Zscan4: a novel gene expressed exclusively in late 2-cell embryos and embryonic stem cells

Abstract

The first wave of transcription, called zygotic genome activation (ZGA), begins during the 2-cell stage in mouse preimplantation development and marks a vital transition from the maternal genetic to the embryonic genetic program. Utilizing DNA microarray data, we looked for genes that are expressed only during ZGA and found Zscan4, whose expression is restricted to late 2-cell stage embryos. Sequence analysis of genomic DNA and cDNA clones revealed nine paralogous genes tightly clustered in 0.85 Mb on mouse chromosome 7. Three genes are not transcribed and are thus considered pseudogenes. Among the six expressed genes named Zscan4a-Zscan4f, three - Zscan4c, Zscan4d, and Zscan4f - encode full-length ORFs with 506 amino acids. Zscan4d is a predominant transcript at the late 2-cell stage, whereas Zscan4c is a predominant transcript in embryonic stem (ES) cells. No transcripts of any Zscan4 genes are detected in any other cell types. Reduction of Zscan4 transcript levels by siRNAs delays the progression from the 2-cell to the 4-cell stage and produces blastocysts that fail to implant or proliferate in blastocyst outgrowth culture. Zscan4 thus seems to be essential for preimplantation development.

Figure 1

(A) Exon-Intron structures of nine Zscan4 paralogs. New gene symbols we proposed are shown in bold italics with the current gene symbols. (B) Putative protein structures of Zscan4 paralogs. Predicted domains are also shown.

Figure 2

(A) Genome structure of Zscan4 locus (encompassing 850 kb on Chromosome 7). A top panel shows genes near Zscan4 locus. A lower panel shows 9 Zscan4 paralogous genes and their characteristic features. Six other genes (LOCs) are predicted in this region, but unrelated to Zscan4. (B) TaqI-, MspI-, or TaqI/MspI-digested DNA fragment sizes predicted from the genome sequences assembled from individual BAC sequences. (C) Southern blot analysis of C57BL/6J genomic DNAs digested with TaqI, MspI, or TaqI/MspI restriction enzymes. Sizes of all DNA fragments hybridized with a Zscan4 probe (containing only exon 3 from cDNA clone C0348C03) matched with those predicted in (B), validating the manually assembled sequences. (D) Examples of sequence alignments of 9 paralogous copies and corresponding electropherogram obtained by sequencing RT-PCR products made from 2-cell embryos. Electropherograms of the position 1 shows that the expression of Zscan4e is very low, if any, in 2-cell embryos, whereas those of the positions 2 and 3 show that Zscan4d is a predominant transcript. (E) Examples of sequence alignments of 9 paralogous copies and corresponding electropherogram obtained by sequencing RT-PCR products made from ES cells. Electropherogram of the position 1 shows that the expression of Zscan4d is very low, if any, in ES cells, whereas electropherogram of the position 2 shows that Zscan4a is expressed at some level and either Zscan4b or Zscan4e (or both) is expressed in ES cells. Electropherogram of the position 3 shows that Zscan4a is expressed in ES cells.

Figure 3

(A) Expression profile of Zscan4 during preimplantation development by whole mount in situ hybridization (WISH). Hybridizations were performed simultaneously under the same experimental conditions for all preimplantation developmental stages. Photos were taken at 200x magnification using phase contrast. Zscan4 shows a transient and high expression in the late 2-cell embryos. Such a high level of expression was not observed in 3-cell (two examples indicated by red arrows) and 4-cell embryos. (B) Expression profile of Zscan4 during preimplantation development by qRT-PCR analysis. Three sets of 10 pooled embryos were collected from each stage (O, oocyte; 1, 1-cell embryo; E2, early 2-cell embryo; L2, late 2-cell embryo; 4, 4-cell embryo; 8, 8-cell embryo; M, morula; and B, blastocyst) and used for qRT-PCR analysis. The expression levels of Zscan4 were normalized by those of Chuk control, and then the averaged expressions at each stage were represented as a fold change compared to the expression level in oocytes.

Figure 4

(A) Three types of siRNA technologies used for the analysis of Zscan4 in preimplantation embryos and their target sequences. (B) The locations of siRNA target sequences in the Zscan4 cDNA. (C) Development of shZscan4-injected embryos. The morphology of representative embryos is shown. Stages of shZscan4-injected and shControl-injected embryos were assessed at 61 hrs, 80 hrs, 98 hrs, and 108 hrs post-hCG injections. (D) Development of shZscan4-injected embryos. shZscan4-injected (gray bars) and shControl-injected (white bars) embryos were staged and counted at 61 hrs, 80 hrs, 98 hrs, and 108 hrs post-hCG injections. M, morula; B, blastocysts. (E) Transcript levels of Zscan4 in shControl-injected and shZscan4-injected 2-cell embryos by qRT-PCR analysis. The expression levels were normalized by Eef1a1.

Figure 5

Development of embryos received shZscan4-injection in the nucleus of one blastomere of early 2-cell embryos. (A-C) The stages of shZscan4- (gray) and shControl- (white) microinjected embryos were assessed at 52 hrs, 74 hrs, and 96 hrs post-hCG injections. (D) The 3-cell embryos had one blastomere remained as a 2-cell stage blastomere size and two smaller blastomeres with the size of 4-cell stage blastomeres. (E) This panel shows 5-cell embryos with one delayed blastomere and four smaller blastomeres with the size of 8-cell blastomeres. These embryos eventually formed blastocyst-like structures (F), but they seemed to be the mixtures of blastocyst-like cell mass and morula-like cell mass. The morula-like cell mass developed from one blastomere received shZscan4 injection, as shown by the presence of GFP, which was carried in the shZscan4 plasmid (G). Magnification: 200 x.

Figure 6

(A) Expressions of Zscan4 and Pou5f1 in the blastocysts, blastocyst outgrowth, and ES cells by the whole mount in situ hybridization. (B) A schematic illustration of the Zscan4 expression patterns.