Midgestation Embryos Research Articles

Fetal growth delay caused by loss of non-canonical imprinting is resolved late in pregnancy and culminates in offspring overgrowth.

Germline epigenetic programming, including genomic imprinting, substantially influences offspring development. Polycomb Repressive Complex 2 (PRC2) plays an important role in Histone 3 Lysine 27 trimethylation (H3K27me3)-dependent imprinting, loss of which leads to growth and developmental changes in mouse offspring. In this study, we show that offspring from mouse oocytes lacking the PRC2 protein Embryonic Ectoderm Development (EED) were initially developmentally delayed, characterised by low blastocyst cell counts and substantial growth delay in mid-gestation embryos. This initial developmental delay was resolved as offspring underwent accelerated fetal development and growth in late gestation resulting in offspring that were similar stage and weight to controls at birth. The accelerated development and growth in offspring from Eed-null oocytes was associated with remodelling of the placenta, which involved an increase in fetal and maternal tissue size, conspicuous expansion of the glycogen-enriched cell population, and delayed parturition. Despite placental remodelling and accelerated offspring fetal growth and development, placental efficiency, and fetal blood glucose levels were low, and the fetal blood metabolome was unchanged. Moreover, while expression of the H3K27me3-imprinted gene and amino acid transporter Slc38a4 was increased, fetal blood levels of individual amino acids were similar to controls, indicating that placental amino acid transport was not enhanced. Genome-wide analyses identified extensive transcriptional dysregulation and DNA methylation changes in affected placentas, including a range of imprinted and non-imprinted genes. Together, while deletion of Eed in growing oocytes resulted in fetal growth and developmental delay and placental hyperplasia, our data indicate a remarkable capacity for offspring fetal growth to be normalised despite inefficient placental function and the loss of H3K27me3-dependent genomic imprinting.

Glycolytic state of aortic endothelium favors hematopoietic transition during the emergence of definitive hematopoiesis.

The first definitive hematopoietic progenitors emerge through the process of endothelial-to-hematopoietic transition in vertebrate embryos. With molecular regulators for this process worked out, the role of metabolic pathways used remains unclear. Here, we performed nano-LC-MS/MS-based proteomic analysis and predicted a metabolic switch from a glycolytic to oxidative state upon hematopoietic transition. Mitochondrial activity, glucose uptake, and glycolytic flux analysis supported this hypothesis. Systemic inhibition of lactate dehydrogenase A (LDHA) increased oxygen consumption rate in the hemato-endothelial system and inhibited the emergence of intra-aortic hematopoietic clusters. These findings were corroborated using Tie2-Cre-mediated deletion of Ldha that showed similar effects on hematopoietic emergence. Conversely, stabilization of HIF-1α via inhibition of oxygen-sensing pathway led to decreased oxidative flux and promoted hematopoietic emergence in mid-gestation embryos. Thus, cell-intrinsic regulation of metabolic state overrides oxygenated microenvironment in the aorta to promote a glycolytic metabolic state that is crucial for hematopoietic emergence in mammalian embryos.

RRNA transcription is integral to phase separation and maintenance of nucleolar structure.

Transcription of ribosomal RNA (rRNA) by RNA Polymerase (Pol) I in the nucleolus is necessary for ribosome biogenesis, which is intimately tied to cell growth and proliferation. Perturbation of ribosome biogenesis results in tissue specific disorders termed ribosomopathies in association with alterations in nucleolar structure. However, how rRNA transcription and ribosome biogenesis regulate nucleolar structure during normal development and in the pathogenesis of disease remains poorly understood. Here we show that homozygous null mutations in Pol I subunits required for rRNA transcription and ribosome biogenesis lead to preimplantation lethality. Moreover, we discovered that Polr1a-/-, Polr1b-/-, Polr1c-/- and Polr1d-/- mutants exhibit defects in the structure of their nucleoli, as evidenced by a decrease in number of nucleolar precursor bodies and a concomitant increase in nucleolar volume, which results in a single condensed nucleolus. Pharmacological inhibition of Pol I in preimplantation and midgestation embryos, as well as in hiPSCs, similarly results in a single condensed nucleolus or fragmented nucleoli. We find that when Pol I function and rRNA transcription is inhibited, the viscosity of the granular compartment of the nucleolus increases, which disrupts its phase separation properties, leading to a single condensed nucleolus. However, if a cell progresses through mitosis, the absence of rRNA transcription prevents reassembly of the nucleolus and manifests as fragmented nucleoli. Taken together, our data suggests that Pol I function and rRNA transcription are required for maintaining nucleolar structure and integrity during development and in the pathogenesis of disease.

Divergent expression of Neurl3 from hemogenic endothelial cells to hematopoietic stem progenitor cells during development

Prior to the generation of hematopoietic stem cells (HSCs) from the hemogenic endothelial cells (HECs) mainly in the dorsal aorta in midgestational mouse embryos, multiple hematopoietic progenitors including erythro-myeloid progenitors and lymphoid progenitors are generated from yolk sac HECs. These HSC-independent hematopoietic progenitors have recently been identified as major contributors to functional blood cell production until birth. However, little is known about yolk sac HECs. Here, combining integrative analyses of multiple single-cell RNA-sequencing datasets and functional assays, we reveal that Neurl3-EGFP, in addition to marking the continuum throughout the ontogeny of HSCs from HECs, can also serve as a single enrichment marker for yolk sac HECs. Moreover, while yolk sac HECs have much weaker arterial characteristics than either arterial endothelial cells in the yolk sac or HECs within the embryo proper, the lymphoid potential of yolk sac HECs is largely confined to the arterial-biased subpopulation featured by the Unc5b expression. Interestingly, the B lymphoid potential of hematopoietic progenitors, but not for myeloid potentials, is exclusively detected in Neurl3-negative subpopulations in midgestational embryos. Taken together, these findings enhance our understanding of blood birth from yolk sac HECs and provide theoretical basis and candidate reporters for monitoring step-wise hematopoietic differentiation.

Nupr1 Negatively Regulates Endothelial to Hematopoietic Transition in the Aorta-Gonad-Mesonephros Region.

In the aorta of mid-gestational mouse embryos, a specialized endothelial subpopulation termed hemogenic endothelial cells (HECs) develops into hematopoietic stem and progenitor cells (HSPCs), through a conserved process of endothelial-to-hematopoietic transition (EHT). EHT is tightly controlled by multiple intrinsic and extrinsic mechanisms. Nevertheless, the molecular regulators restraining this process remain poorly understood. Here, it is uncovered that, one of the previously identified HEC signature genes, Nupr1, negatively regulates the EHT process. Nupr1 deletion in endothelial cells results in increased HSPC generation in the aorta-gonad-mesonephros region. Furthermore, single-cell transcriptomics combined with serial functional assays reveals that loss of Nupr1 promotes the EHT process by promoting the specification of hematopoiesis-primed functional HECs and strengthening their subsequent hematopoietic differentiation potential toward HSPCs. This study further finds that the proinflammatory cytokine, tumor necrosis factor α (TNF-α), is significantly upregulated in Nupr1-deficient HECs, and the use of a specific TNF-α neutralizing antibody partially reduces excessive HSPC generation in the explant cultures from Nupr1-deficient embryos. This study identifies a novel negative regulator of EHT and the findings indicate that Nupr1 is a new potential target for future hematopoietic stem cell regeneration research.

The arginine methyltransferase Carm1 is necessary for heart development.

To discover genes implicated in human congenital disorders, we performed ENU mutagenesis in the mouse and screened for mutations affecting embryonic development. In this work, we report defects of heart development in mice homozygous for a mutation of coactivator-associated arginine methyltransferase 1 (Carm1). While Carm1 has been extensively studied, it has never been previously associated with a role in heart development. Phenotype analysis combining histology and microcomputed tomography imaging shows a range of cardiac defects. Most notably, many affected midgestation embryos appear to have cardiac rupture and hemorrhaging in the thorax. Mice that survive to late gestation show a variety of cardiac defects, including ventricular septal defects, double outlet right ventricle, and persistent truncus arteriosus. Transcriptome analyses of the mutant embryos by mRNA-seq reveal the perturbation of several genes involved in cardiac morphogenesis and muscle development and function. In addition, we observe the mislocalization of cardiac neural crest cells at E12.5 in the outflow tract. The cardiac phenotype of Carm1 mutant embryos is similar to that of Pax3 null mutants, and PAX3 is a putative target of CARM1. However, our analysis does not support the hypothesis that developmental defects in Carm1 mutant embryos are primarily due to a functional defect of PAX3.

Neural defects caused by total and Wnt1-Cre mediated ablation of p120ctn in mice

Backgroundp120 catenin (p120ctn) is an important component in the cadherin-catenin cell adhesion complex because it stabilizes cadherin-mediated intercellular junctions. Outside these junctions, p120ctn is actively involved in the regulation of small GTPases of the Rho family, in actomyosin dynamics and in transcription regulation. We and others reported that loss of p120ctn in mouse embryos results in an embryonic lethal phenotype, but the exact developmental role of p120ctn during brain formation has not been reported.ResultsWe combined floxed p120ctn mice with Del-Cre or Wnt1-Cre mice to deplete p120ctn from either all cells or specific brain and neural crest cells. Complete loss of p120ctn in mid-gestation embryos resulted in an aberrant morphology, including growth retardation, failure to switch from lordotic to fetal posture, and defective neural tube formation and neurogenesis. By expressing a wild-type p120ctn from the ROSA26 locus in p120ctn-null mouse embryonic stem cells, we could partially rescue neurogenesis. To further investigate the developmental role of p120ctn in neural tube formation, we generated conditional p120ctnfl/fl;Wnt1Cre knockout mice. p120ctn deletion in Wnt1-expressing cells resulted in neural tube closure defects (NTDs) and craniofacial abnormalities. These defects could not be correlated with misregulation of brain marker genes or cell proliferation. In contrast, we found that p120ctn is required for proper expression of the cell adhesion components N-cadherin, E-cadherin and β-catenin, and of actin-binding proteins cortactin and Shroom3 at the apical side of neural folds. This region is of critical importance for closure of neural folds. Surprisingly, the lateral side of mutant neural folds showed loss of p120ctn, but not of N-cadherin, β-catenin or cortactin.ConclusionsThese results indicate that p120ctn is required for neurogenesis and neurulation. Elimination of p120ctn in cells expressing Wnt1 affects neural tube closure by hampering correct formation of specific adhesion and actomyosin complexes at the apical side of neural folds. Collectively, our results demonstrate the crucial role of p120ctn during brain morphogenesis.

Embryonic endothelial evolution towards first hematopoietic stem cells revealed by single-cell transcriptomic and functional analyses

Hematopoietic stem cells (HSCs) in adults are believed to be born from hemogenic endothelial cells (HECs) in mid-gestational embryos. Due to the rare and transient nature, the HSC-competent HECs have never been stringently identified and accurately captured, let alone their genuine vascular precursors. Here, we first used high-precision single-cell transcriptomics to unbiasedly examine the relevant EC populations at continuous developmental stages with intervals of 0.5 days from embryonic day (E) 9.5 to E11.0. As a consequence, we transcriptomically identified two molecularly different arterial EC populations and putative HSC-primed HECs, whose number peaked at E10.0 and sharply decreased thereafter, in the dorsal aorta of the aorta-gonad-mesonephros (AGM) region. Combining computational prediction and in vivo functional validation, we precisely captured HSC-competent HECs by the newly constructed Neurl3-EGFP reporter mouse model, and realized the enrichment further by a combination of surface markers (Procr+Kit+CD44+, PK44). Surprisingly, the endothelial-hematopoietic dual potential was rarely but reliably witnessed in the cultures of single HECs. Noteworthy, primitive vascular ECs from E8.0 experienced two-step fate choices to become HSC-primed HECs, namely an initial arterial fate choice followed by a hemogenic fate conversion. This finding resolves several previously observed contradictions. Taken together, comprehensive understanding of endothelial evolutions and molecular programs underlying HSC-primed HEC specification in vivo will facilitate future investigations directing HSC production in vitro.

Developmental genome-wide DNA methylation asymmetry between mouse placenta and embryo

ABSTRACT In early embryos, DNA methylation is remodelled to initiate the developmental program but for mostly unknown reasons, methylation marks are acquired unequally between embryonic and placental cells. To better understand this, we generated high-resolution DNA methylation maps of mouse mid-gestation (E10.5) embryo and placenta. We uncovered specific subtypes of differentially methylated regions (DMRs) that contribute directly to the developmental asymmetry existing between mid-gestation embryonic and placental DNA methylation patterns. We show that the asymmetry occurs rapidly during the acquisition of marks in the post-implanted conceptus (E3.5-E6.5), and that these patterns are long-lasting across subtypes of DMRs throughout prenatal development and in somatic tissues. We reveal that at the peri–implantation stages, the de novo methyltransferase activity of DNMT3B is the main driver of methylation marks on asymmetric DMRs, and that DNMT3B can largely compensate for lack of DNMT3A in the epiblast and extraembryonic ectoderm, whereas DNMT3A can only partially compensate in the absence of DNMT3B. However, as development progresses and as DNMT3A becomes the principal de novo methyltransferase, the compensatory DNA methylation mechanism of DNMT3B on DMRs becomes less effective.

Loss of Ing3 Expression Results in Growth Retardation and Embryonic Death.

The ING3 candidate tumour suppressor belongs to a family of histone modifying proteins involved in regulating cell proliferation, senescence, apoptosis, chromatin remodeling, and DNA repair. It is a stoichiometric member of the minimal NuA4 histone acetyl transferase (HAT) complex consisting of EAF6, EPC1, ING3, and TIP60. This complex is responsible for the transcription of an essential cascade of genes involved in embryonic development and in tumour suppression. ING3 has been linked to head and neck and hepatocellular cancers, although its status as a tumour suppressor has not been well established. Recent studies suggest a pro-metastasis role in prostate cancer progression. Here, we describe a transgenic mouse strain with insertional mutation of an UbC-mCherry expression cassette into the endogenous Ing3 locus, resulting in the disruption of ING3 protein expression. Homozygous mutants are embryonically lethal, display growth retardation, and severe developmental disorders. At embryonic day (E) 10.5, the last time point viable homozygous embryos were found, they were approximately half the size of heterozygous mice that develop normally. µCT analysis revealed a developmental defect in neural tube closure, resulting in the failure of formation of closed primary brain vesicles in homozygous mid-gestation embryos. This is consistent with high ING3 expression levels in the embryonic brains of heterozygous and wild type mice and its lack in homozygous mutant embryos that show a lack of ectodermal differentiation. Our data provide direct evidence that ING3 is an essential factor for normal embryonic development and that it plays a fundamental role in prenatal brain formation.

Nessys: A new set of tools for the automated detection of nuclei within intact tissues and dense 3D cultures.

Methods for measuring the properties of individual cells within their native 3D environment will enable a deeper understanding of embryonic development, tissue regeneration, and tumorigenesis. However, current methods for segmenting nuclei in 3D tissues are not designed for situations in which nuclei are densely packed, nonspherical, or heterogeneous in shape, size, or texture, all of which are true of many embryonic and adult tissue types as well as in many cases for cells differentiating in culture. Here, we overcome this bottleneck by devising a novel method based on labelling the nuclear envelope (NE) and automatically distinguishing individual nuclei using a tree-structured ridge-tracing method followed by shape ranking according to a trained classifier. The method is fast and makes it possible to process images that are larger than the computer’s memory. We consistently obtain accurate segmentation rates of >90%, even for challenging images such as mid-gestation embryos or 3D cultures. We provide a 3D editor and inspector for the manual curation of the segmentation results as well as a program to assess the accuracy of the segmentation. We have also generated a live reporter of the NE that can be used to track live cells in 3 dimensions over time. We use this to monitor the history of cell interactions and occurrences of neighbour exchange within cultures of pluripotent cells during differentiation. We provide these tools in an open-access user-friendly format.

When blood development meets single-cell transcriptomics.

Blood cells arise during embryonic development by three temporally distinct waves. Belonging to the third wave, hematopoietic stem cells (HSCs) are generated from hemogenic endothelium via endothelial-to-hematopoietic transition in mid-gestational embryos. Recently, studies combined with single-cell transcriptomics have provided massive new insights into the molecular evolutions and the underlying mechanisms of distinct waves of hematopoietic specification. In this review, we discuss the current single-cell profiling techniques, the most recent novel findings involved in the generation of distinct waves of blood cells, especially the HSCs, using single-cell transcriptional profiling combined with functional evaluations, and the perspectives to use the accumulating huge single-cell transcriptional data sets to study developmental hematopoiesis.

Moderate maternal folic acid supplementation ameliorates adverse embryonic and epigenetic outcomes associated with assisted reproduction in a mouse model.

Could clinically-relevant moderate and/or high dose maternal folic acid supplementation prevent aberrant developmental and epigenetic outcomes associated with assisted reproductive technologies (ART)? Our results demonstrate dose-dependent and sex-specific effects of folic acid supplementation in ART and provide evidence that moderate dose supplements may be optimal for both sexes. Children conceived using ART are at an increased risk for growth and genomic imprinting disorders, often associated with DNA methylation defects. Folic acid supplementation is recommended during pregnancy to prevent adverse offspring outcomes; however, the effects of folic acid supplementation in ART remain unclear. Outbred female mice were fed three folic acid-supplemented diets, control (rodent daily recommended intake or DRI; CD), moderate (4-fold DRI; 4FASD) or high (10-fold DRI; 10FASD) dose, for six weeks prior to ART and throughout gestation. Mouse ART involved a combination of superovulation, in vitro fertilisation, embryo culture and embryo transfer. Midgestation embryos and placentas (n = 74-99/group) were collected; embryos were assessed for developmental delay and gross morphological abnormalities and embryos and placentas were examined for epigenetic defects. We assessed methylation at four imprinted genes (Snrpn, Kcnq1ot1, Peg1 and H19) in matched midgestation embryos and placentas (n = 31-32/group) using bisulfite pyrosequencing. In addition, we examined genome-wide DNA methylation patterns in placentas (n = 6 normal placentas per sex/group) and embryos (n = 6 normal female embryos/group; n = 3 delayed female embryos/group) using reduced representation bisulfite sequencing (RRBS). Moderate, but not high dose supplementation, was associated with a decrease in the proportion of developmentally delayed embryos. Although moderate dose folic acid supplementation reduced DNA methylation variance at certain imprinted genes in embryonic and placental tissues, high dose supplementation exacerbated the negative effects of ART at imprinted loci. Furthermore, folic acid supplements resolved female-biased aberrant imprinted gene methylation. Supplementation was more effective at correcting ART-induced genome-wide methylation defects in male versus female placentas; however, folic acid supplementation also led to additional methylation perturbations which were more pronounced in males. The RRBS data from this study have been submitted to the NCBI Gene Expression Omnibus under the accession number GSE123143. Although the combination of mouse ART utilised in this study consisted of techniques commonly used in human fertility clinics, there may be species differences. Therefore, human studies, designed to determine the optimal levels of folic acid supplementation for ART pregnancies, and taking into account foetal sex, are warranted. Taken together, our findings support moderation in the dose of folic acid supplements taken during ART. This work was funded by the Canadian Institutes of Health Research (FDN-148425). The authors declare no conflict of interest.

Isolation of High-Molecular-Weight DNA from Mouse Yolk Sacs and the Like.

Often, genotyping of mouse embryos is required, and a small part, such as the yolk sac, can be used for this purpose. Here, DNA samples are prepared from extra-embryonic tissues by digestion with Proteinase K and subsequent extraction. The yolk sac of mid-gestation or later-stage embryos provides a sufficient amount of DNA for Southern analysis. Small tissues of a few hundred cells are used for genotyping early postimplantation- and preimplantation-stage embryos by polymerase chain reaction (PCR).

Rational Targeting of a NuRD Sub-Complex for Fetal Hemoglobin Induction Following Comprehensive in Situ Mutagenesis

Sickle cell disease and β-thalassemia are major hemoglobin disorders for which induction of fetal hemoglobin (HbF) can mitigate disease severity. However, the molecular mechanisms underlying the developmental repression of HbF remain incompletely understood. The nucleosome remodeling and deacetylase (NuRD) complex is a major negative regulator of HbF level. In this study, we sought to identify possible rational therapeutic strategies targeting critical NuRD determinants.We employed comprehensive dense mutagenesis using pooled CRISPR screening in HUDEP-2 human erythroid precursors to disrupt protein coding sequences of all 13 genes of the NuRD complex, including CHD, MTA, GATAD2, HDAC, MBD, and RBBP family members. The custom sgRNA library included 5,038 sgRNAs. We found that only 5 genes, CHD4, MTA2, GATAD2A, HDAC2, and MBD2, were required for HbF repression, suggesting that a non-redundant NuRD sub-complex contributes to HbF silencing. We validated the existence of this NuRD sub-complex by mass spectrometry analysis after immunoprecipitation of CHD4 and MTA2 as well as MTA2-BioID2 mediated proximity labeling. Remarkably, 5 of the 6 NuRD subunit proteins commonly detected by these three methods were identified as functional by CRISPR screening (MTA2, RBBP4, CHD4, GATAD2A, HDAC2).Disruption of CHD4 resulted in the highest HbF induction of any of the NuRD subunits. However, unlike the other NuRD genes, CHD4 disruption also led to cellular toxicity. We observed a small group of sgRNAs within the CHDCT2 domain of CHD4 associated with high HbF induction yet relatively modest negative fitness. We validated by electroporation of Cas9:sgRNA to CD34+ HSPC primary erythroid precursors that in-frame mutations of CHD4 CHDCT2 escape cellular toxicity while inducing HbF. Similarly, we targeted homologous amino acid residues within mouse Chd4 CHDCT2 domain by Cas9 mutagenesis in mouse oocytes. While loss of Chd4 is lethal at the blastocyst stage, homozygous in-frame deletions within the Chd4 CHDCT2 domain are tolerated in mouse embryos and result in increased γ-globin expression in mid-gestation embryos bearing transgenic human β-globin gene clusters.To investigate the mechanism whereby in-frame deletions at CHD4 CHDCT2 impact NuRD, we performed glycerol gradient density sedimentation, which revealed that these in-frame mutations impair the recruitment of CHD4 to the NuRD complex. A recent study demonstrated that the previously poorly characterized CHD4 CHDCT2 domain directly binds to GATAD2 factors (Torrado et al, FEBS J, 2017). We observed a cluster of sgRNAs associated with heightened HbF enrichment scores at the C-terminal region of GATAD2A encompassing a C2C2-type GATA zinc finger. We hypothesized that ectopic expression of this GATAD2A zinc finger might competitively bind to CHD4 and displace CHD4 from NuRD. Overexpression of the GATAD2A zinc finger in both HUDEP-2 and CD34+ HSPC derived primary erythroid precursors led to robust induction of HbF without negatively impacting cellular fitness. Immunoprecipitation of the GATAD2A zinc finger enriched CHD4 but not other endogenous NuRD components, such as GATAD2A or MBD2. Moreover, glycerol gradient density sedimentation showed that the GATAD2A zinc finger co-sedimented with sub-NuRD fractions of CHD4. Together these data suggest that expression of the GATAD2A zinc finger sequesters CHD4 from NuRD, yet spares cytotoxicity.In summary, we show that biochemical disruption of the CHD4-GATAD2A interaction could serve as a rational therapeutic strategy to potently induce HbF for the β-hemoglobin disorders while preventing cellular toxicity associated with complete CHD4 inhibition. DisclosuresNo relevant conflicts of interest to declare.

Spatial transcriptomic survey of human embryonic cerebral cortex by single-cell RNA-seq analysis

The cellular complexity of human brain development has been intensively investigated, although a regional characterization of the entire human cerebral cortex based on single-cell transcriptome analysis has not been reported. Here, we performed RNA-seq on over 4,000 individual cells from 22 brain regions of human mid-gestation embryos. We identified 29 cell sub-clusters, which showed different proportions in each region and the pons showed especially high percentage of astrocytes. Embryonic neurons were not as diverse as adult neurons, although they possessed important features of their destinies in adults. Neuron development was unsynchronized in the cerebral cortex, as dorsal regions appeared to be more mature than ventral regions at this stage. Region-specific genes were comprehensively identified in each neuronal sub-cluster, and a large proportion of these genes were neural disease related. Our results present a systematic landscape of the regionalized gene expression and neuron maturation of the human cerebral cortex.

Early Gestational Hypoxia and Adverse Developmental Outcomes.

Hypoxia is a normal and essential part of embryonic development. However, this state may leave the embryo vulnerable to damage when oxygen supply is disturbed. Embryofetal response to hypoxia is dependent on duration and depth of hypoxia, as well as developmental stage. Early postimplantation rat embryos were resilient to hypoxia, with many surviving up to 1.5 hr of uterine clamping, while most mid-gestation embryos were dead after 1 hour of clamping. Survivors were small and many had a range of defects, principally terminal transverse limb reduction defects. Similar patterns of malformations occurred when embryonic hypoxia was induced by maternal hypoxia, interruption of uteroplacental flow, or perfusion and embryonic bradycardia. There is good evidence that high altitude pregnancies are associated with smaller babies and increased risk of some malformations, but these results are complicated by increased risk of pre-eclampsia. Early onset pre-eclampsia itself is associated with small for dates and increased risk of atrio-ventricular septal defects. Limb defects have clearly been associated with chorionic villus sampling, cocaine, and misoprostol use. Similar defects are also observed with increased frequency among fetuses who are homozygous for thalassemia. Drugs that block the potassium current, whether as the prime site of action or as a side effect, are highly teratogenic in experimental animals. They induce embryonic bradycardia, hypoxia, hemorrhage, and blisters, leading to transverse limb defects as well as craniofacial and cardiovascular defects. While evidence linking these drugs to birth defects in humans is not compelling, the reason may methodological rather than biological. Birth Defects Research 109:1358-1376, 2017.© 2017 Wiley Periodicals, Inc.

Transcript analysis identifies differential uterine gene expression profile beyond the normal implantation window in mice

Uterine receptivity is defined as a state when the uterine milieu is favorable for blastocyst implantation and it can only last for a limited time period. In this study, by utilizing the embryo transfer model, it was observed that a portion of blastocysts could initiate implantation even when transferred beyond the timing of normal uterine receptivity, while their mid-gestational embryo development exhibited severe retardation, suggesting that the uterine status beyond the normal implantation window is unconducive for normal implantation. We further performed microarray analysis to explore the molecular basis that distinguishes the normal and defective uterine receptivity. A total of 229 genes was found to be differentially expressed, and a large amount of them were epithelium-expressing genes and responsive to progesterone signaling.

Oxidative Stress: A Potential Therapeutic Target in Placental Malaria.

Placental malaria, characterized by sequestration of Plasmodium falciparum in the maternal placental blood space and associated inflammatory damage, contributes to poor birth outcomes and ~200,000 infant deaths annually. Specific mechanisms that contribute to placental damage and dysfunction during malaria are not completely understood. To investigate a potential role for oxidative stress, antioxidant genes and markers for oxidative damage were assessed by quantitative PCR and immunohistochemistry in Plasmodium chabaudi AS-infected pregnant mice. Widespread evidence of lipid peroxidation was observed and was associated with higher antioxidant gene expression in conceptuses of infected mice. To assess the extent to which this oxidative damage might contribute to poor birth outcomes and be amenable to therapeutic intervention, infected pregnant mice were treated with N-acetylcysteine, a free radical scavenger, or tempol, an intracellular superoxide dismutase mimetic. The results show that mice treated with N-acetylcysteine experienced malaria induced-pregnancy loss at the same rate as control animals and failed to mitigate placental oxidative damage. In contrast, tempol-treated mice exhibited subtle improvement in embryo survival at gestation day 12. Although lipid peroxidation was not consistently reduced in the placentas of these mice, it was inversely related to embryo viability. Moreover, reduced IFN-γ and CCL2 plasma levels in treated mice were associated with midgestational embryo viability. Thus, although oxidative stress is remarkable in placental malaria and its mitigation by antioxidant therapy may improve pregnancy outcomes, the underlying mechanistic basis and potential therapeutic strategies require additional investigation.

Murine hemogenic endothelial precursors display heterogeneous hematopoietic potential exvivo.

Hematopoietic stem and progenitor cells (HSPCs) sustain life-long hematopoiesis and are first detected in the embryo by transplantation at embryonic day 10.5 (E10.5). HSPCs are mesodermal in origin and ultimately emerge from a subset of arterial endothelium (i.e., hemogenic endothelium [HE]), which is highly concentrated in the aorta-gonad-mesonephros region of the midgestation embryo. Here, we used clonal exvivo assays, in which endothelial cells isolated from the midgestation aorta and vitelline and umbilical arteries are co-cultured on supportive stroma, to show that only about 0.1%, 1.3%, and 0.29% of E9.5, E10.5, and E11.5 endothelium are functional HE, respectively. We further show high phenotypic and functional variability in the hematopoietic potential of individual hemogenic endothelial precursors. Using unique niche stroma capable of providing the signals necessary for definitive hematopoietic stem cell (dHSC) induction, we demonstrate that this variability in HE includes their potential to support phenotypic dHSCs. These data suggest the presence of a continuum of maturing HE with distinct hematopoietic potential or HE representative of a heterogeneous pool of precursors that give rise to HSPCs with disparate hematopoietic potential.