Differentiation (1 990) 45: 76-83
Developmentally regulated expression of embigin, a member of the immunoglobulin superfamily found in embryonal carcinoma cells Ruo-Pan Huang, Masayuki Ozawa, Kenji Kadomatsu, and Takashi Muramatsu* Department of Biochemistry, Faculty of Medicine, Kagoshima University, 1208-1 Usukicho, Kagoshima 890, Japan Accepted in revised form July 19, 1990
Abstract. Embigin is a member of the immunoglobulin superfamily found in embryonal carcinoma cells. The present study deals with embigin gene expression in adult and embryonic cells. By RNA blot analyses, high levels of embigin mRNA were detected in embryonal carcinoma cells with different differentiation potentials, namely F9 and PCC4 cells. Similar or slightly higher levels of the RNA were detected in the embryonic and extraembryonic portions of 9-day mouse embryos. When F9 cells were induced to differentiate by treatment with retinoic acid and dibutyryl cyclic AMP for 2-5 days, the RNA level increased significantly. On the other hand, embigin mRNA dramatically dropped to almost background levels in embryos, from 11 days postcoitus (P.c.). Many organs of adult mice expressed only low levels of embigin mRNA, but considerable amounts of the transcript were found in the ovary and also in the uterus of pregnant mice (4 days p.c. and 12 days P.c.). In situ hybridization experiments revealed large amounts of embigin RNA in the embryo from 7 to 9 days p.c. The visceral endoderm of 7-day embryos, and the brain, visceral yolk sac and presumptive foregut of 9-day embryos were the sites where intense signals of embigin RNA were localized.
portant roles in the differentiation of thymic precursor cells either to helper or cytotoxic T cells [ 5 , 25, 281. Cell adhesion molecules on nerve cells, such as N-CAM [4], L1 [19], MAG [l], and contactin [23], have also been found to be members of the superfamily, and they are involved in the mutual interaction of nerve cells. Most of the members of the immunoglobulin superfamily have not been reported in early embryonic cells. They are usually expressed after certain stages in embryogenesis. So far, only two members of the superfamily have been described as being present in embryonal carcinoma (EC) cells, which are teratocarcinoma stem cells and resemble multipotential cells of early embryos [ 161;one of them is N-CAM [ 111. The other superfamily member expressed in EC cells has a molecular weight of 62000-90000 and has a V-domain-like sequence near the transmembrane region [22]. This glycoprotein, previously called GP-70, will be referred to as embigin (embryo immunoglobulin) in this communication. The embigin protein sequence deduced from the cDNA sequence is distinct from other members of the immunoglobulin superfamily [22]. Because of its expression in EC cells, embigin is expected to be involved in the early stages of embryogenesis. As the first step in analyzing the function of embigin, we examined the pattern of embigin expression in embryonic and adult cells.
+
Introduction Methods The immunoglobulin superfamily comprises diverse kinds of cell-surface and soluble proteins, many of which are involved in cell-cell interactions [lo, 301. The immunoglobulin-like domain present in these proteins is particularly suitable for molecular recognition. The mutual interaction of the superfamily members in the regulation of cell differentiation has been documented in the case of T lymphocytes. T cell receptors, CD3, MHC class 1/11 antigens and CD4/8, all of which are members of the superfamily, interact with each other and exert im-
* To whom offprint requests should be sent
Cell lines and culture techniques. The EC cells F9 [2] and PCC4 [12], PYS-2 parietal endoderm cells [14] and STO fibroblasts [17] were cultured as described in Nomoto et al. [20]. Teratocarcinoma OTT6050 [26] was maintained and grown in 129/Sv mice by intraperitoneal passage. F9 cells were induced to differentiate by treatment with retinoic acid and di-butyryl cyclic AMP essentially as described by Strickland et al. [27] except that the concentration of retinoic acid was raised to 1 p M . Embryos and organs. Embryos were obtained from naturally mated ICR mice. The day when a vaginal plug was observed was taken as day 0 p.c. Unless otherwise specified, all organs were isolated from adult ICR mice.
77 Hybridization probes. For slot-blot hybridization, embigin cDNA probe was prepared from a 590-bp EcoRI-BamHI restriction fragment extending from 792 to 1382 nucleotides in clone p o l 5 as shown in Fig. 4. We called this probe the E/B probe. The probe was labeled with [32P]-dCTP by a random oligonucleotide primer [6]. For the ribonuclease protection assay and in situ hybridization, a 590-bp EcoRI-BumHI fragment was subcloned into SP64 (antisense) and SP65 (sense). Antisense and sense RNA were generated with SP6 polymerase after linearization by EcoRI or BamHI, respectively.
EDTA). After heating at 85" C for 8 min. hybridization was performed at 45" C for 18 h. Then the sample was digested with 40 pg/ ml ribonuclease A (Sigma) and 2 pg/ml ribonuclease T, (Sigma) at 30" C for 30 min. The protected fragment was analyzed on a 4% polyacrylamide/8 M urea sequencing gel.
In situ hybridization. In situ hybridization was performed essentially as described by Wilkinson et al. [29]. Briefly, fixed embryos were embedded in paraffin, and sections of 6 prn thickness were cut. Three sections at 6- to 18-pm intervals were applied for the hybridization with antisense or sense probe and hematoxylin eosin staining. For in situ hybridization, sections were transferred to albumincoated slides and dried overnight at 50°C. The slides were then treated with 20 pg/ml proteinase K (Sigma) and acetic anhydride prior to hybridization with [35S]-UTP-labeled probes ( 800 Ci/ mmole, Amersham) [9]. The [35S]-labeled RNA was degraded to an average length of 100 bases [3] and the specificity was confirmed by Northern blot (data not shown). The hybridization solution consisted of 0.03 ng/pl probe in: 50% formamide; 0.3 M sodium chloride; 20 mM Tris-HCI, pH 8.0; 5 mM EDTA; 10 mM sodium phosphate, pH 8.0; 10% dextran sulfate; 1 x Denhardt's solution; 0.5 mg/rnl yeast tRNA; and 20 mM dithiothreitol. The solution was applied to slides and covered with siliconized baked coverslips. Slides were immersed in prewarmed mineral oil and incubated overnight at 50' C. Slides were washed as described including the wash with 20pg/ml ribonuclease A (Sigma) for 30 min at 37" C [9]. Slides were coated with Konica NR-M2 nuclear emulsion, diluted one to one with distilled water, by the dipping method. These slides were stored under low humidity at 4" C for 2 weeks and developed with Fuji Rendol for 4 min at 20" C.
EDNAcloning and DNA sequence analysis. The Agtl 1 cDNA library constructed from F9 EC cells was screened by the plaque hybridization method [15] using the embigin E/B probe. The library was constructed from cultured F9 cells according to the method described previously [22] by H. Muramatsu in our laboratory. Phage DNA of the positive clones was prepared using the plate lysate method [15]. and the cDNA inserts were cloned into the EcoRl site of pUC8. Following the subcloning of restriction endonuclease fragments into pUC18 or pUC8, DNA sequencing was performed, employing the dideoxy chain termination method [24] adapted for denatured plasmid templates [7].
-
RNA analysis. Total RNA was prepared by the guanidinium isothiocyanate method [ 151. For slot-blot hybridization, serial dilutions of total RNA were applied to nitrocellulose using an apparatus from Schleicher and Schuell Co., followed by hybridization with a [32P]-labeled probe. Filters were washed three times for 15 min with an excess of 2 xO.15 M NaCI, 0.015 M sodium citrate (SSC)/ 0.1 YO sodium dodecyl sulfate (SDS) at 50" C and then washed twice for 30 min with 0.1 x SSC/O.l YO SDS at the same temperature. The degree of hybridization was quantitated by scanning densitometry of each hybridization using an Ultroscan XL (LKB) laser densitometer equipped with an automatic integrator; the values within the linear range were adopted and used for further calculation. For Northern blot analysis, total RNA (30 pg) and poly(A)+RNA (10 pg) were denatured with glyoxal and dimethyl sulfoxide and were separated on 1.O% agarose gel [15]. After transferring to nitrocellulose, RNA was hybridized with the E/B probe. The conditions of hybridization and washing were identical to those employed in slot blotting. The RNase protection assay was performed essentially as described by Zinn et al. [31]. The [32P]-UTP-labeledantisense probe was purified by electrophoresis in a 4% sequencing gel [18]. Hybridization was carried out using 20 pg total RNA along with 2 x lo5 cpm purified [32P]-labeledprobe in 30 p1 hybridization solution (80% formamide, 0.4 M NaCI, 40 mM Pipes pH 6.5, 1 rnM
B
A
._ =
$
Results Expression of embigin niRNA in culture cells and in organs of adult mice
The amount of embigin mRNA was examined by slotblot analysis using a [32P]-labeled E/B probe of embigin cDNA. As reported previously, F9 EC cells contained significant amounts of the mRNA. Slightly higher amounts of embigin mRNA were detected in teratocarcinoma OTT6050 and PCC4 EC cells (Fig. 1 A). In subsequent analyses, RNA from teratocarcinoma OTT6050 was always used as the standard, and we expressed the
3
f
C
Embigin
20 10
t
-
10
RNA(AI~)
c)
-
I
fi-Actin
.-u) I-
D 0
Ln
6
6
-
6
$
0,
! l !
0
c.
Fig. 1A-D. The relative amount of embigin gene transcript in various mouse organs, cultured cells and the embryo proper. Total RNA was serially diluted, spotted on nitrocellulose filters and analyzed by slot hybridization using [32P]-labeled E/B fragment of embigin cDNA or fl-actin cDNA (for control) as probes. These experiments were repeated at least twice and gave the same results. A Teratocarcinoma cells and STO fibroblasts. B Organs of adult mice. C F9 cells induced to differentiate by treatment with retinoic acid and dibutyryl cyclic AMP at day 0 with no treatment and n days after the treatment. D Mouse embryo proper. The no. of days indicates the days of gestation
78
Table 1. Comparison of embigin mRNA level in various cells, mouse embryos and adult organs
Table 1. (continued)
~~
Sources of RNA
Intensity The value normalized of hybridization by the intensity to embigin probe' of hybridization to 8-actin probe
Teratocarcinoma OTT6050
1 .oo
1 .oo
Cells F9 PCC4 PYS-2 STO
0.866 1.15 0.821 0.329
0.630 0.884 0.607 0.335
F9 cells induced to differentiate for 0 days 1 days 2 days 3 days 4 days 5 days
Sources of RNA
5 days 6 days 7 days 8 days 10 days 12 days 18 days
0.196 0.150 0.235 0.210 0.207 0.660 0.389
0.197 0.133 0.201 0.206 0.196 0.513 0.303
0.067
0.074
Placenta from the mice after following days p.c. 15 days 0.1 33 18 days 0.117
0.144 0.107
From mice 5 days after delivery 0.800 0.712 3.01 2.42 2.58 2.26
0.590 0.528 2.22 1.78 1.90 1.67
Embryo proper on the following days of gestation 9 days 1.29 1 1 days
Developmentally regulated expression of embigin, a member of the immunoglobulin superfamily found in embryonal carcinoma cells Ruo-Pan Huang, Masayuki Ozawa, Kenji Kadomatsu, and Takashi Muramatsu* Department of Biochemistry, Faculty of Medicine, Kagoshima University, 1208-1 Usukicho, Kagoshima 890, Japan Accepted in revised form July 19, 1990
Abstract. Embigin is a member of the immunoglobulin superfamily found in embryonal carcinoma cells. The present study deals with embigin gene expression in adult and embryonic cells. By RNA blot analyses, high levels of embigin mRNA were detected in embryonal carcinoma cells with different differentiation potentials, namely F9 and PCC4 cells. Similar or slightly higher levels of the RNA were detected in the embryonic and extraembryonic portions of 9-day mouse embryos. When F9 cells were induced to differentiate by treatment with retinoic acid and dibutyryl cyclic AMP for 2-5 days, the RNA level increased significantly. On the other hand, embigin mRNA dramatically dropped to almost background levels in embryos, from 11 days postcoitus (P.c.). Many organs of adult mice expressed only low levels of embigin mRNA, but considerable amounts of the transcript were found in the ovary and also in the uterus of pregnant mice (4 days p.c. and 12 days P.c.). In situ hybridization experiments revealed large amounts of embigin RNA in the embryo from 7 to 9 days p.c. The visceral endoderm of 7-day embryos, and the brain, visceral yolk sac and presumptive foregut of 9-day embryos were the sites where intense signals of embigin RNA were localized.
portant roles in the differentiation of thymic precursor cells either to helper or cytotoxic T cells [ 5 , 25, 281. Cell adhesion molecules on nerve cells, such as N-CAM [4], L1 [19], MAG [l], and contactin [23], have also been found to be members of the superfamily, and they are involved in the mutual interaction of nerve cells. Most of the members of the immunoglobulin superfamily have not been reported in early embryonic cells. They are usually expressed after certain stages in embryogenesis. So far, only two members of the superfamily have been described as being present in embryonal carcinoma (EC) cells, which are teratocarcinoma stem cells and resemble multipotential cells of early embryos [ 161;one of them is N-CAM [ 111. The other superfamily member expressed in EC cells has a molecular weight of 62000-90000 and has a V-domain-like sequence near the transmembrane region [22]. This glycoprotein, previously called GP-70, will be referred to as embigin (embryo immunoglobulin) in this communication. The embigin protein sequence deduced from the cDNA sequence is distinct from other members of the immunoglobulin superfamily [22]. Because of its expression in EC cells, embigin is expected to be involved in the early stages of embryogenesis. As the first step in analyzing the function of embigin, we examined the pattern of embigin expression in embryonic and adult cells.
+
Introduction Methods The immunoglobulin superfamily comprises diverse kinds of cell-surface and soluble proteins, many of which are involved in cell-cell interactions [lo, 301. The immunoglobulin-like domain present in these proteins is particularly suitable for molecular recognition. The mutual interaction of the superfamily members in the regulation of cell differentiation has been documented in the case of T lymphocytes. T cell receptors, CD3, MHC class 1/11 antigens and CD4/8, all of which are members of the superfamily, interact with each other and exert im-
* To whom offprint requests should be sent
Cell lines and culture techniques. The EC cells F9 [2] and PCC4 [12], PYS-2 parietal endoderm cells [14] and STO fibroblasts [17] were cultured as described in Nomoto et al. [20]. Teratocarcinoma OTT6050 [26] was maintained and grown in 129/Sv mice by intraperitoneal passage. F9 cells were induced to differentiate by treatment with retinoic acid and di-butyryl cyclic AMP essentially as described by Strickland et al. [27] except that the concentration of retinoic acid was raised to 1 p M . Embryos and organs. Embryos were obtained from naturally mated ICR mice. The day when a vaginal plug was observed was taken as day 0 p.c. Unless otherwise specified, all organs were isolated from adult ICR mice.
77 Hybridization probes. For slot-blot hybridization, embigin cDNA probe was prepared from a 590-bp EcoRI-BamHI restriction fragment extending from 792 to 1382 nucleotides in clone p o l 5 as shown in Fig. 4. We called this probe the E/B probe. The probe was labeled with [32P]-dCTP by a random oligonucleotide primer [6]. For the ribonuclease protection assay and in situ hybridization, a 590-bp EcoRI-BumHI fragment was subcloned into SP64 (antisense) and SP65 (sense). Antisense and sense RNA were generated with SP6 polymerase after linearization by EcoRI or BamHI, respectively.
EDTA). After heating at 85" C for 8 min. hybridization was performed at 45" C for 18 h. Then the sample was digested with 40 pg/ ml ribonuclease A (Sigma) and 2 pg/ml ribonuclease T, (Sigma) at 30" C for 30 min. The protected fragment was analyzed on a 4% polyacrylamide/8 M urea sequencing gel.
In situ hybridization. In situ hybridization was performed essentially as described by Wilkinson et al. [29]. Briefly, fixed embryos were embedded in paraffin, and sections of 6 prn thickness were cut. Three sections at 6- to 18-pm intervals were applied for the hybridization with antisense or sense probe and hematoxylin eosin staining. For in situ hybridization, sections were transferred to albumincoated slides and dried overnight at 50°C. The slides were then treated with 20 pg/ml proteinase K (Sigma) and acetic anhydride prior to hybridization with [35S]-UTP-labeled probes ( 800 Ci/ mmole, Amersham) [9]. The [35S]-labeled RNA was degraded to an average length of 100 bases [3] and the specificity was confirmed by Northern blot (data not shown). The hybridization solution consisted of 0.03 ng/pl probe in: 50% formamide; 0.3 M sodium chloride; 20 mM Tris-HCI, pH 8.0; 5 mM EDTA; 10 mM sodium phosphate, pH 8.0; 10% dextran sulfate; 1 x Denhardt's solution; 0.5 mg/rnl yeast tRNA; and 20 mM dithiothreitol. The solution was applied to slides and covered with siliconized baked coverslips. Slides were immersed in prewarmed mineral oil and incubated overnight at 50' C. Slides were washed as described including the wash with 20pg/ml ribonuclease A (Sigma) for 30 min at 37" C [9]. Slides were coated with Konica NR-M2 nuclear emulsion, diluted one to one with distilled water, by the dipping method. These slides were stored under low humidity at 4" C for 2 weeks and developed with Fuji Rendol for 4 min at 20" C.
EDNAcloning and DNA sequence analysis. The Agtl 1 cDNA library constructed from F9 EC cells was screened by the plaque hybridization method [15] using the embigin E/B probe. The library was constructed from cultured F9 cells according to the method described previously [22] by H. Muramatsu in our laboratory. Phage DNA of the positive clones was prepared using the plate lysate method [15]. and the cDNA inserts were cloned into the EcoRl site of pUC8. Following the subcloning of restriction endonuclease fragments into pUC18 or pUC8, DNA sequencing was performed, employing the dideoxy chain termination method [24] adapted for denatured plasmid templates [7].
-
RNA analysis. Total RNA was prepared by the guanidinium isothiocyanate method [ 151. For slot-blot hybridization, serial dilutions of total RNA were applied to nitrocellulose using an apparatus from Schleicher and Schuell Co., followed by hybridization with a [32P]-labeled probe. Filters were washed three times for 15 min with an excess of 2 xO.15 M NaCI, 0.015 M sodium citrate (SSC)/ 0.1 YO sodium dodecyl sulfate (SDS) at 50" C and then washed twice for 30 min with 0.1 x SSC/O.l YO SDS at the same temperature. The degree of hybridization was quantitated by scanning densitometry of each hybridization using an Ultroscan XL (LKB) laser densitometer equipped with an automatic integrator; the values within the linear range were adopted and used for further calculation. For Northern blot analysis, total RNA (30 pg) and poly(A)+RNA (10 pg) were denatured with glyoxal and dimethyl sulfoxide and were separated on 1.O% agarose gel [15]. After transferring to nitrocellulose, RNA was hybridized with the E/B probe. The conditions of hybridization and washing were identical to those employed in slot blotting. The RNase protection assay was performed essentially as described by Zinn et al. [31]. The [32P]-UTP-labeledantisense probe was purified by electrophoresis in a 4% sequencing gel [18]. Hybridization was carried out using 20 pg total RNA along with 2 x lo5 cpm purified [32P]-labeledprobe in 30 p1 hybridization solution (80% formamide, 0.4 M NaCI, 40 mM Pipes pH 6.5, 1 rnM
B
A
._ =
$
Results Expression of embigin niRNA in culture cells and in organs of adult mice
The amount of embigin mRNA was examined by slotblot analysis using a [32P]-labeled E/B probe of embigin cDNA. As reported previously, F9 EC cells contained significant amounts of the mRNA. Slightly higher amounts of embigin mRNA were detected in teratocarcinoma OTT6050 and PCC4 EC cells (Fig. 1 A). In subsequent analyses, RNA from teratocarcinoma OTT6050 was always used as the standard, and we expressed the
3
f
C
Embigin
20 10
t
-
10
RNA(AI~)
c)
-
I
fi-Actin
.-u) I-
D 0
Ln
6
6
-
6
$
0,
! l !
0
c.
Fig. 1A-D. The relative amount of embigin gene transcript in various mouse organs, cultured cells and the embryo proper. Total RNA was serially diluted, spotted on nitrocellulose filters and analyzed by slot hybridization using [32P]-labeled E/B fragment of embigin cDNA or fl-actin cDNA (for control) as probes. These experiments were repeated at least twice and gave the same results. A Teratocarcinoma cells and STO fibroblasts. B Organs of adult mice. C F9 cells induced to differentiate by treatment with retinoic acid and dibutyryl cyclic AMP at day 0 with no treatment and n days after the treatment. D Mouse embryo proper. The no. of days indicates the days of gestation
78
Table 1. Comparison of embigin mRNA level in various cells, mouse embryos and adult organs
Table 1. (continued)
~~
Sources of RNA
Intensity The value normalized of hybridization by the intensity to embigin probe' of hybridization to 8-actin probe
Teratocarcinoma OTT6050
1 .oo
1 .oo
Cells F9 PCC4 PYS-2 STO
0.866 1.15 0.821 0.329
0.630 0.884 0.607 0.335
F9 cells induced to differentiate for 0 days 1 days 2 days 3 days 4 days 5 days
Sources of RNA
5 days 6 days 7 days 8 days 10 days 12 days 18 days
0.196 0.150 0.235 0.210 0.207 0.660 0.389
0.197 0.133 0.201 0.206 0.196 0.513 0.303
0.067
0.074
Placenta from the mice after following days p.c. 15 days 0.1 33 18 days 0.117
0.144 0.107
From mice 5 days after delivery 0.800 0.712 3.01 2.42 2.58 2.26
0.590 0.528 2.22 1.78 1.90 1.67
Embryo proper on the following days of gestation 9 days 1.29 1 1 days
