Cell–cell adhesion defects in <i>Mrj</i> mutant trophoblast cells are associated with failure to pattern the chorion during early placental development

Watson, Erica D.; Hughes, Martha; Simmons, David G.; Natale, David R.C.; Sutherland, Ann; Cross, James C.

doi:10.1002/dvdy.22755

Cited by 26 publications

(18 citation statements)

References 227 publications

(247 reference statements)

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“…mRNA expression was assessed by quantitative reverse transcription (RT)-PCR using the SYBR green method as previously described (Watson et al, 2011). Briefly, 1 mg of total RNA was reverse transcribed using the Quantitect Reverse Transcription Kit for SYBR green quantitative PCR (#205311, Qiagen).…”

Section: Quantitative Real Time-pcrmentioning

confidence: 99%

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

Rai

Cross

2015

Developmental Biology

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

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Section: Quantitative Real Time-pcrmentioning

confidence: 99%

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

Rai

Cross

2015

Developmental Biology

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“…Quantitative PCR mRNA expression was assessed by quantitative PCR (q-PCR) using the SYBR green method as previously described [32,33]. One microgram of total RNA was reverse transcribed using the Quantitect Reverse Transcription Kit for SYBR green q-PCR (Qiagen; No.…”

Section: Reverse Transcription-polymerase Chain Reactionmentioning

confidence: 99%

“…204143), according to the manufacturer's instructions, and thermocycling was conducted on an MJ Research, DNA Engine, Opticon2 thermocycler. Primer sequences for glial cells missing homolog 1 (gcm1) and syncytin A (synA) were previously reported [32]. Primers for ID2, Prl3b1 (Pl2), Tpbpa, and Ctsq were obtained from Qiagen (Quantitect Primer Assays, QT00255157, QT00123060, QT01749181, and QT00119707, respectively).…”

Section: Reverse Transcription-polymerase Chain Reactionmentioning

confidence: 99%

AMPK Knockdown in Placental Trophoblast Cells Results in Altered Morphology and Function

Carey

Albers

Doliboa

et al. 2014

Stem Cells and Development

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The placenta is a transient organ that develops upon the initiation of pregnancy and is essential for embryonic development and fetal survival. The rodent placenta consists of distinct lineages and includes cell types that are analogous to those that make up the human placenta. Trophoblast cells within the labyrinth layer, which lies closest to the fetus, fuse and come in contact with maternal blood, thus facilitating nutrient and waste exchange between the mother and the baby. Abnormalities of the placenta may occur as a result of cellular stress and have been associated with pregnancy-associated disorders: such as preeclampsia, intrauterine growth restriction, and placental insufficiency. Cellular stress has also been shown to alter proliferation and differentiation rates of trophoblast cells. This stress response is important for cell survival and ensures continued placental functionality. AMP-activated protein kinase is an important sensor of cellular metabolism and stress. To study the role of AMPK in the trophoblast cells, we used RNA interference to simultaneously knockdown levels of both the AMPK alpha isoforms, AMPKa1 and AMPKa2. SM10 trophoblast progenitor cells were transduced with AMPKa1/2 shRNA and stable clones were established to analyze the effects of AMPK knockdown on important cellular functions. Our results indicate that a reduction in AMPK levels causes alterations in cell morphology, growth rate, and nutrient transport, thus identifying an important role for AMPK in the regulation of placental trophoblast differentiation.

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“…While no differences in cell count were found between treatments on day 2 or 4, marked reductions in cell counts in both the 0.2% (48% reduction) and 1% EtOH groups (44% reduction) were observed on day 6 (Figure 4.1A). This time point coincides with terminal differentiation of trophoblasts, as expression of stem cell markers are rapidly lost by day 2 of culture and expression of markers of mature cell types is just commencing on day 4 (Watson et al, 2011). This result is consistent with studies that demonstrate reduced proliferation in response to 20-40mM (0.09-0.18%) EtOH in cultured human cytotrophoblasts (first trimester villous explants and BeWo cells) (Lui et al, 2014).…”

Section: Cell Countssupporting

confidence: 81%

The Effects of Maternal Periconceptional Ethanol Exposure on the Development of the Pre-Implantation Embryo, Placenta, and the Influence of the Uterine Environment

Kalisch-Smith¹

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Exposure of the embryo or fetus to perturbations in utero can result in intrauterine growth restriction (IUGR), a primary risk factor for the development of adult disease.One critical window that can result in developmental programming, is the periconceptional period which comprises maturation of the oocyte, conception and the formation of the blastocyst prior to embryo implantation. Alcohol is a common exposure during this period, as it is consumed prior to pregnancy recognition, and often ceases soon after. Maternal periconceptional alcohol exposure (PC-EtOH) in rat dams has been previously shown to cause fetal growth restriction and changes to the late gestation placenta. PC-EtOH also results in the development of adult onset disease, including insulin insensitivity, often in a sexually dimorphic manner. However, the mechanisms by which PC-EtOH can cause programming are relatively unexplored. This thesis aimed to characterise the effects of alcohol on 1. sex-specific pre-implantation development and trophoblast differentiation using in vitro (cell culture) and in vivo (rodent model) techniques, 2. alterations to the early uterine environment and 3. the interactions and communication between the embryo and uterus, and the resultant impacts on placental development across gestation.The direct effect of EtOH on in vitro differentiation was assessed in mouse trophoblast stem cells (Chapter 4). Results demonstrated that doses of 0.2% and 1% EtOH reduced total cell count and expression of trophoblast subtype markers at terminal differentiation (day 6 of culture). Using our in vivo rat model, we also examined basal sex differences in blastocyst and placental development from pre-implantation to lategestation in naturally cycling dams (Chapter 3). Although there were no significant differences between sexes in pre-implantation development (blastocyst cell numbers, trophoblast differentiation), by mid-gestation (E15) placentas from males were heavier and had increased blood space volume and surface area (to both maternal and fetal compartments) than those from females. This likely contributed to the greater fetal body weight in males at E20. This study confirmed the need to examine the effects of PC-EtOH in a sexually dimorphic manner across all of pregnancy.To study the effects of PC-EtOH in vivo, Sprague-Dawley rat dams were given a liquid diet containing either control (0% v/v EtOH) or EtOH (12.5% v/v EtOH) from embryonic (E) day -4 to E4. The following day dams were returned to chow for the remainder of 3 gestation. Dams were sacrificed at E5 to determine cell number of the pre-implantation embryo and its capacity to differentiate into cells required for invasion (Chapter 5).Pre-implantation studies showed PC-EtOH altered inner cell mass count, trophoblast differentiation to trophoblast giant cells (TGCs) and trophoblast behaviour in a sexually dimorphic manner, with females showing the most deleterious outcomes. PC-EtOH females also showed reduced expression of Prl4a1, a gene exclusively expressed by TGCs...

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

Cited by 26 publications

References 227 publications

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

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

AMPK Knockdown in Placental Trophoblast Cells Results in Altered Morphology and Function

The Effects of Maternal Periconceptional Ethanol Exposure on the Development of the Pre-Implantation Embryo, Placenta, and the Influence of the Uterine Environment

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