chondrogenesis, or neurogenesis - Europe PMC

Proc. Natl. Acad. Sci. USA Vol. 89, pp. 12132-12136, December 1992 Genetics

Helix-loop-helix transcription factors E12 and E47 are not

essential for skeletal or cardiac myogenesis, erythropoiesis, chondrogenesis, or neurogenesis (homologous recombinatlon/embryonic stem cels)

YUAN ZHUANG*, CHUL G. KIM*, STEVE BARTELMEZ*t, PEIFENG CHENG*, MARK GROUDINE*, AND HAROLD WEINTRAUBt *Fred Hutchinson Cancer Research Center and *Howard Hughes Medical Institute, 1124 Columbia Street, Seattle, WA 98112 Contributed by Harold Weintraub, September 24, 1992

E12 and E47 are two non-tissue-specific helx-oop-helix (HLH) trnscription factors encoded by the E2A

ABSTRACT

gene. Previous studies suggested that they are Involved in regulation of differentiation in many tissue types Includig muscle, blood, and nerve through direct heterodimer interactions with tissue-specific HL proteins. To gin fther genetic Insight into the functions of E12 and E47 during cell differentiation, we mutated both copies of the E2A gene in mouse embryonic stem (ES) cells and then tested the effect on differentiation in vitro. We find that the ES cells lacking functional E12 and E47 are capable of differentiating into both skeletal and cardiac muscle, erythrocytes, neurons, and cartilage to the same extent as wild-type cells. These results indicate that the E2A gene is not essential for differentiation of these cell types and suggest that redundant genes may control these developmental pathways.

E12 and E47 belong to a large group of transcription factors that share an amphipathic helix-loop-helix (HLH) structure. These HLH proteins preferentially dimerize with each other through the HLH domain, and the dimers are involved in transcriptional activation through binding to specific DNA sequences via a basic region immediately N-terminal to the HLH domain (1, 2). Data from genetic analyses in Drosophila (3) and biochemical studies in mammalian cells have indicated that the interactions between different HLH proteins are important for their regulatory function during cell differentiation. For example, MyoD, a muscle-specific HLH gene, can initiate myogenesis when transfected into a variety of nonmuscle types of tissue-cultured or primary cells (4). Recent studies suggest that the heterodimer formed by MyoD and the ubiquitously expressed E12 and E47 is important for this muscle-specific regulation (5). Thus, E12 and E47, although ubiquitously expressed in a variety of cell types, are likely to play an important role in myogenesis. It has also been proposed that E12 and E47 are involved in hematopoiesis and neurogenesis because they can form dimers with Tal-1 and MASH, hematopoietic and neural-specific HLH proteins, respectively (6, 7). The regulatory role of these HLH proteins is further supported by the fact that Id, a dominant negative HLH protein that lacks a basic region and preferentially pairs with E12 and E47, can suppress the transcriptional activity of MyoD and inhibit differentiation of a variety of other cell types (8, 9). To further understand the function of E12 and E47 in muscle, blood, and nerve differentiation, we used homologous recombination techniques to inactivate E12 and E47 in mouse embryonic stem (ES) cells. Previous studies have shown that all three lineages can be routinely derived from ES cells in

tissue culture (10). Our rationale was to sequentially knock out both copies of the E2A gene in the diploid ES cells and then to study the effect of the mutations on cell differentiation. MATERIALS AND METHODS Cell Lines and Transfection. Several ES cell lines were tested for E2A targeting. CC1.2 was a gift from M. Capecchi (University of Utah); CCE was a gift from E. Robertson (Columbia University); E14-TGa was purchased from the American Type Culture Collection. All the ES cell lines were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and 0.1 mM 2-mercaptoethanol. To keep the cells in the undifferentiated state, CC1.2 and CCE were maintained on irradiated Sto fibroblasts and E14 was maintained in the leukemia inhibitory factor supplemented medium without the feeder cells. All transfections were performed by electroporation with 25 .Ag of linearized plasmid DNA with 2-4 x 107 ES cells. Selection media were applied 24 hr later. Active G418 (125 j.g/ml) was used for neomycin phosphotransferase (Neo); hygromycin B (100 pLg/ml) was used for hygromycin phosphotransferase (Hygro); 120 FM hypoxanthine/0.4 ,uM aminopterine/20 ,AM thymidine was used for hypoxanthine phosphoribosyltransferase (HPRT); 2 ,AM gancyclovir was used for thymidine kinase (TK). PCR and Southern Analyses. PCR was used to screen the targeting events. Individual clones were picked out with cloning cylinders, and half of the cells were used for quick PCR analysis (11). The PCR-positive clones were then further analyzed by Southern blotting. Reverse Transcription (RT)-PCR Assay. PCR was used to determine the expression pattern of the E2A gene in knockout clones. Primers (see Fig. 1) that are specific to certain exons of the E2A gene and the marker genes were used to detect the common as well as the specific splicing patterns of the wild-type and mutant alleles. The assays were done essentially as described (12): Total cellular RNA was made from undifferentiated ES cells grown without feeders; 100 ng of each RNA sample was used for RT with random hexamers; and 1/10th of the resulting cDNAs was amplified with exon-specific primers for 28 cycles. Because a small fraction of radiolabeled nucleotide was included in the PCR, the PCR products were directly analyzed on a 5% polyacrylamide gel. Differentiation and Tumor Assays. In vitro differentiation was carried out as follows: 2 x 106 cells were plated in 15 ml of ES cell culture medium without leukemia inhibitory factor Abbreviations: HLH, helix-oop-helix; ES cells, embryonic stem cells; Neo, neomycin phosphotransferase; Hygro, hygromycin phosphotransferase; HPRT, hypoxanthine phosphoribosyltransferase; TK, thymidine kinase; RT, reverse transcription. tPresent address: Department of Pathology, University of Washington, Seattle, WA 98195.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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Proc. Natl. Acad. Sci. USA 89 (1992)

Zhuang et al.

mutually excluded (i.e., E12 and E47 specific) exons, which encode the entire HLH domain (Fig. 1). Thus, targeting constructs can be 'conveniently made that result in either specific or complete mutations of the gene by altering these two exons. As shown in Fig. 1, the two types of targeting constructs were designed to inactivate E12 and/or E47 after recombination with the E2A locus. To enhance the efficiency of selection of the targeting events, we exploited the fact that the gene is actively transcribed in ES cells and used the promoterless strategy to build most of our targeting constructs. As a result, the targeting frequency was reasonably high, with an average of -30% after a single round of selection (Table 1). Our initial approach was to generate knockouts of the E12-specific exon (with the type I constructs in Fig. 1). Because the protein sequences and the biochemical properties of E12 and E47 are very similar, we presumed that less effect would occur if only one of the proteins was inactivated. An E12 double knockout clone was obtained by two consecutive targeting experiments that resulted in insertions of marker genes in the middle of the E12-specific exon. The disruption of E12 protein and expression of the marker genes were ensured by fusing the coding sequences of the markers into the first helix of the HLH region of the E12 protein. The correct targeting events were screened out by PCR and then confirmed by Southern analysis. Due to the complexity of the splicing pattern of E2A, RT-PCR was used to determine the types of transcripts produced in the double knockouts. As shown in Fig. 2A,

in a 100-mm plate (bacterial grade from Fisher) to allow embryoid bodies to form. The suspension was diluted 1:3 on day 3 and was kept as suspension as long as the experiment required; the medium was changed as needed. To stimulate muscle and nerve formation, embryoid bodies were transferred back onto a tissue culture plate after 1 week to 10 days in suspension, and the medium was changed into low serum condition (2% horse serum plus 5 Iug of insulin per ml and 10 ,ug of transferrin per ml) 2-3 days after cells reattached to the plate. Under the suspension culture condition, erythropoiesis can be observed (by RNA analysis and benzidine staining) starting from day 8; cardiac muscle can be visualized starting from day 10 by rhythmic contraction of the embryoid bodies; nerve cells can be found weeks after plating out on culture plates, and this neurogenic process can be accelerated and augmented by brief treatment of the culture with retinoic acid; skeletal muscles usually appear much later (10-20 days after plating) and at a relatively low frequency. Benzidine staining was done by adding a half volume of staining solution (0.2% benzidine hydrochloride and 0.3% hydrogen peroxide in 3% acetic acid) directly into the suspension culture, and the dark blue staining can be visualized within a few minutes. The tumor assay was carried out by injecting 2-5 x 106 cells subcutaneously into 129/J male mice (The Jackson Laboratories). Solid tumors (10-20 mm) were dissected out 3 weeks later and used for primary culture and histological analysis.

RESULTS AND DISCUSSION The E2A gene encodes E12 and E47 through alternative splicing. The only difference between the two proteins is the a

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Neo and Hygro were used as selection markers in two separate targeting constructs. For type II fusions, which yield a complete knockout of E12 and E47, Neo was used to replace the entire genomic sequence between the two common exons bordering the E12- and E47-spic regions. The coding sequence of Neo was in-frame with the N terminus of E2A proteins. For the type H double-selection construct, HPRT was used as a positive selection marker and TK was used as a negative selection marker. Both markers carried their own promoters. For both types I and II constructs, the total length of the E2A genomic sequence used was -3 kilobases. Sizes of marker genes shown on the map are not in proportion to the E2A genomic sequence. Open boxes, exons of the E2A gene; solid lines between boxes, introns; *, termination site of E2A translation. Positions of PCR primers used in determining the expression pattern of wild-type and mutant mRNAs are also indicated.

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Table 1. Targeting frequencies with types I and II constructs Frequency, Total % Cell type clones Targeted Construct Type I 40 2 5 CC1.2 pEG122-NF 5 45 11 E14-TGa 14 1 7 E14-TGa pEG122-HF 67 4 6 pEG122-NF-ENB CC1.2 25 8 2 CC1.2 Type II 12 5 42 CC1.2 pEG5-NF 4 22 18 CCE 14 21 3 E14-TGa 10 2 20 E14-TGa 25 3 12 E14-TGa pEG5-NF-ENB 1 1 72 pEG5-HPRT-TK E14-TGa pEG122-NF is the type I Neo fusion construct shown in Fig. 1; pEG122-HF is the type I Hygro fusion construct shown in Fig. 1 and used for a second round of E12 targeting. pEG122-NF-ENB is the same as pEG122-NF except that a P-galactosidase marker is inserted immediately downstream of the Neo gene. The P-galactosidase cassette was made with the internal ribosomal entry site from the encephalomyocarditis virus, a nuclear localization signal, and the coding sequence of P-galactosidase. Because transcription of a-galactosidase is determined by the upstream E2A promoter, targeting clones obtained with this type of construct can be used for monitoring expression of the E2A gene (data not shown). pEG5-NF is the type II Neo fusion construct shown in Fig. 1. pEG5-NF-ENB is the same as pEG5-NF except with a (-galactosidase cassette inserted 3' of the Neo coding sequence. pEG5-HPRT-TK is the type II doubleselection construct built with the same backbone as that of pEG5-NF (see Fig. 1). Total clones shown with pEG5-HPRT-TK were after double selection.

wild-type E12 RNA is replaced by two fusion RNAsnamely, a Neo fusion (lane d+i) and a Hygro fusion (lane d+j). Interestingly, the insertion of marker sequences at the E12 exon appears to cause a dramatic reduction in splicing of the downstream E47 exon (lane d+f). Based on the RT-PCR data and Northern analyses (data not shown), it seems that all splicing downstream of the insertion site is severely inhibited. Therefore, the cell line carrying the E12-specific knockout produces no E12 and only a small amount of E47 mRNAs (weakly detected by RT-PCR).

Proc. Natl. Acad. Sci. USA 89 (1992) As the E12 knockout has no obvious effects on growth and differentiation (see below), we next generated double knockout clones of both E12 and E47 exons by using the type II constructs. Both type II Neo fusion and type II Hygro fusion constructs were made and tested. Although the Neo marker worked in this particular fusion context, the Hygro marker did not (data not shown). We then used the conventional double-selection strategy-i.e., positive and negative selections (14). In the type II double-selection construct, the HPRT (15) and TK markers were used for positive and negative selection, respectively (Fig. 1). Although selection with this construct is less efficient, the overall targeting frequency obtained is comparable to the Neo fusion constructs (Table 1). The genotype and the expression pattern of the type II knockouts were analyzed and confirmed by tests similar to those performed with the E12 knockout clone. Clones obtained with consecutive targeting of the type II constructs produce no E12 or E47 mRNAs (see Fig. 2B), yet they grew normally in our culture conditions. Two simple tests were then performed with the wild-type and the mutated ES cells to determine whether the mutations result in any abnormality in differentiation: (i) induction of muscle, blood, and nerve formation in culture, and (ii) analysis of teratomas formed by subcutaneous injection ofES cells into mice (10). The differentiated cell types obtained from these experiments were determined by their morphology, chemical and immunological staining, and RNA expression pattern. To our surprise, we found that these double knockouts, like the parental wild-type ES cells, could still make both skeletal and cardiac muscle, cartilage, nerve, and erythrocytes (Fig. 3). In our assay, 50-90%16 of embryoid bodies formed in suspension culture contain cardiac muscle and erythrocytes, as judged by rhythmic contraction and benzidine staining, respectively; no obvious difference was observed between the parental and double knockout clones. For a detailed quantitative analysis, we also examined the expression of erythrocyte- and neural-specific mRNAs by RT-PCR. No significant changes in either the timing or the level of expression of these markers during differentiation was observed (data not shown). Our results indicate that E12 and E47 are not essential for the terminal differentiations of cardiac and skeletal muscle, erythrocytes, neurons, or cartilage. However, we cannot rule

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Genetics: Zhuang et al.

Proc. Nati. Acad. Sci. USA 89 (1992)

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FIG. 3. Differentiation of E2A knockouts. E2A knockout clones were examined by both in vitro differentiation and tumor assays. Photomicrographs from a double knockout of E12 and E47; similar results were obtained with wild type, single knockout, and double ckout of E12. (A) Phase-contrast micrograph (x50) showing a cluster of skeletal muscle cells derived from in vitro differentiation. (B) Phase-contrast micrograph (x100) showing neurons derived from in vitro differentiation. (C) Benzidine stain showing erythropoiesis inside an embryoid body. (x 160.) (D) MF-20, a myosin heavy-chain-specific antibody, detects myotubes (rhodamine staining) derived from the primary culture oftumors. (x200.) (E) Section of hematoxylin and eosin-stained tumors showing muscle and cartilage. (X100.)

out the possibility that E12 and E47 are still involved in more subtle regulation of these cell types in vivo. The lack of phenotype in the E2A mutations seems contradictory to previous studies (see Introduction). One plausible explanation is that the function of the E2A gene is redundant in mammals. The failure to observe phenotypes with several mammalian genes analyzed by the knockout strategy has been attributed to redundancy (16). In fact, the mammalian

gene products of the ITF-2 and HEB genes have recently been shown to have biochemical and functional properties similar to those of E12 and E47 (17, 18). We would like to thank C. Lanz, K. Thomas, and M. Capecchi for their advice on ES cells and gene targeting. We thank E. Roberson for providing the CCE cells and J. Lee and R. Kopan for help on the tumor assay. We appreciate the help and advice from our colleagues

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Genetics: Zhuang et al.

at Fred Hutchinson Cancer Center. Y.Z. is supported by a Damon Runyon-Walter Winchell Postdoctoral Fellowship; C.G.K. is supported by an award from the Cooley's Anemia Foundation. This work was supported in part by National Institutes of Health Grants CA42506 to H.W. and BL48356 to M.G. 1. Weintraub, H., Davis, R., Tapscott, S., Thayer, M., Krause, M., Benezra, R., Blackwell, T. K., Turner, D., Rupp, R., Hollenberg, S., Zhuang, Y. & Lassar, A. B. (1991) Science 251, 761-766. 2. Olson, E. N. (1990) Genes Dev. 4, 1454-1461. 3. Parkhurst, S. M., Bopp, D. & Ish-Horowicz, D. (1991) Cell 63, 1179-1191. 4. Weintraub, H., Tapscott, S. J., Davis, R. L., Thayer, M., Adam, M. A., Lassar, A. B. & Miller, A. D. (1989) Proc. Nati. Acad. Sci. USA 86, 5434-5438. 5. Lassar, A. B., Davis, R. L., Wright, W. E., Kadesch, T., Murre, C., Voronova, A., Baltimore, D. & Weintraub, H. (1991) Cell 66, 305-315. 6. Hsu, H. L., Cheng, J. T., Chen, Q. & Baer, R. (1991) Mol. Cell. Biol. 11, 3037-3042.

Proc. Nadl. Acad. Sci. USA 89 (1992) 7. Johnson, J. E., Birren, S. J., Saito, T. & Anderson, D. J. (1992) Proc. Natd. Acad. Sci. USA 89, 3596-3600. 8. Benezra, R., Davis, R. L., Lockshon, D., Turner, D. L. & Weintraub, H. (1990) Cell 61, 49-59. 9. Kreider, B. L., Benezra, R., Rovera, G. & Kadesch, T. (1992) Science 255, 1700-1702. 10. Robertson, E. J. (1987) Teratocarcinomas and Embryonic Stem Cells (IRL, Oxford). 11. Kim, H.-S. & Smithies, 0. (1988) Nucleic Acids Res. 16, 8887-8903. 12. Rupp, R. A. & Weintraub, H. (1991) Cell 65, 927-937. 13. Sun, X. H. & Baltimore, D. (1991) Cell 64, 459-470. 14. Mansour, S. L., Thomas, K. R. & Capecchi, M. R. (1988) Nature (London) 33, 348-352. 15. Reid, L. H., Gregg, R. G., Smithies, 0. & Koller, B. H. (1990) Proc. Natl. Acad. Sci. USA 87, 4299-4303. 16. Soriano, P., Montgomery. C., Geske, R. & Bradley, A. (1991) Cell 64, 693-702. 17. Henthorn, P., Kiledjian, M. & Kadesch, T. (1990) Science 247, 467-470. 18. Hu, J. S., Olson, E. & Kinston, R. E. (1992) Mol. Cell. Biol. 12, 1031-1042.