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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Gene Expr Patterns. Author manuscript; available in PMC Feb 1, 2009.
Published in final edited form as:
PMCID: PMC2238805

An in situ hybridization-based screen for heterogeneously expressed genes in mouse ES cells


We previously reported that Zscan4 showed heterogeneous expression patterns in mouse embryonic stem (ES) cells. To identify genes that show similar expression patterns, we carried out high-throughput in situ hybridization assays on ES cell cultures for 244 genes. Most of the genes are involved in transcriptional regulation, and were selected using microarray-based comparisons of gene expression profiles in ES and embryonal carcinoma (EC) cells versus differentiated cell types. Pou5f1 (Oct4, Oct3/4) and Krt8 (EndoA) were used as controls. Hybridization signals were detected on ES cell colonies for 147 genes (60%). The majority (136 genes) of them showed relatively homogeneous expression in ES cell colonies. However, we found that two genes unequivocally showed Zscan4-like spotted expression pattern (spot-in-colony pattern; Whsc2 and Rhox9). We also found that nine genes showed relatively heterogeneous expression pattern (mosaic-in-colony pattern: Zfp42/Rex1, Rest, Atf4, Pa2g4, E2f2, Nanog, Dppa3/Pgc7/Stella, Esrrb, and Fscn1). Among these genes, Zfp42/Rex1 showed unequivocally heterogeneous expression in individual ES cells prepared by the CytoSpin. These results show the presence of different types or states of cells within ES cell cultures otherwise thought to be undifferentiated and homogeneous, suggesting a previously unappreciated complexity in ES cell cultures.

Keywords: ES cells, EC cells, pluripotent stem cells, heterogeneous gene expression, homogeneous gene expression, Zscan4, Pou5f1, Oct4, Oct3/4, Krt8, EndoA, Whsc2, Nelfa, Rhox9, Zfp42, Rex1, Rest, Zfp42, Rex1, Rest, Atf4, Pa2g4, E2f2, Nanog, Dppa3, Pgc7, Stella, Esrrb, Fscn1

1. Results and discussion

1.1. Rationale for the study

Mouse ES cells remain undifferentiated when cultured in the presence of leukemia inhibitory factor (LIF) (Niwa, 2007). The undifferentiated state of ES cells is usually verified by positive staining for expression of alkaline phosphatase and Pou5f1 (aka: Oct4, Oct3/4). It has been thought that these undifferentiated ES cells are relatively homogeneous and retain capacities for pluripotency and self-renewal (Niwa, 2007). Therefore, the analysis of ES cells is typically population-based. For example, we have analyzed the global gene expression profiles of ES cells, comparing populations of ES cells cultured in conditions which promote or prevent cellular differentiation (Aiba et al., 2006; Sharova et al., 2007).

However, the presence of heterogeneous cell populations in undifferentiated ES cell cultures is now increasingly recognized. Recent reports have demonstrated that surface markers, such as Ssea1, Pecam1, and Icam1, are expressed heterogeneously in mouse ES cell cultures, and their expression patterns appear to be modulated by differentiation processes in these cultures (Cui et al., 2004; Li et al., 2005). Furthermore, in the case of Pecam1, overall expression levels and isoform distributions are indicative of ES cell differentiation state (Cui et al., 2004; Li et al., 2005) as well as embryonic developmental potential (Furusawa et al., 2004). The presence of T-positive cells in ES cell colonies has also been reported (Suzuki et al., 2006). Recently, we have reported that a gene named Zscan4, which is expressed exclusively in 2-cell embryos and ES cells, shows heterogeneous expression in ES cells: only up to 10% of ES cells cultured in undifferentiated conditions express Zscan4 by in situ hybridization (Falco et al., 2007). We suspect the existence of other transcription-regulating genes that show similar expression patterns.

The goals of this study are to find additional evidence for heterogeneous cell populations within undifferentiated ES cell cultures by identifying such genes and characterizing their expression patterns. We report here a two-step approach to identify transcription factor genes responsible for heterogeneity in ES cell cultures, possibly with roles in regulating early differentiation.

1.2. Identification of transcription factor genes expressed predominantly in ES/EC cells

First, we used a microarray-based comparison of gene expression profiles in ES and embryonal carcinoma (EC) cells vs. differentiated cell types to identify enriched transcripts in ES and EC cells. Previously published Microarray data sets (Carter et al., 2003; Aiba et al., 2006) generated using the NIA Mouse 22K Microarray (Carter et al., 2003) were processed using the NIA Array Analysis Tool (Sharov et al., 2005b). Expression profiles for undifferentiated cultures of 129/SvEv and R1 ES cells were combined with those of F9 and P19 embryonal carcinoma (EC) cells to represent stem cell gene expression patterns. Expression profiles for trophoblast stem (TS) cells, neural stem/progenitor (NS) cells, differentiated cells (DC) from the NS cells, and E12.5 whole placenta were included to represent gene expression from differentiation-committed or differentiated cells (Figure 1A). Hierarchical clustering of tissues was carried out using the average distance method, and all tissues representing stem cell gene expression were grouped into a single compact branch of the dendrogram, along with TS cells (Figure 1A), suggesting that as a cultured, oligopotent stem cell line, TS cells have a gene expression profile that bears many similarities to EC and ES cell profiles, which are not related to pluripotency. Sets of genes expressed predominantly in ES/EC cells were identified for clusters 10, 12, 13, and 14 as described previously (Sharov et al., 2005b) and these lists were combined to form a list of 541 genes.

Figure 1
(A) Selection of ES/EC-enriched genes

Based on GO annotations (Ashburner et al., 2000), protein motif homology, and literature reports, a list of 2,727 known/putative transcription factor and/or DNA-binding genes was identified, 2,025 of which are identified and annotated in the NIA Mouse Gene Index v5.0 (NMGI5) (Sharov et al., 2005a). This list was used to identify ~300 transcription factor candidates from the ES/EC-enriched gene list. In a similar process, data sets comparing gene expression in ES and TS cells on the NIA Mouse 44K Microarray (Carter et al., 2005), a whole-genome platform which allows estimation of absolute expression levels, were filtered to identify 126 ES-enriched transcription factor candidate genes. In this case, genes with absolute expression levels estimated at ≥1 copy/cell in ES cells and < 1 copy/cell in TS cells were considered “ES-enriched”. Genes predominantly expressed in the inner cell mass (ICM) of mouse blastocysts were previously identified using whole-mount in situ hybridization (Yoshikawa et al., 2006) and 61 of these genes were included in the candidate list. Finally, unpublished gene lists from experiments performed in our laboratory were added, as well as individual genes of interest under study. All gene lists were compiled into a non-redundant master list of 344 candidate genes.

1.3. Clone selection and verification

For each candidate gene that is represented in NMGI4, transcript assembly models (Sharov et al., 2005a) were consulted for manual selection of 319 cDNA clone templates for in situ hybridization probes. When possible, cDNA clones covering 3’UTRs of genes were selected to give higher specificity, and clones with inserts ≤1 Kb in length were preferred. In 38 cases where a given gene was not represented in NMGI4 or a suitable cDNA clone for that gene was not available in NIA libraries, clones were selected from other sources (e.g., Open Biosystems Inc. collections). All bacterial clones were sequence-verified prior to use as ISH probe templates. Finally, we obtained digoxigenin-labeled riboprobes for 254 genes.

1.4. High-throughput in situ protocol

Previously we reported the development of a protocol for a high-throughput whole mount in situ hybridization of preimplantation mouse embryos and the expression patterns of 98 genes in mouse blastocysts (Yoshikawa et al., 2006). We adapted this protocol to mouse ES cell culture (see the detailed protocol in Supplement). We tested Pou5f1, which is known to be expressed in undifferentiated ES cells, and Krt8 (EndoA), which is known to be expressed in trophectoderm and visceral endoderm. The transcripts of Pou5f1 were detected in the undifferentiated colonies of ES cells, whereas the transcripts of Krt8 were detected in the flatter cells that surround undifferentiated colonies (Fig. 2, Fig. 3). To increase the throughput of the procedure, we adapted the method to gelatin-coated 12-well microtiter plates. This particular culture condition was not necessarily best suited to undifferentiated ES cells. In fact, we frequently observed cells with features of differentiation, such as flatter cell morphology and the expression of Krt8, along with undifferentiated cells in typical compact colonies. Therefore, in each plate, the expression patterns of 10 genes were examined, together with Pou5f1 and Krt8 as controls. We also scored in situ hybridization results of cells only within compact ES cell colonies (see Fig. 2 and and33 for examples).

Figure 2Figure 2Figure 2Figure 2Figure 2Figure 2
ES cell in situ hybridization results
Figure 3Figure 3
Magnified view of representative genes

1.5. In situ hybridization screen

We then carried out in situ hybridization of 254 genes (Figure 2). Based on the visual inspection of high-resolution images of these results, we classified the expression patterns of 254 genes by their signal intensities (Table 1). Six categories of signal intensity (non-detectable - 97/40%, very faint – 56/23%, faint – 36/15%, medium – 27/11%, strong – 13/5%, and very strong – 15/6%) were identified, with decreasing membership at higher signal intensities. In fact, almost half of the clones hybridized produced no discernable signal, but this aspect of the results is not surprising, given that many transcription factor/DNA-binding gene products are expressed at low levels, compared to metabolic and structural proteins. Because ISH is not as sensitive as other transcript detection methods, we might expect that some low-abundance transcripts may be missed, and some detection sensitivity is sacrificed for the localization information which is provided by ISH. For example, it is well known that Foxd3 (Hanna et al., 2002) and Klf4 (Nakatake et al., 2006) are expressed in mouse ES cells and play an important function, but we could not detect any hybridization signals for these genes (Figure 3). This is clearly a limitation of the current methodology.

Table 1
A list of all the genes screened and the in situ results

The observed expression patterns grouped the 147 positive genes into three groups (Table 1): (i) 136 genes showed relatively homogeneous expression in ES cell colonies. Magnified images for representative genes are shown in Fig. 3. These expression patterns resembled that of Pou5f1: strong expression in the center of ESC colonies, with reduction or absence of expression at the more differentiated, epithelioid edges of colonies and isolated cells. (ii) 2 genes showed Zscan4-like spotted expression patterns (spot-in-colony pattern: Rhox9 and Whsc2; Fig. 3); (iii) 9 genes showed heterogeneous expression patterns (mosaic-in-colony pattern: Zfp42, Atf4, Dppa3, Esrrb, E2f2, Fscn1, Pa2g4, Nanog, and Rest; Fig. 3). Although the expression patterns of these genes were distinctive compared to those of homogeneously expressed genes (Fig. 3), we note that, except for Zscan4, Rhox9, Whsc2, and Zfp42, these classifications were rather subjective and should be taken with caution. In fact, among 9 genes that showed mosaic-in-colony expression pattern, only Zfp42/Rex1 showed unequivocal heterogeneous expression pattern at the single cell level, when in situ hybridization was carried out on ES cells trypsinized and attached to a glass slide by the CytoSpin (Fig. 4). Some other genes also appeared to be heterogeneous (e.g., Atf4, Dppa3, Nanog, Pa2g4, and Rest), but the further confirmation by more quantitative methods will be required.

Figure 4
In situ hybridization results of CytoSpin-prepared ES cells

These results show the presence of different types or states of cells in ES cell cultures, suggesting the previously unrecognized complexity of ES cell cultures grown under standard, non-differentiating conditions. We will briefly describe the features of these genes.

1.6. Genes with spotted expression patterns (spot-in-colony pattern)

Among genes we examined in this study, only three genes (Zscan4, Rhox9, and Whsc2) showed highly heterogeneous “spotted” expression patterns in undifferentiated ES cell cultures.

Zscan4, which encodes SCAN domain and four zinc-finger domains, was identified previously for the heterogeneous spot-in-colony expression pattern (Falco et al., 2007). Zscan4 is expressed exclusively in mouse 2-cell embryos and ES cells. Reducing the level of Zscan4 transcripts delays the progression from 2-cell embryos to 4-cell embryos.

Probes for Rhox9 (originally identified as Psx2) detected a sparse, heterogeneous expression pattern. Rhox homeobox genes are located in a cluster on the X chromosome and are the result of very recent gene duplications (Maclean et al., 2005; MacLean et al., 2006). As a consequence, Rhox6/Psx1 and Rhox9/Psx2 are virtually identical at the DNA sequence level, and our in situ hybridization probes cannot distinguish between the two genes. Quantitative RT-PCR analysis using primer pairs that can distinguish Rhox6 and Rhox9 showed that Rhox9 constituted 85% of transcripts and Rhox6 constituted 15% of transcripts in undifferentiated ES cells (data not shown). Therefore, transcripts detected by in situ hybridization were most likely Rhox9. Rhox6 is known to be a marker of the trophectoderm lineage (Han et al., 1998; Chun et al., 1999), but Rhox9 is expressed specifically in female germ cells (Takasaki et al., 2001).

Whsc2/NelfA mRNA was strongly expressed in a sparse heterogeneous pattern (two to four non-adjacent cells per colony). Whsc2 is a mammalian homolog of the Drosophila NELFA gene (Wright et al., 1999; Wu et al., 2005), which complexes with other Nelf proteins and can repress transcription by pausing RNA Polymerase II elongation (Wu et al., 2005). In Drosophila and mammals, it has been reported to be expressed ubiquitously (Wright et al., 1999; Yamaguchi et al., 1999; Mariotti et al., 2000). NELFA is involved in the regulation of immediate early expression of JunB in HepG2 cells (Aida et al., 2006).

1.7. Genes with heterogeneous expression patterns (mosaic-in-colony pattern)

Zfp42/Rex1 has been identified as a marker for undifferentiated EC cells (Hosler et al., 1989) and often called a pluripotency marker gene. The expression of Zfp42 is much lower in ES and embryonic germ (EG) cells derived from the C57BL/6 mouse strain than in those derived from the 129 mouse strain (Sharova et al., 2007). The mosaic expression pattern of Zfp42 has also been shown recently (Yayoi Toyooka and Hitoshi Niwa, personal communication).

RE1-silencing transcription factor (Rest) negatively regulates many neuronal genes in stem and progenitor cells (Chong et al., 1995). The expression of this gene was not detected in either ICM or TE in blastocysts by whole mount in situ hybridization (Yoshikawa et al., 2006). It has been shown that REST binds to the promoter region of Pou5f1 in ES cells (Boyer et al., 2005; Loh et al., 2006) and its expression is suppressed when Pou5f1 was repressed in mouse ES cells (Matoba et al., 2006). Reduction of Rest transcripts by siRNA in mouse ES cells does not seem to affect the undifferentiated state of ES cells (Loh et al., 2006).

Activating transcription factor 4 (Atf4) is a member of ATF/CREB (activating transcription factor/cyclic AMP response element binding protein) family of basic region-leucine zipper (bZip) transcription factors (Ameri and Harris, 2007). Atf4 is induced by oxidative and other stresses and known to be involved in multiple processes, including hematopoiesis, lens and skeletal development, and fertility (Ameri and Harris, 2007).

E2F transcription factor 2 (E2f2) is one of the eight E2F family member genes, E2f1-E2f8 (DeGregori and Johnson, 2006). E2Fs have diverse target genes and functions, including cell cycle regulation, apoptosis, and development (DeGregori and Johnson, 2006).

Nanog was originally isolated as a gene whose overexpression maintains the undifferentiated state of ES cells in the absence of LIF and has been used as a marker for pluripotency (Chambers et al., 2003; Mitsui et al., 2003). Heterogeneous expression of Nanog in mouse ES cells has been shown recently (Singh et al., 2007).

Dppa3/Pgc7/Stella was originally isolated as a marker for early germ cell differentiation (Saitou et al., 2002; Sato et al., 2002; Bortvin et al., 2003). Using a GFP marker under the regulation of the Stella promoter, it has been shown that GFP was expressed in a heterogeneous manner, i.e., a “salt-pepper” fashion (Payer et al., 2006).

Estrogen related receptor beta (Esrrb) plays an important role in the development of primordial germ cells (PGCs) (Mitsunaga et al., 2004). Recently it has been shown that Esrrb is one of the pluripotency-related genes (Ivanova et al., 2006; Loh et al., 2006).

Fascin homolog 1, actin bundling protein (Fscn1) has been shown to be expressed in neural and mesenchymal derivative cells (De Arcangelis et al., 2004).

1.8. Genes with homogeneous expression patterns within ES cell colonies

These genes will provide useful markers for undifferentiated ES cells. It will be important to study how these gene expression patterns change during the differentiation of ES cell cultures.

2. Experimental Procedures

2.1. Candidate gene/clone list assembly

Gene expression microarray data from previously published studies (Carter et al., 2005; Aiba et al., 2006) was analyzed using the NIA Microarray Analysis Tool (Sharov et al., 2005b) to generate lists of genes enriched in ES cell cultures grown in non-differentiating conditions. Official gene symbols and NIA Mouse Gene Index 5.0 (NMGI5) identifiers (Sharov et al., 2005a) were assigned to each gene, and NMGI5 locus and transcript models were used to select cDNA clones to be used as probe templates. Preference was given to NIA cDNA clones covering 3’UTRs, with an insert length ≤ 1 kb. In cases where suitable NIA cDNA clones were not available for a given gene, clones were selected and ordered from Open Biosystems Incorporated (OBI).

2.2. RNA probe preparation

cDNA clones were arrayed and cultured in 96-well microtiter plates. Inserts were PCR amplified directly from 2 μl bacterial culture in 100 μl PCR reactions (5 units RedTaq DNA polymerase in 10 mM Tris-HCl, pH 8.3; 50 mM KCl; 1.1 mM MgCl2; 0.01% gelatin; 200 μM each dNTP; 0.2 μM each primer). Reactions were cycled as follows: 30 cycles of denaturation at 95 °C for 30 seconds, annealing at 47 °C for 30 seconds, extension at 72 °C for 4 minutes, followed by the final extension at 72 °C for 10 minutes. NIA cDNA clones (in pSPORT-based backbones) were amplified using M13 forward (-20) (5’-GTAAAACGACGGCCAGT-3’) and M13 reverse (5’-GGAAACAGCTATGACCATG-3’) primers. OBI cDNA clones in backbones without M13 forward and reverse sites were amplified using T7 (5’-GTAATACGACTCACTATAGGGC-3’) and T3 (5’-AATTAACCCTCACTAAAGGG-3’) primers. PCR products were purified using a QIAquick PCR purification kit (Qiagen), eluted in 100 μl of buffer, and quantitated using a ND1000 spectrophotometer (NanoDrop Technologies Inc.). Digoxigenin-labeled RNA probes were transcribed from the PCR product templates using DIG RNA Labeling Mix (Roche) and the appropriate RNA polymerase. Ethanol-precipitated probes were re-suspended in water and quantitated by agarose gel electrophoresis or by running an RNA 6000 Nano Assay on a 2100 Bioanalyzer (Agilent Technologies).

2.3. Cell culture

ES cells (line 129.3, derived from 129Sv/J strain mice) were cultured at 37°C in 5% CO2 without feeders on gelatin-coated 12-well plastic plates using standard ES cell culture medium (DMEM, 15%FBS, 1 mM sodium pyruvate, 0.1 mM non-essential amino acids, 2 mM glutamate, 0.1 mM β-mercaptoethanol, 50U/ml penicillin, 50 μg/ml streptomycin,1,000 U/ml ESGRO LIF). Cells were seeded at 2×104 cells/well and cultured for 3 days before ISH processing.

2.4. In situ hybridization

Cells were fixed in 4% PFA/PBS at 4°C overnight. After digestion with proteinase K, cells were hybridized overnight with 10 μl Digoxigenin-labeled riboprobe at 62°C overnight. Cells were then washed, blocked, incubated with alkaline phosphatase-conjugated anti-Digoxigenin antibody, and incubated with NBT/BCIP detection buffer for 30 minutes. See supplemental data for full protocol details.

2.5. Cytospin Cell Preparation

Cells were cultured as described in the section 2.3. The cells were harvested with 1X accutase and centrifuged at 1000 rpm for 5 minutes, washed with PBS, and fixed overnight in 4% PFA in PBS at 4 °C. The cells were washed twice with PBS, counted, and resuspended in PBS at a concentration of 1×106 cells/ml. Cells (100,000) were attached to coated microscope slides (Shandon, cat#5991056) in a Shandon, Cytospin3 centrifuge at 500 rpm (low acceleration) for 5 min and dried overnight on a slide warmer at 37 °C. ISH was performed on the slides as described in the section 2.4.

2.6. Image acquisition and processing

ISH preparations were imaged under bright-field optics on an inverted microscope with DIC/phase-contrast optics. Cells were photographed at 4x, 10x, and 20x magnification. TIFF images were imported into an Open Microscopy Environment (OME) (Goldberg et al., 2005) server and manually annotated. Images were divided into 5 categories based on apparent signal strength, and four categories based on the type of staining pattern observed (Table 1). All images are available at the Jackson Laboratory Gene Expression Database (GXD) (http://www.informatics.jax.org) and the public OME site (http://ome.grc.nia.nih.gov).

Supplementary Material


Supplemental Materials:

Supplemental Text S1: ES cell in situ protocol


We would also like to thank Yayoi Toyooka and Hitoshi Niwa (RIKEN Center for Developmental Biology, Kobe, Japan) for sharing the information about heterogeneous expression of Zfp42/Rext1 gene in mouse ES cells. We would like to thank Michal Zalzman, Yulan Piao, Yong Qian, and Dawood Dudekula for technical assistance and discussion. We would also like to thank Ilya Goldberg and Harry Hochheiser for help in making data available through the OME. This research was supported by the Intramural Research Program of the NIH, National Institute on Aging.


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