RESUMEN
In Brief: Advanced maternal age is associated with a higher rate of pregnancy complications that are unrelated to karyotypic abnormalities of the oocyte. This study shows that the murine uterine stroma undergoes profound epigenetic changes affecting active and repressive histone modification profiles that are associated with impaired endometrial functionality and underpin the decline in reproductive performance of aged females. Abstract: Decidualization describes the transformation of the uterine stroma in response to an implanting embryo, a process critical for supporting the development of the early embryo, for ensuring normal placentation and ultimately for a healthy reproductive outcome. Maternal age has been found to impede the progression of decidualization, heightening the risk of reproductive problems. Here, we set out to comprehensively characterize this deficit by pursuing transcriptomic and epigenomic profiling approaches specifically in the uterine stromal cell (UtSC) compartment of young and aged female mice. We find that UtSCs from aged females are globally far less responsive to the decidualization stimulus triggered by exposure to the steroid hormones estrogen and progesterone. Despite an overall transcriptional hyperactivation of genes that are differentially expressed as a function of maternal age, the hormonally regulated genes specifically fail to be activated in aged UtSCs. Moreover, even in their unstimulated 'ground' state, UtSCs from aged females are epigenetically distinct, as determined by genomic enrichment profiling for the active and repressive histone marks H3K4me3 and H3K9me3, respectively. We find that many hormone-inducible genes exhibit a profound lack of promoter-associated H3K4me3 in aged UtSCs, implying that a significant enrichment of active histone marks prior to gene stimulation is required to enable the elicitation of a rapid transcriptional response. With this combination of criteria, our data highlight specific deficits in epigenetic marking and gene expression of ion channels and vascular markers. These results point to fundamental defects in muscle-related and perivascular niche functions of the uterine stroma with advanced maternal age.
Asunto(s)
Envejecimiento , Decidua , Epigénesis Genética , Células del Estroma , Femenino , Animales , Ratones , Células del Estroma/metabolismo , Decidua/metabolismo , Decidua/patología , Código de Histonas , Histonas/metabolismo , Útero/metabolismo , Útero/patología , Embarazo , Reproducción , Ratones Endogámicos C57BL , Edad MaternaRESUMEN
The glycosylphosphatidylinositol (GPI) biosynthetic pathway in the endoplasmic reticulum (ER) is crucial for generating GPI-anchored proteins (GPI-APs), which are translocated to the cell surface and play a vital role in cell signaling and adhesion. This study focuses on two integral components of the GPI pathway, the PIGL and PIGF proteins, and their significance in trophoblast biology. We show that GPI pathway mutations impact on placental development impairing the differentiation of the syncytiotrophoblast (SynT), and especially the SynT-II layer, which is essential for the establishment of the definitive nutrient exchange area within the placental labyrinth. CRISPR/Cas9 knockout of Pigl and Pigf in mouse trophoblast stem cells (mTSCs) confirms the role of these GPI enzymes in syncytiotrophoblast differentiation. Mechanistically, impaired GPI-AP generation induces an excessive unfolded protein response (UPR) in the ER in mTSCs growing in stem cell conditions, akin to what is observed in human preeclampsia. Upon differentiation, the impairment of the GPI pathway hinders the induction of WNT signaling for early SynT-II development. Remarkably, the transcriptomic profile of Pigl- and Pigf-deficient cells separates human patient placental samples into preeclampsia and control groups, suggesting an involvement of Pigl and Pigf in establishing a preeclamptic gene signature. Our study unveils the pivotal role of GPI biosynthesis in early placentation and uncovers a new preeclampsia gene expression profile associated with mutations in the GPI biosynthesis pathway, providing novel molecular insights into placental development with implications for enhanced patient stratification and timely interventions.
Asunto(s)
Diferenciación Celular , Glicosilfosfatidilinositoles , Placentación , Trofoblastos , Trofoblastos/metabolismo , Trofoblastos/citología , Femenino , Embarazo , Animales , Humanos , Ratones , Placentación/genética , Glicosilfosfatidilinositoles/metabolismo , Glicosilfosfatidilinositoles/biosíntesis , Placenta/metabolismo , Placenta/citología , Vía de Señalización Wnt , Preeclampsia/metabolismo , Preeclampsia/genética , Preeclampsia/patología , Retículo Endoplásmico/metabolismo , Vías Biosintéticas/genética , Respuesta de Proteína Desplegada , Sistemas CRISPR-CasRESUMEN
The placenta is the organ that dictates the reproductive outcome of mammalian pregnancy by supplying nutrients and oxygen to the developing fetus to sustain its normal growth. During early mammalian development, trophoblast cells are the earliest cell type to differentiate with multipotent capacity to generate the trophoblast components of the placenta. The isolation and use of mouse trophoblast stem cells (mTSCs) to model in vitro trophoblast differentiation, in combination with CRISPR/Cas9 genome editing technology, has provided tremendous insight into the molecular mechanisms governing early mouse placentation. By knocking out a specific gene of interest in mTSCs, researchers are shedding light onto the molecular pathways involved in normal placental development and pregnancy disorders associated with abnormal placentation. In this chapter, we provide a detailed protocol for the genetic modification of mTSCs by using CRISPR/Cas9 genome editing system.
Asunto(s)
Sistemas CRISPR-Cas , Placenta , Embarazo , Femenino , Animales , Ratones , Ratones Noqueados , Trofoblastos , Diferenciación Celular/genética , Células Madre , MamíferosRESUMEN
Normal developmental progression relies on close interactions between the embryonic and extraembryonic lineages in the pre- and peri-gastrulation stage conceptus. For example, mouse epiblast-derived FGF and NODAL signals are required to maintain a stem-like state in trophoblast cells of the extraembryonic ectoderm, while visceral endoderm signals are pivotal to pattern the anterior region of the epiblast. These developmental stages also coincide with the specification of the first heart precursors. Here, we established a robust differentiation protocol of mouse embryonic stem cells (ESCs) into cardiomyocyte-containing embryoid bodies that we used to test the impact of trophoblast on this key developmental process. Using trophoblast stem cells (TSCs) to produce trophoblast-conditioned medium (TCM), we show that TCM profoundly slows down the cardiomyocyte differentiation dynamics and specifically delays the emergence of cardiac mesoderm progenitors. TCM also strongly promotes the retention of pluripotency transcription factors, thereby sustaining the stem cell state of ESCs. By applying TCM from various mutant TSCs, we further show that those mutations that cause a trophoblast-mediated effect on early heart development in vivo alter the normal cardiomyocyte differentiation trajectory. Our approaches provide a meaningful deconstruction of the intricate crosstalk between the embryonic and the extraembryonic compartments. They demonstrate that trophoblast helps prolong a pluripotent state in embryonic cells and delays early differentiative processes, likely through production of leukemia inhibitory factor (LIF). These data expand our knowledge of the multifaceted signaling interactions among distinct compartments of the early conceptus that ensure normal embryogenesis, insights that will be of significance for the field of synthetic embryo research.
RESUMEN
The placenta is a unique organ system that functionally combines both maternal and fetal cell types with distinct lineage origins. Normal placentation is critical for developmental progression and reproductive success. Although the placenta is best known for its nutrient supply function to the fetus, genetic experiments in mice highlight that the placenta is also pivotal for directing the proper formation of specific fetal organs. These roles underscore the importance of the placenta for pregnancy outcome and lifelong health span, which makes it essential to better understand the molecular processes governing placental development and function and to find adequate models to study it. In this review, we provide an overview of placental development and highlight the instructional role of the epigenome in dictating cell fate decisions specifically in the placental trophoblast cell lineage. We then focus on recent advances in exploring stem cell and organoid models reflecting the feto-maternal interface in mice and humans that provide much-improved tools to study events in early development. We discuss stem cells derived from the placenta as well as those artificially induced to resemble the placenta, and how they can be combined with embryonic stem cells and with endometrial cell types of the uterus to reconstitute the early implantation site. We then allude to the exciting prospects of how these models can be harnessed in biomedicine to enhance our understanding of the pathological underpinnings of pregnancy complications in a patient-specific manner, and ultimately to facilitate therapeutic approaches of tissue- and organ-based regenerative medicine.
Asunto(s)
Placenta , Trofoblastos , Embarazo , Femenino , Humanos , Animales , Ratones , Placenta/metabolismo , Trofoblastos/metabolismo , Trofoblastos/patología , Placentación , Diferenciación Celular , Epigénesis GenéticaRESUMEN
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.
Asunto(s)
Cardiopatías , Trofoblastos , Femenino , Embarazo , Animales , Ratones , Placenta , Corazón , Células Epiteliales , Ratones TransgénicosRESUMEN
DNA methylation is a repressive epigenetic modification that is essential for development, exemplified by the embryonic and perinatal lethality observed in mice lacking de novo DNA methyltransferases (DNMTs). Here we characterise the role for DNMT3A, 3B and 3L in gene regulation and development of the mouse placenta. We find that each DNMT establishes unique aspects of the placental methylome through targeting to distinct chromatin features. Loss of Dnmt3b results in de-repression of germline genes in trophoblast lineages and impaired formation of the maternal-foetal interface in the placental labyrinth. Using Sox2-Cre to delete Dnmt3b in the embryo, leaving expression intact in placental cells, the placental phenotype was rescued and, consequently, the embryonic lethality, as Dnmt3b null embryos could now survive to birth. We conclude that de novo DNA methylation by DNMT3B during embryogenesis is principally required to regulate placental development and function, which in turn is critical for embryo survival.
Asunto(s)
Metilación de ADN , ADN Metiltransferasa 3A , Embarazo , Femenino , Animales , Ratones , Placentación , Placenta/metabolismo , ADN (Citosina-5-)-Metiltransferasas/genética , Epigénesis GenéticaRESUMEN
Histone citrullination is a relatively poorly studied epigenetic modification that involves the irreversible conversion of arginine residues into citrulline. It is conferred by a small family of enzymes known as protein arginine deiminases (PADIs). PADI function supports the pluripotent state of embryonic stem cells, but in other contexts, also promotes efficient cellular differentiation. In the current study, we sought to gain deeper insights into the possible roles of PADIs in mouse trophoblast stem cells (TSCs). We show that Padi2 and Padi3 are the most highly expressed PADI family members in TSCs and are rapidly down-regulated upon differentiation. Padi2/3 double knockout (DKO) TSCs express lower levels of stem cell transcription factors CDX2 and SOX2 and are prone to differentiate into extremely large trophoblast giant cells, an effect that may be mediated by centrosome duplication defects. Interestingly, Padi2/3 DKO TSCs display alterations to their epigenomic landscape, with fewer H3K9me3-marked chromocentric foci and globally reduced 5-methylcytosine levels. DNA methylation profiling identifies that this effect is specifically evident at CpG islands of critical trophoblast genes, such as Gata3, Peg3, Socs3 and Hand1. As a consequence of the hypomethylated state, these factors are up-regulated in Padi2/3 DKO TSCs, driving their premature differentiation. Our data uncover a critical epigenetic role for PADI2/3 in safeguarding the stem cell state of TSCs by modulating the DNA methylation landscape to restrict precocious trophoblast differentiation.
Asunto(s)
Epigenómica , Células Madre Embrionarias de Ratones , Arginina Deiminasa Proteína-Tipo 2/metabolismo , Arginina Deiminasa Proteína-Tipo 3/metabolismo , Trofoblastos , Animales , Diferenciación Celular/genética , Ratones , Desiminasas de la Arginina Proteica/genética , Desiminasas de la Arginina Proteica/metabolismo , Trofoblastos/metabolismoRESUMEN
Complex morphological traits are the product of many genes with transient or lasting developmental effects that interact in anatomical context. Mouse models are a key resource for disentangling such effects, because they offer myriad tools for manipulating the genome in a controlled environment. Unfortunately, phenotypic data are often obtained using laboratory-specific protocols, resulting in self-contained datasets that are difficult to relate to one another for larger scale analyses. To enable meta-analyses of morphological variation, particularly in the craniofacial complex and brain, we created MusMorph, a database of standardized mouse morphology data spanning numerous genotypes and developmental stages, including E10.5, E11.5, E14.5, E15.5, E18.5, and adulthood. To standardize data collection, we implemented an atlas-based phenotyping pipeline that combines techniques from image registration, deep learning, and morphometrics. Alongside stage-specific atlases, we provide aligned micro-computed tomography images, dense anatomical landmarks, and segmentations (if available) for each specimen (N = 10,056). Our workflow is open-source to encourage transparency and reproducible data collection. The MusMorph data and scripts are available on FaceBase ( www.facebase.org , https://doi.org/10.25550/3-HXMC ) and GitHub ( https://github.com/jaydevine/MusMorph ).
Asunto(s)
Bases de Datos Factuales , Ratones , Animales , Encéfalo , Ratones/anatomía & histología , Microtomografía por Rayos XRESUMEN
A distinct age-related alteration in the uterine environment has recently been identified as a prevalent cause of the reproductive decline in older female mice. However, the molecular mechanisms that underlie age-associated uterine adaptability to pregnancy are not known. Sirtuin 1 (SIRT1), a multifunctional NAD+-dependent deacetylase that regulates cell viability, senescence and inflammation during aging, is reduced in aged decidua. Thus, we hypothesize that SIRT1 plays a critical role in uterine adaptability to pregnancy and that uterine-specific ablation of Sirt1 gene accelerates premature uterine aging. Female mice with uterine ablation of Sirt1 gene using progesterone receptor Cre (PgrCre) exhibit subfertility and signs of premature uterine aging. These Sirt1-deficient mothers showed decreases in litter size from their 1st pregnancy and became sterile (25.1 ± 2.5 weeks of age) after giving birth to the third litter. We report that uterine-specific Sirt1 deficiency impairs invasion and spacing of blastocysts, and stromal cell decidualization, leading to abnormal placentation. We found that these problems traced back to the very early stages of hormonal priming of the uterus. During the window of receptivity, Sirt1 deficiency compromises uterine epithelial-stromal crosstalk, whereby estrogen, progesterone and Indian hedgehog signaling pathways are dysregulated, hampering stromal cell priming for decidualization. Uterine transcriptomic analyses also link these causes to perturbations of histone proteins and epigenetic modifiers, as well as adrenomedullin signaling, hyaluronic acid metabolism, and cell senescence. Strikingly, our results also identified genes with significant overlaps with the transcriptome of uteri from aged mice and transcriptomes related to master regulators of decidualization (e.g. Foxo1, Wnt4, Sox17, Bmp2, Egfr and Nr2f2). Our results also implicate accelerated deposition of aging-related fibrillar Type I and III collagens in Sirt1-deficient uteri. Collectively, SIRT1 is an important age-related regulator of invasion and spacing of blastocysts, as well as decidualization of stromal cells.
Asunto(s)
Decidua , Sirtuina 1 , Envejecimiento , Animales , Blastocisto , Decidua/metabolismo , Implantación del Embrión/fisiología , Femenino , Proteínas Hedgehog/metabolismo , Ratones , Embarazo , Sirtuina 1/genética , Sirtuina 1/metabolismo , Células del Estroma/metabolismo , Útero/metabolismoRESUMEN
Advanced maternal age (AMA) poses the single greatest risk to a successful pregnancy. Apart from the impact of AMA on oocyte fitness, aged female mice often display defects in normal placentation. Placental defects in turn are tightly correlated with brain and cardiovascular abnormalities. It therefore follows that placenta, brain and heart development may be particularly susceptible to the impact of AMA. In the current study, we compared global transcriptomes of placentas, brains, hearts, and facial prominences from mid-gestation mouse conceptuses developed in young control (7-13 wks) and aging (43-50 wks) females. We find that AMA increases transcriptional heterogeneity in all tissues, but particularly in fetal brain. Importantly, even overtly normally developed embryos from older females display dramatic expression changes in neurodevelopmental genes. These transcriptomic alterations in the brain are likely induced by defects in placental development. Using trophoblast stem cells (TSCs) as a model, we show that exposure to aging uterine stromal cell-conditioned medium interferes with normal TSC proliferation and causes precocious differentiation, recapitulating many of the defects observed in placentas from aged females. These data highlight the increased risk of AMA on reproductive outcome, with neurodevelopment being the most sensitive to such early perturbations and with potential for lifelong impact.
Asunto(s)
Placenta , Placentación , Embarazo , Femenino , Animales , Ratones , Placenta/metabolismo , Edad Materna , Trofoblastos/metabolismo , EncéfaloRESUMEN
KEY POINTS: Endoplasmic reticulum (ER) stress promotes placental dysmorphogenesis and is associated with poor pregnancy outcomes. We show that unfolded protein response signalling pathways located in the ER drive differentiation of mouse trophoblast stem cells into trophoblast subtypes involved in development of the placental labyrinth zone and trophoblast invasion. In a mouse model of chronic ER stress (Eif2s1tm1RjK ), higher ER stress in homozygous blastocysts is accompanied by reduced trophectoderm cell number and developmental delay and also is associated with an increased incidence of early pregnancy loss. Administration of the chemical chaperone, tauroursodeoxycholic acid, to Eif2s1+/tm1RjK heterozygous females during pregnancy alleviated ER stress in the mutant placenta, restored normal trophoblast populations and reduced the frequency of early pregnancy loss. Our results suggest that alleviation of intrauterine ER stress could provide a potential therapeutic target to improve pregnancy outcome in women with pre-gestational metabolic or gynaecological conditions. ABSTRACT: Women with pre-gestational health conditions (e.g. obesity, diabetes) or gynaecological problems (e.g. endometriosis) are at increased risk of adverse pregnancy outcomes including miscarriage, pre-eclampsia and fetal growth restriction. Increasing evidence suggests that unfavourable intrauterine conditions leading to poor implantation and/or defective placentation are a possible causative factor. The endoplasmic reticulum (ER) unfolded protein response (UPRER ) signalling pathways are a convergence point of various physiological stress stimuli that can be triggered by an unfavourable intrauterine environment. Therefore, we explored the impact of ER stress on mouse trophoblast differentiation in vitro, mouse blastocyst formation and early placenta development in the Eif2s1tm1RjK mutant mouse model of chronic ER stress. Chemically-manipulated ER stress or activation of UPRER pathways in a mouse trophoblast stem cell line promoted lineage-specific differentiation. Co-treatment with specific UPRER pathway inhibitors rescued this effect. Although the inner cell mass was unaffected, the trophectoderm of homozygous Eif2s1tm1RjK blastocysts exhibited ER stress associated with a reduced cell number. Furthermore, one-third of Eif2s1tm1RjK homozygous blastocysts exhibited severe developmental defects. We have previously reported a reduced trophoblast population and premature trophoblast differentiation in Eif2s1tm1RjK homozygous placentas at mid-gestation. Here, we demonstrate that treatment of Eif2s1+/tm1RjK heterozygous pregnant females with the chemical chaperone tauroursodeoxycholic acid alleviated ER stress, restored the trophoblast population and reduced the frequency of embryonic lethality. Our data suggest that therapeutic targeting of ER stress may improve pregnancy outcome in women with pre-gestational metabolic or gynaecological conditions.
Asunto(s)
Aborto Espontáneo , Placentación , Animales , Diferenciación Celular , Estrés del Retículo Endoplásmico , Femenino , Humanos , Ratones , Placenta , Embarazo , TrofoblastosRESUMEN
Normal function of the placenta depends on the earliest developmental stages when trophoblast cells differentiate and invade into the endometrium to establish the definitive maternal-fetal interface. Previously, we identified the ubiquitously expressed tumour suppressor BRCA1-associated protein 1 (BAP1) as a central factor of a novel molecular node controlling early mouse placentation. However, functional insights into how BAP1 regulates trophoblast biology are still missing. Using CRISPR/Cas9 knockout and overexpression technology in mouse trophoblast stem cells, here we demonstrate that the downregulation of BAP1 protein is essential to trigger epithelial-mesenchymal transition (EMT) during trophoblast differentiation associated with a gain of invasiveness. Moreover, we show that the function of BAP1 in suppressing EMT progression is dependent on the binding of BAP1 to additional sex comb-like (ASXL1/2) proteins to form the polycomb repressive deubiquitinase (PR-DUB) complex. Finally, both endogenous expression patterns and BAP1 overexpression experiments in human trophoblast stem cells suggest that the molecular function of BAP1 in regulating trophoblast differentiation and EMT progression is conserved in mice and humans. Our results reveal that the physiological modulation of BAP1 determines the invasive properties of the trophoblast, delineating a new role of the BAP1 PR-DUB complex in regulating early placentation.
Asunto(s)
Transición Epitelial-Mesenquimal/genética , Proteínas Represoras/genética , Proteínas Supresoras de Tumor/genética , Ubiquitina Tiolesterasa/genética , Animales , Diferenciación Celular/genética , Regulación de la Expresión Génica , Humanos , Ratones , Proteínas Represoras/metabolismo , Trofoblastos/fisiología , Proteínas Supresoras de Tumor/metabolismo , Ubiquitina Tiolesterasa/metabolismoRESUMEN
Advancing maternal age causes a progressive reduction in fertility. The decline in developmental competence of the oocyte with age is likely to be a consequence of multiple contributory factors. Loss of epigenetic quality of the oocyte could impair early developmental events or programme adverse outcomes in offspring that manifest only later in life. Here, we undertake joint profiling of the transcriptome and DNA methylome of individual oocytes from reproductively young and old mice undergoing natural ovulation. We find reduced complexity as well as increased variance in the transcriptome of oocytes from aged females. This transcriptome heterogeneity is reflected in the identification of discrete sub-populations. Oocytes with a transcriptome characteristic of immature chromatin configuration (NSN) clustered into two groups: one with reduced developmental competence, as indicated by lower expression of maternal effect genes, and one with a young-like transcriptome. Oocytes from older females had on average reduced CpG methylation, but the characteristic bimodal methylation landscape of the oocyte was preserved. Germline differentially methylated regions of imprinted genes were appropriately methylated irrespective of age. For the majority of differentially expressed transcripts, the absence of correlated methylation changes suggests a post-transcriptional basis for most age-related effects on the transcriptome. However, we did find differences in gene body methylation at which there were corresponding changes in gene expression, indicating age-related effects on transcription that translate into methylation differences. Interestingly, oocytes varied in expression and methylation of these genes, which could contribute to variable competence of oocytes or penetrance of maternal age-related phenotypes in offspring.
Asunto(s)
Envejecimiento/genética , Envejecimiento/metabolismo , Metilación de ADN , Oocitos/metabolismo , Transcriptoma , Envejecimiento/patología , Animales , Senescencia Celular/genética , Senescencia Celular/fisiología , Cromatina/genética , Cromatina/metabolismo , Epigénesis Genética , Femenino , Edad Materna , Ratones , Ratones Endogámicos C57BL , Oocitos/crecimiento & desarrollo , Oocitos/patología , RNA-Seq , Análisis de la Célula IndividualRESUMEN
Mutations in the SNX14 gene cause spinocerebellar ataxia, autosomal recessive 20 (SCAR20) in both humans and dogs. Studies implicating the phenotypic consequences of SNX14 mutations to be consequences of subcellular disruption to autophagy and lipid metabolism have been limited to in vitro investigation of patient-derived dermal fibroblasts, laboratory engineered cell lines and developmental analysis of zebrafish morphants. SNX14 homologues Snz (Drosophila) and Mdm1 (yeast) have also been conducted, demonstrated an important biochemical role during lipid biogenesis. In this study we report the effect of loss of SNX14 in mice, which resulted in embryonic lethality around mid-gestation due to placental pathology that involves severe disruption to syncytiotrophoblast cell differentiation. In contrast to other vertebrates, zebrafish carrying a homozygous, maternal zygotic snx14 genetic loss-of-function mutation were both viable and anatomically normal. Whilst no obvious behavioural effects were observed, elevated levels of neutral lipids and phospholipids resemble previously reported effects on lipid homeostasis in other species. The biochemical role of SNX14 therefore appears largely conserved through evolution while the consequences of loss of function varies between species. Mouse and zebrafish models therefore provide valuable insights into the functional importance of SNX14 with distinct opportunities for investigating its cellular and metabolic function in vivo.
Asunto(s)
Viabilidad Fetal/genética , Metabolismo de los Lípidos/genética , Placenta/anomalías , Nexinas de Clasificación/genética , Ataxias Espinocerebelosas/genética , Animales , Animales Modificados Genéticamente , Diferenciación Celular/genética , Desarrollo Embrionario/genética , Femenino , Humanos , Ratones , Ratones Endogámicos C57BL , Modelos Animales , Fenotipo , Fosfolípidos/sangre , Embarazo , Trofoblastos/citología , Pez CebraRESUMEN
BCOR is a critical regulator of human development. Heterozygous mutations of BCOR in females cause the X-linked developmental disorder Oculofaciocardiodental syndrome (OFCD), and hemizygous mutations of BCOR in males cause gestational lethality. BCOR associates with Polycomb group proteins to form one subfamily of the diverse Polycomb repressive complex 1 (PRC1) complexes, designated PRC1.1. Currently there is limited understanding of differing developmental roles of the various PRC1 complexes. We therefore generated a conditional exon 9-10 knockout Bcor allele and a transgenic conditional Bcor expression allele and used these to define multiple roles of Bcor, and by implication PRC1.1, in mouse development. Females heterozygous for Bcor exhibiting mosaic expression due to the X-linkage of the gene showed reduced postnatal viability and had OFCD-like defects. By contrast, Bcor hemizygosity in the entire male embryo resulted in embryonic lethality by E9.5. We further dissected the roles of Bcor, focusing on some of the tissues affected in OFCD through use of cell type specific Cre alleles. Mutation of Bcor in neural crest cells caused cleft palate, shortening of the mandible and tympanic bone, ectopic salivary glands and abnormal tongue musculature. We found that defects in the mandibular region, rather than in the palate itself, led to palatal clefting. Mutation of Bcor in hindlimb progenitor cells of the lateral mesoderm resulted in 2/3 syndactyly. Mutation of Bcor in Isl1-expressing lineages that contribute to the heart caused defects including persistent truncus arteriosus, ventricular septal defect and fetal lethality. Mutation of Bcor in extraembryonic lineages resulted in placental defects and midgestation lethality. Ubiquitous over expression of transgenic Bcor isoform A during development resulted in embryonic defects and midgestation lethality. The defects we have found in Bcor mutants provide insights into the etiology of the OFCD syndrome and how BCOR-containing PRC1 complexes function in development.
Asunto(s)
Catarata/congénito , Embrión de Mamíferos , Defectos de los Tabiques Cardíacos , Microftalmía , Complejo Represivo Polycomb 1 , Proteínas Represoras , Animales , Catarata/embriología , Catarata/genética , Catarata/patología , Embrión de Mamíferos/embriología , Embrión de Mamíferos/patología , Defectos de los Tabiques Cardíacos/embriología , Defectos de los Tabiques Cardíacos/genética , Defectos de los Tabiques Cardíacos/patología , Ratones , Microftalmía/embriología , Microftalmía/genética , Microftalmía/patología , Complejo Represivo Polycomb 1/genética , Complejo Represivo Polycomb 1/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismoRESUMEN
The ten-eleven translocation factor TET1 and its conferred epigenetic modification 5-hydroxymethylcytosine (5hmC) have important roles in maintaining the pluripotent state of embryonic stem cells (ESCs). We previously showed that TET1 is also essential to maintain the stem cell state of trophoblast stem cells (TSCs). Here, we establish an integrated panel of absolute 5hmC levels, genome-wide DNA methylation and hydroxymethylation patterns, transcriptomes, and TET1 chromatin occupancy in TSCs and differentiated trophoblast cells. We show that the combined presence of 5-methylcytosine (5mC) and 5hmC correlates with transcriptional activity of associated genes. Hypoxia can slow down the global loss of 5hmC that occurs upon differentiation of TSCs. Notably, unlike in ESCs and epiblast cells, most TET1-bound regions overlap with active chromatin marks and TFAP2C binding sites and demarcate putative trophoblast enhancer regions. These chromatin modification and occupancy patterns are highly informative to identify novel candidate regulators of the TSC state.
Asunto(s)
Metilación de ADN , Proteínas de Unión al ADN/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Células Madre/metabolismo , Trofoblastos/metabolismo , Animales , Hipoxia de la Célula , Proteínas de Unión al ADN/genética , Elementos de Facilitación Genéticos , Ratones , Proteínas Proto-Oncogénicas/genética , Células Madre/citología , Factor de Transcripción AP-2/genética , Factor de Transcripción AP-2/metabolismo , Trofoblastos/citologíaRESUMEN
Reproductive decline in older female mice can be attributed to a failure of the uterus to decidualise in response to steroid hormones. Here, we show that normal decidualisation is associated with significant epigenetic changes. Notably, we identify a cohort of differentially methylated regions (DMRs), most of which gain DNA methylation between the early and late stages of decidualisation. These DMRs are enriched at progesterone-responsive gene loci that are essential for reproductive function. In female mice nearing the end of their reproductive lifespan, DNA methylation fidelity is lost at a number of CpG islands (CGIs) resulting in CGI hypermethylation at key decidualisation genes. Importantly, this hypermethylated state correlates with the failure of the corresponding genes to become transcriptionally upregulated during the implantation window. Thus, age-associated DNA methylation changes may underlie the decidualisation defects that are a common occurrence in older females. Alterations to the epigenome of uterine cells may therefore contribute significantly to the reproductive decline associated with advanced maternal age.
Asunto(s)
Envejecimiento/genética , Implantación del Embrión/genética , Epigénesis Genética/fisiología , Reproducción/fisiología , Animales , Células Cultivadas , Islas de CpG/genética , Metilación de ADN/fisiología , Decidua/fisiología , Embrión de Mamíferos , Femenino , Masculino , Edad Materna , Ratones , Ratones Endogámicos C57BL , Embarazo , Reproducción/genéticaRESUMEN
The original version of this article unfortunately contained a mistake. In reviewing the phenotype associated with Mapk14 (p38alpha MAPK) mutation as evaluated by Adams et al. (2000) using tetraploid aggregation chimeric embryos, the authors mistakenly stated that rescue of embryo lethality was short-lived and that embryos died two days later of non-placenta-related causes. In fact, as reported by Adams et al. (2000), when the placental defect of global null embryos was rescued, p38alpha(-/-) embryos developed to term and were normal in appearance. The authors apologize for the error.