In VitroCell.Dev.Biol.28A:61-66,January1992 © t992TissueCultureAssociation 0883-8364/92 $01.50+0.00
EXPRESSION OF FIBROBLAST GROWTH FACTOR RECEPTORS BY EMBRYONAL CARCINOMA CELLS AND EARLY MOUSE EMBRYOS WENDY J. CAMPBELL, KEITH A. MILLER, TODD M. ANDERSON, JAMES D. SHULL, ANDANGIE RIZZINOt Eppley Institutefor Cancer Research and Allied Diseases,Departmentof Pathology and Microbiology (A. R., K. A. M.), University of Nebraska Medical Center, 42nd and DeweyAvenue, Omaha, Nebraska 68198 (Accepted 7 October 1991)
SUMMARY We have previously shown that differentiation of embryonal carcinoma (EC) cells leads to both increased binding of FGF (fibroblast growth factor) and suppression of k-FGF expression. In the current study, we examined the expression of FGF receptors by EC ceils, EC-derived differentiated cells and early mammalian embryos using the technique of reverse transcfiption-polymerase chain reaction (RT-PCR). We determined that both mouse, F9, and human, NT2/D1, EC cells as well as their differentiated counterparts express transcripts for two forms of FGF receptors, bek (bacterially expressed kinase) and fig (fins-like gene). In addition, we determined that mouse blastocysts express fig transcripts. The presence of FGF receptor transcripts in early embryos and the previous finding of FGF-related activity in medium conditioned by mouse blastocysts argue that the FGF family plays important roles during early mammalian development. Key words: fibroblast growth factor receptors; embryonal carcinoma cells; mammalian embryogenesis; polymerase chain reaction. INTRODUCTION
1991; Haub and Goldfarb, 1991) and int-2 (Jakobovits et al., 1986) are expressed at later developmental stages. These findings and the finding that one or more forms of FGF play a role during mesoderm formation (Slack et al., 1987; Amaya et al., 1991) argue that the FGF family of growth factors plays multiple roles during early mammalian development. In contrast to the situation for FGFs, little is known about the expression of FGF receptors during early mammalian development. Several years ago, EC ceils and EC cells induced to differentiate with retinoic acid (RA) were shown to express FGF receptors (Rizzino et al., 1988). Scatchard analysis of basic FGF binding to F9 EC cells and their differentiated cells determined that F9 EC cells exhibit 7- to 10-fold fewer FGF receptors than their differentiated counterparts; whereas, differentiation induced no discernable change in the apparent dissociation constants. In addition, EC-derived differentiated ceils, but not the parental EC cells, exhibit increased cell proliferation in response to basic FGF (Rizzino et al., 1988). Genes for FGF receptors appear to exhibit a complexity similar to that of the FGF family of growth factors. Several different genes for FGF receptors have been cloned (Kornbluth et al., 1988; Lee et al., 1989; Ruta et al., 1988; Reid et al., 1990; Safran et al., 1990) and receptor complexity appears to be enhanced by alternative mRNA splicing (Hou et al., 1991; Fasel et al., 1991; Reid et al., 1990; Hattori et al., 1990; Houssaint et al., 1990; Mansukhani et al., 1990). Two of the first FGF receptor genes to be isolated, bek (bacterially expressed kinase) and fig (fms-like gene), are expressed
Embryonal carcinoma (EC) cells, which mimic important stages of early mammalian development, have been used extensively to assist in the identification of growth factors produced during early development (reviewed in Rizzino, 1989). In the case of the fibroblast growth factor (FGF) family, more than one form appears to be produced by EC cells and their differentiated ceils. The fibroblast growth factor family consists of at least seven members, basic FGF, acidic FGF, k-FGF, int-2, FGF-5, FGF-6 and KGF (reviewed in Baird and Klagsburn, 1991). Existing data argue that EC cells produce k-FGF (Rizzino et al., 1988; Yoshida et al., 1988; Tiesman and Rizzino, 1989; Peters et al., 1989) and possibly FGF-5 (H~bert et al., 1990). Whereas, EC cells induced to differentiate do not produce k-FGF (Rizzino et al., 1988; Tiesman and Rizzino, 1989; Velcich et al., 1989) and express transcripts for int-2 (Jakobovits et al., 1986), FGF-5 (H~bert et al., I990), basic FGF (H~bert et al., 1990; Braunhut et al., 1989) and acidic FGF (Braunhut et al., 1989). Pre- and early post-implantation mouse embryos appear to exhibit a pattern of FGF expression similar to that observed in EC cells and their differentiated ceils. Mouse blastocysts, which produce a growth factor immunologicaily related to FGF (Rizzino et al., 1990), express transcripts for k-FGF (Rappolee et al., 1990), whereas basic FGF (Rappolee et al., 1988), FGF-5 (H~bert et al., l To whom correspondenceshould be addressed. 61
62
CAMPBELL ET AL.
F9 m
-m
M
u
because it could be used to examine the expression of FGF receptors by mouse embryos at early stages of development. In this study, we report the expression of both fig and bek transcripts by F9 EC ceils, N T 2 / D 1 EC cells, and their respective differentiated counterparts. Furthermore, we report that mouse blastocysts express transcripts for fig.
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FIo. 2. Expression of transcripts for bek by human NT2/D1 EC cells and their differentiated cells as detected by RT-PCR. Primers were synthesized as described for detection of bek in the case of F9 cells (Figure 1). RT-PCR was performed in a 50 #1 reaction volume containing 20 #M primer mix with the following thermal cycler conditions: for 30 cycles, samples were heated to 94 ° C for 3 rain to denature DNA, ramped for 1 rain to 61° C and maintained at 61 o C for 2 rain to allow primer annealing, and heated to 72 ° C for 1 min to extend primers. RT-PCR products were subjected to electrophoresis and restriction endonuclease digestions as described in Figure 1. The appropriate uncut (523 bp), Bgl II (371 + 152 bp)- and Eco RI (344 + 179bp)-digested fragments were generated.
63
DEVELOPMENT AND FGF RECEPTORS
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EXPRESSION OF FIBROBLAST GROWTH FACTOR RECEPTORS BY EMBRYONAL CARCINOMA CELLS AND EARLY MOUSE EMBRYOS WENDY J. CAMPBELL, KEITH A. MILLER, TODD M. ANDERSON, JAMES D. SHULL, ANDANGIE RIZZINOt Eppley Institutefor Cancer Research and Allied Diseases,Departmentof Pathology and Microbiology (A. R., K. A. M.), University of Nebraska Medical Center, 42nd and DeweyAvenue, Omaha, Nebraska 68198 (Accepted 7 October 1991)
SUMMARY We have previously shown that differentiation of embryonal carcinoma (EC) cells leads to both increased binding of FGF (fibroblast growth factor) and suppression of k-FGF expression. In the current study, we examined the expression of FGF receptors by EC ceils, EC-derived differentiated cells and early mammalian embryos using the technique of reverse transcfiption-polymerase chain reaction (RT-PCR). We determined that both mouse, F9, and human, NT2/D1, EC cells as well as their differentiated counterparts express transcripts for two forms of FGF receptors, bek (bacterially expressed kinase) and fig (fins-like gene). In addition, we determined that mouse blastocysts express fig transcripts. The presence of FGF receptor transcripts in early embryos and the previous finding of FGF-related activity in medium conditioned by mouse blastocysts argue that the FGF family plays important roles during early mammalian development. Key words: fibroblast growth factor receptors; embryonal carcinoma cells; mammalian embryogenesis; polymerase chain reaction. INTRODUCTION
1991; Haub and Goldfarb, 1991) and int-2 (Jakobovits et al., 1986) are expressed at later developmental stages. These findings and the finding that one or more forms of FGF play a role during mesoderm formation (Slack et al., 1987; Amaya et al., 1991) argue that the FGF family of growth factors plays multiple roles during early mammalian development. In contrast to the situation for FGFs, little is known about the expression of FGF receptors during early mammalian development. Several years ago, EC ceils and EC cells induced to differentiate with retinoic acid (RA) were shown to express FGF receptors (Rizzino et al., 1988). Scatchard analysis of basic FGF binding to F9 EC cells and their differentiated cells determined that F9 EC cells exhibit 7- to 10-fold fewer FGF receptors than their differentiated counterparts; whereas, differentiation induced no discernable change in the apparent dissociation constants. In addition, EC-derived differentiated ceils, but not the parental EC cells, exhibit increased cell proliferation in response to basic FGF (Rizzino et al., 1988). Genes for FGF receptors appear to exhibit a complexity similar to that of the FGF family of growth factors. Several different genes for FGF receptors have been cloned (Kornbluth et al., 1988; Lee et al., 1989; Ruta et al., 1988; Reid et al., 1990; Safran et al., 1990) and receptor complexity appears to be enhanced by alternative mRNA splicing (Hou et al., 1991; Fasel et al., 1991; Reid et al., 1990; Hattori et al., 1990; Houssaint et al., 1990; Mansukhani et al., 1990). Two of the first FGF receptor genes to be isolated, bek (bacterially expressed kinase) and fig (fms-like gene), are expressed
Embryonal carcinoma (EC) cells, which mimic important stages of early mammalian development, have been used extensively to assist in the identification of growth factors produced during early development (reviewed in Rizzino, 1989). In the case of the fibroblast growth factor (FGF) family, more than one form appears to be produced by EC cells and their differentiated ceils. The fibroblast growth factor family consists of at least seven members, basic FGF, acidic FGF, k-FGF, int-2, FGF-5, FGF-6 and KGF (reviewed in Baird and Klagsburn, 1991). Existing data argue that EC cells produce k-FGF (Rizzino et al., 1988; Yoshida et al., 1988; Tiesman and Rizzino, 1989; Peters et al., 1989) and possibly FGF-5 (H~bert et al., 1990). Whereas, EC cells induced to differentiate do not produce k-FGF (Rizzino et al., 1988; Tiesman and Rizzino, 1989; Velcich et al., 1989) and express transcripts for int-2 (Jakobovits et al., 1986), FGF-5 (H~bert et al., I990), basic FGF (H~bert et al., 1990; Braunhut et al., 1989) and acidic FGF (Braunhut et al., 1989). Pre- and early post-implantation mouse embryos appear to exhibit a pattern of FGF expression similar to that observed in EC cells and their differentiated ceils. Mouse blastocysts, which produce a growth factor immunologicaily related to FGF (Rizzino et al., 1990), express transcripts for k-FGF (Rappolee et al., 1990), whereas basic FGF (Rappolee et al., 1988), FGF-5 (H~bert et al., l To whom correspondenceshould be addressed. 61
62
CAMPBELL ET AL.
F9 m
-m
M
u
because it could be used to examine the expression of FGF receptors by mouse embryos at early stages of development. In this study, we report the expression of both fig and bek transcripts by F9 EC ceils, N T 2 / D 1 EC cells, and their respective differentiated counterparts. Furthermore, we report that mouse blastocysts express transcripts for fig.
F9-Diff. m
Ilg
--
m
u
1~
, g , , ,¢0
1353-~> 1078-1:>
MATERIALSANDMETHODS
872-[;> 603--I>
281,271--I:> 234--[;> 194-{:>
344
1078-(> 872--t:> 603-1;>
281,271-1;:> 234-1:>
194--[:>
~.. 179 152
FIo. 2. Expression of transcripts for bek by human NT2/D1 EC cells and their differentiated cells as detected by RT-PCR. Primers were synthesized as described for detection of bek in the case of F9 cells (Figure 1). RT-PCR was performed in a 50 #1 reaction volume containing 20 #M primer mix with the following thermal cycler conditions: for 30 cycles, samples were heated to 94 ° C for 3 rain to denature DNA, ramped for 1 rain to 61° C and maintained at 61 o C for 2 rain to allow primer annealing, and heated to 72 ° C for 1 min to extend primers. RT-PCR products were subjected to electrophoresis and restriction endonuclease digestions as described in Figure 1. The appropriate uncut (523 bp), Bgl II (371 + 152 bp)- and Eco RI (344 + 179bp)-digested fragments were generated.
63
DEVELOPMENT AND FGF RECEPTORS
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m m ,mmm
O'J o
""
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M
U
Q)
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