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Loss of DUX causes minor defects in zygotic genome activation and is compatible with mouse development

Nature Genetics volume 51, pages 947–951 (2019)Cite this article

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Abstract

How maternal factors in oocytes trigger zygotic genome activation (ZGA) is a long-standing question in developmental biology. Recent studies in 2-cell-like embryonic stem cells (2C-like cells) suggest that transcription factors of the DUX family are key regulators of ZGA in placental mammals1,2. To characterize the role of DUX in ZGA, we generated Dux cluster knockout (KO) mouse lines. Unexpectedly, we found that both Dux zygotic KO (Z-KO) and maternal and zygotic KO (MZ-KO) embryos can survive to adulthood despite showing reduced developmental potential. Furthermore, transcriptome profiling of the MZ-KO embryos revealed that loss of DUX has minimal effects on ZGA and most DUX targets in 2C-like cells are normally activated in MZ-KO embryos. Thus, contrary to the key function of DUX in inducing 2C-like cells, our data indicate that DUX has only a minor role in ZGA and that loss of DUX is compatible with mouse development.

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Fig. 1: Loss of DUX is compatible with mouse development.
Fig. 2: Loss of DUX causes minor defects in ZGA.
Fig. 3: The majority of DUX targets identified in 2C-like cells are activated normally in Dux MZ-KO 2-cell embryos.

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Data availability

All RNA-seq data sets that were generated in this study have been deposited in the Gene Expression Omnibus under accession number GSE121746. Oocyte and 1-cell RNA-seq data were obtained from a previous publication23. HA-DUX ChIP–seq data and Dux overexpression RNA-seq data in mouse ES cells were downloaded from a previous report1.

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Acknowledgements

We would like to thank A. Inoue for training Z.C. to manipulate mouse embryos and for advice on generating the Dux KO mouse lines. We acknowledge X. Fu, C. Zhang, X. Wu and W. Zhang for helpful discussion. We thank N. Djekidel for his advice on bioinformatic analyses. This project was supported by the US National Institutes of Health (NIH) (R01HD092465) and Howard Hughes Medical Institute. Y.Z. is an Investigator of the Howard Hughes Medical Institute.

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Authors and Affiliations

  1. Howard Hughes Medical Institute, Boston Children’s Hospital, Boston, MA, USA

    Zhiyuan Chen & Yi Zhang

  2. Program in Cellular and Molecular Medicine, Boston Children’s Hospital, Boston, MA, USA

    Zhiyuan Chen & Yi Zhang

  3. Division of Hematology/Oncology, Department of Pediatrics, Boston Children’s Hospital, Boston, MA, USA

    Zhiyuan Chen & Yi Zhang

  4. Department of Genetics, Harvard Medical School, Boston, MA, USA

    Yi Zhang

  5. Harvard Stem Cell Institute, Boston, MA, USA

    Yi Zhang

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  1. Zhiyuan Chen
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Contributions

Z.C. and Y.Z. conceived the project. Z.C. designed and performed experiments. Z.C. analyzed sequencing datasets. Z.C. and Y.Z. interpreted the data and wrote the manuscript.

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Correspondence to Yi Zhang.

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Integrated supplementary information

Supplementary Figure 1 Dux zygotic CRISPR-Cas9 injection does not impair mouse pre-implantation development.

a) Schematic view of the genotyping primer design (not drawn in scale). b) Blastocyst rate of embryos following Cas9/sgRNA injection at 1-cell stage. The concentration of Cas9, and sgRNA1/2 are 100, 50, 50 ng/µl, respectively. Numbers in the parentheses are the embryos analyzed for each group. Two independent zygotic CRIPSR-Cas9 injections were performed. BDF1 strain was used for this experiment. c) An agarose gel image illustrating the nested PCR-based genotyping assay of the F0 blastocysts. Genotyping results of 13 embryos are shown in this image. Black and red arrows point to the embryos with mono-allelic and bi-allelic deletion of the Dux cluster, respectively. A full scan of the gel can be found in Supplementary Figure 6. d) Summary of the Dux cluster genotyping results of F0 blastocysts (n = 36).

Supplementary Figure 2 Generation of Dux F0 mice and summary of the genotyping results.

a) Summary of CRISPR/Cas9-mediated Dux cluster KO in mice. b) An agarose gel illustrating the genotyping assay used for Dux mouse colonies. A full scan of the gel can be found in Supplementary Figure 6. c) Summary of the Dux mice crosses. Litter size is presented as mean ± SD.

Supplementary Figure 3 Loss of DUX does not impair ovulation and pre-implantation development.

a) Number of ovulated oocytes and 1-cell embryos (IVF) collected from WT/Het and KO female mice. Each grey dot represents a single female mouse analyzed (n = 9 for each group). Female mice for both groups were subject to superovulation. For 1-cell embryos, oocytes from WT/Het and KO females were fertilized with WT and KO sperm, respectively. Measure of center and error bar indicate mean and SD, respectively. Two-tailed student’s t-test was used to compare the numbers of oocytes and 1-cell embryos between Het/WT and KO groups (oocyte p = 0.97; 1-cell p = 0.85). b) Percentage of IVF-derived embryos that reached the 2-cell (~ 28 hpi), 4-cell (~ 48 hpi), morula (~ 78 hpi), and blastocyst (~ 120 hpi) stages. The IVF experiments were performed four times (denoted as dots) and in total 235 (from 13 mice) and 223 (from 11 mice) embryos were analyzed for control and KO group, respectively. Measure of center and error bar indicate mean and SD, respectively. Fisher’s exact test (two-sided) was used to compare blastocyst stage embryos between the two groups (p = 0.45). c) Representative images of WT/Het and MZ-KO embryos at ~ 120 hpi. The IVF experiments were performed four times. Scale bar, 100 μm.

Supplementary Figure 4 Scatter plots showing high reproducibility of the RNA-seq replicates.

Pearson correlation was indicated for each pair of replicates. Two and three RNA-seq replicates were performed for one-cell and two-cell embryos, respectively.

Supplementary Figure 5 The expression levels of the majority of known DUX targets were not affected by Dux MZ-KO in 2-cell embryos.

a) Scatter plot comparing the gene expression levels between 2C-like (MERVL::GFP-positive) and non-2C cells (MERVL::GFP-negative). Two RNA-seq replicates (Hendrickson P.G. et al., Nat Genet. 49, 941–945, 2017) were used for differential gene expression analyses. Genes expressed higher in 2C-like cells and higher in non-2C cells are colored with red and blue, respectively (FC > 2 and FDR < 0.05). b) Genome browser views illustrating the expression levels of known DUX targets in 2C-like cells and 2-cell embryos. HA-DUX ChIP-seq track and RNA-seq tracks of non-2C (2C-) and 2C-like (2C+) cells were obtained from a previous study (Hendrickson P.G. et al., Nat Genet. 49, 941–945, 2017). Only uniquely aligned reads were used to generate the ChIP-seq and RNA-seq tracks.

Supplementary Figure 6 Full scans of gel images presented in Supplementary Figures 1 and 2.

Black boxes indicate the cropped areas.

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Chen, Z., Zhang, Y. Loss of DUX causes minor defects in zygotic genome activation and is compatible with mouse development. Nat Genet 51, 947–951 (2019). https://doi.org/10.1038/s41588-019-0418-7

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