In vitro culture of mouse blastocysts beyond the implantation stages

Bedzhov, Ivan; Leung, Chuen Yan; Bialecka, Monika; Zernicka‐Goetz, Magdalena

doi:10.1038/nprot.2014.186

Cited by 153 publications

(150 citation statements)

References 18 publications

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“…When cultured in Matrigel, blastocyst inner mass cells generate rosettes and a polarized outer endoderm layer (Bedzhov et al., 2014). In these conditions, ESC-derived rosettes form this PE layer, albeit at much lower efficiency, as defined by GATA4 and DIDO1 expression and OCT4 downregulation (Figure S7A).…”

Section: Resultsmentioning

confidence: 99%

DIDO as a Switchboard that Regulates Self-Renewal and Differentiation in Embryonic Stem Cells

et al. 2017

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SummaryTransition from symmetric to asymmetric cell division requires precise coordination of differential gene expression. We show that embryonic stem cells (ESCs) mainly express DIDO3 and that their differentiation after leukemia inhibitory factor withdrawal requires DIDO1 expression. C-terminal truncation of DIDO3 (Dido3ΔCT) impedes ESC differentiation while retaining self-renewal; small hairpin RNA-Dido1 ESCs have the same phenotype. Dido3ΔCT ESC differentiation is rescued by ectopic expression of DIDO3, which binds the Dido locus via H3K4me3 and RNA POL II and induces DIDO1 expression. DIDO1, which is exported to cytoplasm, associates with, and is N-terminally phosphorylated by PKCiota. It binds the E3 ubiquitin ligase WWP2, which contributes to cell fate by OCT4 degradation, to allow expression of primitive endoderm (PE) markers. PE formation also depends on phosphorylated DIDO3 localization to centrosomes, which ensures their correct positioning for PE cell polarization. We propose that DIDO isoforms act as a switchboard that regulates genetic programs for ESC transition from pluripotency maintenance to promotion of differentiation.

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Section: Resultsmentioning

confidence: 99%

DIDO as a Switchboard that Regulates Self-Renewal and Differentiation in Embryonic Stem Cells

et al. 2017

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“…After female mice were stimulated by injecting PMSG and hCG (Ningbo Second Hormone Factory), metaphase II (MII) oocytes were collected in M2 medium from oviducts after hCG 13 h; 1-cell, 2-cell embryos, and morulas were flushed from oviducts at E0.5, E1.5, and E2.5 days; and blastocysts were collected from uteruses at E3.5. Blastocysts were cultured in 1066 medium supplemented with 10% FBS, 1 M sodium pyruvate, 2 M L-glutamine, and 50 g/ml of penicillin/streptomycin at 37°C in 5% CO 2 (50,51). The culture dishes for blastocysts were pre-treated with 0.2% gelatin.…”

Section: Methodsmentioning

confidence: 99%

Trp-Asp (WD) Repeat Domain 1 Is Essential for Mouse Peri-implantation Development and Regulates Cofilin Phosphorylation

Xiao

Wan

et al. 2017

Journal of Biological Chemistry

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Edited by Xiao-Fan WangTrp-Asp (WD) repeat domain 1 (WDR1) is a highly conserved actin-binding protein across all eukaryotes and is involved in numerous actin-based processes by accelerating Cofilin severing actin filament. However, the function and the mechanism of WDR1 in mammalian early development are still largely unclear. We now report that WDR1 is essential for mouse periimplantation development and regulates Cofilin phosphorylation in mouse cells. The disruption of maternal WDR1 does not obviously affect ovulation and female fertility. However, depletion of zygotic WDR1 results in embryonic lethality at the periimplantation stage. In WDR1 knock-out cells, we found that WDR1 regulates Cofilin phosphorylation. Interestingly, WDR1 is overdosed to regulate Cofilin phosphorylation in mouse cells. Furthermore, we showed that WDR1 interacts with Lim domain kinase 1 (LIMK1), a well known phosphorylation kinase of Cofilin. Altogether, our results provide new insights into the role and mechanism of WDR1 in physiological conditions.The actin cytoskeleton is a highly dynamic structure that regulates cell motility, adhesion, division, and growth and has a fundamental function in embryonic development (1-3). In physiological conditions, actin filaments are continually assembled and disassembled in response to varied cellular activities (4, 5). The dynamics of F-actin is well orchestrated by a large number of actin-binding proteins (5, 6). Actin-depolymerizing factor Cofilin plays critical roles in the modulation of actin cytoskeleton dynamics (7). In vitro, Cofilin severs actin filament at low concentrations, whereas it fully decorates actin filaments and suppresses the severing activity at high concentrations (8, 9). In addition, Cofilin severing F-actin is not explained well for how the filaments of actin cytoskeleton can be rapidly disassembled in physiological conditions (4). Thus, the function of Cofilin in actin dynamics depends not only on its relative concentration to actin, but also on other regulatory proteins in vivo. Dysfunction of these regulating proteins results in aberrant actin cytoskeleton in various cellular and developmental processes (10 -12).Cofilin directly binds with actin to function as a regulator of the actin dynamics (13). However, the phosphorylation of Cofilin at Ser-3 blocks its binding site to actin (14, 15). Thus, the activity of Cofilin is controlled by phosphorylation and dephosphorylation processes, which are regulated by cascade reactions of several protein kinases and phosphatases. Lim domain kinases (LIMKs) 4 and testis-specific protein kinases (TESKs) are responsible for the phosphorylation of Cofilin, whereas slingshot (SSH) family protein phosphatases and pyridoxal phosphatase (PDXP) catalyze the dephosphorylation reaction (16 -20). These kinases and phosphatases are also regulated by their phosphorylation or localization in cytoplasm to maintain the level of Cofilin phosphorylation in response to extracellular stimuli (21-23).The activity of Cofilin on actin disassembly is gr...

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“…Self-assembly/ organization has also been observed directly in attached mammalian blastocysts in vitro. These studies established that human (Deglincerti et al, 2016a;Shahbazi et al, 2016) and mouse (Bedzhov et al, 2014b;Morris et al, 2012) embryos can be cultured ex vivo in the absence of maternal tissues, showing important selforganizing events. Although these assays are currently ethically limited to studying human embryos for only up to 14 days of development, they provide a complementary approach to embryoid and gastruloid experiments for quantitatively studying early human development and are likely to become increasingly popular in the coming years.…”

Section: Introductionmentioning

confidence: 93%

Embryoids, organoids and gastruloids: new approaches to understanding embryogenesis

2017

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Cells have an intrinsic ability to self-assemble and self-organize into complex and functional tissues and organs. By taking advantage of this ability, embryoids, organoids and gastruloids have recently been generated in vitro, providing a unique opportunity to explore complex embryological events in a detailed and highly quantitative manner. Here, we examine how such approaches are being used to answer fundamental questions in embryology, such as how cells selforganize and assemble, how the embryo breaks symmetry, and what controls timing and size in development. We also highlight how further improvements to these exciting technologies, based on the development of quantitative platforms to precisely follow and measure subcellular and molecular events, are paving the way for a more complete understanding of the complex events that help build the human embryo.

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In vitro culture of mouse blastocysts beyond the implantation stages

Cited by 153 publications

References 18 publications

DIDO as a Switchboard that Regulates Self-Renewal and Differentiation in Embryonic Stem Cells

DIDO as a Switchboard that Regulates Self-Renewal and Differentiation in Embryonic Stem Cells

Trp-Asp (WD) Repeat Domain 1 Is Essential for Mouse Peri-implantation Development and Regulates Cofilin Phosphorylation

Embryoids, organoids and gastruloids: new approaches to understanding embryogenesis

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