Neuronal and mesodermal differentiation of P19 embryonal carcinoma cells is characterized by expression of specific marker genes and modulated by activin and fibroblast growth factors

We have used the P19 embryonal carcinoma (EC) aggregation system as a model for early mouse development to study induction and modulation of mesodermal and neuronal differentiation. By studying the expression of marker genes for differentiated cells in this model we have shown that there is a good correlation between the differentiation direction induced in P19 EC aggregates and the expression of these genes. Expression of the neuronal gene midkine is exclusively upregulated when P19 EC cells are induced to form neurons while expression of early mesodermal genes such as Brachyury T, evx-1 , goosecoid and nodal is elevated after induction to the mesodermal pathway. In the present study we have further shown that activin A blocks the different directions of differentiation of P19 EC cells induced by retinoic acid (RA) in a dose-dependent way. To understand the mechanism behind this inhibitory action of activin A the expression of several RA-responsive genes, including the three RA receptor genes (RARα, RARβ and RARγ) was determined. Since activin has no clear effect on the expression and activity of the RAR it is very likely that this factor acts downstream of these receptors. In addition to activin, fibroblast growth factors (FGF) were shown to modulate P19 EC cell differentiation. However, in contrast to activin, FGF exclusively blocks the mesodermal differentiation of P19 EC cells by either 10⁻⁹mol/L RA or a factor produced by visceral endoderm-like cells (END-2 factor). The FGF effect is dose-independent. These results suggest an important function for RA and the END-2 factor in the induction and for activin and FGF in the modulation of specific differentiation processes in murine development.     

Follistatin is a developmentally regulated cytokine in neural differentiation.

Activin acts mitogenically on P19 cells as well as being inhibitory of the differentiation of retinoic acid-treated P19 cells and some neuroblastoma cell lines. Here, we show some lines of evidence that follistatin, an activin-binding protein, is also involved in neural differentiation. Counteracting the activity of activin, addition of follistatin suppresses the anchorage-independent growth of P19 cells in soft agar and stimulates neurite outgrowth of a neuroblastoma cell line, IMR-32 cells. While activin does not seem to be expressed significantly, follistatin is demonstrated in the conditioned medium of these cells. Furthermore, the expression of follistatin in P19 cells is subject to dynamic fluctuations in response to retinoic acid treatment. These neural cells may produce follistatin in a cell stage-specific manner in order to interact with exogenously derived activin.     

Differentiation of aggregated murine P19 embryonal carcinoma cells is induced by a novel visceral endoderm-specific FGF-like factor and inhibited by activin A

Aggregation of P19 embryonal carcinoma cells in the presence of a factor, secreted by the visceral endoderm-like cell line END-2, induces differentiation to cell types including visceral endoderm, mesoderm-derived muscle tissue and neurons. This factor is different from activin A, type beta transforming growth factors (TGF beta) and fibroblast growth factors (FGF) although its acid- and heat-lability and its stability in the presence of reducing agents resemble the properties of the FGFs. The END-2 factor is completely inhibited in its action by activin A. This inhibitory effect of activin A is not specific for the END-2 factor as retinoic acid (RA)-induced differentiation of aggregated P19 EC cells into neurons (10(-8) M RA) or mesoderm-derived muscle tissue (10(-9) M RA) is also completely inhibited by activin A. The results of this study suggest that the END-2 activity and activin A are intimately involved in the induction and regulation, respectively, of early differentiation processes in vertebrate embryogenesis.     

Differential expression of inhibin subunits and follistatin, but not of activin receptor type II, during early murine embryonic development.

Activins are known to be potentially important regulators of early developmental processes in amphibians, birds, and mammalians. In this study we report the expression of the inhibin subunits, including those that make up activin, the activin-binding protein follistatin, and activin receptor type II in several in vitro systems that model early murine embryonic development, namely embryonic stem (ES) cells, embryonal carcinoma (EC) cells, and their differentiated derivatives. In addition, we examine the expression pattern of these factors in different stages of the mouse embryo itself. Expression of inhibin alpha and beta A subunits is restricted to certain differentiated cell types, while beta B subunits are expressed in both differentiated and undifferentiated cells. Our results further indicate a change in the expression pattern of inhibin subunits during early development from beta B at the blastocyst stage largely to beta A in postgastrulation embryos. This is similar to the expression pattern at equivalent stages of Xenopus and chick development. Expression of the activin-binding protein follistatin is altered by the induction of differentiation of P19 EC and ES cells by several factors, including retinoic acid. In contrast to the inhibin subunits and follistatin, activin receptor levels are not influenced by differentiation in these cell types. The results of this study demonstrate that the inhibin subunits and follistatin, but not the activin receptor type II, are differentially expressed during early murine development and suggest that the different forms of activin/inhibin are involved in the regulation of different developmental processes.     

The Activin A-Dependent Proliferation of PCC3/A/1 Embryonal Carcinoma Cells in Serum-Free Medium

Examination of the growth requirements of murine embryonal carcinoma cells (EC cells) or embryonic stem cells (ES cells) in serum-free medium revealed that PCC3 EC cells required activin A to grow and/or survive in such medium. In the absence of activin A, PCC3 cells began to disintegrate within 3 days under any serum-free conditions examined. P19 and AT805 EC cells grew even in serum-free medium without activin A but their growth rates were slightly facilitated by its addition. F9 EC cells also grew in the medium without activin A and its addition somewhat inhibited their growth rate. Three independently isolated ES cell lines and feeder-dependent PSA-1 EC cells also grew in serum-free medium without activin A if leukemia inhibitory factor (LIF) was supplemented. The addition of activin A had little effect on their growth rates. These findings suggest that PCC3 EC cells are a sort of nutritional mutant requiring activin A, thus making them useful in stidies on the growth regulatory mechanisms of EC/ES cells and/or the action of activin on EC/ES cells.     

Identification of the two types of specific receptor for activin/EDF expressed on Friend leukemia and embryonal carcinoma cells

Activin and inhibin are polypeptide factors which control the release of follicle stimulating hormone(FSH) from pituitary cells. The recent finding that erythroid differentiation factor(EDF) is identical to activin showed the multifunctional feature of this protein. To identify the specific receptor for activin, the binding of 125I-labeled activinA was investigated for a number of culture cell lines. Friends leukemia cell, which can be differentiated by activin, and embryonal carcinoma(EC) cells(PCC3, P19 and F9), were found to express 3500-20,000 per cell of activin receptors. Scatchard plot analysis of the binding data shows that the receptors on those cells could be divided into two groups with different Kd values. The Kd values of high and low affinity receptors are 0.15-0.4 nM and 1.5-3.0 nM respectively. The proportion of the number of the high and low affinity receptors was varied in each cell. Inhibin was able to compete for activin binding to both types of receptors, although the binding affinity was about 50-200 fold lower than that of activinA. Transforming growth factor-beta had no binding ability to the activin receptors.     

Inhibitory control of neural differentiation in mammalian cells

 In Xenopus embryos, a truncated type II activin receptor (Δ1XAR1), capable of blocking signals by several transforming growth factor (TGF)-β family members, can induce neural tissue suggesting neural fate is under inhibitory control. Activin and bone morphogenetic protein 4 (BMP4) can act as neural inhibitors but only BMP4 can induce epidermis in Xenopus ectodermal cells. We have used the pluripotent mouse embryonal carcinoma cell line P19 to examine whether the mechanisms of ectodermal cell fate decisions are conserved among vertebrates. We show that a P19 cell line expressing Δ1XAR1 will differentiate into neurons. In addition, BMP4 inhibits retinoic acid (RA)-induced neural differentiation of P19 cells and induces keratin expression. These results suggest that in mammals as in amphibians neural fate is under inhibitory control and BMP4 can alter ectodermal differentiation. Received: 23 September 1996 / Accepted: 8 January 1997     

Isolation and characterization of activin receptor from mouse embryonal carcinoma cells. Identification of its serine/threonine/tyrosine protein kinase activity.

The activin receptor protein was isolated from the mouse embryonal carcinoma (EC) cell line P19 by three cycles of affinity chromatography on an activin A-immobilized column. The purified receptor had a specific and high affinity for activins A, AB, and B (Kd = 345 pM), but not for transforming growth factor beta. The purified activin receptor was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and ligand blotting analysis as a single protein of 70 kDa. The amino acid sequence of the first 18 NH2-terminal residues revealed that the receptor is a member of the activin receptor family. The purified receptor phosphorylated itself and exogenous substrate proteins on serine, threonine, and tyrosine residues, indicating that the activin receptor is a transmembrane serine/threonine/tyrosine protein kinase. These results suggest that signal transduction of activin employs a novel pathway via a new class of cellular receptor in EC P19 cells.     

Neural Epidermal Growth Factor-Like Like Protein 2 (NELL2) Promotes Aggregation of Embryonic Carcinoma P19 Cells by Inducing N-Cadherin Expression

NELL2 was first identified as a mammalian homolog of chick NEL (Neural EGF-like) protein. It is almost exclusively expressed in neurons of the rat brain and has been suggested to play a role in neural differentiation. However, there is still no clear evidence for the detailed function of NELL2 in the differentiation of neurons. In this study, we identified NELL2 function during neural differentiation of mouse embryonic carcinoma P19 cells. Endogenous expression of NELL2 in the P19 cells increased in parallel with the neuronal differentiation induced by retinoic acid (RA). We found that the mouse NELL2 promoter contains RA response elements (RAREs) and that treatment with RA increased NELL2 promoter activity. Transfection of P19 cells with NELL2 expression vectors induced a dramatic increase in cell aggregation, resulting in the facilitation of neural differentiation. Moreover, NELL2 significantly increased N-cadherin expression in the P19 cell. These data suggest that NELL2 plays an important role in the regulation of neuronal differentiation via control of N-cadherin expression and cell aggregation.     

Activin synergistically increased c-jun mRNA in P19 embryonal carcinoma cells in the presence of retinoic acid.

Activin and retinoids, which are involved in the induction and regulation of the early differentiation process in vertebrate embryogenesis, synergistically increased the amount of c-jun mRNA in P19 embryonal carcinoma cells, but activin alone had no effect. Among the retinoids, all-trans-retinoic acid most effectively increased c-jun mRNA. Activin (lng/ml) was sufficient to induce the synergistic increase of c-jun mRNA in P19 EC cells with all-trans-retinoic acid. The synergistic increase of the amount of c-jun mRNA by their cooperative action may be important in vertebrate development.     

过量表达Wnt-1基因诱导P19细胞的神经分化

Ｗｎｔ－１基因在小鼠神经发育过程中起着重要的作用。该基因在胚胎性癌细胞Ｐ１９细胞经分化过程中存在瞬时性表达。利用克隆到的Ｗｎｔ－１基因转染Ｐ１９细胞,可使细胞不经视黄酸诱导,自发向神经细胞方向分化。     

Directed differentiation of mouse P19 embryonal carcinoma cells to neural cells in a serum- and retinoic acid-free culture medium

P19 embryonal carcinoma cells (EC-cells) provide a simple and robust culture system for studying neural development. Most protocols developed so far for directing neural differentiation of P19 cells depend on the use of culture medium supplemented with retinoic acid (RA) and serum, which has an undefined composition. Hence, such protocols are not suitable for many molecular studies. In this study, we achieved neural differentiation of P19 cells in a serum- and RA-free culture medium by employing the knockout serum replacement (KSR) supplement. In the KSR-containing medium, P19 cells underwent predominant differentiation into neural lineage and by day 12 of culture, neural cells were present in 100% of P19-derived embryoid bodies (EBs). This was consistently accompanied by the increased expression of various neural lineage-associated markers during the course of differentiation. P19-derived neural cells comprised of NES⁺ neural progenitors (~?46%), TUBB3⁺ immature neurons (~?6%), MAP2⁺ mature neurons (~?2%), and GFAP⁺ astrocytes (~?50%). A heterogeneous neuronal population consisting of glutamatergic, GABAergic, serotonergic, and dopaminergic neurons was generated. Taken together, our study shows that the KSR medium is suitable for the differentiation of P19 cells to neural lineage without requiring additional (serum and RA) supplements. This stem cell differentiation system could be utilized for gaining mechanistic insights into neural differentiation and for identifying potential neuroactive compounds.     

Inhibition of differentiation by leukemia inhibitory factor distinguishes two induction pathways in P19 embryonal carcinoma cells

The ability of leukemia inhibitory factor (LIF) to block differentiation of P19 embryonal carcinoma (EC) cells under a variety of induction conditions was determined. LIF inhibits differentiation under several conditions which lead to endodermal and mesodermal cell lineages including skeletal and cardiac muscle. In contrast, LIF does not block differentiation when cells are induced under conditions which lead to neuro-ectodermal cell types including neurons and astroglial cells. These studies demonstrate that P19 EC cell differentiation can be divided into LIF sensitive and insensitive pathways which correlate with differentiation of endodermal/mesodermal and neuro-ectodermal cell types, respectively. The effect of LIF on mRNA levels for several genes which have previously been implicated in mediating differentiation in P19 EC cells was determined. LIF has no effect on the mRNA levels for retinoic acid receptor (RAR) alpha, RAR beta, RAR gamma, jun A, jun D, c-fos, or fra-1. In contrast LIF stimulates jun B mRNA expression by a factor of four to six under all induction conditions.     

巢蛋白在P19神经元分化过程中的表达

小鼠巢蛋白（ｎｅｓｔｉｎ）基因编码了一个中等纤维骨架蛋白,该基因在小鼠中枢神经系统发育过程中的瞬时性表达,为了推测该基因的神经发育过程中可能的功能,我们分析了该基因在ＲＡ诱导的Ｐ１９胚胎性癌细胞体外神经分化过程中的表达规律,结果显示,在上述过程中,巢蛋白基因的表达早于神经前体细胞（ｎｅｕｒａｌｐｒｅｃｕｓｏｒｃｅｌｌ）中表达的ＢＭＰ４,以及在成熟神经元特异表达的标分子神经线（ＮＦ１６０）,表明巢蛋     

Cell-cell interaction can influence drug-induced differentiation of murine embryonal carcinoma cells

When cultured in the presence of either retinoic acid (RA) or dimethyl sulfoxide (DMSO), aggregates of the P19 line of mouse embryonal carcinoma (EC) cells differentiate and the spectrum of cell types formed depends on the drug dose. It is shown here the EC cells rapidly lose their colony-forming ability when cultured as aggregates in the presence of DMSO. This loss of plating efficiency (PE) also occurs rapidly following RA treatment. Loss of PE has been used as a quantitative procedure for assessing the rate of drug-induced differentiation. The relationship between drug dose and loss of PE is much steeper for DMSO than for RA, suggesting that these two drugs affect different stages of the differentiation decision-making apparatus. Mutant EC cell lines (D3 and RAC65) do not differentiate in the presence of drug-inducers (DMSO and RA, respectively). Neither differentiation-deficient mutant has an altered ability to form gap junctions. When D3 and P19 cells were mixed within the same DMSO-treated aggregates, the D3 cells remained undifferentiated and the P19 cells differentiated much less efficiently than if they were cultured in the absence of the D3 cells. When RAC65 and P19 cells were mixed in RA-treated aggregates, each cell responded to the drug as though the other were absent. Thus RA behaves as a cell-autonomous inducer of differentiation, whereas DMSO-induced differentiation seems to be mediated by interactions between neighboring cells.     

Differentiation of mouse p19 embryonic carcinoma stem cells injected into an empty zebrafish egg chorion in a microfluidic device

Mouse P19 embryonic carcinoma (EC) stem cells were xenotransplanted into the emptied chorion, the transparent envelope of a fertilized zebrafish egg (rather than mouse native zona pellucida) combined with a microfluidic device to study P19 EC cell differentiation in the chorion biomaterial. A distilled-water jet was used to remove the innate yolk and perivitelline inner mass from the chorion. P19 EC cells were injected into the emptied chorion using a micropipette, and they were subsequently cultured until the inner space of the chorion became completely occupied by cells. A simple microfluidic device was used for handling convenience and effective experiment. At d15, we found neural cells in the outer layer of the cell mass and beating cardiomyocytes in the inner layer of the large embryoid body. We propose that even though the species are different, the external innate membranes developed for embryo protection represent a useful type of ECM.