Expression of Dux family genes in early preimplantation embryos

Abstract

After fertilization, the zygotic genome is activated through two phases, minor zygotic activation (ZGA) and major ZGA. Recently, it was suggested that DUX is expressed during minor ZGA and activates some genes during major ZGA. However, it has not been proven that Dux is expressed during minor ZGA and functions to activate major ZGA genes, because there are several Dux paralogs that may be expressed in zygotes instead of Dux. In this study, we found that more than a dozen Dux paralogs, as well as Dux, are expressed during minor ZGA. Overexpression of some of these genes induced increased expression of major ZGA genes. These results suggest that multiple Dux paralogs are expressed to ensure a sufficient amount of functional Dux and its paralogs which are generated during a short period of minor ZGA with a low transcriptional activity. The mechanism by which multiple Dux paralogs are expressed is discussed.

Figure 1

Expression of the Dux family, Zfp352, and Zscan4d during preimplantation development. Fifty MII stage oocytes and embryos at the 1-, 2-(early, mid and late), 4-cell, and morula stages were collected at 13, 16, 24, 32, 40 and 70 h post insemination (hpi), respectively, and subjected to reverse transcription polymerase chain reaction (RT-PCR). Two independent experiments were conducted and similar results were obtained. (A) Electrophoresis images of the Dux family, Zfp352, Zscan4d, and rabbit α-globin (external control) PCR products. (B) The band densities in (A) were quantified using ImageJ. The band densities of the three genes are relative to that of rabbit α-globin. The values at the early 2-cell stage were set to 1 and the relative values were calculated. The average value from two experiments is shown.

Figure 2

Effect of inhibition of DNA replication on the suppression of Dux family expression during the 2-cell stage. Fifty MII stage oocytes and embryos at the 1-, 2-(early, mid and late), 4-cell, and morula stages were collected at 13, 16, 24, 32 and 40 hpi, respectively. The expression levels of Dux family genes were examined by RT-PCR. Rabbit α-globin was used as an external control. The expressions of Dux family genes are relative to that of rabbit α-globin. Gene expression from the 1-cell stage was set to 1 and the relative values were calculated. Two independent experiments were conducted and similar results were obtained. The average value from two experiments is shown.

Figure 3

Effects of Dux family overexpression on the change in gene expression pattern in the early 2- and 4-cell stages. cRNA encoding Dux, DuxG1, DuxG9, or firefly luciferase (Luc, control) were microinjected into a blastomere of a 2-cell stage embryo at 16–20 hpi and cultured in vitro. Cells were collected and RT-PCR was conducted to amplify Dux family, Zfp352, Zscan4d, Galk1, Wsb1 and rabbit α-globin (external control) at 40 hpi. MII stage oocytes and non injected embryos at 16 and 40 hpi were also collected for RT-PCR. Thirty cells were used for each sample. (A) Electrophoresis images of PCR products. (B–F) The band densities in (A) were quantified using image J. The band density of each gene is relative to that of rabbit α-globin. The values at the early 2-cell stage (16 hpi) were set to 1 and the relative values were calculated. Five (Dux family, Zfp352 and Zscan4d) and three (Galk1 and Wsb1) independent experiments were conducted. Error bars represent SE. Asterisks indicate significant difference (p < 0.05) by paired Student t-test.

Figure 4

Effect of overexpression of the Dux family on the development of 2-cell stage embryos. A blastomere of a 2-cell stage embryo was microinjected with cRNA encoding DuxG1 or Dux G9 and then cultured until 40 hpi in vitro. Control embryos were not injected. (A,B) Schematic diagrams of the experiments. Photograph of embryos that were injected with (A) or without (B) DuxG1 or DuxG9 cRNA. Scale bar = 100 μm. (C) The developmental stages of the embryos at 40 hpi. Parentheses indicate the number of embryos observed at 40 hpi. (D) DuxG1 and RFP cRNA were simultaneously microinjected into a blastomere of 2-cell stage embryos and cultured until 45 hpi in vitro. Then the embryos were immunostained with RFP antibody at 45 hpi. DNA was detected by DAPI staining. Scale bar = 20 μm.

Figure 5

DUX4 expression in human FSHD myocytes and Dux family expression in mouse embryos. In human FSHD, only DUX4, which is located at the end of tandem repeats, is expressed in muscle, whereas in early mouse embryos, a large number of Dux paralogs are expressed during minor ZGA. The red and blue triangles represent active and inactive Dux family genes, respectively.