Development Of Extraembryonic Tissues Research Articles

Disruption of maternal vascular remodeling by a fetal endoretrovirus-derived gene in preeclampsia

BackgroundPreeclampsia, one of the most lethal pregnancy-related diseases, is associated with the disruption of uterine spiral artery remodeling during placentation. However, the early molecular events leading to preeclampsia remain unknown.ResultsBy analyzing placentas from preeclampsia, non-preeclampsia, and twin pregnancies with selective intrauterine growth restriction, we show that the pathogenesis of preeclampsia is attributed to immature trophoblast and maldeveloped endothelial cells. Delayed epigenetic reprogramming during early extraembryonic tissue development leads to generation of excessive immature trophoblast cells. We find reduction of de novo DNA methylation in these trophoblast cells results in selective overexpression of maternally imprinted genes, including the endoretrovirus-derived gene PEG10 (paternally expressed gene 10). PEG10 forms virus-like particles, which are transferred from the trophoblast to the closely proximate endothelial cells. In normal pregnancy, only a low amount of PEG10 is transferred to maternal cells; however, in preeclampsia, excessive PEG10 disrupts maternal vascular development by inhibiting TGF-beta signaling.ConclusionsOur study reveals the intricate epigenetic mechanisms that regulate trans-generational genetic conflict and ultimately ensure proper maternal–fetal interface formation.

Extraembryonic tissues: exploring concepts, definitions and functions across the animal kingdom.

Extraembryonic tissues: exploring concepts, definitions and functions across the animal kingdom.

Mouse embryo model derived exclusively from embryonic stem cells undergoes neurulation and heart development.

Several invitro models have been developed to recapitulate mouse embryogenesis solely from embryonic stem cells (ESCs). Despite mimicking many aspects of early development, they fail to capture the interactions between embryonic and extraembryonic tissues. To overcome this difficulty, we have developed a mouse ESC-based invitro model that reconstitutes the pluripotent ESC lineage and the two extraembryonic lineages of the post-implantation embryo by transcription-factor-mediated induction. This unified model recapitulates developmental events from embryonic day 5.5 to 8.5, including gastrulation; formation of the anterior-posterior axis, brain, and a beating heart structure; and the development of extraembryonic tissues, including yolk sac and chorion. Comparing single-cell RNA sequencing from individual structures with time-matched natural embryos identified remarkably similar transcriptional programs across lineages but also showed when and where the model diverges from the natural program. Our findings demonstrate an extraordinary plasticity of ESCs to self-organize and generate a whole-embryo-like structure.

Tracing the emergence of primordial germ cells from bilaminar disc rabbit embryos and pluripotent stem cells.

Rabbit embryos develop as bilaminar discs at gastrulation as in humans and most other mammals, whereas rodents develop as egg cylinders. Primordial germ cells (PGCs) appear to originate during gastrulation according to many systematic studies on mammalian embryos. Here, we show that rabbit PGC (rbPGC) specification occurs at the posterior epiblast at the onset of gastrulation. Using newly derived rabbit pluripotent stem cells, we show robust and rapid induction of rbPGC-like cells invitro with WNT and BMP morphogens, which reveals SOX17 as the critical regulator of rbPGC fate as in several non-rodent mammals. We posit that development as a bilaminar disc is a crucial determinant of the PGC regulators, regardless of the highly diverse development of extraembryonic tissues, including the amnion. We propose that investigations on rabbits with short gestation, large litters, and where gastrulation precedes implantation can contribute significantly to advances in early mammalian development.

Molecular events in the Wharton’s jelly and blood vessels of human umbilical cord

The umbilical cord is seen as the main junction between the developing embryo or fetus and placenta. We studied the antimicrobial response, the presence of undifferentiated cells and TGF-α, as well as tissue degeneration and compensatory remodelling processes in the cells of human umbilical cord.Seven umbilical cord tissue samples obtained during premature and full term births were stained with hematoxylin and eosin and by immunohistochemistry for human beta defensin 2 (hBD-2), hematopoietic progenitor cell antigen CD34, matrix metalloproteinase 2 (MMP-2), the tissue inhibitor of matrix metalloproteinase 2 (TIMP-2), nestin and transforming growth factor alpha (TGF-α). The intensity of staining was graded semiquantitatively.Antimicrobial response was more prominent in the Wharton’s jelly – we found numerous hBD-2-containing cells, while hBD-2 positive cells in the walls of arteries and vein varied from moderate to abundance. Numerous cells in the Wharton’s jelly contained CD34, while in the walls of blood vessels few to moderate stained positive for CD34. MMP-2, TIMP-2 and nestin positive cells were found in all the tissue samples and varied from numerous in the walls of blood vessels to abundance in the Wharton’s jelly and the inflammation region. The abundance of TGF-α-containing cells was found in Wharton’s jelly and moderate to numerous cells of blood vessels contained TGF-α.Conclusions: The human umbilical cord possesses antimicrobial activity and shows the presence of undifferentiated cells. The striking distribution and the expression of MMP-2, TIMP-2 and nestin suggest their role in the very effective and extensive tissue degeneration and compensatory remodelling processes. TGF-α seems to be an important growth factor for extra-embryonic tissue development.

Loss of function of mouse Pax‐Interacting Protein 1‐associated glutamate rich protein 1a (Pagr1a) leads to reduced Bmp2 expression and defects in chorion and amnion development

Human PAX-Interacting Protein 1 (PAXIP1)-associated glutamate rich protein 1 (PAGR1, also known as PA1) originally was discovered as part of a complex containing PAXIP1 and histone H3K4 methyltransferases MLL3 and MLL4, suggesting a role in epigenetic gene regulation. Further in vitro studies suggested additional functions in DNA damage repair and transcription. However, in vivo analysis of PAGR1 function has been lacking. Here we show that expression of the cognate mouse gene Pagr1a is found predominately in the extraembryonic and chorionic ectoderm from pregastrulation stages and is up-regulated within the embryo proper after gastrulation. Embryos with a germ line deletion of Pagr1a establish the anterior-posterior axis, and show normal neuroectodermal, mesodermal, and endodermal patterning, but fail to develop beyond the four- to five-somite stage or to undergo axial rotation. Pagr1a(-) (/) (-) embryos also show abnormal development of extraembryonic tissues with defects seen in the amnion, chorion and visceral yolk sac. At the molecular level, Pagr1a(-) (/) (-) embryos have reduced expression of BMP2, a known regulator of extraembryonic development. Loss of mouse Pagr1a function leads to defective extraembryonic development, likely due at least in part to altered BMP signaling, contributing to developmental arrest.

A Staging Scheme for the Development of the Moth Midge Clogmia albipunctata

Model organisms, such as Drosophila melanogaster, allow us to address a wide range of biological questions with experimental rigour. However, studies in model species need to be complemented by comparative studies if we are to fully understand the functional properties and evolutionary history of developmental processes. The establishment of new model organisms is crucial for this purpose. One of the first essential steps to establish a species as an experimental model is to carefully describe its life cycle and development. The resulting staging scheme serves as a framework for molecular studies, and allows us to homologise developmental processes between species. In this paper, we have characterised the life cycle and development of an emerging non-drosophilid dipteran model system: the moth midge Clogmia albipunctata. In particular, we focus on early embryogenesis (cleavage and blastoderm cycles before gastrulation), on formation and retraction of extraembryonic tissues, and on formation of the germ line. Considering the large evolutionary distance between the two species (approximately 250 million years), we find that the development of C. albipunctata is remarkably conserved compared to D. melanogaster. On the other hand, we detect significant differences in morphology and timing affecting the development of extraembryonic tissues and the germ line. Moreover, C. albipunctata shows several heterochronic shifts, and lacks head involution and associated processes during late stages of development.

Contribution of Maternal NODAL to Term Pregnancy

Contribution of Maternal NODAL to Term Pregnancy

Essential role of the CUL4B ubiquitin ligase in extra-embryonic tissue development during mouse embryogenesis

Mutations of the CUL4B ubiquitin ligase gene are causally linked to syndromic X-linked mental retardation (XLMR). However, the pathogenic role of CUL4B mutations in neuronal and developmental defects is not understood. We have generated mice with targeted disruption of Cul4b, and observed embryonic lethality with pronounced growth inhibition and increased apoptosis in extra-embryonic tissues. Cul4b, but not its paralog Cul4a, is expressed at high levels in extra-embryonic tissues post implantation. Silencing of CUL4B expression in an extra-embryonic cell line resulted in the robust accumulation of the CUL4 substrate p21(Cip1/WAF) and G2/M cell cycle arrest, which could be partially rescued by silencing of p21(Cip1/WAF). Epiblast-specific deletion of Cul4b prevented embryonic lethality and gave rise to viable Cul4b null mice. Therefore, while dispensable in the embryo proper, Cul4b performs an essential developmental role in the extra-embryonic tissues. Our study offers a strategy to generate viable Cul4b-deficient mice to model the potential neuronal and behavioral deficiencies of human CUL4B XLMR patients.

Effects of simvastatin on miRNA profile of multipotent mesenchymal stromal cells from human amniotic membrane

Effects of simvastatin on miRNA profile of multipotent mesenchymal stromal cells from human amniotic membrane

The mouse KRAB zinc-finger protein CHATO is required in embryonic-derived tissues to control yolk sac and placenta morphogenesis

Yolk sac and placenta are required to sustain embryonic development in mammals, yet our understanding of the genes and processes that control morphogenesis of these extraembryonic tissues is still limited. The chato mutation disrupts ZFP568, a Krüppel-Associated-Box (KRAB) domain Zinc finger protein, and causes a unique set of extraembryonic malformations, including ruffling of the yolk sac membrane, defective extraembryonic mesoderm morphogenesis and vasculogenesis, failure to close the ectoplacental cavity, and incomplete placental development. Phenotypic analysis of chato embryos indicated that ZFP568 does not control proliferation or differentiation of extraembryonic lineages but rather regulates the morphogenetic events that shape extraembryonic tissues. Analysis of chimeric embryos showed that Zfp568 function is required in embryonic-derived lineages, including the extraembryonic mesoderm. Depleting Zfp568 affects the ability of extraembryonic mesoderm cells to migrate. However, explanted Zfp568 mutant cells could migrate properly when plated on appropriate extracellular matrix conditions. We show that expression of Fibronectin and Indian Hedgehog are reduced in chato mutant yolk sacs. These data suggest that ZFP568 controls the production of secreted factors required to promote morphogenesis of extraembryonic tissues. Our results support previously undescribed roles of the extraembryonic mesoderm in yolk sac morphogenesis and in the closure of the ectoplacental cavity and identify a novel role of ZFP568 in the development of extraembryonic tissues.

Essential role for the Pak4 protein kinase in extraembryonic tissue development and vessel formation

Pak4 is a member of the group B family of Pak serine/threonine kinases, originally identified as an effector protein for the Rho GTPase Cdc42. Pak4 knockout mice are embryonic lethal and do not survive past embryonic day 11.5. Previous work on Pak4 knockout mice has focused on studying the phenotype of the embryo. Abnormalities in the extraembryonic tissue, however, are common causes of early embryonic death in knockout mice. Extraembryonic tissue associated with the Pak4-null embryos was therefore examined. Abnormalities in both yolk sacs and placentas resulted when Pak4 was deleted. These included a lack of vasculature throughout the extraembryonic tissue, as well as an abnormally formed labyrinthine layer of the placenta. Interestingly, epiblast-specific deletion of Pak4 using a conditional knockout system, did not rescue the embryonic lethality. In fact, it did not even rescue the extraembryonic tissue defects. Our results suggest that the extraembryonic tissue abnormalities are secondary to defects that occur in response to epiblast abnormalities. More detailed analysis suggests that abnormalities in vasculature throughout the extraembryonic tissue and the epiblast may contribute to the death of the Pak4-null embryos.

E2F4 cooperates with pRB in the development of extra-embryonic tissues

The retinoblastoma gene, RB-1, was the first identified tumor suppressor. Rb −/− mice die in mid-gestation with defects in proliferation, differentiation and apoptosis. The activating E2F transcription factors, E2F1–3, contribute to these embryonic defects, indicating that they are key downstream targets of the retinoblastoma protein, pRB. E2F4 is the major pRB-associated E2F in vivo, yet its role in Rb −/− embryos is unknown. Here we establish that E2f4 deficiency reduced the lifespan of Rb −/− embryos by exacerbating the Rb mutant placental defect. We further show that this reflects the accumulation of trophectoderm-like cells in both Rb and Rb;E2f4 mutant placentas. Thus, Rb and E2f4 play cooperative roles in placental development. We used a conditional mouse model to allow Rb −/−;E2f4 −/− embryos to develop in the presence of Rb wild-type placentas. Under these conditions, Rb −/−;E2f4 −/− mutants survived to birth. These Rb −/−;E2f4 −/− embryos exhibited all of the defects characteristic of the Rb and E2f4 single mutants and had no novel defects. Taken together, our data show that pRB and E2F4 cooperate in placental development, but play largely non-overlapping roles in the development of many embryonic tissues.

Genomic evolution of the placenta using co-option and duplication and divergence

The invention of the placenta facilitated the evolution of mammals. How the placenta evolved from the simple structure observed in birds and reptiles into the complex organ that sustains human life is one of the great mysteries of evolution. By using a timecourse microarray analysis including the entire lifetime of the placenta, we uncover molecular and genomic changes that underlie placentation and find that two distinct evolutionary mechanisms were utilized during placental evolution in mice and human. Ancient genes involved in growth and metabolism were co-opted for use during early embryogenesis, likely enabling the accelerated development of extraembryonic tissues. Recently duplicated genes are utilized at later stages of placentation to meet the metabolic needs of a diverse range of pregnancy physiologies. Together, these mechanisms served to develop the specialized placenta, a novel structure that led to expansion of the eutherian mammal, including humankind.

Loss of Parp-1 affects gene expression profile in a genome-wide manner in ES cells and liver cells.

BackgroundMany lines of evidence suggest that poly(ADP-ribose) polymerase-1 (Parp-1) is involved in transcriptional regulation of various genes as a coactivator or a corepressor by modulating chromatin structure. However, the impact of Parp-1-deficiency on the regulation of genome-wide gene expression has not been fully studied yet.ResultsWe employed a microarray analysis covering 12,488 genes and ESTs using mouse Parp-1-deficient (Parp-1-/-) embryonic stem (ES) cell lines and the livers of Parp-1-/- mice and their wild-type (Parp-1+/+) counterparts. Here, we demonstrate that of the 9,907 genes analyzed, in Parp-1-/- ES cells, 9.6% showed altered gene expression. Of these, 6.3% and 3.3% of the genes were down- or up-regulated by 2-fold or greater, respectively, compared with Parp-1+/+ ES cells (p < 0.05). In the livers of Parp-1-/- mice, of the 12,353 genes that were analyzed, 2.0% or 1.3% were down- and up-regulated, respectively (p < 0.05). Notably, the number of down-regulated genes was higher in both ES cells and livers, than that of the up-regulated genes. The genes that showed altered expression in ES cells or in the livers are ascribed to various cellular processes, including metabolism, signal transduction, cell cycle control and transcription. We also observed expression of the genes involved in the pathway of extraembryonic tissue development is augmented in Parp-1-/- ES cells, including H19. After withdrawal of leukemia inhibitory factor, expression of H19 as well as other trophoblast marker genes were further up-regulated in Parp-1-/- ES cells compared to Parp-1+/+ ES cells.ConclusionThese results suggest that Parp-1 is required to maintain transcriptional regulation of a wide variety of genes on a genome-wide scale. The gene expression profiles in Parp-1-deficient cells may be useful to delineate the functional role of Parp-1 in epigenetic regulation of the genomes involved in various biological phenomena.

Concise Review: Regulation of Embryonic Stem Cell Lineage Commitment by Mitogen-Activated Protein Kinases

Embryonic stem (ES) cells can give rise, in vivo, to the ectodermal, endodermal, and mesodermal germ layers and, in vitro, can differentiate into multiple cell lineages, offering broad perspectives in regenerative medicine. Understanding the molecular mechanisms governing ES cell commitment is an essential challenge in this field. The mitogen-activated protein kinase (MAPK) pathways extracellular signal-regulated kinase (ERK), c-Jun amino-terminal kinase (JNK), and p38MAPK are able to regulate ES commitment from early steps of the process to mature differentiated cells. Whereas the ERK pathway inhibits the self-renewal of ES cells, upon commitment this pathway is involved in the development of extraembryonic tissues, in early mesoderm differentiation, and in the formation of mature adipocytes; p38MAPK displays a large spectrum of action from neurons to adipocytes, and JNK is involved in both ectoderm and primitive endoderm differentiations. Furthermore, for a given pathway, several of these effects are isoform-dependent, revealing the complexity of the cellular response to activation of MAPK pathways. Regarding tissue regeneration, the potential outcome of systematic analysis of the function of different MAPKs in different ES cell differentiation programs is discussed. Disclosure of potential conflicts of interest is found at the end of this article.

Selective overexpression of the hypoxia-inducible transcription factor, HIF-2alpha, in placentas from women with preeclampsia.

Transcription factors orchestrate the development of extraembryonic tissues. Because placental hypoxia likely plays an important role in both normal and abnormal placentation, we have been investigating the hypoxia-inducible transcription factors (HIFs) in the human placenta. In this report, we focus on the placentas from women with preeclampsia. Because the placenta is a large, heterogeneous organ, we employed a systematic and unbiased approach to placental sampling, and our results are based on the analyses of eight biopsy sites per placenta. We observed no significant differences in HIF-1alpha or -2alpha mRNA expression between normal term and preeclamptic placentas. Nor was HIF protein expression significantly different, with the notable exception of HIF-2alpha, which, on average, was increased by 1.7-fold in the preeclamptic placentas (P: < 0.03 vs. normal term placentas). Considering all 48 paired placental biopsy sites (eight sites each for six normal term and six preeclamptic placentas), HIF-2alpha protein levels in the preeclamptic placentas exceeded those in the normal term placentas in 39, or 81%, of the paired sites (P: < 0.0013). The HIF-2alpha immunoreactivity was mainly located in the nuclei of the syncytiotrophoblast and fetoplacental vascular endothelium in the preeclamptic villous placenta. To control for the earlier gestational age of the preeclamptic placentas, an additional group of placentas from preterm deliveries without preeclampsia were also evaluated. The HIF protein expression was comparable in these preterm specimens and the normal term placentas. We conclude that protein expression of HIF-2alpha, but not of HIF-1alpha or -1beta, is selectively increased in the preeclamptic placenta. The molecular mechanism(s) of this abnormality as well as the genes affected downstream are currently under investigation. To our knowledge, this is the first report of abnormal HIF-2alpha expression in human disease other than cancer.

Establishment of Imprinted Methylation Patterns during Development

Genomic imprinting is a classic example of epigenetic control of gene expression in mammals. It represents a process that marks the parental origin of certain genes, resulting in their allele-specific expression. Imprinting, which is implicated in the inequality of the maternal and paternal genomes in mammals, results in the developmental failure of isoparental embryos to develop properly (Surani 1993). An imbalance of specific parental chromosomes in the embryo or aberrant expression of the imprinted genes results in a number of genetic disorders in man and may also contribute to tumor development (Feinberg 1993; Nicholls 1993). Several model explanations have been proposed for the evolutionary acquisition of genomic imprinting as a developmental control mechanism in mammals (Moore and Haig 1991; Barlow 1993; Varmuza and Mann 1994). One of these models (Moore and Haig 1991) suggests that imprinting might have evolved in mammals because of the conflicting “interests” of maternal and paternal genes within the embryo. This model proposes that paternally expressed genes promote embryonic growth, whereas maternal genes act to restrain the use of maternal resources. A suitable example that corroborates this argument is the paternally expressed Igf2 gene and its counterpart, maternally expressed receptor (Igf2r). The different contribution to the embryonic phenotype of paternal and maternal genes had in fact been demonstrated in parthenogenetic and androgenetic embryos. Although the development of extraembryonic tissues in parthenogenetic embryos harboring two copies of the maternal genome is impaired, the development of the embryo proper is normal. In contrast, the embryo proper in androgenones...

Use of triple tissue blastocyst reconstitution to study the development of diploid parthenogenetic primitive ectoderm in combination with fertilization-derived trophectoderm and primitive endoderm

Diploid mouse conceptuses lacking a paternal genome can form morphologically normal but small fetuses of up to 25 somites, but they invariably fail to develop beyond mid-gestation. Such conceptuses differ from normal most notably in the poor development of extra-embryonic tissues which are largely of trophectodermal and primitive endodermal origin. However, it is not clear whether the demise of diploid parthenogenetic (P) or gynogenetic (G) conceptuses is attributable entirely to the defective development of these two tissues or whether differentiation of the primitive ectoderm, the precursor of the foetus, extra-embryonic mesoderm and amnion, is also impaired by the absence of a paternal genome. Therefore, a new blastocyst reconstitution technique was used which enabled primitive ectoderm from P blastocysts to be combined with primitive endoderm and trophectoderm from fertilization-derived (F) blastocysts. One third of the 'triple tissue' reconstituted blastocysts that implanted yielded foetuses. However, all foetuses recovered on the 11th or 12th day of gestation were small and, with one exception, either obviously retarded or arrested in development. The exception was a living 44 somite specimen which is the most advanced P foetus yet recorded. Foetuses were invariably degenerating in conceptuses recovered on the 13th day. In contrast, at least 16% of control reconstituted blastocysts with primitive ectoderm as well as primitive endoderm and trophectoderm of F origin developed normally on the 13th day of gestation or to term. Hence, the presence of a paternal genome seems to be essential for normal differentiation of all 3 primary tissues of the mouse blastocyst. The P foetuses that developed from reconstituted blastocysts were so closely invested by their membranes that they often showed abnormal flexure of the posterior region of the body. Several also showed a deficiency of allantoic tissue. Therefore, the possibility that the defect in development of P primitive ectoderms resided in their extra-embryonic tissues was investigated by analysing a series of chimaeras produced by injecting them into intact F blastocysts. The foregoing anomalies were not discernible even when P cells made a large contribution to the extra-embryonic mesoderm or amnion plus umbilical cord. Furthermore, selection against P cells was no greater in extra-embryonic derivatives of the primitive ectoderm than in the foetus itself.

Parental origin effects in mice

ABSTRACT Nuclear transplantation experiments in mice, reviewed elsewhere in this Symposium, have clearly demonstrated that the maternal and paternal genomes from which the embryo is formed are not functionally equivalent. The paternal genome appears to be essential for the normal development of extraembryonic tissues and the maternal genome for some stage of embryonic development. These findings provide some explanation for the observations that in mammals diploid parthenotes possessing two maternal genomes fail to survive (Markert, 1982) and that, in man, embryos with two paternal chromosome sets are inviable, forming hydatidiform moles (Kajii &amp; Ohama, 1977). It has been proposed that a specific ‘imprinting’ of the paternal genomes occurs during gametogenesis so that the presence of both a female and male pronculeus is essential in an egg for full-term development (Barton, Surani &amp; Norris, 1984; McGrath &amp; Solter, 1984a; Surani, Barton &amp; Norris, 1984).