Project description:Although early developmental processes involve cell fate decisions that define the body axes and establish progenitor cell pools, development does not cease once cells are specified. Instead, most cells undergo specific maturation events where changes in the cell transcriptome ensure that the proper gene products are expressed to carry out unique physiological functions. Pancreatic acinar cells mature post-natally to handle an extensive protein synthetic load, establsih organized apical-basal polarity for zymogen granule trafficking, and assemble gap-junctions to perimt efficient cell-cell communication. Despite significant progress in defining transcriptional networks that control initial acinar cell specification and differentiation decisions, little is know regarding the role of transcription factors in the specification and maintenance of maturation events. One candidate maturation effector is MIST1, a secretory cell-restricted transcription factor that has been implicated in controlling regulated exocytosis events in a number of cell types. Embryonic knock-out of MIST1 generates acinar cells that fail to establish an apical-basal organization, fail to properly localize zymogen granule and fail to communicate intra-cellularly, making the exocrine organ highly suceptible to pancreatic diseases. In an effort to identify the gene expression differences responsible for MIST1 regulating mature acinar properties. We generated a tamoxifen-inducible mouse model where MIST1 expression could be activated in vivoand performed gene expression arrays on wildtype, MIST1-null, and induced MIST1 pancreatic RNA. RNA was isolated from pancreata of 8 week old mice using the Qiagen RNeasy Midi kit. Pancreta of wildtype, MIST1-null, and MIST1-null with a tamoxifen inducible MIST1-expressing transgene were harvested 36 hours post-tamoxifen administration. Therefore, this experiment provides information on steady-state gene expression differences between wildtype and MIST1-null mice as well as immediate gene expression changes induced by MIST1 expression.
Project description:Gene loci that are hypermethylated and repressed in embryonic (ESCs) but hypomethylated and expressed in trophoblast (TSCs) stem cells are very rare and may have particularly important roles in early developmental cell fate decisions, as previously shown for Elf5. Here, we assessed another member of this small group of genes, Placenta Expressed Transcript 1 (Plet1), for its function in establishing trophoblast lineage identity and modulating trophoblast differentiation. We find that Plet1 is tightly repressed by DNA methylation in ESCs but expressed on the cell surface of TSCs and trophoblast giant cells. In hypomethylated ESCs that are prone to acquire some trophoblast characteristics, Plet1 is required to confer a trophoblast-specific gene expression pattern, including up-regulation of Elf5. Plet1 displays an unusual biphasic expression profile during TSC differentiation and thus may be pivotal in balancing trophoblast self-renewal and differentiation. Furthermore, overexpression and CRISPR/Cas9-mediated knockout in TSCs showed that high Plet1 levels favour differentiation towards the trophoblast giant cell lineage, whereas lack of Plet1 preferentially induces syncytiotrophoblast formation. Thus, the endogenous dynamics of Plet1 expression establish important patterning cues within the trophoblast compartment by promoting differentiation towards the syncytiotrophoblast or giant cell pathway in Plet1-low and Plet1-high cells, respectively.
Project description:Large-scale phenotyping efforts have demonstrated that approximately 25-30% of mouse gene knockouts cause intrauterine lethality. Analysis of these mutants has largely focused on the embryo and not the placenta, despite the crucial role of this extraembryonic organ for developmental progression. Here we screened 103 embryonic lethal and sub-viable mouse knockout lines from the Deciphering the Mechanisms of Developmental Disorders program for placental phenotypes. We found that 68% of knockout lines that are lethal at or after mid-gestation exhibited placental dysmorphologies. Early lethality (embryonic days 9.5-14.5) is almost always associated with severe placental malformations. Placental defects correlate strongly with abnormal brain, heart and vascular development. Analysis of mutant trophoblast stem cells and conditional knockouts suggests that a considerable number of factors that cause embryonic lethality when ablated have primary gene function in trophoblast cells. Our data highlight the hugely under-appreciated importance of placental defects in contributing to abnormal embryo development and suggest key molecular nodes that govern placenta formation.
Project description:Trophoblast stem cells (TSCs) give rise to specialized cell types within the placenta. However, the regulatory mechanisms that guide trophoblast cell fate decisions during placenta development remain ill defined. Here we exploited ATAC-seq and transcriptional profiling strategies to describe dynamic changes in gene expression and chromatin accessibility during TSC differentiation. We detect significantly increased chromatin accessibility at key genes upregulated as TSCs exit from the stem cell state. However, downregulated gene expression is not simply due to the loss of chromatin accessibility in proximal regions. Additionally, transcriptional targets recognized by the zinc finger transcriptional repressor Prdm1/Blimp1, an essential regulator of placenta development, were identified in ChIP-seq experiments. Comparisons with previously reported ChIP-seq datasets for primordial germ cell-like cells and E18.5 small intestine, combined with functional annotation analysis revealed that Blimp1 has broadly shared as well as cell type-specific functional activities unique to the trophoblast lineage. Importantly, Blimp1 not only silences TSC gene expression but also prevents aberrant activation of divergent developmental programmes. Overall the present study provides new insights into the chromatin landscape and Blimp1-dependent regulatory networks governing trophoblast gene expression.
Project description:The ZFP36L3 protein is a rodent-specific, placenta- and yolk sac-specific member of the tristetraprolin (TTP) family of CCCH tandem zinc finger proteins. These proteins bind to AU-rich elements in target mRNAs, and promote their deadenylation and decay. We addressed the hypotheses that the absence of ZFP36L3 would result in the accumulation of target transcripts in placenta and/or yolk sac, and that some of these would be important for female reproductive physiology and overall fecundity. Mice deficient in ZFP36L3 exhibited decreased neonatal survival rates, but no apparent morphological changes in the placenta or surviving offspring. We found Zfp36l3 to be paternally imprinted, with profound parent-of-origin effects on gene expression. The protein was highly expressed in the syncytiotrophoblast cells of the labyrinth layer of the placenta, and the epithelial cells of the yolk sac. RNA-Seq of placental mRNA from Zfp36l3 knockout (KO) mice revealed many significantly upregulated transcripts, whereas there were few changes in KO yolk sacs. Many of the upregulated placental transcripts exhibited decreased decay rates in differentiated trophoblast stem cells derived from KO blastocysts. Several dozen transcripts were deemed high probability targets of ZFP36L3; these include proteins known to be involved in trophoblast and placenta physiology. Type 1 transferrin receptor mRNA was unexpectedly decreased in KO placentas, despite an increase in its stability in KO stem cells. This receptor is crucial for placental iron uptake, and its decrease was accompanied by decreased iron stores in the KO fetus, suggesting that this intrauterine deficiency might have deleterious consequences in later life.
Project description:The trophectoderm layer of the blastocyst-stage embryo is the precursor for all trophoblast cells in the placenta. Human trophoblast stem (TS) cells have emerged as an attractive tool for studies on early trophoblast development. However, the use of TS cell models is constrained by the limited genetic diversity of existing TS cell lines and restrictions on using human fetal tissue or embryos needed to generate additional lines. Here we report the derivation of two distinct stem cell types of the trophectoderm lineage from human pluripotent stem cells. Analogous to villous cytotrophoblasts in vivo, the first is a CDX2<sup>-</sup> stem cell comparable with placenta-derived TS cells-they both exhibit identical expression of key markers, are maintained in culture and differentiate under similar conditions, and share high transcriptome similarity. The second is a CDX2<sup>+</sup> stem cell with distinct cell culture requirements, and differences in gene expression and differentiation, relative to CDX2<sup>-</sup> stem cells. Derivation of TS cells from pluripotent stem cells will significantly enable construction of in vitro models for normal and pathological placental development.
Project description:Trophoblasts are the first cell type to be specified during embryogenesis, and they are essential for placental morphogenesis and function. Trophoblast stem (TS) cells are the progenitor cells for all trophoblast lineages; control of TS cell differentiation into distinct trophoblast subtypes is not well understood. Mice lacking the transcription factor OVO-like 2 (OVOL2) fail to produce a functioning placenta, and die around embryonic day 10.5, suggesting that OVOL2 may be critical for trophoblast development. Therefore, our objective was to determine the role of OVOL2 in mouse TS cell fate. We found that OVOL2 was highly expressed in mouse placenta and differentiating TS cells. Placentas and TS cells lacking OVOL2 showed poor trophoblast differentiation potential, including increased expression of stem-state associated genes (Eomes, Esrrb, Id2) and decreased levels of differentiation-associated transcripts (Gcm1, Tpbpa, Prl3b1, Syna). Ectopic OVOL2 expression in TS cells elicited precocious differentiation. OVOL2 bound proximate to the gene encoding inhibitor of differentiation 2 (ID2), a dominant negative helix-loop-helix protein, and directly repressed its activity. Overexpression of ID2 was sufficient to reinforce the TS cell stem state. Our findings reveal a critical role of OVOL2 as a regulator of TS cell differentiation and placental development, in-part by coordinating repression of ID2.
Project description: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.
Project description:This study characterized the effect of the reduced utero-placental perfusion pressure (RUPP) model of placental insufficiency on placental morphology and trophoblast differentiation at mid-late gestation (E14.5). Altered trophoblast proliferation, reduced syncytiotrophoblast gene expression, increased numbers of sinusoidal trophoblast giant cells, decreased Vegfa and decreased pericyte presence in the labyrinth were observed in addition to changes in maternal blood spaces, the fetal capillary network and reduced fetal weight. Further, the junctional zone was characterized by reduced spongiotrophoblast and glycogen trophoblast with increased trophoblast giant cells. Increased Hif-1? and TGF-?-3 in vivo with supporting hypoxia studies in trophoblast stem (TS) cells in vitro, support hypoxia as a contributing factor to the RUPP placenta phenotype. Together, this study identifies altered cell populations within the placenta that may contribute to the phenotype, and thus support the use of RUPP in the mouse as a model of placenta insufficiency. As such, this model in the mouse provides a valuable tool for understanding the phenotypes resulting from genetic manipulation of isolated cell populations to further understand the etiology of placenta insufficiency and fetal growth restriction. Further this study identifies a novel relationship between placental insufficiency and pericyte depletion in the labyrinth layer.