Gene Expression in Visceral Endoderm: a Comparison of Mutant and Wild-type F9 Embryonal Carcinoma Cell Differentiation Melissa B. Rogers,* S i m o n C. Watkins,~ a n d Lorraine J. G u d a s * *Department of Biological Chemistry and Molecular Pharmacology and the ~Structural Molecular Biology Department, Harvard Medical School, and the Dana-Farber Cancer Institute, Boston, Massachusetts 02115
Abstract. We have examined the abundance and cell specificity of several mRNAs that are regulated during the retinoic acid (RA)-induced differentiation of F9 embryonal carcinoma cells to visceral endoderm. The experiments confirmed the multistep nature of this process by demonstrating the expression of the ERA1/Hox 1.6 message within 6 h after RA addition; the expression of messages specific for the extracellular matrix proteins laminin B1 and B2, and collagen IV(or) between days 4 and 12; and the expression of two visceral endoderm markers, ~fetoprotein (AFP) and H19, by days 8-15. In situ hybridization experiments revealed that the collagen IV(t~l) mRNA is restricted to the outer cell layer of F9 cell aggregates regardless of the presence or absence of RA. Laminin B1 and B2 mRNAs are concentrated in the outer cell layer of RA-treated aggregates although significant levels of message are also observed within the interior cells of the aggregates. Unexpectedly, AFP mRNA is detectable in only a subset of the outer cells of F9 cell aggregates grown 15 d in the presence of RA. The results obtained from wild-type F9 cells were
T
n~ first cell types to differentiate from the inner cell mass of the mouse blastocyst are the parietal and visceral endoderm (VE). t In the developing mouse embryo, VE lines the inner cell mass and eventually forms the visceral yolk-sac, while parietal endoderm cells migrate onto the inner surface of the trophectoderm and lay down a thick extracellular matrix (ECM) known as Reichert's membrane. While the question of how a subset of undifferentiated cells becomes the highly specialized extraembryonic endoderm is of great interest, the small size and inaccessibility of the mouse embryo makes the in vivo study of this problem difficult. Fortunately, the murine embryonal carcinoma cell line F9 can be induced to differentiate into cells resembling either parietal endoderm or VE in vitro, thus facilitating the study of this developmental question. 1. Abbreviations used in this paper: AFP, ¢-fetoprotein; ECM, extracellular matrix; RA, retinoic acid; VE, visceral endoderm.
© The Rockefeller University Press, 0021-9525/90/05/1767111 $2.00 The Journal of Cell Biology, Volume 110, May 1990 1767-1777
compared with those from a mutant F9 cell line, RA5-1, which was previously shown to synthesize collagen IV containing six- to ninefold less 4-hydroxyproline than that in wild-type F9 cells. RA-5-1 cells exhibit four- to sixfold less of the mRNAs encoding two visceral endoderm proteins, AFP and H19, than wildtype F9 cells after RA treatment of RA-5-1 aggregates. RA-5-1 cells, however, do exhibit an RA-associated increase in the level of ERA-1/Hox 1.6 mRNA within 6 h after adding RA. Although the collagen IV protein level is similar in wild-type F9 and RA-5-1 aggregates, the collagen IV(t~l) message level is 6-20-fold greater in aggregates of mutant cells than in aggregates of wild-type cells. Moreover, in situ hybridizations showed that this message is evenly distributed throughout the RA-5-1 aggregates rather than restricted to the outer cell layers as it is in wild-type F9 aggregates. These results suggest that abnormal collagen IV expression and localization are associated with decreased expression of the visceral endoderm markers, AFP and H19, in RA-5-1 cell aggregates.
F9 embryonal carcinoma ceils are the malignant stem cells of a mouse teratocarcinoma. Monolayer cultures of F9 embryonal carcinoma cells grown in the presence of the vitamin A derivative, retinoic acid (RA), differentiate into nonmalignant cells resembling the primitive endoderm of the mouse blastocyst. If the media is further supplemented with dibutyryl cAMP and theophylline, the cells become biochemically indistinguishable from parietal endoderm. As in the mouse embryo, these cells are characterized by the increased production of numerous proteins including the ECM proteins laminin and collagen IV (Strickland et al., 1980). One of the earliest known molecular responses to RA is an increase in the abundance of a Hox 1.6 homeobox containing transcript (LaRosa and Gudas, 1988b). The induction of the ERA-I/Hox 1.62mRNA within 2 h after RA treatment of F9 2. The gene referred to as ERA-1 in LaRosa and Gudas (1988a) was subsequently shown to contain the Hox 1.6 homeobox sequence (Baron et al., 1987; LaRosa and Gudas, 1988b) and will be referred to as ERA-1/Hox 1.6 in this paper.
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cell monolayer cultures has been shown to be independent o f protein synthesis, suggesting that this gene is a primary target of RA (LaRosa and Gudas, 1988a). Alternatively, F9 cells grown in suspension in the presence o f RA aggregate and, within 1 wk, form embryoid bodies consisting of stem cells surrounded by an outer layer of cells that resemble mouse VE (Hogan et al., 1981). The differentiation of F9 stem cells into VE has been shown to be a multistep process of which some crucial features are aggregation, epithelium and basement membrane formation, and finally secretion of VE-specific differentiation products such as o~-fetoprotein (AFP) and H19 (Grover et al., 1983b; Pacbnis et al., 1984). These messages, which are coordinately regulated during murine embryogenesis (Pachnis et al., 1984), may be detected in RA-treated F9 embryoid bodies by 5 d of culture (Grover et al., 1983a; Young and Tilghman, 1984). The importance o f a contiguous basement membrane in attaining maximal secretion of A F P was demonstrated by the inability of F9 stem cells to differentiate into VE when subjected to experimental treatments that alter basement membrane formation, such as enrichment or depletion of exogenous laminin (Grover et al., 1983a) or tunicamycin treatment (Grabel and Martin, 1983). To analyze further the role of the E C M in the formation of VE, we have characterized the differentiation of a mutant F9 cell line RA-5-1. Monolayer cultures of RA-5-1 cells exhibit a six- to ninefold reduction in the posttranslational hydroxylation of collagen IV, consistent with the observation that the RA-5-1 prolyl-4-hydroxylase has an abnormally high Km for collagen IV hydroxylation (Wang and Gudas, 1984; Wang et al., 1989). The RA-5-1 line was isolated by virtue of its ability to grow in soft agar in the presence of RA; wildtype F9 cells differentiate in the presence of RA and are no longer able to grow in soft agar. The RA-5-1 cells only partially differentiate into parietal endoderm in monolayer culture, but whether the abnormal collagen IV synthesis also inhibited differentiation of the cells into VE was unknown. We first characterized the expression and localization patterns of several gene transcripts whose abundance is regulated during differentiation of wild-type F9 cells into VE. The deposition patterns for many of these proteins has been previously described (Grover et al., 1983b; Grabel and Casanova, 1986); however, since the E C M proteins and A F P are secreted, these immunocytochemical studies at the light level do not allow definition of the cells synthesizing these proteins. Consequently, we used in situ hybridization to cellular R N A , since m R N A localization clearly identifies the cells in the aggregates that express these genes. A comparison of these results and those obtained from the mutant cell line RA-5-1 indicates that, in addition to its inability to differentiate fully into parietal endoderm, this cell line is unable to differentiate fully into VE. Moreover, the regulation of collagen I V ( a l ) message levels is perturbed in this cell line. The data presented here suggest that abnormal expression of collagen IV may prevent normal differentiation of F9 embryonal carcinoma cells into VE.
were dispersed with trypsin into single cells and cultured as aggregates in untreated Petri plates at a density of 1 x 10s cetls/ml in the presence of 50 nM RA. The media from the suspended cultures was replaced by fresh media and RA every 48 h. The F9 mutant stem cell line SYW-F9-M-RA-5-1was isolated after mutagenesis of F9 wild-type stem cells by exposure for 4 h to 5 ~g/ml of N-methyl-NLnitronitmso-guaKIdine. Survivingcells weregrown under nonselective conditions for 7 d to allow phenotypic expression, followedby plating in 0.34% agamse containing RA over a mouse embryo fibroblastfeeder layer. From the mntagen-treatedpopulation of F9 cells, we obtained mutant lines that formed colonies in agarose in the presence of 5 ~M RA at a frequency of 0.5 -2 X 10-5. The frequency of obtaining spontaneous mutants was
Abstract. We have examined the abundance and cell specificity of several mRNAs that are regulated during the retinoic acid (RA)-induced differentiation of F9 embryonal carcinoma cells to visceral endoderm. The experiments confirmed the multistep nature of this process by demonstrating the expression of the ERA1/Hox 1.6 message within 6 h after RA addition; the expression of messages specific for the extracellular matrix proteins laminin B1 and B2, and collagen IV(or) between days 4 and 12; and the expression of two visceral endoderm markers, ~fetoprotein (AFP) and H19, by days 8-15. In situ hybridization experiments revealed that the collagen IV(t~l) mRNA is restricted to the outer cell layer of F9 cell aggregates regardless of the presence or absence of RA. Laminin B1 and B2 mRNAs are concentrated in the outer cell layer of RA-treated aggregates although significant levels of message are also observed within the interior cells of the aggregates. Unexpectedly, AFP mRNA is detectable in only a subset of the outer cells of F9 cell aggregates grown 15 d in the presence of RA. The results obtained from wild-type F9 cells were
T
n~ first cell types to differentiate from the inner cell mass of the mouse blastocyst are the parietal and visceral endoderm (VE). t In the developing mouse embryo, VE lines the inner cell mass and eventually forms the visceral yolk-sac, while parietal endoderm cells migrate onto the inner surface of the trophectoderm and lay down a thick extracellular matrix (ECM) known as Reichert's membrane. While the question of how a subset of undifferentiated cells becomes the highly specialized extraembryonic endoderm is of great interest, the small size and inaccessibility of the mouse embryo makes the in vivo study of this problem difficult. Fortunately, the murine embryonal carcinoma cell line F9 can be induced to differentiate into cells resembling either parietal endoderm or VE in vitro, thus facilitating the study of this developmental question. 1. Abbreviations used in this paper: AFP, ¢-fetoprotein; ECM, extracellular matrix; RA, retinoic acid; VE, visceral endoderm.
© The Rockefeller University Press, 0021-9525/90/05/1767111 $2.00 The Journal of Cell Biology, Volume 110, May 1990 1767-1777
compared with those from a mutant F9 cell line, RA5-1, which was previously shown to synthesize collagen IV containing six- to ninefold less 4-hydroxyproline than that in wild-type F9 cells. RA-5-1 cells exhibit four- to sixfold less of the mRNAs encoding two visceral endoderm proteins, AFP and H19, than wildtype F9 cells after RA treatment of RA-5-1 aggregates. RA-5-1 cells, however, do exhibit an RA-associated increase in the level of ERA-1/Hox 1.6 mRNA within 6 h after adding RA. Although the collagen IV protein level is similar in wild-type F9 and RA-5-1 aggregates, the collagen IV(t~l) message level is 6-20-fold greater in aggregates of mutant cells than in aggregates of wild-type cells. Moreover, in situ hybridizations showed that this message is evenly distributed throughout the RA-5-1 aggregates rather than restricted to the outer cell layers as it is in wild-type F9 aggregates. These results suggest that abnormal collagen IV expression and localization are associated with decreased expression of the visceral endoderm markers, AFP and H19, in RA-5-1 cell aggregates.
F9 embryonal carcinoma ceils are the malignant stem cells of a mouse teratocarcinoma. Monolayer cultures of F9 embryonal carcinoma cells grown in the presence of the vitamin A derivative, retinoic acid (RA), differentiate into nonmalignant cells resembling the primitive endoderm of the mouse blastocyst. If the media is further supplemented with dibutyryl cAMP and theophylline, the cells become biochemically indistinguishable from parietal endoderm. As in the mouse embryo, these cells are characterized by the increased production of numerous proteins including the ECM proteins laminin and collagen IV (Strickland et al., 1980). One of the earliest known molecular responses to RA is an increase in the abundance of a Hox 1.6 homeobox containing transcript (LaRosa and Gudas, 1988b). The induction of the ERA-I/Hox 1.62mRNA within 2 h after RA treatment of F9 2. The gene referred to as ERA-1 in LaRosa and Gudas (1988a) was subsequently shown to contain the Hox 1.6 homeobox sequence (Baron et al., 1987; LaRosa and Gudas, 1988b) and will be referred to as ERA-1/Hox 1.6 in this paper.
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cell monolayer cultures has been shown to be independent o f protein synthesis, suggesting that this gene is a primary target of RA (LaRosa and Gudas, 1988a). Alternatively, F9 cells grown in suspension in the presence o f RA aggregate and, within 1 wk, form embryoid bodies consisting of stem cells surrounded by an outer layer of cells that resemble mouse VE (Hogan et al., 1981). The differentiation of F9 stem cells into VE has been shown to be a multistep process of which some crucial features are aggregation, epithelium and basement membrane formation, and finally secretion of VE-specific differentiation products such as o~-fetoprotein (AFP) and H19 (Grover et al., 1983b; Pacbnis et al., 1984). These messages, which are coordinately regulated during murine embryogenesis (Pachnis et al., 1984), may be detected in RA-treated F9 embryoid bodies by 5 d of culture (Grover et al., 1983a; Young and Tilghman, 1984). The importance o f a contiguous basement membrane in attaining maximal secretion of A F P was demonstrated by the inability of F9 stem cells to differentiate into VE when subjected to experimental treatments that alter basement membrane formation, such as enrichment or depletion of exogenous laminin (Grover et al., 1983a) or tunicamycin treatment (Grabel and Martin, 1983). To analyze further the role of the E C M in the formation of VE, we have characterized the differentiation of a mutant F9 cell line RA-5-1. Monolayer cultures of RA-5-1 cells exhibit a six- to ninefold reduction in the posttranslational hydroxylation of collagen IV, consistent with the observation that the RA-5-1 prolyl-4-hydroxylase has an abnormally high Km for collagen IV hydroxylation (Wang and Gudas, 1984; Wang et al., 1989). The RA-5-1 line was isolated by virtue of its ability to grow in soft agar in the presence of RA; wildtype F9 cells differentiate in the presence of RA and are no longer able to grow in soft agar. The RA-5-1 cells only partially differentiate into parietal endoderm in monolayer culture, but whether the abnormal collagen IV synthesis also inhibited differentiation of the cells into VE was unknown. We first characterized the expression and localization patterns of several gene transcripts whose abundance is regulated during differentiation of wild-type F9 cells into VE. The deposition patterns for many of these proteins has been previously described (Grover et al., 1983b; Grabel and Casanova, 1986); however, since the E C M proteins and A F P are secreted, these immunocytochemical studies at the light level do not allow definition of the cells synthesizing these proteins. Consequently, we used in situ hybridization to cellular R N A , since m R N A localization clearly identifies the cells in the aggregates that express these genes. A comparison of these results and those obtained from the mutant cell line RA-5-1 indicates that, in addition to its inability to differentiate fully into parietal endoderm, this cell line is unable to differentiate fully into VE. Moreover, the regulation of collagen I V ( a l ) message levels is perturbed in this cell line. The data presented here suggest that abnormal expression of collagen IV may prevent normal differentiation of F9 embryonal carcinoma cells into VE.
were dispersed with trypsin into single cells and cultured as aggregates in untreated Petri plates at a density of 1 x 10s cetls/ml in the presence of 50 nM RA. The media from the suspended cultures was replaced by fresh media and RA every 48 h. The F9 mutant stem cell line SYW-F9-M-RA-5-1was isolated after mutagenesis of F9 wild-type stem cells by exposure for 4 h to 5 ~g/ml of N-methyl-NLnitronitmso-guaKIdine. Survivingcells weregrown under nonselective conditions for 7 d to allow phenotypic expression, followedby plating in 0.34% agamse containing RA over a mouse embryo fibroblastfeeder layer. From the mntagen-treatedpopulation of F9 cells, we obtained mutant lines that formed colonies in agarose in the presence of 5 ~M RA at a frequency of 0.5 -2 X 10-5. The frequency of obtaining spontaneous mutants was
