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NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2009 Feb 1.
Published in final edited form as: Gene Expr Patterns. 2007 Nov 4;8(3):181–198. doi: 10.1016/j.gep.2007.10.009

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

Mark G Carter 1,*,#, Carole A Stagg 1,*, Geppino Falco 1, Toshiyuki Yoshikawa 1, Uwem C Bassey 1, Kazuhiro Aiba 1, Lioudmila V Sharova 1, Nabeebi Shaik 1, Minoru SH Ko 1,&
PMCID: PMC2238805  NIHMSID: NIHMS39384  PMID: 18178135

Abstract

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.

Figure 1

Using the NIA Microarray Analysis Tool (lgsun.grc.nia.nih.gov/ANOVA/), data were compiled from 30 two-channel fluorescent hybridizations of linearly amplified cRNA target mixtures on NIA/Agilent 22K v1.1 60-mer oligo arrays, representing 10 separate tissues and cell cultures in triplicate against a common universal reference. Nodes 12, 13, and 14 on the hierarchical clustering tree above represent transcripts which are enriched in ES cells only, ES cells and F9 EC cells, and ES and EC cells, respectively, relative to TS and differentiated tissues. Of these 541 gene transcripts, approximately 300 encode known or putative transcription factors, DNA binding proteins, or contain motifs associated with DNA-binding and/or transcription-regulating activity. (ES_129a, ES_129b = 129Sv-derived ES cells cultured in two different conditions as described (Aiba et al., 2006); ES_R1 = R1 ES cell line; EC_F9, EC_P19 = F9 and P19 embryonic carcinoma cells; TS = trophoblast stem cells; NS1, NS5 = neural stem/progenitor cells cultured for 1 and 5 days; DC = differentiated neural stem cells; PL = E12.5 whole placenta.

(B) ES cell culture in situ hybridization screening workflow. Clones were identified in NIA cDNA libraries representing genes from our list of approximately 300 known or putative transcription factors. For genes not represented, clones were obtained from Open Biosystems. All clones were single-colony purified, PCR amplified with M13 forward and reverse primers, and PCR products were sequenced for verification. Digoxigenin-labeled antisense RNA probes were synthesized from PCR products and hybridized to fixed ES cells grown on gelatin-coated plastic in LIF-supplemented medium for 3 days. Probes producing extremely dark and/or non-specific signals were examined for repeat element sequences, and gene-specific primers were designed to amplify repeat-free probe templates, which were used for subsequent hybridizations.

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 3 for examples).

Figure 2. ES cell in situ hybridization results.

Figure 2

Figure 2

Figure 2

Figure 2

Figure 2

Figure 2

Distribution of gene expression intensity and pattern in ES cell cultures by in situ hybridization In situ hybridized ES cells were photographed under standardized conditions, and images from 252 different clones which were successfully isolated, sequence verified, PCR-amplified, transcribed into DIG-labeled probes, and hybridized were classified according to expression intensity and pattern type (Table 1). High-resolution images are available at the public OME site (http://ome.grc.nia.nih.gov).

Figure 3. Magnified view of representative genes.

Figure 3

Figure 3

In situ hybridization results of representative genes are selected from Fig. 2 and magnified to show the detailed staining patterns. See the text for the interpretation of results.

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

Gene TF? cDNA clone IDs Signal Intensity Expression Pattern in ES colonies
Pou5f1 (Oct3/4, Oct4) Yes H3028H01 strong Homogeneous
Krt8 (EndoA) No H3031C01 strong Peripheral
Zscan4 (Gm397) Yes C0407B02 strong Spot-in-Colony
A730008L03Rik (Nelfb) Yes K0109B03 v faint Homogeneous
Actl6a (Baf53a) Yes H3080C12 faint Homogeneous
Aire Yes EMM1002-7515871 strong Homogeneous
Akp2 Yes C0257F12 med Homogeneous
Ankhd1 Yes K0400H03 v faint Homogeneous
Ankrd10 Yes K0122H01 v faint Homogeneous
Ankrd25 Yes H3123C11 v faint Homogeneous
Arid1a (Baf250a) Yes H3023A03 strong Homogeneous
Arid2 Yes K0917G07 n/d n/d
Arid3b Yes C0326H11 v faint Homogeneous
Ash2l Yes H3147F02 v faint Homogeneous
Atf1 Yes H4067F10 v strong Homogeneous
Atf4 Yes H3124E06 faint Mosaic-in-Colony
Atm Yes H8200G06 n/d n/d
Bat4 Yes H3059D08 n/d n/d
Bbx Yes H3026G07 faint Homogeneous
BC024969 (Adnp2) Yes C0866H11 v faint Homogeneous
BC031441 Yes H8230G02 v strong Homogeneous
BC068171 (Dkc1) No H3066D04 v faint Homogeneous
Blimp1 (Prdm1) Yes EMM1032-597986 n/d n/d
Bnc2 Yes H3065E10 v faint Homogeneous
Bpnt1 Yes H3057C10 n/d n/d
Bteb1 (Klf9) Yes H3027A04 v faint Homogeneous
Cbfa2t1h (Runx1t1) Yes H3125F02 v faint Homogeneous
Cbfa2t2h (Cbfa2t2) Yes H3091A05 v faint Homogeneous
Cbx8 Yes H4002D08 n/d n/d
Cct4 No H3027F12 faint Homogeneous
Cdk7 Yes H3002H05 n/d n/d
Cpe No H4028H11 n/d n/d
Crsp3 (Med23) No H4006A08 n/d n/d
Csda Yes C0202D02 faint Homogeneous
Csen (Kcnip3) Yes K0906H01 n/d n/d
Cth No H3103D05 n/d n/d
Dazap2 Yes A0813H07 n/d n/d
Dctn4 No H4070C12 n/d n/d
Ddx4 (Mvh) No H3134G04 n/d n/d
Dedd2 Yes H4061C06 med Homogeneous
Dek Yes H3138A12 faint Homogeneous
Dll1 No MMM1013-9202259 n/d n/d
Dpf2 (Req) Yes C0818B07 n/d n/d
Dppa3 (Stella, Pgc7) No L0289B08 strong Mosaic-in-Colony
E130016E03Rik (Rad54b) Yes H3116F06 n/d n/d
E2f2 Yes EMM1002-7498698 med Mosaic-in-Colony
E2f5 Yes H3072H12 n/d n/d
Egr1 (Krox24) Yes B0267A11 v faint Homogeneous
Eif5b No H3053D03 med Homogeneous
Elf3 Yes H3015C12 v faint Homogeneous
Ell3 Yes E0754G01 n/d n/d
Eno3 No H4027G12 strong Homogeneous
Esrrb Yes H4053E01 med Mosaic-in-Colony
Esx1 Yes EMM1002-6760787 med Homogeneous
Etv5 Yes C0282F03 med Homogeneous
Ezh1 Yes H3154F11 n/d n/d
Fem1a Yes C0407G11 v faint Homogeneous
Fem1b Yes H3029F04 n/d n/d
Fhl2 Yes H3033C07 n/d n/d
Fosl2 Yes H3110A12 n/d n/d
Foxd3 (Genesis) Yes EMM1002-1111636 n/d n/d
Fscn1 No H3006D08 strong Mosaic-in-Colony
Gabpa Yes C0260F10 v faint Homogeneous
Gata1 Yes H3038A09 n/d n/d
Gata2 Yes H3009F04 faint Homogeneous
Gata3 Yes H3049E04 med Homogeneous
Gcdh Yes C0328C11 med Homogeneous
Gli1 Yes A0243G01 faint Homogeneous
Gli2 Yes K0239G06 faint Homogeneous
Gm1739 No H3128A09 v strong Homogeneous
Gmeb1 Yes K0502H03 n/d n/d
Gtf2a1lf Yes EMM1002-4195952 n/d n/d
Gtf2e1 Yes H3105C05 n/d n/d
Gtf2e2 Yes H3144B01 faint Homogeneous
Gtf2h3 Yes H8201G03 strong Homogeneous
Gtf2i Yes H3140H12 med Homogeneous
Hes6 Yes MMM101365220 v strong Homogeneous
Hmga1 Yes H3029B11 strong Homogeneous
Hmgn3 Yes B0224F03 v faint Homogeneous
Hoxa1 Yes EMM1032-608139 n/d n/d
Hoxb1 Yes H3031H08 strong Homogeneous
Hoxc10 Yes H3120B02 strong Homogeneous
Hoxc6 Yes EMM1002-25304 n/d n/d
Hoxc8 Yes H4041F10 med Homogeneous
Hoxd13 Yes EMM1002-6823925 faint Homogeneous
Hsf2bp Yes H3125B05 n/d n/d
Icsbp1 (Irf8) Yes H3072F09 n/d n/d
Ilf2 Yes H3029E05 med Homogeneous
Ing3 Yes H4072A09 n/d n/d
Irx2 Yes EMM1002-5626242 med Homogeneous
Irx3 Yes H3010C07 strong Homogeneous
Irx6 Yes EMM1002-1856631 v faint Homogeneous
Jarid1b Yes H3041C04 faint Homogeneous
Jmjd2c Yes H4053F10 n/d n/d
Jub No H3024A03 v strong Homogeneous
Jun Yes H3058C09 faint Homogeneous
Khdrbs1 Yes A0501F06 n/d n/d
Klf2 Yes EMM1002-7515994 v strong Homogeneous
Klf4 Yes H3015B01 n/d n/d
Klf5 Yes H3102C04 v faint Homogeneous
L3mbtl2 Yes A0354D01 v faint Homogeneous
Lamb1-1 (Lamb1) No H3147C04 n/d n/d
Lass4 (Trh1) Yes H3129C07 n/d n/d
Lmyc1 (Mycl1) Yes H3024G07 med Homogeneous
Mcm3 Yes C0403F03 strong Homogeneous
Mef2d Yes C0261E02 v faint Homogeneous
Mga Yes K0110B04 v faint Homogeneous
Miz1 (Pias2) Yes K0232F02 n/d n/d
Mll3 Yes H3057C11 n/d n/d
Mnat1 Yes C0025B02 n/d n/d
Mrpl49 No H3039A04 v faint Homogeneous
Mtf2 Yes H3048E07 faint Homogeneous
Mxd4 Yes H3131B07 v faint Homogeneous
Mxi1 Yes H4077G09 n/d n/d
Mybl2 (Bmyb) Yes H3144E01 faint Homogeneous
Mycn (Nmyc1) Yes H4041A05 v strong Homogeneous
Myod1 (MyoD) Yes EMM1002-1452165 v strong Homogeneous
Myst2 Yes H3107B07 faint Homogeneous
Nanog Yes H3050A07 faint Mosaic-in-Colony
Nanos1 No K0409A10 n/d n/d
Ncoa1 Yes A0400A03 v faint Homogeneous
Neurod1 Yes H4035E12 n/d n/d
Nf1 No H4070B01 n/d n/d
Nfatc2ip Yes H3046E09 n/d n/d
Nfe2l2 Yes C0819F04 n/d n/d
Nfkbie Yes MMM1013-7512133 faint Homogeneous
Nfyb Yes H3146B03 n/d n/d
Nkx2-6 Yes EMM1002-1148995 v faint Homogeneous
Nkx6-2 Yes EMM1002-21216 n/d n/d
Notch3 Yes H4028C12 strong Homogeneous
Notch4 Yes EMM1002-6752438 v strong Homogeneous
Npr1 No L0927H03 v faint Homogeneous
Nr2f1 (COUPTF1) Yes H3096D07 n/d n/d
Nr5a2 (LRH1) Yes H3087G01 v faint Homogeneous
Nr6a1 (Gcnf) Yes H3026F12 faint Homogeneous
Nrg1 Yes EMM1032-582702 n/d n/d
Nrg2/LOC381149 No EMM1002-5539961 v strong Homogeneous
Nrl Yes EMM1002-4982597 n/d n/d
Nufip1 Yes H3009B11 v faint Homogeneous
Otx2 Yes H3030H12 v faint Homogeneous
Pa2g4 Yes H3024B11 med Mosaic-in-Colony
Papolg Yes J0235C01 n/d n/d
Pcdhb16 Yes K0997C06 v faint Homogeneous
Pfdn1 Yes H3157C01 n/d n/d
Phf17 Yes H3127B05 v faint Homogeneous
Pitx2 Yes E0473B09 n/d n/d
Pml Yes C0208F04 med Homogeneous
Polr2i Yes H3089E01 n/d n/d
Polr3k Yes H3083H08 n/d n/d
Prkcbp1 Yes H3027G06 n/d n/d
Prrx1 Yes K0403B09 n/d n/d
Rab2b Yes A0363E07 v strong Homogeneous
Rab35 Yes H3136E02 n/d n/d
Rad18 Yes C0407D04 n/d n/d
Rai14 Yes K0417A05 n/d n/d
Ranbp1 No H3104D06 faint Homogeneous
Rbak Yes H3091E03 n/d n/d
Rbpsuh (Rbpj) Yes H4069C09 faint Homogeneous
Rcor1 Yes H4030A11 n/d n/d
Rdbp No H3025E10 faint Homogeneous
Rest Yes H3153C03 med Mosaic-in-Colony
Rfx2 Yes H8188D01 n/d n/d
Rhox9 (Psx2) Yes L0042E08 faint Spot-in-Colony
Rpo1-2 Yes H8164D08 med Homogeneous
Rpo1-3 Yes H3121B06 v faint Homogeneous
Ruvbl1 Yes H3016C12 v faint Homogeneous
Sall1 Yes H4055D01 med Homogeneous
Sin3b Yes C0281D12 faint Homogeneous
Sirt1 Yes H3119B12 v faint Homogeneous
Sirt2 Yes C0285H01 faint Homogeneous
Sirt3 Yes A0352G08 n/d n/d
Sitpec (Ecsit) Yes H4049D06 v faint Homogeneous
Six4 Yes H4056D02 v faint Homogeneous
Skd3 (Clpb) Yes C0341A10 med Homogeneous
Skiip (Snw1) Yes C0218H03 v faint Homogeneous
Slc4a10 Yes H4035E11 n/d n/d
Smarca1 (Snf2l) Yes H4004G06 n/d n/d
Smarca4 (Brg1) Yes C0402B01 faint Homogeneous
Sox13 Yes L0928B04 med Homogeneous
Sox15 Yes H3081C12 n/d n/d
Sox2 Yes C0403H11 faint Homogeneous
Sp1 Yes H3016H10 n/d n/d
Sp5 Yes EMM1002-5883694 n/d n/d
Sqrdl No H3122H06 n/d n/d
Stat4 Yes H4077D08 n/d n/d
Suv39h1 Yes H3141G04 v faint Homogeneous
T (Brachyury) Yes H3029B05 n/d n/d
Taf5 Yes K0278H01 n/d n/d
Taf5l Yes B0410D11 v faint Homogeneous
Taf7 Yes H3071E08 v faint Homogeneous
Tbp Yes K0240H01 v faint Homogeneous
Tcea3 Yes H4005B08 faint Homogeneous
Tcerg1 Yes H3128B05 faint Homogeneous
Tcf15 Yes EMM1002-1464550 n/d n/d
Tcf7 Yes H4003H11 n/d n/d
Tcf7l2 Yes H4028E05 n/d n/d
Tcfap2e Yes H3100H07 n/d n/d
Tcfap4 Yes MMM1013-9200312 v faint Homogeneous
Tfam Yes H8244A10 med Homogeneous
Tfdp2 Yes H4061E07 v faint Homogeneous
Th1l Yes C0208E04 faint Homogeneous
Thrap2 (Med13l) Yes H3137F02 faint Homogeneous
Tle4 Yes H3103G12 n/d n/d
Tox Yes K0748A03 n/d n/d
Trib3 Yes H3035B01 n/d n/d
Trp63 Yes E0485F10 v faint Homogeneous
Trps1 Yes A0909F05 n/d n/d
Trpv6 Yes H8162C12 v faint Homogeneous
Ttbk1 No H3137A12 n/d n/d
Ube2d3 No H3082D12 v faint Homogeneous
Ureb1 No H3129D11 v faint Homogeneous
Usp39 No H3005G05 v faint Homogeneous
Whsc2 (NelfA) Yes C0288C12 med Spot-in-Colony
Yeats4 Yes H4002F05 v faint Homogeneous
Zfp105 No H3154D12 v faint Homogeneous
Zfp111 Yes K0957E07 n/d n/d
Zfp143 Yes L0949H07 n/d n/d
Zfp206 (Zscan10) Yes H4040D10 n/d n/d
Zfp219 No H3014D06 med Homogeneous
Zfp239 Yes H4060F01 med Homogeneous
Zfp278 (Patz1) Yes K0852A06 v strong Homogeneous
Zfp286 Yes C0919G04 n/d n/d
Zfp296 No H3086D06 med Homogeneous
Zfp354b Yes EMM1032-6904991 n/d n/d
Zfp42 (Rex1) Yes H3036F04 strong Mosaic-in-Colony
Zfp445 Yes K0508E08 v faint Homogeneous
Zfp51 Yes H3122C02 n/d n/d
Zfp553 Yes C0405D09 v strong Homogeneous
Zfp62 Yes B0129B06 n/d n/d
Zfp82 Yes H4054E06 faint Homogeneous
Zic3 Yes B0716B04 faint Homogeneous
1110005A23Rik Yes H3148B10 v faint Homogeneous
1110025L05Rik (Bola2) Yes H3085B11 v faint Homogeneous
1110051B16Rik Yes E0702B01 n/d n/d
1110054H05Rik (Foxk2) Yes C0202E10 faint Homogeneous
1700012H05Rik (Rbmxl2) No EMM1002-7533455 v faint Homogeneous
1810007M14Rik Yes H4079B04 faint Homogeneous
2410081M15Rik No H3093G04 v strong Homogeneous
2610014H22Rik (Wbp7) Yes H3159D08 n/d n/d
2810405K07Rik (Zfp661) Yes K0914F08 faint Homogeneous
4921520G13Rik Yes MMM1013-9335122 n/d n/d
4930504E06Rik No K0251E12 v faint Homogeneous
4930548G07Rik Yes L0924F06 med Homogeneous
4933406J07Rik (Syce1) No H4051E05 v strong Homogeneous
5830417I10Rik Yes H3038H08 n/d n/d
6720457D02Rik Yes B0142C08 n/d n/d
9030612M13Rik Yes H3010E12 n/d n/d

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.

Figure 4

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

01. Supplemental Materials.

Supplemental Text S1: ES cell in situ protocol

02

Acknowledgments

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.

Footnotes

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Supplementary Materials

01. Supplemental Materials.

Supplemental Text S1: ES cell in situ protocol

02

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