Mutation in folate metabolism causes epigenetic instability and transgenerational effects on development.

The importance of maternal folate consumption for normal development is well established, yet the molecular mechanism linking folate metabolism to development remains poorly understood. The enzyme methionine synthase reductase (Mtrr) is necessary for utilization of methyl groups from the folate cycle. We found that a hypomorphic mutation of the mouse Mtrr gene results in intrauterine growth restriction, developmental delay, and congenital malformations, including neural tube, heart, and placental defects. Importantly, these defects were dependent upon the Mtrr genotypes of the maternal grandparents. Furthermore, we observed widespread epigenetic instability associated with altered gene expression in the placentas of wild-type grandprogeny of Mtrr-deficient maternal grandparents. Embryo transfer experiments revealed that Mtrr deficiency in mice lead to two distinct, separable phenotypes: adverse effects on their wild-type daughters' uterine environment, leading to growth defects in wild-type grandprogeny, and the appearance of congenital malformations independent of maternal environment that persist for five generations, likely through transgenerational epigenetic inheritance.

Blastocyst transfer in mice alters the placental transcriptome and growth.

Assisted reproduction technologies (ARTs) are becoming increasingly common. Therefore, how these procedures influence gene regulation and foeto-placental development are important to explore. Here, we assess the effects of blastocyst transfer on mouse placental growth and transcriptome. C57Bl/6 blastocysts were transferred into uteri of B6D2F1 pseudopregnant females and dissected at embryonic day 10.5 for analysis. Compared to non-transferred controls, placentas from transferred conceptuses weighed less even though the embryos were larger on average. This suggested a compensatory increase in placental efficiency. RNA sequencing of whole male placentas revealed 543 differentially expressed genes (DEGs) after blastocyst transfer: 188 and 355 genes were downregulated and upregulated, respectively. DEGs were independently validated in male and female placentas. Bioinformatic analyses revealed that DEGs represented expression in all major placental cell types and included genes that are critical for placenta development and/or function. Furthermore, the direction of transcriptional change in response to blastocyst transfer implied an adaptive response to improve placental function to maintain foetal growth. Our analysis revealed that CpG methylation at regulatory regions of two DEGs was unchanged in female transferred placentas and that DEGs had fewer gene-associated CpG islands (within ~20 kb region) compared to the larger genome. These data suggested that altered methylation at proximal promoter regions might not lead to transcriptional disruption in transferred placentas. Genomic clustering of some DEGs warrants further investigation of long-range, cis-acting epigenetic mechanisms including histone modifications together with DNA methylation. We conclude that embryo transfer, a protocol required for ART, significantly impacts the placental transcriptome and growth.

Three-dimensional cultures of trophoblast stem cells autonomously develop vascular-like spaces lined by trophoblast giant cells.

The maternal blood space in the mouse placenta is lined not by endothelial cells but rather by various subtypes of trophoblast giant cells (TGCs), defined by their location and different patterns of gene expression. While TGCs invade the spiral arteries to displace the maternal endothelium, the rest of the vascular space is created de novo but the mechanisms are not well understood. We cultured mouse trophoblast stem (TS) cells in suspension and found that they readily form spheroids (trophospheres). Compared to cells grown in monolayer, differentiating trophospheres showed accelerated expression of TGC-specific genes. Morphological and gene expression studies showed that cavities form within the trophospheres that are primarily lined by Prl3d1/Pl1α-positive cells analogous to parietal-TGCs (P-TGCs) which line the maternal venous blood within the placenta. Lumen formation in trophospheres and in vivo was associated with cell polarization including CD34 sialomucin deposition on the apical side and cytoskeletal rearrangement. While P-TGCs preferentially formed in trophospheres at atmospheric oxygen levels (19%), decreasing oxygen to 3% shifted differentiation towards Ctsq-positive sinusoidal and/or channel TGCs. These studies show that trophoblast cells have the intrinsic ability to form vascular channels in ways analogous to endothelial cells. The trophosphere system will be valuable for assessing mechanisms that regulate specification of different TGC subtypes and their morphogenesis into vascular spaces.

A positive feedback loop involving Gcm1 and Fzd5 directs chorionic branching morphogenesis in the placenta.

Chorioallantoic branching morphogenesis is a key milestone during placental development, creating the large surface area for nutrient and gas exchange, and is therefore critical for the success of term pregnancy. Several Wnt pathway molecules have been shown to regulate placental development. However, it remains largely unknown how Wnt-Frizzled (Fzd) signaling spatiotemporally interacts with other essential regulators, ensuring chorionic branching morphogenesis and angiogenesis during placental development. Employing global and trophoblast-specific Fzd5-null and Gcm1-deficient mouse models, combining trophoblast stem cell lines and tetraploid aggregation assay, we demonstrate here that an amplifying signaling loop between Gcm1 and Fzd5 is essential for normal initiation of branching in the chorionic plate. While Gcm1 upregulates Fzd5 specifically at sites where branching initiates in the basal chorion, this elevated Fzd5 expression via nuclear ß-catenin signaling in turn maintains expression of Gcm1. Moreover, we show that Fzd5-mediated signaling induces the disassociation of cell junctions for branching initiation via downregulating ZO-1, claudin 4, and claudin 7 expressions in trophoblast cells at the base of the chorion. In addition, Fzd5-mediated signaling is also important for upregulation of Vegf expression in chorion trophoblast cells. Finally, we demonstrate that Fzd5-Gcm1 signaling cascade is operative during human trophoblast differentiation. These data indicate that Gcm1 and Fzd5 function in an evolutionary conserved positive feedback loop that regulates trophoblast differentiation and sites of chorionic branching morphogenesis.

Placental origins of adverse pregnancy outcomes: potential molecular targets: an Executive Workshop Summary of the Eunice Kennedy Shriver National Institute of Child Health and Human Development.

Although much progress is being made in understanding the molecular pathways in the placenta that are involved in the pathophysiology of pregnancy-related disorders, a significant gap exists in the utilization of this information for the development of new drug therapies to improve pregnancy outcome. On March 5-6, 2015, the Eunice Kennedy Shriver National Institute of Child Health and Human Development of the National Institutes of Health sponsored a 2-day workshop titled Placental Origins of Adverse Pregnancy Outcomes: Potential Molecular Targets to begin to address this gap. Particular emphasis was given to the identification of important molecular pathways that could serve as drug targets and the advantages and disadvantages of targeting these particular pathways. This article is a summary of the proceedings of that workshop. A broad number of topics were covered that ranged from basic placental biology to clinical trials. This included research in the basic biology of placentation, such as trophoblast migration and spiral artery remodeling, and trophoblast sensing and response to infectious and noninfectious agents. Research findings in these areas will be critical for the formulation of the development of future treatments and the development of therapies for the prevention of a number of pregnancy disorders of placental origin that include preeclampsia, fetal growth restriction, and uterine inflammation. Research was also presented that summarized ongoing clinical efforts in the United States and in Europe that has tested novel interventions for preeclampsia and fetal growth restriction, including agents such as oral arginine supplementation, sildenafil, pravastatin, gene therapy with virally delivered vascular endothelial growth factor, and oxygen supplementation therapy. Strategies were also proposed to improve fetal growth by the enhancement of nutrient transport to the fetus by modulation of their placental transporters and the targeting of placental mitochondrial dysfunction and oxidative stress to improve placental health. The roles of microRNAs and placental-derived exosomes, as well as messenger RNAs, were also discussed in the context of their use for diagnostics and as drug targets. The workshop discussed the aspect of safety and pharmacokinetic profiles of potential existing and new therapeutics that will need to be determined, especially in the context of the unique pharmacokinetic properties of pregnancy and the hurdles and pitfalls of the translation of research findings into practice. The workshop also discussed novel methods of drug delivery and targeting during pregnancy with the use of macromolecular carriers, such as nanoparticles and biopolymers, to minimize placental drug transfer and hence fetal drug exposure. In closing, a major theme that developed from the workshop was that the scientific community must change their thinking of the pregnant woman and her fetus as a vulnerable patient population for which drug development should be avoided, but rather be thought of as a deprived population in need of more effective therapeutic interventions.

Adaptability and potential for treatment of placental functions to improve embryonic development and postnatal health.

For an organ that is so critical for life in eutherian mammals, the placenta hardly gets the attention that it deserves. The placenta does a series of remarkable things, including implanting the embryo in the uterus, negotiating with the mother for nutrients but also protecting her health during pregnancy, helping establish normal metabolic and cardiovascular function for life postnatally (developmental programming) and initiating changes that prepare the mother to care for and suckle her young after birth. Different lines of evidence in experimental animals suggest that the development and function of the placenta are adaptable. This means that some of the changes observed in pathological pregnancies may represent attempts to mitigate the impact of fetal growth and development. Key and emerging concepts are reviewed here concerning how we may view the placenta diagnostically and therapeutically in pregnancy complications, focusing on information from experimental studies in mice, sheep and cattle, as well as association studies from humans. Hundreds of different genes have been shown to underlie normal placental development and function, some of which have promise as tractable targets for intervention in pregnancies at risk for poor fetal growth.

Development of the hemochorial maternal vascular spaces in the placenta through endothelial and vasculogenic mimicry.

The maternal vasculature within the placenta in primates and rodents is unique because it is lined by fetal cells of the trophoblast lineage and not by maternal endothelial cells. In addition to trophoblast cells that invade the uterine spiral arteries that bring blood into the placenta, other trophoblast subtypes sit at different levels of the vascular space. In mice, at least five distinct subtypes of trophoblast cells have been identified which engage maternal endothelial cells on the arterial and venous frontiers of the placenta, but which also form the channel-like spaces within it through a process analogous to formation of blood vessels (vasculogenic mimicry). These cells are all large, post-mitotic trophoblast giant cells. In addition to assuming endothelial cell-like characteristics (endothelial mimicry), they produce dozens of different hormones that are thought to regulate local and systemic maternal adaptations to pregnancy. Recent work has identified distinct molecular pathways in mice that regulate the morphogenesis of trophoblast cells on the arterial and venous sides of the vascular circuit that may be analogous to specification of arterial and venous endothelial cells.

The transcriptional co-repressor Grg3/Tle3 promotes pancreatic endocrine progenitor delamination and ß-cell differentiation.

Pancreatic ß-cells arise from Ngn3(+) endocrine progenitors within the trunk epithelium of the embryonic pancreas. The emergence of endocrine cells requires E-cadherin downregulation, but the crucial steps that elicit such are not clear, yet probably important for ultimately being able to efficiently generate ß-cells de novo from stem cells. Grg3 (groucho-related gene 3, also known as Tle3), encodes a member of the Groucho/TLE family of co-repressors and its function in various cell contexts is mediated by recruitment to target genes by different transcription factors. Grg proteins broadly regulate the progression of progenitor cells to differentiated cell types, but specific developmental mechanisms have not been clear. We find that Grg3 is expressed in most ß-cells and a subset of other endocrine cell types in the pancreas. Grg3 is highly expressed in Ngn3(+) endocrine progenitor descendants just after transient Ngn3 expression. Grg3-null embryos die at E14.5, which is associated with placental defects, so we explanted E12.5 pancreata to allow endocrine differentiation to occur in culture. Grg3 knockout explants displayed a drastic decrease in the differentiation of all endocrine cell types owing to defects in the delamination of early endocrine progenitors from the trunk epithelium. We find that Grg3 normally suppresses E-cadherin gene expression, thereby allowing delamination of endocrine cells from the trunk epithelium and revealing how this transcriptional co-repressor modulates this crucial step of ß-cell development.

Pregnancy Hyperglycemia in Prolactin Receptor Mutant, but Not Prolactin Mutant, Mice and Feeding-Responsive Regulation of Placental Lactogen Genes Implies Placental Control of Maternal Glucose Homeostasis.

Pregnancy is often viewed as a conflict between the fetus and mother over metabolic resources. Insulin resistance occurs in mothers during pregnancy but does not normally lead to diabetes because of an increase in the number of the mother's pancreatic beta cells. In mice, this increase is dependent on prolactin (Prl) receptor signaling but the source of the ligand has been unclear. Pituitary-derived Prl is produced during the first half of pregnancy in mice but the placenta produces Prl-like hormones from implantation to term. Twenty-two separate mouse genes encode the placenta Prl-related hormones, making it challenging to assess their roles in knockout models. However, because at least four of them are thought to signal through the Prl receptor, we analyzed Prlr mutant mice and compared their phenotypes with those of Prl mutants. We found that whereas Prlr mutants develop hyperglycemia during gestation, Prl mutants do not. Serum metabolome analysis showed that Prlr mutants showed other changes consistent with diabetes. Despite the metabolic changes, fetal growth was normal in Prlr mutants. Of the four placenta-specific, Prl-related hormones that have been shown to interact with the Prlr, their gene expression localizes to different endocrine cell types. The Prl3d1 gene is expressed by trophoblast giant cells both in the labyrinth layer, sitting on the arterial side where maternal blood is highest in oxygen and nutrients, and in the junctional zone as maternal blood leaves the placenta. Expression increases during the night, though the increase in the labyrinth is circadian whereas it occurs only after feeding in the junctional zone. These data suggest that the placenta has a sophisticated endocrine system that regulates maternal glucose metabolism during pregnancy.

The transcriptional co-repressor TLE3 regulates development of trophoblast giant cells lining maternal blood spaces in the mouse placenta.

TLE3 is a transcriptional co-repressor that interacts with several DNA-binding repressors, including downstream effectors of the Notch signaling pathway. We generated Tle3-deficient mice and found that they die in utero and their death is associated with abnormal development of the placenta with major defects in the maternal vasculature. In the normal placenta, maternal blood spaces are lined, not as usual in the mammalian circulation by endothelial cells, but rather by specialized embryo-derived cells of the trophoblast cell lineage named trophoblast giant cells (TGC). Tle3 mRNA is expressed in those specialized TGC and Tle3 mutants show severe defects in differentiation of TGC-lined channels and lacunar spaces that take blood out of the labyrinth zone of the placenta and into the uterine veins. The mutants also show somewhat milder defects on the arterial-side of the maternal vascular circuit in spiral arteries and canals that take blood into the labyrinth. Notch2 and Tle3 expression patterns overlap in several TGC subtypes and we found that Tle3 and Notch2 mutants have some overlapping features. However, they also show differences implying that TLE3 may mediate some but not all of the effects of Notch2 signaling during placenta development. Therefore, formation of the different types of maternal blood spaces by different TGC subtypes is regulated by distinct molecular mechanisms.

The basic helix-loop-helix transcription factor Hand1 regulates mouse development as a homodimer.

Hand1 is a basic helix-loop-helix transcription factor that is essential for development of the placenta, yolk sac and heart during mouse development. While Hand1 is essential for trophoblast giant cell (TGC) differentiation, its potential heterodimer partners are not co-expressed in TGCs. To test the hypothesis that Hand1 functions as homodimer, we generated knock-in mice in which the Hand1 gene was altered to encode a tethered homodimer (TH). Some Hand1(TH/-) conceptuses in which the only form of Hand1 is Hand1(TH) are viable and fertile, indicating that homodimer Hand1 is sufficient for mouse survival. ~2/3 of Hand1(TH/-) and all Hand1(TH/TH) mice died in utero and displayed severe placental defects and variable cardial and cranial-facial abnormalities, indicating a dosage-dependent effect of Hand1(TH). Meanwhile, expression of the Hand1(TH) protein did not have negative effects on viability or fertility in all Hand1(TH/+) mice. These data imply that Hand1 homodimer plays a dominant role during development and its expression dosage is critical for survival, whereas Hand1 heterodimers can be either dispensable or play a regulatory role to modulate the activity of Hand1 homodimer in vivo.

Defects in placental syncytiotrophoblast cells are a common cause of developmental heart disease.

Placental abnormalities have been sporadically implicated as a source of developmental heart defects. Yet it remains unknown how often the placenta is at the root of congenital heart defects (CHDs), and what the cellular mechanisms are that underpin this connection. Here, we selected three mouse mutant lines, Atp11a, Smg9 and Ssr2, that presented with placental and heart defects in a recent phenotyping screen, resulting in embryonic lethality. To dissect phenotype causality, we generated embryo- and trophoblast-specific conditional knockouts for each of these lines. This was facilitated by the establishment of a new transgenic mouse, Sox2-Flp, that enables the efficient generation of trophoblast-specific conditional knockouts. We demonstrate a strictly trophoblast-driven cause of the CHD and embryonic lethality in one of the three lines (Atp11a) and a significant contribution of the placenta to the embryonic phenotypes in another line (Smg9). Importantly, our data reveal defects in the maternal blood-facing syncytiotrophoblast layer as a shared pathology in placentally induced CHD models. This study highlights the placenta as a significant source of developmental heart disorders, insights that will transform our understanding of the vast number of unexplained congenital heart defects.

Ablation of Tpbpa-positive trophoblast precursors leads to defects in maternal spiral artery remodeling in the mouse placenta.

The placenta is composed of multiple trophoblast cell types that have diverse endocrine, vascular and nutrient transport functions. We have developed a transgenic system to investigate the developmental and functional roles of specific cell types using conditional expression of a cytotoxin to induce cell ablation in transgenic mice. The Tpbpa gene is expressed in ectoplacental cone cells starting between embryonic days (E) 7.5 and 8.5, and later in the spongiotrophoblast layer of the mature placenta. Tpbpa-positive cells are progenitors of many trophoblast subtypes including three subtypes of trophoblast giant cells (TGCs) and glycogen trophoblast cells. We used a Cre recombinase transgene driven by the Tpbpa promoter to irreversibly activate a diphtheria toxin A (DTA) transgene. Cre/DTA double transgenic placentas showed dramatic reduction of Tpbpa-positive spongiotrophoblast cells by E10.5 and conceptuses died by ~E11.5. The number of cells associated with maternal blood spaces, spiral artery TGCs (SpA-TGCs) and canal TGCs, and glycogen trophoblast cells were reduced. The loss of these specific trophoblast subtypes, especially SpA-TGCs, was correlated with a decrease in maternal spiral artery diameters, indicating a critical role of these cells in modulating the maternal vasculature. In contrast, parietal TGCs were not significantly reduced by progenitor cell ablation, suggesting that there is compensatory growth of this population and indeed a population of Ascl2 (Mash2)-positive/Tpbpa-negative cells was increased in the spongiotrophoblast layer in the Cre/DTA double transgenics. Our work demonstrates that the Tpbpa-positive lineage is essential for placental function and particularly critical for maternal vasculature remodeling.

Cell-cell adhesion defects in Mrj mutant trophoblast cells are associated with failure to pattern the chorion during early placental development.

Early placental development in mice involves patterning of the chorion into distinct layers, though little is understood regarding the interactions that regulate its organization. Here we demonstrate that keratin aggregates found in Mrj(-/-) chorionic trophoblast cells are associated with abnormal cell morphology, collapse of the actin cytoskeleton, E-cadherin and ß-catenin misexpression and extracellular matrix (ECM) disorganization. Accordingly, Mrj(-/-) trophoblast cells in vitro are nonadherent and display erratic migratory behavior. These cells also fail to differentiate into syncytiotrophoblast cells since Rhox4b expression, a marker of syncytiotrophoblast progenitors, was maintained and Gcm1, Synb, and Syna expression failed to increase. This differentiation defect was not solely attributable to E-cadherin misexpression or ECM disorganization. However, plating Mrj-deficient cells on exogenous laminin-511 normalized their cell behavior. Lastly, we show that Mrj(-/-) chorions at embryonic day 8.5 have expanded Rhox4b expression domains and do not form normal layers of gene expression suggesting that chorion patterning requires Mrj.

Exploration of genetic factors resulting in abnormal disease in cattle experimentally challenged with bovine spongiform encephalopathy.

Since the discovery of bovine spongiform encephalopathy (BSE), researchers have orally challenged cattle with infected brain material to study various aspects of disease pathogenesis. Unlike most other pathogens, oral BSE challenge does not always result in the expected clinical presentation and pathology. In a recent study, steers were challenged orally with BSE and all developed clinical signs and were sacrificed and tested. However, despite a similar incubation and clinical presentation, one of the steers did not have detectable PrPSc in its brain. Samples from this animal were analysed for genetic differences as well as for the presence of in vitro PrPSc seeding activity or infectivity to determine the BSE status of this animal and the potential reasons that it was different. Seeding activity was detected in the brainstem of the abnormal steer but it was approximately one million times less than that found in the normal BSE positive steers. Intra-cranial challenge of bovinized transgenic mice resulted in no transmission of disease. The abnormal steer had different genetic sequences in non-coding regions of the PRNP gene but detection of similar genotypes in Canadian BSE field cases, that showed the expected brain pathology, suggested these differences may not be the primary cause of the abnormal result. Breed composition analysis showed a higher Hereford content in the abnormal steer as well as in two Canadian atypical BSE field cases and several additional abnormal experimental animals. This study could point towards a possible impact of breed composition on BSE pathogenesis.

Activin promotes differentiation of cultured mouse trophoblast stem cells towards a labyrinth cell fate.

Prolonged maintenance of trophoblast stem (TS) cells requires fibroblast growth factor (FGF) 4 and embryonic fibroblast feeder cells or feeder cell-conditioned medium. Previous studies have shown that TGF-beta and Activin are sufficient to replace embryonic fibroblast-conditioned medium. Nodal, a member of the TGF-beta superfamily, is also known to be important in vivo for the maintenance of TS cells in the developing placenta. Our current studies indicate that TS cells do not express the Nodal co-receptor, Cripto, and do not respond directly to active Nodal in culture. Conversely, Activin subunits and their receptors are expressed in the placenta and TS cell cultures, with Activin predominantly expressed by trophoblast giant cells (TGCs). Differentiation of TS cells in the presence of TGC-conditioned medium or exogenous Activin results in a reduction in the expression of TGC markers. In line with TGC-produced Activin representing the active component in TGC-conditioned medium, this differentiation-inhibiting effect can be reversed by the addition of follistatin. Additional experiments in which TS cells were differentiated in the presence or absence of exogenous Activin or TGF-beta show that Activin but not TGF-beta results in the maintenance of expression of TS cell markers, prolongs the expression of syncytiotrophoblast markers, and significantly delays the expression of spongiotrophoblast and TGC markers. These results suggest that Activin rather than TGF-beta (or Nodal) acts directly on TS cells influencing both TS cell maintenance and cell fate, depending on whether the cells are also exposed to FGF4.

Homozygous missense N629D hERG (KCNH2) potassium channel mutation causes developmental defects in the right ventricle and its outflow tract and embryonic lethality.

Loss-of-function mutations in the human ERG1 potassium channel (hERG1) frequently underlie the long QT2 (LQT2) syndrome. The role of the ERG potassium channel in cardiac development was elaborated in an in vivo model of a homozygous, loss-of-function LQT2 syndrome mutation. The hERG N629D mutation was introduced into the orthologous mouse gene, mERG, by homologous recombination in mouse embryonic stem cells. Intact homozygous embryos showed abrupt cessation of the heart beat. N629D/N629D embryos die in utero by embryonic day 11.5. Their developmental defects include altered looping architecture, poorly developed bulbus cordis, and distorted aortic sac and branchial arches. N629D/N629D myocytes from embryonic day 9.5 embryos manifested complete loss of I(Kr) function, depolarized resting potential, prolonged action potential duration (LQT), failure to repolarize, and propensity to oscillatory arrhythmias. N629D/N629D myocytes manifest calcium oscillations and increased sarcoplasmic reticulum Ca(+2) content. Although the N629D/N629D protein is synthesized, it is mainly located intracellularly, whereas +/+ mERG protein is mainly in plasmalemma. N629D/N629D embryos show robust apoptosis in craniofacial regions, particularly in the first branchial arch and, to a lesser extent, in the cardiac outflow tract. Because deletion of Hand2 produces apoptosis, in similar regions and with a similar final developmental phenotype, Hand2 expression was evaluated. Robust decrease in Hand2 expression was observed in the secondary heart field in N629D/N629D embryos. In conclusion, loss of I(Kr) function in N629D/N629D cardiovascular system leads to defects in cardiac ontogeny in the first branchial arch, outflow tract, and the right ventricle.

Transcriptional repressor erf determines extraembryonic ectoderm differentiation.

Extraembryonic ectoderm differentiation and chorioallantoic attachment are fibroblast growth factor (FGF)- and transforming growth factor beta-regulated processes that are the first steps in the development of the placenta labyrinth and the establishment of the fetal-maternal circulation in the developing embryo. Only a small number of genes have been demonstrated to be important in trophoblast stem cell differentiation. Erf is a ubiquitously expressed Erk-regulated, ets domain transcriptional repressor expressed throughout embryonic development and adulthood. However, in the developing placenta, after 7.5 days postcoitum (dpc) its expression is restricted to the extraembryonic ectoderm, and its expression is restricted after 9.5 dpc in a subpopulation of labyrinth cells. Homozygous deletion of Erf in mice leads to a block of chorionic cell differentiation before chorioallantoic attachment, resulting in a persisting chorion layer, a persisting ectoplacental cone cavity, failure of chorioallantoic attachment, and absence of labyrinth. These defects result in embryo death by 10.5 dpc. Trophoblast stem cell lines derived from Erf(dl1/dl1) knockout blastocysts exhibit delayed differentiation and decreased expression of spongiotrophoblast markers, consistent with the persisting chorion layer, the expanded giant cell layer, and the diminished spongiotrophoblast layer observed in vivo. Our data suggest that attenuation of FGF/Erk signaling and consecutive Erf nuclear localization and function is required for extraembryonic ectoderm differentiation, ectoplacental cone cavity closure, and chorioallantoic attachment.

Neural stem cell self-renewal requires the Mrj co-chaperone.

The Mrj co-chaperone is expressed throughout the mouse conceptus, yet its requirement for placental development has prohibited a full understanding of its embryonic function. Here, we show that Mrj(-/-) embryos exhibit neural tube defects independent of the placenta phenotype, including exencephaly and thin-walled neural tubes. Molecular analyses revealed fewer proliferating cells and a down-regulation of early neural progenitor (Pax6, Olig2, Hes5) and neuronal (Nscl2, SCG10) cell markers in Mrj(-/-) neuroepithelial cells. Furthermore, Mrj(-/-) neurospheres are significantly smaller and form fewer secondary neurospheres indicating that Mrj is necessary for self-renewal of neural stem cells. However, the molecular function of Mrj in this context remains elusive because Mrj does not colocalize with Bmi-1, a self-renewal protein. Furthermore, unlike in Mrj(-/-) placentas, intermediate filament-containing aggregates do not accumulate in Mrj(-/-) neuroepithelium, ruling out nestin as a substrate for Mrj. Regardless, Mrj plays an important role in neural stem cell self-renewal.