Archives

  • 2018-07
  • 2018-10
  • 2018-11
  • 2019-04
  • 2019-05
  • 2019-06
  • 2019-07
  • 2019-08
  • 2019-09
  • 2019-10
  • 2019-11
  • 2019-12
  • 2020-01
  • 2020-02
  • 2020-03
  • 2020-04
  • 2020-05
  • 2020-06
  • 2020-07
  • 2020-08
  • 2020-09
  • 2020-10
  • 2020-11
  • 2020-12
  • 2021-01
  • 2021-02
  • 2021-03
  • 2021-04
  • 2021-05
  • 2021-06
  • 2021-07
  • 2021-08
  • 2021-09
  • 2021-10
  • 2021-11
  • 2021-12
  • 2022-01
  • 2022-02
  • 2022-03
  • 2022-04
  • 2022-05
  • 2022-06
  • 2022-07
  • 2022-08
  • 2022-09
  • 2022-10
  • 2022-11
  • 2022-12
  • 2023-01
  • 2023-02
  • 2023-03
  • 2023-04
  • 2023-05
  • 2023-06
  • 2023-08
  • 2023-09
  • 2023-10
  • 2023-11
  • 2023-12
  • 2024-01
  • 2024-02
  • 2024-03
  • 2024-04
  • 2024-05

    • Discussion We previously reported that the conventional gene

    2018-11-06

    Discussion We previously reported that the conventional genetic depletion of Pelo in mouse results in an embryonic lethality at early post-implantation stages (Adham et al., 2003). Here, we report that Pelo-null ESCs are continuously propagated and retained their capacity to form undifferentiated colonies at clonal densities, but fail to differentiate into ExEn lineage in EBs. Conversely, overexpression of Pelo in ESCs led to down-regulation of pluripotency-related genes and a preferential activation of genes that regulate the differentiation of ESCs into an ExEn cell lineage. Upon the aggregation of ES cells in suspension, the outer layer of developing EBs differentiates into ExEn, which deposits extracellular matrix into the underlying basement membrane (BM). Inside the BM, a primitive ectoderm layer is developed, and cavitation is formed in the core of the EBs (Niwa, 2010). The developmental process of EBs mimics the early embryonic stages of late elastase inhibitor to egg cylinder (E4.5–E6.5). Both formation of ExEn and cavitation have been shown to be regulated by the BMP signaling pathway in mouse embryos and EBs. Thus, inhibition of BMP signaling by expression of a dominant-negative BMP receptor, down-regulation of Bmp6 expression in ectodermal cells or addition of the BMP antagonist Noggin in culture prevents the development of ExEn in EBs (Coucouvanis and Martin, 1999; Conley et al., 2007; Rong et al., 2012). A significant decrease in the expression levels of BMP-targeted genes, phosphorylated Smad1/5, and the overexpression of Noggin in Pelo-deficient EBs suggest that PELO regulates differentiation toward ExEn lineage through the activation of BMP signaling. These results were supported by the observations of restored ExEn development in mutant EBs grown in medium supplemented with BMP4. Further, the negative effect of conditioned medium collected from mutant EBs on the ExEn formation in wild-type EBs and the significant decrease of luciferase activity in the BMP responsive reporter cell line indicate that mutant EBs secrete extracellular modulators which attenuate the BMP signaling activity. Several factors regulate the activity of BMP signaling at intracellular and extracellular levels. The responsiveness of wild-type and mutant cells to Noggin and BMP4 treatment excludes the role of PELO in regulation of the intracellular modulators of BMP signaling. Extracellular modulators such as Noggin and Chordin antagonize the BMP signal (Piccolo et al., 1996; Zimmerman et al., 1996). Acute overexpression of Noggin in Pelo-null EBs led to suggest that PELO regulates the BMP signaling by negatively regulating Noggin expression at either transcriptional or post-transcriptional levels. Cytoplasmic localization of PELO in human and Drosophila cells (Xi et al., 2005; Burnicka-Turek et al., 2010) rules out that PELO directly regulates Noggin at the transcriptional level. In addition, the fact that the conserved role of PELO in NGD and deletion of PELO do not affect the stability of Noggin transcripts suggests that PELO indirectly down-regulates Noggin through controlling stability of transcription factors regulating Noggin expression. Taken together, these results led us to conclude that the reduced BMP signaling in Pelo-null EBs accounts for the observed defect in ExEn differentiation. In support of our results, impaired ExEn development as a result of the affected BMP signaling was also shown in Smad4-deficient EBs (Sirard et al., 1998). Moreover, BMP-mediated MET activation was shown to be essential for the induction of pluripotency in somatic cells (Li et al., 2010; Samavarchi-Tehrani et al., 2010). The failure of Pelo-deficient MEFs to activate BMP signaling during reprogramming reinforces that PELO is an indispensable component for the activation of BMP signaling during the establishment of pluripotency and ExEn cell lineage commitment. The failure of Pelo-null ESCs to undergo ExEn differentiation was accompanied by a significant decrease in the expression levels of the transcription factors Gata4, Gata6 and Hnf4, which are involved in differentiation and functions of ExEn lineage. Similarly, ESCs lacking either Gata4 or Gata6 failed to form the endodermal outer layer (Soudais et al., 1995; Morrisey et al., 1998). Like the overexpression of Gata4 and Gata6 (Fujikura et al., 2002), forced expression of Pelo directs differentiation of ExEn lineage in ESCs. Remarkably, the expression levels of the Pelo transcript were approximately 1.7-fold higher than that of wild-type ESCs. However, this modest change was sufficient to induce differentiation of ESCs into cells positive for ExEn markers, suggesting a quantitative effect of Pelo on ESC differentiation.