Very early in development, an embryo must begin using its own genome instead of relying only on RNA and proteins inherited from the egg. This transition is known as zygotic genome activation, or ZGA, and it is a critical step in early embryonic development.

Transposable elements, or TEs, are repetitive DNA sequences that can become active during this period. Although they were once viewed mainly as genomic passengers, growing evidence suggests that some transposable elements help regulate gene activity.

Researchers at Tongji University in Shanghai and colleagues developed a new single-cell RNA sequencing method called single-cell TimeLapse sequencing, or scTL-seq, to examine newly produced RNA in individual mouse embryos.

scTL-seq reveals nascent transcription in mouse early embryos 

a Schematic diagram of the scTimeLapse-seq (scTL-seq) procedure. 4sU, 4-thiouridine. TFEA, 2,2,2-trifluoroethylamine. C*, cytidine substitution. b Nucleotide mutation rates after TimeLapse-seq using 2 μg, 100 ng, and 10 ng 293T RNAs. Unconversion, 10 ng 293T RNAs were treated without T-to-C reaction. c Dot plot showing a positive correlation between T-to-C mutation rates in 10 ng TLseq and log2(IP/input) values from 4sU-seq. Red dots represent significant nascent genes identified in 4sU-seq (IP > input and FDR < 0.05). d Venn plot showing the overlap of nascent genes identified by different methods. e Nucleotide mutation rates analyzed in single morula embryos and pooled morulae (25 embryos per replicate, with three biological replicates). s-1 to s-5 represent single morula embryos 1–5. f Venn plot showing the overlap of nascent genes between single morula and pooled morulae. g Number of nascent genes and total genes identified by scTL-seq in embryos during ZGA. h Principal component analysis (PCA) plot showing the differences in nascent RNAs of ZGA genes (gene set is nascent genes from PN3 to 4C) across embryonic stages. i Pol II binding signals at promoter regions (defined as 2 kb upstream and downstream of TSSs) of nascent genes and non-nascent genes (other) at each stage. j LET-seq defined EU-labeled RNA signals of nascent genes and non-nascent genes (other) at each stage. k Number of nascent TEs and total TEs identified by scTL-seq in embryos during ZGA. l PCA plot showing the differences in nascent RNAs of TE (TE set is nascent TE copies from PN3 to 4C) across embryonic stages. m Pol II binding signals at nascent TE regions and random TE regions (10,000 TEs randomly selected from non-nascent TEs) at each stage. n LET-seq defined EU-labeled RNA signals of nascent TE and random TE (10,000 TEs randomly selected from non-nascent TEs) at each stage. Source data are provided as a Source Data file. For b, e, extremely small P values are reported as P < 2.2 × 10−16 owing to the computational precision limit of R. For b, e, i, j, m, n, P values from two-sided Wilcoxon rank-sum tests are shown, and the exact sample sizes are provided in Supplementary Data 5. In the box plots, the centre line indicates the median; the box bounds represent the first (Q1, 25th percentile) and third (Q3, 75th percentile) quartiles; and the whiskers extend to Q3 + 1.5 × IQR (upper) and Q1 − 1.5 × IQR (lower), where IQR = Q3 − Q1.

Measuring newly made RNA

Studying transcription during the earliest stages of development is difficult because embryos contain large amounts of RNA inherited from the mother. These older RNA molecules can make it difficult to determine which transcripts are being produced by the embryo itself.

scTL-seq was designed to distinguish newly synthesized RNA from existing maternal RNA and to determine which parental copy of a gene produced each transcript.

Using the method, the researchers created a detailed map of gene and transposable element activity during mouse zygotic genome activation. They found that transcription during the pronuclear stage was still largely associated with the maternal genome.

MTA retrotransposons have an important role

One group of transposable elements stood out, Mouse Transcripts type-A, or MTA, elements.

MTA elements were already active in oocytes and remained highly active during the pronuclear stage. When researchers reduced MTA activity, normal zygotic gene activation was disrupted, hundreds of other transposable element families showed altered activity, and embryos stopped developing at the one-cell stage.

These results suggest that MTA activity is not simply a byproduct of early embryonic transcription. Instead, it appears to contribute directly to the regulatory processes required for development to continue.

Transposable elements can help control developmental genes

The researchers also identified 662 chimeric transcripts in which transcription was driven by transposable elements. Some of these events were associated with important developmental genes, including Nfya, Setd2, and Tead4.

This finding highlights one way transposable elements can influence gene regulation. By providing transcriptional starting points or regulatory sequences, they can affect when and how nearby genes are expressed.

The team also identified maternal transcription factors, including NOBOX, as possible regulators of MTA activation.

A clearer view of early embryonic transcription

By focusing specifically on newly synthesized RNA at the single-cell level, scTL-seq provides a more detailed view of transcription during the earliest stages of development.

The results show that MTA retrotransposons are closely integrated with zygotic genome activation and help regulate genes required for normal development. More broadly, the work illustrates how single-cell RNA sequencing can reveal transient regulatory events that are difficult to detect when maternal and newly synthesized RNA are measured together.