Single-cell TimeLapse-seq reveals MTA is an essential retrotransposon orchestrating mouse zygotic genome activation

Transposable elements (TEs) are temporally activated during mammalian early embryogenesis and are closely linked to zygotic genome activation (ZGA), yet their nascent transcriptional dynamics remain incompletely resolved owing to limited sensitivity and maternal RNA background. Here, we develop single-cell TimeLapse sequencing (scTL-seq), enabling allele-resolved detection of nascent RNA in individual embryos. Applying scTL-seq to mouse ZGA, we generate a refined transcriptional atlas of genes and TEs and identify the pronuclear (PN) stage activated genes and TEs. We found that transcription at PN stage is largely contributed by the maternal genome and enriched for genes highly transcribed in germinal vesicle oocytes. Similarly, Mouse Transcripts type-A (MTA) elements are transcribed in oocytes and highly active in PN stage. MTA knockdown disrupts zygotic gene activation and dysregulates hundreds of TE subfamilies, resulting in 1-cell embryo arrest. Using nascent transcripts, we further identify 662 TE-driven chimeric transcripts and show that TEs are essential for regulating key developmental genes, including Nfya, Setd2 and Tead4. Finally, we identify maternal transcription factors, including NOBOX, as potential regulators of MTA activation. Together, our study provides a high-resolution nascent transcriptome landscape of mouse ZGA and reveals vital regulatory roles of MTA retrotransposon in orchestrating early embryonic development. This study establishes scTimeLapse-seq to redefine mouse ZGA into three transcriptional waves. It unveils MTA-MaLR as the earliest activated retrotransposon, which acts as a key regulator of ZGA initiation and early embryonic development.