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. Author manuscript; available in PMC: 2007 Sep 27.
Published in final edited form as: Dev Biol. 2007 May 8;307(2):539–550. doi: 10.1016/j.ydbio.2007.05.003

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

Geppino Falco 1,2, Sung-Lim Lee 1,2, Ilaria Stanghellini 1, Uwem C Bassey 1, Toshio Hamatani 1,3, Minoru S H Ko 1,*
PMCID: PMC1994725  NIHMSID: NIHMS27351  PMID: 17553482

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.

Keywords: Preimplantation embryos, ES cells, Expression Profiling, Zygotic Genome Activation, Stage-specific gene expression, Paralogs, mouse genome

Introduction

After fertilization, the maternal genetic program governed by maternally stored RNAs and proteins must be switched to the embryonic genetic program governed by de novo transcription, called zygotic genome activation (ZGA) (DePamphilis et al., 2002; Latham and Schultz, 2001). ZGA is one of the first and most critical events in animal development. Earlier reports have established that ZGA begins during the 1-cell stage by BrUTP incorporation assays (Aoki et al., 1997) and expression assays of plasmid-borne reporter gene (Nothias et al., 1995; Ram and Schultz, 1993). However, global gene expression profiling by DNA microarrays has recently revealed that nearly all genes identified for their increase of expression at the 1-cell stage were insensitive to inhibition by alpha-amanitin, which blocks RNA polymerase II (Hamatani et al., 2004a; Zeng and Schultz, 2005). Therefore, de novo transcription of zygotic genome seems to begin during the 2-cell stage of mouse development (Hamatani et al., 2004a; Zeng and Schultz, 2005). Furthermore, the major burst of ZGA occurs only in the late 2-cell stage (Hamatani et al., 2004a).

Arrest of development at the 2-cell stage has been reported for loss-of-function mutants in Mater/Nalp5 (Tong et al., 2000), Mhr6a/Ube2a (Roest et al., 2004), and Brg1/Smarca4 (Bultman et al., 2006). Although the timing of the developmental arrest coincides with that of the ZGA, these genes are expressed during oogenesis and stored in oocytes, and are not themselves transcribed in the 2-cell stage. Therefore, these maternal effect genes are not suitable for the study of the ZGA. Previously, ZGA has been studied using either exogenous plasmid-borne reporter genes (Nothias et al., 1995) or endogenous but rather ubiquitously expressed genes such as Hsp70.1 (Christians et al., 1995), eIF-4C (Davis et al., 1996), and Xist (Zuccotti et al., 2002) (see (DePamphilis et al., 2002) for additional genes). Although TEAD-2/TEF-4 (Kaneko et al., 1997) and Pou5f1/Oct4 (Palmieri et al., 1994) are considered as transcription factors selectively expressed at ZGA (DePamphilis et al., 2002), these genes are known to be expressed in cells other than 2-cell embryos. It is thus important to identify and study individual ZGA genes, especially any expressed exclusively at the 2-cell stage.

Large-scale EST projects (Ko et al., 2000; Okazaki et al., 2002; Solter et al., 2002) and DNA microarray studies (Hamatani et al., 2004a; Wang et al., 2004; Wang et al., 2005; Zeng et al., 2004) have revealed many novel genes expressed during ZGA. Here, we mined these data to identify a novel gene, Zscan4, which encodes a SCAN domain and four zinc finger domains. Zscan4 is the first example of a gene that is expressed exclusively in late 2-cell embryos and embryonic stem (ES) cells. Loss-of-function study by siRNA technology indicates the important function of Zscan4 genes for the progression from 2- to 4-cell stages and subsequent embryo development.

Results

Identification of 2-cell-specific genes during preimplantation development

Previously we carried out global gene expression profiling of preimplantation embryos and identified a group of genes that showed transient spike-like expression in the 2-cell embryo (Hamatani et al., 2004a). By examining the expression of these genes in the public expressed sequence tag (EST) database (NCBI/NIH; Wheeler et al., 2007), we found a novel gene represented by only 29 cDNA clones out of 4.7 million mouse ESTs. These cDNA clones have been isolated from cDNA libraries derived from ES cells and preimplantation embryos (Supplemental Fig. S1). Furthermore, our previous DNA microarray data showed that the expression of this gene is detected in ES cells but not in embryonal carcinoma (EC) cells (F9 and P19), trophoblast stem (TS) cells, or neural stem/progenitor (NS) cells (Aiba et al., 2006).

Structures and expressions of Zscan4 paralogous genes

One cDNA clone produced and sequenced in our large-scale mouse EST project (clone number C0348C03; (Sharov et al., 2003)) was completely sequenced by the Mammalian Gene Collection (MGC) project (Gen Bank Accession number BC050218; (Gerhard et al., 2004)). The full-length cDNA sequence (BC050218) of 2292 bp was organized into 4 exons, encoding a protein of 506 amino acids (a.a.) (Fig. 1A). Because this cDNA clone was isolated from a cDNA library made from ES cells (Sharov et al., 2003), we isolated another cDNA clone by performing RT-PCR on RNAs isolated from late 2 cell-stage embryos and sequenced it (Genbank accession number: EF143406). This 2268 bp cDNA clone also encoded a protein of 506 amino acids. However, DNA and protein sequences clearly showed that these two cDNAs (BC050218; EF143406) were two different genes with close similarity. Domain prediction analysis (Letunic et al., 2006; http://smart.embl.de/) revealed a SCAN (Leucine Rich Element) domain and four zinc finger domains at the N- and C-terminals, respectively (Fig. 1B). By searching the UCSC Genome Browser (Kuhn et al., 2007), we also found a hypothetical human ortholog - zinc finger and SCAN domain containing 4 (ZSCAN4) that shares 45% of amino acid sequence similarity with the high conservation in SCAN (50%) and zinc finger domains (59%) (Supplemental Fig. S2).

Figure 1.

Figure 1

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(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.

Alignment of full-length cDNA sequences (BC050218 and EF143406) to the mouse genome sequence (Kuhn et al., 2007; Waterston et al., 2002; Wheeler et al., 2007) revealed multiple hits in the proximal region of Chromosome 7 – the region syntenic with human ZSCAN4 (Supplemental Fig. S3). One notable feature of this genome region is the repetition of a similar sequence segment. In fact, the sequences of each copy of Zscan4 and its environs were very similar one to another, leaving the assembled genome sequences of this region less accurate than other regions. To understand the genomic structure of this region better, we manually reassembled individual BAC clone sequences from this region into ∼850 kb of genome sequence contigs (Fig. 2A). Because it was difficult to find a hybridization probe or oligonucleotides to distinguish each copy, we relied on restriction enzymes to distinguish small sequence differences among gene copies. We carried out Southern blot analysis by digesting C57BL/6J mouse genomic DNAs with TaqI alone, MspI alone, or TaqI/MspI (Fig. 2B and C). The detected DNA fragments confirmed the presence of 9 paralogous Zscan4 genes predicted from the assembled genome sequences.

Figure 2.

Figure 2

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(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.

We then aligned the full-length cDNA sequence (BC050218) to the assembled genome sequence and found 9 gene copies, all of which had a multiexon gene organization (Fig. 1, 2A). Because we found no evidence that they were transcribed based on available EST information and sequencing analysis of RT-PCR products, three gene copies appeared to be pseudogenes (see below for more details). Therefore, we named them Zscan4-ps1, Zscan4-ps2, and Zscan4-ps3, according to the conventions of mouse gene nomenclature. Because the remaining 6 gene copies were transcribed and encoded ORFs, we named them Zscan4a, Zscan4b, Zscan4c, Zscan4d, Zscan4e, and Zscan4f. Three genes - Zscan4a, Zscan4b, and Zscan4e – encoded ORFs with 360 a.a., 195 a.a., and 195 a.a., respectively, which included the SCAN domain but not all four zinc finger domains (Fig. 1B).

Three genes - Zscan4c, Zscan4d, and Zscan4f - encoded full-length ORFs (506 a.a.) with > 94% similarities (see Supplement 2). The main features of these genes are summarized in Fig. 2A. Zscan4c corresponds to the cDNA clone isolated from ES cells (C0348C03; BC050218; Gm397). Zscan4d corresponds to the cDNA clone isolated from 2-cell embryos (EF143406). Zscan4f corresponds to a gene predicted from the genome sequence (XM_145358). Similarities of both ORFs and mRNAs between these three genes were very high (Supplemental Fig. S2). It is thus most likely that these three genes have the same function. To measure the expression levels of each paralog, DNA sequences of the nine Zscan4 paralogs were analyzed by the Clustal X multiple-sequence alignment program, which showed the presence of sequence differences specific to each paralog (data not shown). To examine the expression levels of each gene in 2-cell embryos and ES cells, we sequenced cDNA fragments amplified by RT-PCR from 2-cell embryos (Supplemental Fig. 4) and ES cells (Supplemental Fig. 5). We estimated the expression level of each paralog based on the amplitudes of each nucleotide at polymorphic sites (see Fig. 2D for examples of 2-cell embryos, Fig. 2E for examples of ES cells, Materials and Methods for details). The results are summarized in Fig. 2A. In 2-cell embryos Zscan4d was a predominant transcript (90%). In contrast, in ES cells Zscan4c was a predominant transcript (40%), although Zscan4f showed fewer but significant numbers of transcripts (24%). These results were consistent with the origin of each cDNA clone: Zscan4c cDNA clone from the ES cell cDNA library and Zscan4d cDNA clone from the 2-cell embryo library.

Analysis of Zscan4 expression in preimplantation embryos using Whole Mount In Situ Hybridization (WISH)

Whole mount in situ hybridization (WISH) using the cDNA clone (C0348C03) as a probe detected high-level transcripts in late 2-cell embryos (Fig. 3A). The transcript was not detected in unfertilized eggs and embryos in other preimplantation stages, including 3-cell embryos, suggesting a high specificity of gene expression at the late 2-cell stage and a relatively short half-life of the transcripts. Quantitative reverse-transcriptase PCR (qRT-PCR) analysis confirmed the WISH results (Fig. 3B). Previous microarray analyses showed that the expression of this gene at the late 2-cell stage was suppressed in embryos treated with α-amanitin (a blocker of RNA pol II-based transcription) (Hamatani et al., 2004a), confirming that this gene is transcribed de novo during the major burst of ZGA. The transient expression pattern was observed both in in vitro cultured embryos and in freshly isolated in vivo embryos (Hamatani et al., 2004a). Although individual genes among the 9 paralogous Zscan4 genes could not be distinguished by WISH and qRT-PCR, the expression is mainly Zscan4d, because 90% of transcripts in 2-cell embryos were attributed to Zscan4d (Fig. 2A).

Figure 3.

Figure 3

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(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.

Function of Zscan4 in preimplantation development

As a first step to characterize the function of Zscan4 genes, we focused on preimplantation development. We initially explored standard gene targeting, but found it difficult for three reasons. First, sequences of Zscan4 paralogs and surrounding genomic regions are too similar to design targeting constructs for specific genes. Second, it is highly likely that a Zscan4d−/− phenotype can be compensated functionally by other Zscan4 paralogs, because in addition to predominantly-expressed Zscan4d, at least 3 other similar copies (Zscan4a, Zscan4e, and Zscan4f) were also transcribed in 2-cell embryos. Third, the presence of other predicted genes, though not annotated as genes yet, within the ∼850 kb Zscan4 locus makes it difficult to delete the entire Zscan4 locus without interfering with other genes. Therefore, we decided to employ RNA interference technology. Although RNAi and siRNA technology has been successfully used to block the expression of specific genes in preimplantation embryos (Kim et al., 2002; Stein et al., 2005), widely recognized off-target effects are generally a major concern (Jackson et al., 2006; Scacheri et al., 2004; Semizarov et al., 2003). To increase the confidence in effects of Zscan4 knockdown, we used three independent RNA interference technologies: an oligonucleotide-based siRNA (denoted here siZscan4 and obtained from Invitrogen), a vector-based shRNA (denoted here shZscan4 and obtained from Genscript), and a mixture of oligonucleotide siRNAs (denoted here plus-siZscan4 and obtained from Dharmacon) (Fig. 4A, B). Oligonucleotide sequences used for siZscan4, shZscan4, plus-siZscan4 matched 100% with cDNA sequences of Zscan4a, Zscan4b, Zscan4c, Zscan4d, Zscan4e, and Zscan4f, except for shZscan4, which had 2 bp mismatches with Zscan4b and Zscan4e (Fig. 4A, B).

Figure 4.

Figure 4

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(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.

We microinjected an shZscan4 vector into the male pronucleus of zygotes at 21-23 hours after the hCG injection and followed embryos during preimplantation development (Fig. 4C and D). At 61 hours post-hCG, when the majority (58.8%) of shControl-injected embryos already reached the 4-cell stage, the majority (78.8%) of shZscan4-injected embryos remained at the 2-cell stage. By 98 hours post-hCG, when the majority (70.0%) of shControl-injected embryos reached blastocyst stage, the majority (52.5%) of shZscan4-injected embryos reached only morula stage. Significant reduction (∼95%) of Zscan4 RNA levels was confirmed by qRT-PCR analysis (Fig. 4E). Taken together, these results indicate that the development of shZscan4-injected embryos was delayed for about 24 hrs between the 2- and 4-cell stages, followed by progression to later stages at a speed comparable to that of shControl-injected embryos. Essentially the same results were obtained using two different siRNA technologies: siZscan4 (Supplemental Fig. S6) and plus-siZscan4 (Supplemental Fig. S7).

We noticed that siZscan4-injected embryos formed normal looking blastocysts at 3.5 days post coitum (d.p.c.), but often failed to become expanded and hatched blastocysts at 4.5 d.p.c. For example, 45% of siZscan4-injected embryos failed to hatch, but only 6% of siControl-injected embryos failed to hatch (Supplemental Fig. S6B). To test whether these blastocysts were compromised even at 3.5 d.p.c., we transferred shZscan4-injected blastocysts to the uterus of pseudopregnant mice and found that none of the shZscan4-injected blastocysts implanted successfully, whereas most shControl-injected embryos implanted (Table 1). We also carried out in vitro blastocyst outgrowth experiments and found that cells of shZscan4-injected blastocysts failed to proliferate in in vitro culture (Table 1). These results demonstrated that transient expression of Zscan4 at the late 2-cell stage is important for the development of functional blastocysts.

Table 1.

Blastocyst outgrowth (A) and post-implantation development (B) of embryos received pronuclear injection of shZscan4 or shControl

A. Blastocyst Outgrowth No. of tested blastocysts No. of successful outgrowth
shZscan4 16 0
shControl 17 7
B. Embryo Transfer No. of blastocysts transferred to pseudopregnant mother No. of pups born
shZscan4 8 0
shControl 10 4
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Note. An shZscan4 or shControl vector was microinjected into the male pronucleus of zygotes at 21-23 hours after the hCG injection. Early blastocysts (3.5 d.p.c.) formed from these embryos were subjected to tests of blastocysts outgrowth (A) and embryo transfer (B). In the outgrowth assay, the presence of proliferating cells after 6 days in culture was considered to be successful outgrowth (see Figure 6 for example of successful outgrowth and see Materials and Methods for details of experimental procedures).

The notion that the reduction of Zscan4 expression level delays the development of preimplantation embryos at the 2-cell stage was further supported by the fact that when shZscan4 was injected into one of the blastomeres of early 2-cell stage embryos, ∼28% of embryos became 3-cell embryos (Fig. 5A). One blastomere that received shZscan4 by injection remained a 2-cell blastomere, whereas the other blastomere cleaved into two smaller blastomeres with the size of 4-cell blastomeres (Fig. 5D). Subsequently, these embryos (24%) became unevenly cleaved embryos, typically 5-cell embryos, with one 2-cell-sized blastomere and four 8-cell-sized blastomeres (Figure 5B, E). These embryos eventually formed blastocyst-like structures, but they seemed to be mixtures of blastocyst-like cell mass and morula-like cell mass (Fig. 5C, F). Because an shZscan4 plasmid contains a GFP-expression unit, the fate of a blastomere received an shZscan4 by injection can be traced by the presence of a GFP. As expected, morula-like cell mass was often GFP-positive (Fig. 5G), indicating about 1 day-delay in development of an shZscan4-injected blastomere. In contrast, when shControl was injected into one of the blastomeres at the early 2-cell stage, nearly all embryos cleaved normally (Fig. 5A, B, C).

Figure 5.

Figure 5

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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.

To investigate the effect of prolonged Zscan4d expression on preimplantation development, we overexpressed Zscan4d by microinjecting a Zscan4d-expressing plasmid into the male pronucleus of zygotes. Although the Zscan4d plasmid-injected embryos showed a rate of development similar to control plasmid-injected embryos, the blastocysts failed to accomplish outgrowth (Table 2A) and failed to implant (Table 2B). The results suggest that timely downregulation of Zscan4d is also important for the proper development of blastocysts.

Table 2.

Blastocyst outgrowth (A) and post-implantation development (B) of embryos received pronuclear injection of a Zscan4d-expressing plasmid or a control plasmid

A. Blastocyst Outgrowth No. of tested blastocysts No. of successful outgrowth
Zscan4d-expressing plasmid 10 2
Control plasmid 15 11
B. Embryo Transfer No. of blastocysts transferred to pseudopregnant mother No. of pups
Zscan4d-expressing plasmid 10 0
Control plasmid 14 5
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Note. A plasmid vector expressing constitutively Zscan4d gene or control empty vector was microinjected into the male pronucleus of zygotes at 21-23 hours after the hCG injection. Early blastocysts (3.5 d.p.c.) formed from these embryos were subjected to the same tests as described in Table 1.

Analysis of Zscan4 expression in ES cells using Whole Mount In Situ Hybridization (WISH)

One intriguing aspect of the expression pattern of Zscan4 is its exclusive expression in late 2-cell embryos and ES cells. This seems counterintuitive, because ES cells are derived from the ICM and many genes that are expressed in ES cells are also expressed in the ICM (e.g., (Yoshikawa et al., 2006)). We therefore examined the expression of Zscan4 in blastocysts, blastocyst outgrowth, and ES cells using the WISH. We confirmed that expression of Zscan4 was not detected anywhere in blastocysts, including the ICM and the early blastocyst outgrowth (Fig. 6A). However, the expression of Zscan4 began to be detected in a small fraction of cells by the day 6 of the outgrowth. Unexpectedly, the strong expression of Zscan4 was detected in only a small fraction of ES cells in undifferentiated colonies. In contrast, the expression of Pou5f1 (Oct3/4) – well-known marker for pluripotency – was detected in the ICM of blastocysts, in a large fraction of cells in the blastocyst outgrowth, and in the majority of ES cells in undifferentiated colonies (Fig. 6A). Because of the close similarity of cDNA sequences, we could not distinguish Zscan4 paralogs by WISH, but expression analysis by sequencing RT-PCR products mentioned above indicates that Zscan4c and Zscan4f were the genes detected in the subpopulation of cells in blastocyst outgrowth and ES cells by WISH.

Figure 6.

Figure 6

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(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.

Discussion

We report the identification and analysis of Zscan4, which shows transient and specific expression at the late 2-cell stage, as summarized in Fig. 6B. To the best of our knowledge, this is the only gene that is expressed uniquely during the first wave of de novo transcription – ZGA. Therefore, the gene provides a potentially useful marker for ZGA and facilitates further studies of ZGA.

While this manuscript was being prepared, 3-4-cell specific expression of a Zscan4-like gene was reported (Zhang et al., 2006). Although the Genbank accession numbers (XM_142517, XM_159658, XM_142890; representative transcript AK141250) were listed for Zscan4-like gene, in the latest mouse genome sequences, the XM_142517 was changed to Gm397 (Zscan4c in our nomenclature), the XM_159658 and XM_142890 were removed, and AK141250 showed the best match to LOC632758 (Zscan4-ps3 in our nomenclature). The authors have reported only three zinc finger domains for Zscan4-like gene and 3-4-cell specific expression (Zhang et al., 2006). In contrast, our data show four zinc finger domains and late 2-cell specific expression. The reason for the discrepancy is unclear at this point.

Because of the repetitive content of the Zscan4 locus, with 9 paralogous genes, we have employed siRNA technologies to examine the loss-of-function phenotype of Zscan4 genes. The siRNA studies indicate that Zscan4 transcripts are required for the transition from 2-cell embryos to 4-cell embryos. The reduction of Zscan4 transcript levels by siRNAs causes transient developmental arrest at the 2-cell stage for ∼24 hrs. Embryos then resume their development and reach the blastocyst stage. However, these blastocysts are not functionally normal, based on the following observations: (i) the blastocysts failed to expand; (ii) blastocyst outgrowth did not proliferate in in vitro culture; and (iii) the blastocysts failed to implant. The reason for the resumption of development after the transient 2-cell arrest is not clear at this point. It could be caused by the incomplete repression of Zscan4 expression by siRNA methods. In fact, when 2-cell arrest occurs in mutant embryos with the disruption of maternal Mater/Nalp5 (Tong et al., 2000), Mhr6a/Ube2a (Roest et al., 2004), and Brg1/Smarca4 (Bultman et al., 2006), it is a terminal phenotype, and mutant embryos do not resume development. On the other hand, the disruption of maternal genes does not necessarily cause 2-cell arrest: loss of some genes leads to arrest at the 1-cell stage (Npm2 (Burns et al., 2003), Zar1 (Wu et al., 2003), Hsf1 (Christians et al., 2000)); and others, to arrest at later preimplantation stages (Stella (Bortvin et al., 2004; Payer et al., 2003), Pms2 (Gurtu et al., 2002)).

Although we have shown the importance of Zscan4 in the progression of 2-cell to 4-cell embryos, the mechanism of Zscan4 function remains unknown. The structural information of a protein sometimes provides hints about mechanism. The SCAN domain of ZSCAN4 is a highly conserved motif that is specific to vertebrate (Edelstein and Collins, 2005). The C2H2 zinc finger domains have a primary role in binding to specific DNA segments and interacting with other cellular factors to control the transcription of target genes. Although the function of most SCAN-ZFPs is unknown, some of these proteins have been implicated in the transcriptional regulation of growth factors as well as of other genes involved in cell survival and differentiation (Edelstein and Collins, 2005). It is thus possible that Zscan4 is also a transcription factor, and if so, it may be a key regulator involved in the waves of gene activation during preimplantation development (Hamatani et al., 2004a).

Human ortholog ZSCAN4 exists as a single copy gene, whereas mouse Zscan4 has expanded to 9 paralogous copies, three of which encode proteins with the same amino acids length. As pointed out in the review of SCAN gene family members (Edelstein and Collins, 2005), Zscan4 paralogs are located within the pericentromeric region, which frequently undergoes rapid gene duplications and often carries olfactory receptors (Young et al., 2005). Indeed, some olfactory receptors are located in Zscan4 paralogs region (Fig. 2A). Similarly, several Nalp gene family members, which show maternal expression patterns and play an essential role during preimplantation development (Hamatani et al., 2004b), are also located in this region (Fig. 2A). It is important to point out that Zscan4a, Zscan4b, and Zscan4e encode truncated proteins, but are also expressed at a very low level in late 2-cell embryos and ES cells. The function of these truncated proteins, if any, remains to be investigated.

Interestingly, Zscan4 shows a peculiar mosaic expression in ES cells: only a few percent of ES cells in undifferentiated colonies express Zscan4 (Fig. 6A, B). However, the ICM – the putative in vivo counterpart of ES cells – does not express Zscan4. Therefore, the expression of Zscan4d is turned off after the late 2-cell stage, and Zscan4c (and to some extent Zscan4f) is turned on during blastocyst outgrowth for ES cell derivation. Zscan4 seems likely to play an important role in the in vitro derivation of ES cells from the ICM.

Materials and Methods

Identification and cloning of the mouse Zscan4d gene

Using our DNA microarray data of preimplantation embryos (Hamatani et al., 2004a), we identified a Zscan4d gene for its specific expression in 2-cell embryos and found a corresponding cDNA clone (no. C0348C03; BC050218; R1 ES cells, 129 strain) in our mouse cDNA collection (Sharov et al., 2003). Based on this full-length cDNA sequence, we designed a primer pair (5′-cctccctgggcttcttggcat-3′; 5′-agctgccaaccagaaagacactgt-3′), which was used to PCR-amplify the full-length cDNA sequence of this gene from 2-cell embryos (B6D2F1 mouse). In brief, mRNA was extracted from 2-cell embryos and treated with DNAase (DNA-free, Ambion). The mRNA was annealed with an oligo-dT primer and reverse-transcribed into cDNA with ThermoScript Reverse Transcriptase (Invitrogen). A full-length cDNA clone was PCR-amplified with Ex Taq Polymerase (Takara Mirus Bio, Madison, WI), purified with the Wizard SV Gel and PCR Clean-Up System (Promega Biosciences, San Luis Obispo, CA), cloned into a pENTR plasmid vector with the Directional TOPO Cloning Kit (Invitrogen), and completely sequenced using BigDye Terminator kit (PE Applied Biosystems, Foster City, CA) and DyeEX 96 Kit (Qiagen Valencia, CA) on ABI 3100 Genetic Analyzer (PE Applied Biosystems). The sequence was submitted to Genbank (accession number: EF143406).

We used the WU-BLAST (http://www.ensembl.org/Multi/blastview) and UCSC genome browser to obtain Zscan4 orthologs in the human genome sequence. Open reading frames (ORF) were deduced by ORF finder (http://www.ncbi.nlm.nih.gov/gorf/gorf.html) and protein domains were identified by Pfam HMM database (http://pfam.wustl.edu/hmmsearch.shtml). Orthologous relationships were assessed with the phylogenetic tree of amino acid sequences determined by a sequence distance method and the Neighbor Joining (NJ) algorithm (Saitou and Nei, 1987) using Vector NTI software (Invitrogen, Carlsbad, CA).

All gene names and gene symbols were consulted with and approved by the mouse gene nomenclature committee (personal communication by Dr. Lois Maltais at The Jackson Laboratory, Bar Harbor, Maine, USA).

Southern blot analysis

Southern blot analysis was carried out to validate the genome sequence of Zscan4 locus assembled using individual BAC clone sequences downloaded from the public database (RPCI-23 and RPCI-24 BAC libraries: C57BL/6J strain). A probe, containing the exon 3, was designed and amplified from mouse DNA extracted from ES cells (C57BL/6) using a primer pair (5′-gcattcctacataccaatta-3′; 5′-gatttaatttagctgggctg-3′). The PCR product was purified using GFX PCR DNA and Gel band purification kit (GE Healthcare). Fifteen μg of mouse genomic DNAs extracted from ES cells (BL6.9 line derived from C57BL/6 strain) were digested overnight with restriction enzymes (MspI, TaqI, and MspI/TaqI, see Fig. 2B), fractionated on a 1% (w/v) agarose gel, transferred and immobilized onto nitrocellulose membranes. Blots were hybridized with random-primed 32P-labeled DNA probes under the standard condition. Membranes were subjected to 3x washes of 30 min each (2xSSC/0.1% (w/v) SDS at room temperature, 0.5xSSC/0.1% (w/v) SDS at 42°C, and 0.1xSSC/0.1% (w/v) SDS at room temperature) and autoradiographed for 48 hours at −80°C.

Measurement of gene expression levels

To measure the transcript levels of very similar mRNA species, we employed a well-established method that utilizes DNA sequence traces (Qiu et al., 2003). First, we synthesized cDNAs from ES cells (129.3 ES cells purchased from the Transgenic Core Laboratory of the Johns Hopkins University School of Medicine, Baltimore, MD) and 2-cell embryos (B6D2F1 mice). We then amplified Zscan4 cDNA fragments by using Zscan4-specific primer pair (Zscan4For:5′-cagatgccagtagacaccac-3′; Zscan4Rev5′-gtagatgttccttgacttgc-3′), which 100%-matched to all Zscan4 paralogs. These cDNA fragments were sequenced by using the following primers: Zscan4_For, 5′-cagatgccagtagacaccac-3′; Zscan4_400Rev, 5′-ggaagtgttatagcaattgttc-3′; Zscan4Rev, 5′-gtagatgttccttgacttgc-3′; and Zscan4_300Rev, 5′-gtgttatagcaattgttcttg-3′. Electropherograms of these sequence reads, all of which are shown in Supplemental Fig. 4 and 5, were used to calculate the relative expression levels of 9 paralogous copies of Zscan4 in the following manner. Based on sequence information of transcripts (either predicted from the genome sequence or determined by sequencing cDNA clones), we first identified nucleotide positions, where one or a few paralogous copies can be distinguished based on the nucleotide mismatches. We then used phred base calling program (version 0.020425.c (Ewing et al., 1998)) and obtained the amplitudes of all 4 bases in the electropherogram for those nucleotide sites. After subtracting the noise level (i.e., the average of amplitudes of the bases that are not present in any of the 9 paralogous copies), the amplitudes of each base (A, T, G, C) were obtained. The expression levels of each paralogous copies were calculated by the least square fitting, which found the expression levels that are most consistent with all mismatched nucleotide positions.

Collection and manipulation of embryos

Four- to six-week old B6D2F1 mice were superovulated by injecting 5 IU pregnant mare serum gonadotropin (PMS; Sigma, St Louis, MO, USA) and 5 IU human chorionic gonadotropin (HCG; Sigma) after 46–47 h (Protocol#220MSK-Mi approved by the National Institute on Aging Animal Care and Use Committee). Unfertilized eggs were harvested at 21 h post-HCG with the standard method (Nagy et al., 2003). After removing cumulus cells by incubation in M2 medium (MR-015-D) supplemented with bovine testicular hyaluronidase (HY, 0.1% (w/v), 300 Umg-1), unfertilized eggs were thoroughly washed, selected for good morphology, and collected. Fertilized eggs (1-cell embryos) were also harvested from mated superovulated mice in the same way as unfertilized eggs. Fertilized eggs (1-cell embryos) were cultured in synthetic oviductal medium enriched with potassium (KSOMaa MR-121-D) at 37°C in an atmosphere of 5% CO2. For the embryo transfer procedure, we transferred 3.5 d.p.c. blastocysts into the uteri of 2.5 d.p.c. pseudopregnant ICR female mice.

To synchronize in vitro embryo development, we first selected embryos with two pronuclei (PN). When some of these 1-cell stage embryos started to cleave, we selected these early 2-cell stage embryos and transferred them to another microdrop culture. We cultured these early 2-cell stage embryos until some of them started 2nd cleavage and collected the embryos that are still at the 2-cell stage. These embryos are synchronized at the late 2-cell stage.

DNAs were microinjected into embryos in the following manners.

(1) Pronuclear injection: Plasmid vectors expressing constitutively an siRNA against mouse Zscan4 were constructed by inserting the following 21-mer target sequences in a pRNAT-U6.1/Neo vector (GenScript Corp., Scotch Plains, NJ, USA): shZscan4, gagtgaattgctttgtgtc; and siControl (randomized 21-mer), agagacatagaatcgcacgca. This vector contains a green fluorescence protein (GFP) marker under a cytomegalovirus (CMV) promoter. For RNA interference experiments, 1-2 pl (2-3 ng/μl) of a linearized vector DNA (shZscan4 or shControl) was microinjected into either the male pronucleus of zygotes or the nucleus of a single blastomere in 2-cell stage embryos. A plasmid vector expressing constitutively Zscan4d gene was constructed by cloning the CDS of Zscan4d into a plasmid pPyCAGIP (Chambers et al., 2003). For overexpression experiments, 1-2 pl (2-3 ng/l) of plasmid DNA (Zscan4d-inserted or no insert pPyCAGIP vector) linearized by ScaI was microinjected into the male pronucleus of zygotes.

(2) Cytoplasmic injection: Transient RNA interference experiments were carried out by microinjecting ∼10 pl (5 ng/μl) of oligonucleotides (siZscan4, plus-siZscan4, and siControl) into the cytoplasm of zygotes. The optimal amount of siRNAs was determined by testing different concentrations of siRNAs (4, 20, and 100 ng/μl).

All siRNAs were resuspended and diluted with the microinjection buffer (Specialty Media). The transfer of cultured blastocysts into pseudopregnant recipients was done according to the standard protocol (Nagy et al., 2003). All media were purchased from the Specialty Media (Phillipsburg, NJ).

Culture of ES cells and blastocyst outgrowth

A mouse ES cell line (129.3 line derived from strain 129 and purchased from The Transgenic Core Laboratory of the Johns Hopkins University School of Medicine, Baltimore, MD, USA) was first cultured for two passages into gelatin-coated culture dish in the presence of leukemia inhibitory factor (LIF) to remove contaminating feeder cells. Cells were then seeded on gelatin coated 6-well plates at the density of 1-2 × 105/well (1-2 × 104/cm2) and cultured for 3 days with the complete ES medium: DMEM, 15% FBS; 1000 U/ml ESGRO (mLIF; Chemicon, Temecula, CA); 1 mM sodium pyruvate; 0.1 mM NEAA, 2 mM glutamate, 0.1 mM beta-mercapto ethanol, and penicillin/streptomycin (50 U/50 μg per ml).

Blastocyst outgrowth experiments were carried out according the standard procedure (Nagy et al., 2003). In brief, blastocysts at 3.5 d.p.c. were cultured individually in the DMEM (Gibco catalog no. 10313-021) supplemented with 15% fetal bovine serum, 15 mM HEPES buffer, 100 units/ml of penicillin, 100 μg/ml of streptomycin, 100 μM nonessential amino acids, 4.5 mM of L-glutamine, and 100 μM of ß-mercapto ethanol on gelatinized chamber slides at 37°C in 5% CO2. The cultures were examined and photographed daily. Most blastocysts received an siControl injection attached to a gelatin-coated surface, hatched from zona pellucida, and started to expand the ICM and to form outgrowths with a typical appearance of migrating trophoblastic cells. Most blastocysts received an siZscan4 injection also attached to a gelatin-coated surface and from zona pellucida. However, by day 3 after a slight expansion, the outgrowths stopped proliferating and degenerated. In sum, even after a few days in culture the outgrowths of the siZscan4-blastocysts were dramatically different from those of the siControl-blastocysts.

Whole Mount In situ hybridization (WISH)

A plasmid DNA (clone C0348C03) was digested with SalI/NotI and transcribed in vitro into digoxigenin-labeled antisense and sense probe as control. Embryos obtained from young (7 weeks old) B6D2F1/J mice were fixed in 4% paraformaldehyde and used to perform whole mount in situ hybridization (WISH) according to the previously described protocol (Yoshikawa et al., 2006). WISH was also carried out on cultured ES cells according to the same protocol (Yoshikawa et al., 2006).

Quantitative reverse transcriptase PCR

Embryos for quantitative reverse transcriptase (qRT)-PCR experiments were collected as described above and harvest at 23, 30, 43, 55, 66, 80, and 102 hours post-hCG for 1-cell, early 2 cell, late 2-cell, 4-cell, 8-cell, morula and blastocyst embryos, respectively. Three subsets of 10 synchronized and intact embryos were transferred in PBT 1X (PBS supplemented 0.1% Tween X20) and stored in liquid nitrogen. These pools of embryos were mechanically ruptured by a freeze/thaw step and directly used as a template for cDNA preparations. The Ovation system (NuGen technologies, San Carlos, CA, USA) was used to synthesize cDNAs from each pool. The cDNAs were then diluted to 1:25 in a total of 1000 μl and 2 μl were used as a template for qPCR. The qPCR was performed on the ABI 7900HT Sequence Detection System (Applied Biosystems, Foster City, CA, USA) as previously described (Falco et al., 2006) and data were normalized by Chuk and H2afz with the ΔΔCt method (Falco et al., 2006; Livak and Schmittgen, 2001). Embryos subjected to RNA interference experiments were analyzed in the same way as described above for the normal preimplantation embryos

Supplementary Material

01

Supplemental Figure S1. Number of expressed sequence tags (ESTs) found for Zscan4 in the public EST database (NCBI Mouse UniGene Database, Build: #161: http://www.ncbi.nlm.nih.gov/UniGene/UGOrg.cgi?TAXID=10090).

02

Supplemental Figure S2. Comparison of nucleotide and amino acid sequence similarity (percent identity) among human ZSCAN4, mouse Zscan4c, Zscan4d, and Zscan4f genes. (A) cDNAs (nucleotides). (B) Putative protein coding regions (nucleotides). (C) Putative proteins (amino acids). (D) a SCAN domain (amino acids). (E) ZFP domains (amino acids).

03

Supplemental Figure S3. Zscan4 syntenic regions of mouse and human genomes.

04

Supplemental Figure S4. Electropherograms of Zscan4 cDNA sequences amplified from 2-cell embryos. Asterisks indicate nucleotide positions used to calculate the expression levels of each paralogous copies of Zscan4 (see Materials and Methods for the detail).

05

Supplemental Figure S5. Electropherograms of Zscan4 cDNA sequences amplified from ES cells. Asterisks indicate nucleotide positions used to calculate the expression levels of each paralogous copies of Zscan4 (see Materials and Methods for the detail).

06

Supplemental Figure S6. Development of embryos received siZscan4-injection in cytoplasm. (A) Stages of embryos were recorded for siControl-injected embryos (white bar) and siZscan4-injected embryos (gray bar) at 2.0, 3.5 and 4.0 d.p.c. (B) Assessment of blastocyst-expansion and hatching at 4.5 d.p.c. in siControl-injected embryos (gray bar; a photograph (a)) and siZscan4-injected embryos (black bar; a photograph (b)).

07

Supplemental Figure S7. Development of embryos received plus-siZscan4-injection in cytoplasm. (A) Stages of embryos were recorded for siControl-injected embryos (white bar) and plus-siZscan4-injected embryos (gray bar) at 2.0, 2.2, 3.0, and 4.0 d.p.c. (B-D) The transcript levels of Zscan4 in siControl-injected embryos and plus-siZscan4-injected embryos, measured by qRT-PCR analysis and normalized by Chuk (B) and H2afz (C). (D) The raw data of 3 biological replications of qRT-PCR analysis. †, mean value of a cycle threshold for each biological replicate; ‡, the standard deviation.

Acknowledgments

We would like to thank Dawn Philips for superovulation of mice, Lois Maltais for helping to assign gene nomenclature, Yong Qian for helping sequence analysis and data submission, Toshiyuki Yoshikawa for helping to do whole mount in situ hybridization, Mark G. Carter for helping quantitative PCR analysis, Lioudmila V. Sharova for helping ES cell culture, Alexei A. Sharov for discussion, and David Schlessinger for critical reading of the manuscript. We would also like to thank Ian Chambers for providing a pPyCAGIP plasmid DNA. This research was supported by the Intramural Research Program of the NIH, National Institute on Aging.

Footnotes

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

01

Supplemental Figure S1. Number of expressed sequence tags (ESTs) found for Zscan4 in the public EST database (NCBI Mouse UniGene Database, Build: #161: http://www.ncbi.nlm.nih.gov/UniGene/UGOrg.cgi?TAXID=10090).

NIHMS27351-supplement-01.ppt (39.5KB, ppt)
02

Supplemental Figure S2. Comparison of nucleotide and amino acid sequence similarity (percent identity) among human ZSCAN4, mouse Zscan4c, Zscan4d, and Zscan4f genes. (A) cDNAs (nucleotides). (B) Putative protein coding regions (nucleotides). (C) Putative proteins (amino acids). (D) a SCAN domain (amino acids). (E) ZFP domains (amino acids).

NIHMS27351-supplement-02.ppt (74.5KB, ppt)
03

Supplemental Figure S3. Zscan4 syntenic regions of mouse and human genomes.

NIHMS27351-supplement-03.ppt (62.5KB, ppt)
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Supplemental Figure S4. Electropherograms of Zscan4 cDNA sequences amplified from 2-cell embryos. Asterisks indicate nucleotide positions used to calculate the expression levels of each paralogous copies of Zscan4 (see Materials and Methods for the detail).

NIHMS27351-supplement-04.ppt (280KB, ppt)
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Supplemental Figure S5. Electropherograms of Zscan4 cDNA sequences amplified from ES cells. Asterisks indicate nucleotide positions used to calculate the expression levels of each paralogous copies of Zscan4 (see Materials and Methods for the detail).

NIHMS27351-supplement-05.ppt (155KB, ppt)
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Supplemental Figure S6. Development of embryos received siZscan4-injection in cytoplasm. (A) Stages of embryos were recorded for siControl-injected embryos (white bar) and siZscan4-injected embryos (gray bar) at 2.0, 3.5 and 4.0 d.p.c. (B) Assessment of blastocyst-expansion and hatching at 4.5 d.p.c. in siControl-injected embryos (gray bar; a photograph (a)) and siZscan4-injected embryos (black bar; a photograph (b)).

NIHMS27351-supplement-06.ppt (128KB, ppt)
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Supplemental Figure S7. Development of embryos received plus-siZscan4-injection in cytoplasm. (A) Stages of embryos were recorded for siControl-injected embryos (white bar) and plus-siZscan4-injected embryos (gray bar) at 2.0, 2.2, 3.0, and 4.0 d.p.c. (B-D) The transcript levels of Zscan4 in siControl-injected embryos and plus-siZscan4-injected embryos, measured by qRT-PCR analysis and normalized by Chuk (B) and H2afz (C). (D) The raw data of 3 biological replications of qRT-PCR analysis. †, mean value of a cycle threshold for each biological replicate; ‡, the standard deviation.

NIHMS27351-supplement-07.ppt (168KB, ppt)

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