Mouse Xist expression begins at zygotic genome activation and is timed by a zygotic clock

Abstract

The imprinted mouse Xist (X-inactive specific transcript) gene is involved in the initiation of X-chromosome inactivation. Only the paternal Xist is expressed in preimplantation development beginning from the 4-cell stage, preceding and in correlation with paternal X-inactivation in the extraembryonic lineage of the blastocyst. To better understand the mechanisms regulating Xist expression in early development, we investigated the precise timing of its onset. We set up a single-cell RT-PCR for the simultaneous analysis on single embryos of Xist and Hprt (internal control) cDNAs and a Y-chromosome specific DNA sequence, Zfy (for embryo sexing). Applying this procedure, we demonstrate that Xist expression begins at the G2-phase of 2-cell female embryos, earlier than previously reported and at the same time of the major wave of zygotic genome activation (ZGA). We then examined, if Xist expression at the 2-cell stage is dependent on the lapse of time spent since fertilization, as previously reported for zygotic genes. One-cell embryos at the G2-phase of the first cell-cycle were cultured with cytochalasin D (inhibitor of cytokinesis but not of DNA synthesis or nuclear progression) for a time equivalent to the 4-cell stage in control, untreated embryos. We show that Xist activation occurs at a scheduled time following fertilization despite the embryos being blocked at the 1-cell stage, suggesting the existence of a zygotic clock involved in the regulation of the transcription of this imprinted gene.

Ftx is dispensable for imprinted X-chromosome inactivation in preimplantation mouse embryos

“…It would be of interest to examine whether these adjacent genes are also affected in female cells. We were also interested in whether Ftx is involved in the onset of the imprinted Xist upregulation during early preimplantation stages because previous studies have shown that Xist RNA upregulation is detected from the two- to four-cell stage onward1415. We used q-PCR to examine the Ftx expression at preimplantation stages and found that the expression begins at the blastocyst stage (Figure S1).…”

Follicle microenvironment-associated alterations in gene expression in the mouse oocyte and its polar body

“…Single oocytes or PB in 0.5 µL PBS were transferred to the bottom of 0.5-mL thin-wall Eppendorf tubes containing 1.5 µL lysis buffer (0.8% Igepal, 1 U RNAsin/µL, 5 nM DTT) (24). The specimens were stored at −80°C.…”

Melatonin modulates the expression of BCL-xl and improve the development of vitrified embryos obtained by IVF in mice

“…Simultaneous RNA extraction and cDNA synthesis Preimplantation embryos at blastocyst stages of development were transferred to Eppendorf tube containing 1.5 μl lysis buffer [33]. 2 μl random hexamer and 5 μl water was added to each 2 μl embryo sample.…”