DUX is a non-essential synchronizer of zygotic genome activation

Abstract

Some of the earliest transcripts produced in fertilized human and mouse oocytes code for DUX, a double homeodomain protein that promotes embryonic genome activation (EGA). Deleting Dux by genome editing at the one- to two-cell stage in the mouse impairs EGA and blastocyst maturation. Here, we demonstrate that mice carrying homozygous Dux deletions display markedly reduced expression of DUX target genes and defects in both pre- and post-implantation development, with, notably, a disruption of the pace of the first few cell divisions and significant rates of late embryonic mortality. However, some Dux^-/- embryos give rise to viable pups, indicating that DUX is important but not strictly essential for embryogenesis.

Keywords: DUX; Embryonic development; Zygotic genome activation.

Fig. 1. DUX promotes embryonic development but is not necessary for it.

(A) Schematics of CRISPR/Cas9 depletion of Dux alleles. sgRNAs targeting the flanking region of the Dux repeat recruit Cas9 nucleases for the excision of the allele. Dux and Gm4981 are two isoforms of the Dux gene repeated in tandem in the Dux locus. Smpdl3a and Gcc2 are the genes flanking the Dux locus. The nucleotide sequence represents the exact junction of deletion determined by Sanger sequence. The sequences in blue and green represent, respectively, the DNA sequence upstream and downstream of the Dux deletion. (B) Generation of Dux^−/− transgenic mice. Zygotes were injected in the pronucleus with plasmids encoding for Cas9 nuclease and the specific sgRNAs, transferred to a pseudopregnant mother and the transgenic pups were finally screened for the null alleles. (C) Expression of Dux normalized to β-actin in testes from adult Dux^+/+ and Dux^−/− mice. (D) WT or Dux KO parents were crossed and litter size was quantified. ***P≤0.001, two-tailed, unpaired t-test. (E) Dux^−/− males and females were bred and the number of born pups was quantified. The same animals were bred again and embryos were quantified at E18.5. *P≤0.05, two-tailed, unpaired t-test. (F) Dux^−/− females were crossed with Dux^−/− or Dux^+/+ males and litter size was quantified. ***P≤0.001, two-tailed, unpaired t-test. (G) Dux^+/+ females were crossed with Dux^−/− or Dux^+/+ males and litter size was quantified. In D-F, horizontal lines represent the average and error bars the s.d.

Fig. 2. Dux promotes both pre-implantation development and later stages.

(A) Zygotes from Dux^+/+ (n=3) or Dux^−/− (n=5) parents were monitored every 12 h for their ability to differentiate ex vivo from E1.5 to E4.5. The average percentage of Dux^+/+ (n=27) or Dux^−/− (n=42) embryos reaching a specific embryonic stage at each time point is represented. (B) Dux^−/− embryos were monitored every hour for 68 h from zygote to morula. The brightfield images represent the unusual transition from 2C to 4C with a 3C intermediate. E3.5 embryos from WT (n=30) or Dux KO (n=28) parents were collected. (C) The average percentage of embryos reaching the late-blastocyst stages (white) or failing to differentiate (delayed embryos, gray; dead embryos, black) was quantified. (D) Brightfield images of the E3.5 embryos.

Fig. 3. Genes and TEs activated by DUX in mESCs are expressed at a low level in 2C embryos depleted of DUX.

(A-C) RNA sequencing analysis of 15 2C embryos generated by mating three Dux^+/− males with three Dux^+/− females. Transcription levels of (A) three alternative transcripts of Dux, (B) 2C-specific genes dependent or not on DUX expression in mESCs compared with a random set of genes, and (C) MERVL-int. In red are the putative Dux^−/− embryos selected for the absence of expression of the three alternative transcripts of Dux. (D-F) RNA sequencing analysis of two embryos generated by mating Dux^+/+ mice and two embryos generated by mating Dux^−/− mice. Transcription levels of (D) three alternative transcripts of Dux, (E) putative DUX-dependent genes compared with a random set of genes, and (F) MERVL-int. Dots in B, C, E and F represents the mean expression (log2 normalized counts) of each gene in all embryos with the same genotype. Box limits, 25th and 75th percentiles; lines in the boxes, median. Whiskers are shown as implemented in the ggplot2 package of R. The upper whisker extends from the hinge to the largest value, no further than 1.5× the interquartile range (IQR) from the hinge. The lower whisker extends from the hinge to the smallest value, at most 1.5× the IQR of the hinge. P-value, two-tailed, unpaired t-test.

Fig. 4. Not all DUX target genes are downregulated in 2C embryos in the absence of DUX.

(A,B) Comparative expression of Dux, early ZGA genes [Zscan4, Eif1a, Usp17la, B020004J07Rik (Rik), Tdpoz4, Cml2, Duxbl, Sp110, Zfp352], a 2C-restricted TE (MERVL), normalized to Zbed3, a gene stably expressed during pre-implantation embryonic development, in 2C-stage embryos derived from (A) Dux^+/− breeding (n=4) or (B) Dux^+/+ (n=2) and Dux^−/− (n=3) breeding. Green and blue dots in A represent the mRNA levels of embryos expressing high or low levels of Dux, respectively. Different shades of green or blue in B represent embryos collected from different mothers (975 and 960 are Dux^+/+ mothers, 965, 992 and 994 are Dux^−/− mothers). Horizontal black lines indicate average. *P≤X.XXX, **P≤0.01, ***P≤0.001, two-tailed, unpaired t-test.