Mouse Xist expression begins at zygotic genome activation and is timed by a zygotic clock.
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.
Zuccotti
Maurizio
M
Dipartimento di Medicina Sperimentale, Sezione di Istologia ed Embriologia Generale, Universita' degli Studi di Parma, Parma, Italy. maurizio.zuccotti@unipv.it
Boiani
Michele
M
Ponce
Ruben
R
Guizzardi
Stefano
S
Scandroglio
Renato
R
Garagna
Silvia
S
Redi
Carlo Alberto
CA
D000818:Animals; D001683:Biological Clocks / Q000502:physiology*; D015638:Cytochalasin D / Q000494:pharmacology; D004622:Embryo, Mammalian / Q000187:drug effects / Q000378:metabolism; D005306:Fertilization / Q000235:genetics / Q000502:physiology*; D005307:Fertilization in Vitro; D016195:G2 Phase; D018507:Gene Expression Regulation, Developmental; D016678:Genome; D007041:Hypoxanthine Phosphoribosyltransferase / Q000378:metabolism; D051379:Mice; D010312:Parthenogenesis / Q000235:genetics; D062085:RNA, Long Noncoding; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D022661:RNA, Untranslated / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors; D014157:Transcription Factors / Q000235:genetics*; D014960:X Chromosome / Q000235:genetics; D015053:Zygote / Q000187:drug effects / Q000378:metabolism*
D062085:RNA, Long Noncoding; D012333:RNA, Messenger; D022661:RNA, Untranslated; D014157:Transcription Factors; C077885:XIST non-coding RNA; D015638:Cytochalasin D; D007041:Hypoxanthine Phosphoribosyltransferase