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Oncogene (1998) 17, 2287 ± 2293 ã 1998 Stockton Press All rights reserved 0950 ± 9232/98 $12.00 http://www.stockton-press.co.uk/onc AML1(7/7) embryos do not express certain hematopoiesis-related gene transcripts including those of the PU.1 gene Hitoshi Okada1, Toshio Watanabe2, Masaru Niki2, Hiroshi Takano1, Natsuko Chiba2, Nobuaki Yanai3, Kenzaburo Tani4, Hitoshi Hibino4, Shigetaka Asano4, Michael L Mucenski5, Yoshiaki Ito6, Tetsuo Noda1 and Masanobu Satake2 1 Department of Cell Biology, Cancer Institute, Toshima-ku, Tokyo 170; 2Department of Molecular Immunology; 3Department of Cell Biology, Institute of Development, Aging and Cancer, Tohoku University, Seiryo-machi, Aoba-ku, Sendai 980; 4Department of Hematology-Oncology, The Institute of Medical Science, The University of Tokyo, Shirokanedai, Minato-ku, Tokyo 108, Japan; 5 Human Genome Sciences, Inc., 9410 Key West Avenue, Rockville, Maryland 20850, USA; 6Department of Viral Oncology, Institute for Virus Research, Kyoto University, Shogoin, Sakyo-ku, Kyoto 606, Japan The AML1 and PEBP2b/CBFb genes encode the DNAbinding and non-binding subunits, respectively, of the heterodimeric transcription factor, PEBP2/CBF. Targeting each gene results in an almost identical phenotype, namely the complete lack of de®nitive hematopoiesis in the fetal liver on embryonic day 11.5 (E11.5). We examined and compared the expression levels of various hematopoiesis-related genes in wild type embryos and in embryos mutated for AML1 or PEBP2b/CBFb. The RNAs were prepared from the yolk sacs of E9.5 embryos, from the aorta-gonad- mesonephros regions of E11.5 embryos and from the livers of E11.5 embryos and RT ± PCR was performed to detect various gene transcripts. Transcripts were detected for most of the hematopoiesis-related genes that encode transcription factors, cytokines and cytokine receptors, even in tissues from homozygously targeted embryos. On the other hand, PU.1 transcripts were never detected in any tissue of AML1(7/7) or PEBP2b/CBFb(7/7) embryos. In addition, transcripts for the Vav, ¯k-2/¯t-3, M-CSF receptor, G-CSF receptor and c-Myb genes were not detected in certain tissues of the (7/7) embryos. The results suggest that the expression of a particular set of hematopoiesis-related genes is closely correlated with the PEBP2/CBF function. Keywords: transcription factor; hematopoiesis; AML1; PEBP2b/CBFb; PU.1; RT ± PCR; gene targeting Introduction The AML1 gene encodes the DNA binding subunit of the heterodimeric transcription factor, PEBP2/CBF (Bae et al., 1993, 1994; Kagoshima et al., 1993, 1996; Meyers et al., 1993; Ogawa et al., 1993b). The gene, also known as PEBP2aB or CBFA2, belongs to the mammalian gene family, runt, all of which contain the conserved Runt domain which is responsible for the DNA binding activity of AML1. Several dierent approaches have been used to study the biological signi®cance of the AML1 gene. Firstly, AML1 is located at the chromosomal breakpoints associated Correspondence: M Satake, Department of Molecular Immunology, Institute of Development, Aging and Cancer, Tohoku University, Seiryo-machi, Aoba-ku, Sendai 980-8575, Japan Received 14 April 1998; revised 22 May 1998; accepted 26 May 1998 with several subtypes of human acute myeloid or lymphoblastic leukemia (Erickson et al., 1992; Golub et al., 1995; Mitani et al., 1994; Miyoshi et al., 1991, 1993; Nucifora et al., 1993; Romana et al., 1995; Shurtle et al., 1995). The chimeric genes generated between AML1 and counterpart genes by chromosomal translocations are considered to be responsible for leukemogenesis. Secondly, recent gene targeting studies have shown that the function of AML1 is indispensable for the development of de®nitive hematopoiesis in the murine fetal liver on embryonic day 11.5 (E11.5) (Okuda et al., 1996; Wang et al., 1996a). In AML1(7/7) fetal livers, no hematopoietic cells of any lineage are generated and CFU-C activity cannot be detected. The defect caused by homozygous disruption of AML1 is considered to be intrinsic to multipotent hematopoietic progenitors. This notion is based on the analysis of chimeric mice using AML1 (7/7) embryonic stem (ES) cells. Primitive hematopoiesis which originates in the yolk sac on E7.5 is apparently unaected by AML1 targeting. However, AML1(7/7) embryos start to die on E12.5 due to massive hemorrhage of primitive erythrocytes in the central nervous system by a mechanism that remains to be elucidated. Interestingly, almost identical phenotypes have been reported for targeting of the gene, PEBP2b/CBFb (Niki et al., 1997; Sasaki et al., 1996; Wang et al., 1996b). PEBP2b/ CBFb encodes the non-DNA binding subunit of PEBP2/CBF (Ogawa et al., 1993a; Wang et al., 1993). Therefore, gene targeting studies have demonstrated that the two subunits of the transcription factor PEBP2/CBF, namely AML1 and PEBP2b/CBFb, function as one entity for the generation of hematopoietic progenitors of the de®nitive type. There exist a number of reports of target genes regulated by the transcription factor, PEBP2/CBF. These include the GM-CSF receptor (Frank et al., 1995; Takahashi et al., 1995), M-CSF receptor (Zent et al., 1996; Zhang et al., 1994, 1996), IL-3 (Nimer et al., 1996), myeloperoxidase, neutrophil elastase (Nuchprayoon et al., 1994) and T cell receptors genes (Giese et al., 1995; Hernandez-Munain and Krangel, 1994, 1995; Sun et al., 1995; Wotton et al., 1994 and see a review by Leiden, 1993). The above claims, however, mostly rely on studies using cell lines transfected by reporter plasmids whose transcription is driven by the promoter of a suspected PEBP2/CBF regulated gene. In this study we have taken an PU.1 expression in AML1 targeted embryos H Okada et al 2288 alternative approach by examining genes whose expression is aected in embryonic tissues with an AML1 (7/7) or PEBP2b/CBFb (7/7) background. The expression of genes encoding transcription factors, cytokines and cytokine receptors, which are all known to be involved in hematopoiesis, was examined. The RNAs used for RT ± PCR analysis were prepared from the yolk sac, the embryonic aorta-gonad-mesonephros (AGM) region and the fetal liver. The AGM region has recently been shown to be the bona®de origin of de®nitive hematopoiesis in the murine embryo (Medvinsky and Dzierzak, 1996; Medvinsky et al., 1993; MuÈller et al., 1994). Multipotent hematopoietic progenitors develop ®rst in the AGM region and then settle in the liver through circulation or by direct migration. The transcripts whose expression was aected by mutation of AML1 or PEBP2b/CBFb include those of c-Myb, ¯k-2/¯t-3, M-CSF receptor, GCSF receptor and Vav. In particular, PU.1 transcripts were never detected in any tissue of homozygously targeted embryos. These ®ndings are discussed in respect to gene regulation by PEBP2/CBF. a b Figure 1 Targeting the AML1 gene by homologous recombination. (a) The targeting strategy used to delete the fourth exon of AML1. In the targeting vector the transcriptional direction of neo is opposite to those of the AML1 and DTA genes. E3 and E4 indicate the third and fourth exons of AML1. Probes A, B, C and D whose locations are as indicated were used for Southern hybridization as in panel (b). The sense primers a or c and the antisense primer b were used for PCR to genotype F2 embryos. Abbreviations used for the restriction sites were B for BamHI and X for XbaI. (b) Southern blot analysis of F2 embryos. Genomic DNAs of E11.5 F2 embryos were digested by XbaI in the cases of probes A, C and D or by BamHI in the case of probe B and gel electrophoresed. The blotted ®lters were hybridized with probes A, B, C or D as indicated. The genotypes of AML1 locus in each embryo are indicated. The numbers alongside the ®lters indicate the size of the detected bands in kilobases PU.1 expression in AML1 targeted embryos H Okada et al Results Generation of mouse embryos homozygous for the AML1 disruption The fourth exon of the AML1 genome encodes the central portion of the Runt domain in the AML1 polypeptide (Miyoshi et al., 1995). This was replaced by the neo gene derived from the targeting vector (Figure 1, panel a). By screening the ES cells transfected with the targeting vector, 18 homologous recombinants were obtained. Using two ES clones, we obtained two lines of F1 mice which were heterozygous for the AML1 mutation. Two lines of F2 osprings having the AML1(7/7) genotype displayed identical phenotypes like those described in the Introduction and remained alive until E12.5. The results of Southern blot analysis of the genotypes of the F2 embryos are presented in panel b. Genomic DNA was prepared from E11.5 embryos. Probe A was derived from the genomic sequence and located 3' to the homologous sequence used in the vector. In the case of the heterozygous (+/7) genotype, both the 13.2 kb band of the wild type and the 7.5 kb band from the mutated allele were detected in XbaI-digested DNA. Only the 13.2 and 7.5 kb bands were found for the (+/+) and (7/7) genotypes, respectively. Probe B was located within the 5' homologous sequence used in the vector. This probe detected both the wild type 4.0 kb band and the mutated 2.0 kb band in BamHI-digested DNA prepared from the heterozygous (+/7) embryo. Probes C and D are derived from sequences of the AML1 fourth exon and neo gene, respectively. In XbaIdigested genomic DNA, probe C hybridized with the 13.2 kb band for the wild type (+/+) and heterozygous (+/7) embryos but not for the (7/7) homozygote. On the other hand, the 6.9 kb band was detected by probe D for the (+/7) heterozygote and the (7/7) homozygote but not for the wild type (+/ +) embryo. Thus, the overall results of Southern blot analysis con®rmed the expected pattern of homologous recombination. Histological examination of the yolk sac The embryonic hematopoietic tissues were examined histologically. Figure 2 shows sections of the yolk sacs from E9.5 embryos. At this stage, numerous primitive erythrocytes with nuclei were identi®ed for both the heterozygously and homozygously mutated embryos. This indicates that AML1 is dispensable for primitive hematopoiesis. Expression of hematopoiesis-related genes in AML1-mutated embryos Figure 2 Histological examination of the yolk sacs from AML1(+/7) and (7/7) embryos. Sections of the yolk sacs were prepared from E9.5 embryos in utero and stained with hematoxylin and eosin. The genotypes of AML1 are (+/7) and (7/7) as indicated The E12.5 embryos of AML1 (7/7) were devoid of hematopoietic cells of the de®nitive type in the liver (data not shown). We next examined whether the expression of hematopoiesis-related genes was aected by the AML1 mutation. We selected three hematopoietic tissues, namely the yolk sac of an E9.5 embryo, the AGM region of an E11.5 embryo and the liver of an E11.5 embryo. RNAs were prepared from each tissue and gene expression was examined by the RT ± PCR method. Adequateness of PCR-ampli®cation was checked by examining the size and restriction enzyme digestion pattern of the PCR products. At least four embryos obtained from two dierent litters were used for one genotype. In addition, we also examined embryos mutated for PEBP2b/CBFb, whose generation we reported previously (Niki et al., 1997). Table 1 summarizes the results of RT ± PCR analysis which was performed for each transcript, for each tissue and for each genotype of AML1 or PEBP2b/CBFb. Since the PCR method employed was not very quantitative, the results are simply expressed as being positive or negative. Therefore, even if a transcript was found be present at a lower level in the tissue of one genotype compared to that of same tissue of another genotype, it was still considered to be positive. The AML1 transcript was not detected in any tissue of AML1(7/7) embryos but was detected in the liver of PEBP2b/CBFb(7/7) embryos. On the contrary, the PEBP2b/CBFb transcript was not detected in the liver of PEBP2b/CBFb(7/7) embryos but was detected in all the tissues of AML1(7/7) embryos. 2289 PU.1 expression in AML1 targeted embryos H Okada et al 2290 were prepared from the livers of E11.5 embryos and hybridized with the radiolabeled antisense RNA probe. After digestion by RNase, the samples were run on a sequencing gel (Figure 4). The probe used was a mixture of antisense RNAs which allowed simultaneous detection of both PU.1 and glyceraldehyde -3-phosphate dehydrogenase (GAPDH) transcripts. GAPDH transcripts were detected to a similar extent irrespective of the AML1 genotype. On the other hand, PU.1 transcripts were only evident for the AML1(+/+) and (+/7) genotypes but not for the (7/7) genotype. Each lane in Figure 4 represents an RNA sample prepared from an individual embryo. Thus, the combined results of Figures 3 and 4 con®rm that there was, indeed, no PU.1 transcript expressed in the tissues of AML1(7/7) or PEBP2b/CBFb(7/7) embryos. Absence of PU.1 transcript in the AML1(7/7) embryos The PCR products generated from the PU.1 transcripts were processed for Southern blot analysis (Figure 3). The radiolabeled probe used corresponds to the oligonucleotide sequence located between the sense and antisense primers. No apparent band was detected for the yolk sac, the AGM region or the liver of AML1(7/7) embryos, whereas discrete, positive signals were detected for the tissues of AML1(+/+) and (+/7) embryos (see the upper panel). It is particularly noteworthy that no PU.1 transcripts were detected in the E9.5 yolk sac of AML1(7/7) embryos that contained numerous primitive erythrocytes. From the RNA samples described in the upper panel, similar amounts of PCR products were generated when the primers were set for the hypoxanthine phosphoribosyl transferase (HPRT) cDNA. The absence of the PU.1 transcript in the liver RNA of PEBP2b/CBFb(7/7) embryos was also con®rmed by Southern blot analysis of the RT ± PCR products (data not shown). The expression of the PU.1 transcript was also examined by an RNase protection assay. The RNAs Other genes whose expression was aected by the AML1 mutation In addition to PU.1, several other hematopoiesisrelated genes are aected in their expression by mutation of AML1 and PEBP2b/CBFb (Table 1). The transcripts of the M-CSF receptor and Vav were Table 1. Expression of various genes in AML1-, PEBP2b/CBFb- or c-Myb- mutated embryos as examined by RT±PCR Genotype of AML1 Genotype of PEBP2 b/ Genotype of c-Myb CBF b E9.5 Yolk sac E11.5 AGM E11.5, Liver E11.5, Liver E11.5, Liver (+/+) (+/±) (±/±) (+/+) (+/±) (±/±) (+/+) (+/±) (±/±) (+/+) (+/±) (±/±) (+/+) (+/±) (±/±) AML1 PEBP2 b/CBF b PEBP2 aA GATA1 GATA2 Ikaros Scl/tal1 PU.1 NFE2 c-Myb ¯k-1 ¯k-2/¯t-3 ¯t-1 SCF c-Kit Epo Epo receptor M-CSF M-CSF receptor G-CSF G-CSF receptor Vav b-actin HPRT + + + + + + + + + + + ± + + + + + + + + + + + + + + + + + + + + + + + ± + + + + + + + + + + + + ± + + + + + + ± + + + ± + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + ± + + + + + + ± + + + ± + + + + + + ± + + ± + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + ± + + + + + + ± + ± + ± + + + + + + ± + ± ± + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + ± + + + + + ± + ± + ± + + + + + + ± + ± ± + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + Figure 3 RT ± PCR analysis of PU.1 and HPRT transcripts. PCR was performed on the cDNA templates prepared from the E9.5yolk sacs, the E11.5-AGM regions and the E11.5-livers, using the appropriate primers to detect PU.1 or HPRT transcripts. The PCR products were gel electrophoresed and the blotted ®lters were processed for Southern hybridization. 32P-labeled probes used in hybridization are described in Material and methods. Each lane represents a sample from an individual embryo and the AML1 genotypes of each embryo are as indicated + + + + + + + + + 7 + + + + + + + + + + + + + + PU.1 expression in AML1 targeted embryos H Okada et al to the paucity of hematopoietic cells in the AML1 (7/ 7) or PEBP2b/CBFb (7/7) embryos. Discussion Figure 4 Expression of PU.1 and GAPDH transcripts as assayed by RNase protection. The cytoplasmic RNAs prepared from the livers of E11.5 embryos were hybridized with 32P-labeled RNA probes harboring the antisense sequences of PU.1 or GAPDH. After digestion with RNase, the samples were gel electrophoresed and processed for image analysis. The locations of undigested RNA are indicated by bars. The genotypes of AML1 in each embryos are (+/+), (+/7) or (7/7), as indicated neither detected in the AGM nor in the liver of AML1 (7/7) embryos, but were detected in the yolk sac. The expression of c-Myb and the G-CSF receptor was undetectable in the liver of these embryos but was detectable in the AGM and yolk sac. The AML1 (7/7) embryos did not express ¯k-2/¯t-3 transcripts in the AGM or liver. As for the yolk sac, the eect of the AML1 mutation on ¯k-2/¯t-3 expression cannot be evaluated, since corresponding transcripts were not detected in the yolk sac, even in the case of wild type embryos. This result, however, con®rms that the expression of ¯k-2/¯t-3 is strictly correlated with the development of de®nitive type hematopoiesis (Matthews et al., 1991). It should be noted that homozygous mutations of AML1 or PEBP2b/CBFb did not necessarily cause complete shut down of expression of the hematopoiesis-related genes other than mentioned above. Comparison with the c-Myb targeted embryos Homozygous mutations of ¯k-2/¯t-3 (Mackarehtschian et al., 1995), c-Myb (Mucenski et al., 1991) or PU.1 (McKercher et al., 1996; Scott et al., 1994), all impair hematopoiesis of the de®nitive type, although only in certain cell lineages and to dierent extents. The phenotype that best resembles the case of AML1 (7/ 7) or PEBP2b/CBFb (7/7) is that associated with gene targeting of c-Myb. In the liver of c-Myb (7/7) embryos, no hematopoietic cells with the exception of megakaryocytes can be observed and no CFU-C activities can be detected. Therefore, we examined in parallel what kind of genes are aected in their expression in the c-Myb (7/7) embryonic liver. Interestingly, transcripts of the PU.1, ¯k-2/¯t-3, Vav, G-CSF receptor and M-CSF receptor genes were detected by the RT ± PCR method (Table 1). This indicates that the absence of de®nitive hematopoiesis in the c-Myb (7/7) embryos did not result in the complete lack of expression of the above mentioned transcripts. Thus, the failure to detect a particular species of transcript does not appear to be simply due Of particular interest in the present study is the observation that AML1 (7/7) and PEBP2b/CBFb (7/7) embryos displayed identical loss of certain transcripts of the liver. This is in good agreement with the notion that deletion of even one of the genes of the heterodimeric transcription factor PEBP2/CBF is sucient to abolish its function. It is therefore likely that the absence of certain transcripts in the homozygously mutated embryos is somehow related to the loss of PEBP2/CBF function. Recently, the culture system of AGM in which hematopoietic cells can be generated in vitro has been developed (Mukouyama et al., 1998). We subjected the AGM regions of the AML1 (7/7), c-Myb (7/7) as well as wild type E11.5 embryos to this culture, according to the procedure described. Neither from the AML1 (7/7) or from the c-Myb (7/7) culture, hematopoietic cells were produced at all (details of these cultures will be described elsewhere). Interestingly, transcripts of PU.1 and Vav were not detected in the AML1 (7/7) culture but detected in the c-Myb (7/7) as well as the wild type cultures by the RT ± PCR method (unpublished results). This indicates that AML1 is indeed indispensable for the expression of PU.1 and Vav irrespective of the presence or absence of hematopoiesis, at least in the in vitro culture of AGM. The expression of M-CSF receptor has been reported to be regulated by AML1 (Zhang et al., 1996). The AML1 and C/EBP transcription factors can associate with each other, bind to their adjacent sites together on the promoter of the M-CSF receptor and transactivate it synergistically. This was revealed by transfection studies using various cell lines. Thus, it is noteworthy that the present study employing gene targeted embryos identi®ed the M-CSF receptor as a candidate gene whose expression is regulated by PEBP2/CBF. The transcripts which behaved similarly to the M-CSF receptor in the targeted embryos include those of PU.1, ¯k-2/¯t-3 and Vav. None of these transcripts were detected in the AGM or liver of AML1 (7/7) or PEBP2b/CBFb (7/7) embryos. The possibility that the promoters of PU.1 and Vav are likewise regulated by PEBP2/CBF remains to be elucidated, since survey of their promoter regions (7330 nt through to +1 nt of the PU.1 promoter, Chen et al., 1995 and 7870 nt through to +1 nt of the Vav promoter, Ogilvy et al., 1998) revealed multiple candidate sequences therein, though not typical, for the PEBP2/CBF binding (unpublished observation). In the case of the PU.1 promoter, several transcription factors such as Oct1/2, Spl and PU.1 itself have been reported to be important for its transactivation in myeloid and B lymphoid cell lines (Chen et al., 1995, 1996). Overall, the present study has shown that a particular set of hematopoiesis-related genes are signi®cantly aected in their expression by mutations of AML1 and PEBP2b/CBFb. These observations should help clarify the relationship between the gene expression and the PEBP2/CBF transcription factor in exact molecular and cellular terms. 2291 PU.1 expression in AML1 targeted embryos H Okada et al 2292 Materials and methods Targeting vector and homologous recombination The targeting vector for AML1 replaces a 0.6-kb region that encompasses the fourth exon of AML1 with the neo cassette containing the neo gene regulated by the promoter and the poly(A)+ addition sequence of the murine phosphoglycerol kinase gene (Figure 1a). The neo gene was ¯anked by a 6.3 kb SmaI-EcoRI fragment and a 1.3 kb SmaI-BamHI fragment, each corresponding to 5' and 3' genomic sequences of AML1 surrounding the fourth exon. In addition, the loxP sequences were added to both ends of the neo cassette. The gene for the diphtheria toxin A subunit (DTA) was incorporated into the 3' end of the vector. The vector was linearized with NotI and electroporated into J1 ES cells. Two hundred and thirty G418 resistant ES cell colonies were picked up, expanded as described previously (Nakai et al., 1995) and screened for homologous recombination by Southern blotting using probe A depicted in Figure 1a. Eighteen ES cell clones were identi®ed as heterozygous for the AML1 disruption and two clones were injected into blastocysts derived from C57BL/6J mice. Chimeric mice were bred against C57BL/ 6J female mice. Southern blot analysis of tail DNA of F1 mice showed germline transmission of the mutant AML1 allele. F1 mice heterozygous for the mutation were intercrossed to generate F2 ospring. For genotyping F2 mouse embryos routinely, PCR was performed on genomic DNA as a template. The locations of primer a, 5'CAGGTTTGTCGGGCGGAGCGGTAGA-3', primer b, 5'-CCAAGTCCATCACAGCTGTGCGTT-3' and primer c, 5'-GCTAAAGCGCATGCTCCAGACTGCCTTG-3', are depicted in Figure 1a. PCR ampli®cation (PerkinElmer) was performed for 35 cycles (948C, 1 min; 608C, 2 min; 728C, 2 min). The size of the PCR products were 211 bp from the wild type allele and 340 bp from the mutated allele. All the presented data was obtained using F2 mouse embryos. Targeting the PEBP2b/CBFb and cMyb genes was described previously (Mucenski et al., 1991; Niki et al., 1997). Histological examination The E9.5 embryos in utero were ®xed in formaldehyde solution for 6 h. The yolk sacs were then removed and embedded in paran. Transverse sections with a 10 mm thickness were mounted on slide glasses and stained with hematoxylin and eosin. RT ± PCR analysis E9.5 and E11.5 live embryos were selected on the basis of the presence of a heart beat. Total RNA samples were prepared from the yolk sacs, the AGM regions or the livers of embryos using ISOGEN (Nippon Gene). RNA (2 mg) was reverse transcribed at 428C for 60 min using a cDNA cycle kit (Invitrogen) and the products were dissolved in 10 ml of distilled water. PCR ampli®cation of the cDNAs (0.2 ml each for one reaction) was performed for 35 cycles. One cycle was 948C, 1 min; 608C, 2 min and 728C, 2 min. The PCR products were analysed by 2% (wt/vol) agarose gel electrophoresis. All the primer sets for the genes listed in Table 1 were as described previously (Keller et al., 1993; Koopman et al., 1989; McClanahan et al., 1993; Takahashi et al., 1997; Weiss et al., 1994). For the PCR products of PU.1, c-Myb and HPRT, Southern blot analysis was also carried out. The blotted ®lters were hybridized with 32Pend-labeled oligonucleotide harboring the sequences of PU.1, 5'-CTATCGCCACATGGAGCTGGAACAG-3', cMyb, 5'-TCTGGGGAACAGATGGGCAGAGAT C - 3' or HPRT, 5'-TACGAGGAGTCCTGTTGATGTTGCC-3'. The hybridization was done at 428C for 12 h in 66SSPE, 56Denhardt's solution, 1% (wt/vol) SDS and 0.1 mg/ml of denatured salmon sperm DNA. The blotted ®lters were washed twice at 608C for 15 min in 0.26SSC and 0.1% (wt/vol) SDS and processed for autoradiography. RNase protection analysis The antisense RNA probes were prepared as follows. The cDNA sequence of PU.1 (nt 20 through to nt 571 in the coding region) was subcloned into the PstI-SacI sites of pBluescript. Two mg of the plasmid DNA was linearized by digestion with HindIII and used as a template. Transcription was performed by T7 RNA polymerase and [a-32P]UTP was included in the reaction. Total radioactivity incorporated into RNA was 26107 c.p.m. The antisense RNA probe for GAPDH was prepared in a similar way. The 32P-labeled RNA probes (16105 c.p.m. for PU.1 and 16103 c.p.m. for GAPDH) were mixed with 10 mg of the cytoplasmic RNA prepared from the livers of E11.5 embryos. Hybridization and RNase digestion were performed using a RPAII Ribonuclease Assay Kit (Ambion Inc.). The ®nal sample was run on an 8% (wt/vol) polyacrylamide-8M urea sequencing gel. The dried gel was processed for image analysis. Acknowledgements We thank I Imamura for secretarial assistance. 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