assay, chromatin immunoprecipitation, transcriptional assays, and mutagenesis, we demonstrate that Etv2 binds to the Lmo2 enhancer and transactivates its expression. Collectively, our studies demonstrate that Etv2 is expressed during and required for yolk sac hematoendothelial development, and that Lmo2 is one of the downstream targets of Etv2.

Development of yolk sac inversion in Galea spixii and Cavia porcellus (Rodentia, Caviidae)

Caviomorph development includes an inverted yolk sac. Since principle processes are not understood, we investigated its differentiation in Galea and re-examined material from the guinea pig. Galea showed the typical caviomorph conditions in blastocyst development and the nature of the definitive yolk sac, formed of the visceral layer that became villous, proliferative, vascularized and attached to the uterus and placenta. In contrast to what was known before, in both species parts of the parietal yolk sac and a yolk sac cavity were temporarily present. Data suggest that early yolk sac development in caviomorphs is more complex than thought before.

Efficient and simultaneous generation of hematopoietic and vascular progenitors from human induced pluripotent stem cells

The hematopoietic and vascular lineages are intimately entwined as they arise together from bipotent hemangioblasts and hemogenic endothelial precursors during human embryonic development. In vitro differentiation of human pluripotent stem cells toward these lineages provides opportunities for elucidating the mechanisms of hematopoietic genesis. We previously demonstrated the stepwise in vitro differentiation of human embryonic stem cells (hESC) to definitive erythromyelopoiesis through clonogenic bipotent primitive hemangioblasts. This system recapitulates an orderly hematopoiesis similar to human yolk sac development via the generation of mesodermal-hematoendothelial progenitor cells that give rise to endothelium followed by embryonic primitive and definitive hematopoietic cells. Here, we report that under modified feeder-free endothelial culture conditions, multipotent CD34⁺ CD45⁺ hematopoietic progenitors arise in mass quantities from differentiated hESC and human induced pluripotent stem cells (hiPSC). These hematopoietic progenitors arose directly from adherent endothelial/stromal cell layers in a manner resembling in vivo hematopoiesis from embryonic hemogenic endothelium. Although fibroblast-derived hiPSC lines were previously found inefficient in hemato-endothelial differentiation capacity, our culture system also supported robust hiPSC hemato-vascular differentiation at levels comparable to hESC. We present comparative differentiation results for simultaneously generating hematopoietic and vascular progenitors from both hESC and fibroblast-hiPSC. This defined, optimized, and low-density differentiation system will be ideal for direct single-cell time course studies of the earliest hematopoietic events using time-lapse videography, or bulk kinetics using flow cytometry analyses on emerging hematopoietic progenitors.

The Chromatin-Remodeling Enzymes BRG1 and CHD4 Antagonistically Regulate Vascular Wnt Signaling

Canonical Wnt signaling plays an important role in embryonic and postnatal blood vessel development. We previously reported that the chromatin-remodeling enzyme BRG1 promotes vascular Wnt signaling. Vascular deletion of Brg1 results in aberrant yolk sac blood vessel morphology, which is rescued by pharmacological stimulation of Wnt signaling with lithium chloride (LiCl). We have now generated embryos lacking the chromatin-remodeling enzyme Chd4 in vascular endothelial cells. Unlike Brg1 mutants, Chd4 mutant embryos had normal yolk sac vascular morphology. However, concomitant deletion of Chd4 and Brg1 rescued vascular abnormalities seen in Brg1 mutant yolk sacs to the same extent as LiCl treatment. We hypothesized that Wnt signaling was upregulated in Chd4 mutant yolk sac vasculature. Indeed, we found that Chd4 deletion resulted in upregulation of the Wnt-responsive transcription factor Tcf7 and an increase in Wnt target gene expression in endothelial cells. Furthermore, we identified one Wnt target gene, Pitx2, that was downregulated in Brg1 mutant endothelial cells but was rescued following LiCl treatment and in Brg1 Chd4 double mutant vasculature, suggesting that PITX2 helps to mediate the restoration of yolk sac vascular remodeling under both conditions. We conclude that BRG1 and CHD4 antagonistically modulate Wnt signaling in developing yolk sac vessels to mediate normal vascular remodeling.

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.

Influence of salinity on embryogenesis, survival, growth and oxygen consumption in embryos and yolk-sac larvae of the Nile tilapia

The Nile tilapia is a euryhaline species that offers considerable potential for culture in low-salinity waters. In order to investigate the ability of this species to be reared during the hatchery stages in brackish water, the effects of varying low salinities (0, 7.5, 15, 20 and 25ppt) on hatchability, survival, growth and energetic parameters until yolk-sac absorption were assessed. Salinity up to 20ppt was tolerable, although reduced hatching rates at 15 and 20ppt suggest that these salinities may be less than optimal. Optimum timing of transfer of eggs from freshwater to elevated salinities was 3–4h post-fertilisation, following manual stripping and fertilisation of eggs, however increasing incubation salinity lengthened the time taken to hatch. Dry body weight was related to salinity, with larvae in salinities greater than 15ppt displaying, at hatch, a significantly (GLM: ρ<0.05) lower body weight but containing greater yolk reserves than those in freshwater or lower salinities. Survival at yolk-sac absorption displayed a significant (GLM; ρ<0.05) inverse relationship with increasing salinity and mortalities were particularly heavy in the higher salinities of 15, 20 and 25ppt. Mortalities occurred primarily during early yolk-sac development yet stabilised from day 5 post-hatch onwards. Salinity had a negative effect on yolk absorption efficiency (YAE). Salinity-related differences in oxygen consumption rates were not detectable until day 3 post-hatch. Oxygen consumption rates of larvae in freshwater between 3 and 6days post-hatch were always significantly higher (GLM: ρ<0.05) than those in 7.5, 15, 20 and 25ppt, however, on day 9 post-hatch this pattern was reversed and freshwater larvae had a significantly lower QO2 than those in elevated salinities. Salinity had a significant inverse effect on larval standard length, with elevated salinities producing shorter larvae from hatch until day 6 post-hatch, after which time there was no significant differences between treatments. Salinity had a significant effect on whole larval dry weight, with heavier larvae in elevated salinities throughout the yolk-sac period (GLM; ρ<0.05).

EDD Inhibits ATM-mediated Phosphorylation of p53

The EDD (E3 identified by differential display) gene, first identified as a progestin-induced gene in T-47D breast cancer cells, encodes an E3 ubiquitin ligase with a HECT domain. It was reported that EDD is involved in the G(2)/M progression through ubiquitination of phospho-katanin p60. Previous study has also shown that EDD can act as a transcription cofactor independently of its E3 ligase activity. In this study, we uncover a new role for EDD during cell cycle progression in an E3 ligase-independent manner. We demonstrate that EDD can physically interact with p53 and that this interaction blocks the phosphorylation of p53 by ataxia telangiectasia mutated (ATM). Silencing of EDD induces phosphorylation of p53 at Ser(15) and activates p53 target genes in fibroblasts and some transformed cells without activation of DNA damage response. The G(1)/S arrest induced by EDD depletion depends on p53. On the other hand, overexpression of EDD inhibits p53-Ser(15) phosphorylation and suppresses the induction of p53 target genes during DNA damage, and this effect does not require its E3 ligase activity. Thus, through binding to p53, EDD actively inhibits p53 phosphorylation by ATM and plays a role in ensuring smooth G(1)/S progression.

Factors Affecting the Expression of Differentiation Marker Genes for the Primitive Endoderm Lineage in a Mouse Extra-Embryonic Endoderm Stem Cell Line, XEN26 Cells

ABSTRACTThe extra-embryonic endoderm lineages, visceral endoderm (VE) and parietal endoderm (PE), comprise part of the yolk sac and support embryonic development. Mouse extra-embryonic endoderm stem (XEN) cells were established from primitive endoderm (PrE), the founder cells of VE and PE, from preimplantation blastocysts. The differentiation of XEN cells is usually induced in adherent conditions without medium conditioned by mouse embryonic fibroblasts (MEFCM), which maintains self-renewal of the cells. In this study, we analyzed the effects of suspension culture and retinoic acid (RA) treatment on the differentiation of XEN26 cells, a mouse XEN cell line, toward PrE derivatives. The suspension culture without both MEFCM and RA promoted the expression of α-fetoprotein (Afp) mRNA, a specific marker for VE, at 3- to 4-fold higher levels (P < 0.05) than those in adherent culture. In these conditions, the cellular content of hepatocyte nuclear factor-4α (HNF4α) protein, a crucial transcription factor involving Afp expression, increased, while that of HNF1α was relatively constant. On the other hand, RA treatment induced upregulation of the mRNA for tissue plasminogen activator (Plat), a specific marker for PE, in both adherent and suspension culture conditions. From these results, our new attempts, including the application of RA treatment and suspension culture, for the induction of differentiation in XEN26 cells led to significant upregulation of Plat and Afp, respectively.

The KRAB Zinc Finger Protein ZFP568 is Required in the Extraembryonic Mesoderm for Yolk Sac and Placental Development

The KRAB Zinc Finger Protein ZFP568 is Required in the Extraembryonic Mesoderm for Yolk Sac and Placental Development