High expression of stathmin in multipotential teratocarcinoma and normal embryonic cells versus their early differentiated derivatives

Differentiation (1992) 50: 89-96 Ontogeny, Neoplasia and Differentiation Therapy

0 Springer-Verlag 1992

High expression of stathmin in multipotential teratocarcinoma and normal embryonic cells versus their early differentiated derivatives Valkie Doye’**,Odile Kellermann ’, Marie-Helene Buc-Caron 2, and Andre Sobel’

’ INSERM U153

- CNRS URA614, 17 rue du Fer Moulin, F-75005 Paris, France Institut Pasteur, CNRS URAll48, 25 rue du Docteur Roux, F-75724 Paris Cedex 15, France

Accepted in revised form March 12, 1992

Abstract. Stathmin is a ubiquitous cytoplasmic protein, phosphorylated in response to agents regulating the proliferation, the differentiation and the specialized functions of cells, in a way possibly integrating the actions of diverse concomitant regulatory signals. Its expression is also regulated in relation with cell proliferation and differentiation and reaches a peak at the neonatal stage. To assess the possible role of stathmin at earlier stages of development, we examined its expression and regulation in embryonal carcinoma (EC) and derived cell lines as well as in the early mouse embryo. Interestingly, stathmin is highly abundant in the undifferentiated, multipotential cells of the F9, 1003 and 1009 EC cell lines. Its high expression markedly decreased, both at the protein and mRNA levels, when F9 cells were induced to differentiate into endodermal-like cells with retinoic acid and dibutyryl-CAMP. Stathmin was also much less abundant in differentiated cell lines such as the trophectodermal line TDM-1, as well as in several F9- and 1003-derived cell lines commited to differentiate towards the mesodermal and neuroectodermal lineages but still proliferating. Therefore, the observed decrease of stathmin expression is not related to the reduced proliferation rate but rather to the differentiation of the multipotential EC cells. The immunocytochemical pattern of stathmin expression during early mouse development indicated that stathmin is also highly abundant in the multipotential cells of the inner cell mass of the blastula, whereas it is much lower in the differentiated trophectodermal cells. These results confirm the physiological relevance of the observations with EC cells, and suggest that stathmin, in addition to its high expression at later stages of development and in the adult nervous system, may be considered as a new marker of the multipotential cells of the early mouse embryo.

* To whom offprint requests should be sent

Introduction Stathmin [50, 511 is a ubiquitous cytoplasmic phosphoprotein - also designated P19 [47], pp17 or prosolin [6], p18 [23], 19-K [22], and pp2O-pp21-pp23 [43] - whose phosphorylation has been correlated with the regulation by extracellular effectors of the differentiated functions, the proliferation, or the differentiation of cells in many biological systems (reviewed in [50]). It is a ubiquitous, highly conserved [30, 381 member of a gene family [48, 541 that also includes the neural-specific, growth-associated protein SCGlO [48, 541. Its two isoforms [3, 171 and their numerous phosphorylated states most likely provide the molecular support for the proposed general role of stathmin as an intracellular relay integrating the various second messenger pathways triggered by diverse extracellular signals [50]. In addition to the regulation of its phosphorylation [ 6 , 18, 43, 591, stdthmin is also regulated at the expression level, in relation with cell proliferation and differentiation. A link between stathmin expression and cell proliferation was demonstrated in T lymphocytes, where stdthmin is present at higher levels in proliferating than in resting T cells [15], and in acute leukemia cells by comparison with their normal counterparts [23]. A decrease in stathmin expression was also described during the differentiation of the proliferating myoblasts into functional myotubes [52]. Stathmin is expressed in almost all adult tissues, being most abundant in brain [30,47, 511 where it is essentially concentrated in neurons [12], and in testis [30,47], mostly in germ cells in their meiotic phase [l]. At the neonatal stage, stathmin expression is higher than in the adult in all tissues, both at the protein and mRNA levels [30]. Together with the above-mentioned link of stathmin expression with the regulation of cell proliferation and differentiation, this observation suggested that stathmin, in addition to its “ functional” role in differentiated cells, may play a role in “developmental” regulations as well. Such regulations play a major role also at the very early steps of embryonic development, when the pluripo-

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tent cells arising from the fertilized egg become progressively restricted to a defined lineage. The establishment of clonal cell lines derived from teratocarcinoma provides convenient model systems for the analysis of molecular mechanisms involved in regulations of early mammalian development. Embryonal carcinoma (EC) (teratocarcinoma) cells are indeed analogous in many respects to the pluripotent cells of the inner cell mass (ICM) or of the embryonic ectoderm of the mouse embryo. For example, in the presence of physiological concentrations of retinoic acid (RA), F9 EC cells give rise to a homogeneous population of primitive endodermlike cells [55] that further differentiate into parietal or visceral endoderm-like cells in the presence of both RA and dibutyryl-CAMP [57]. The in vitro differentiation of F9 EC cells is thus homologous to the differentiation of the extraembryonic endoderm from the ICM [24]. However, while EC cells are malignant, most of their differentiated derivatives are not, and cease proliferating. Therefore few cell lines corresponding to early stages of differentiation of the embryo have been established so far. Transformation of EC cells by the pK4 plasmid carrying the SV40 early genes under the control of the adenovirus El A promoter enabled us to immortalize stable clonal cell lines which have features of the precursor cells of the neuroectodermal, mesodermal or endodermal lineages [ 10,28,29]. The commited cells have a homogeneous and stable phenotype and, although expressing the SV40 T antigen, are able to give rise to differentiated derivatives which retain their potential for proliferation. Since stathmin phosphorylation and expression seem related to the regulation of cell differentiation and of development, and since few markers have been described to date that characterize the early steps of mammalian development [34], we examined the expression of stathmin and its regulation when pluripotent cells become progressively restricted to a defined lineage. We show here’ that stathmin is highly abundant in EC cells, and that the expression of both the protein and its mRNA strongly decrease with the first differentiations towards both extraembryonic and embryonic early derivatives. We show further that the observations in cell lines faithfully reflect the pattern of stathmin expression in the mouse blastula.

Methods Cells. EC cell lines 1009 [44], F9 [4], F9K4b2 [27], 1003 [39] and 1003pK4 [29] have been described previously. Both F9K4b2 and 1003pK4 cells have integrated the immortalizing vector pK4, 1003pK4 cells expressing the SV40 T antigen at thc EC cell stage whereas RA induction is required for the expression of the T antigen in the case of F9K4b2. F9K4b2 derivatives include the H7 endodermal stem cell clone which is able to differentiate into parietal and visceral endoderm-like cells expressing cr-fetoprotein [28], and the 1C11 clone of immature stem cells commited to serotoninergic differentiation and acquiring a neuron-like phenotype after induction [10]. 1003pK4 derivatives include the mesoblastic clone 11B (unpublished) and another mesoblastic clone, C1, selected for its ability to form bone both in vivo and in vitro [29]. 3-TDMl Part of this work has been presented elsewhere in abstract form [I81

(TDM) is a trophectodermal cell line expressing the trophectoderma1 marker Endo A cytokeratin [S, 411. Homogeneous clonal cell lines were cultured as previously described [27]. F9 cells were plated on gelatin-coated plastic dishes and their endodermal differentiation was induced as described [57]. Briefly, all-trans retinoic acid (Sigma, St. Louis, MO. USA) was added at lo-’ M (final concentration) on day 0 and, in some experiments, M dibutyryl cyclic AMP (Sigma) was added two days later, the cells being refed with fresh medium and pharmacological effectors every two days. Embryos. Mouse blastocysts were recovered from prepubertal, standardly superovulated F2 C57B1/6 x DBA/2 females (the day a vaginal plug was recovered was taken as day 0 of development) and seeded in Dulbecco modified Eagle’s medium (DME) supplemented with 10% fetal bovine serum (FBS) on gelatin-coated labtek chambers (Nunc, Naperville, Ill., USA). Immunocytochemical experiments were performed 3 days later. Imrnunocytochemistry. Indirect immunofluorescence was carried out as described [9]. In brief, fixation and permeabilization were performed using 4% paraformaldehyde (Aldrich, Darmstadt, Germany) followed by incubation with the antibodies diluted in phosphate buffered saline (PBS) containing 0.1 YOgelatin (Merck, Strasbourg, France) and 0.2% Triton X100. Species-specific secondary antisera coupled to fluorophores (1 :100, Nordic, Tilberg, The Netherlands) were used to visualize sites of primary antibody binding. Stathmin immunoreactivity was revealed using polyclonal antiserum c , raised against a C-terminal peptide (peptide C [30]), specific to stathmin as compared to its related protein SCGlO [48, 541. Identical results were obtained with antiserum I, raised against a peptide from the N-terminal region of stathmin (peptide I [30]). Preimmune serum and rat monoclonal antibodies TROMA 1 recognizing the cytokeratin Endo A [8] were used as controls.

Wesfern blot analysis. Cells from 10 cm diameter dishes were washed twice with PBS, collected in 500 p1 of homogenization buffer (10 m M TRIS HCI pH 7.4, 0.02% NaN,, 10 pg/ml leupeptin, 25 pg/ml aprotinin, 10 pg/ml pepstatin (Sigma), 1 mM EDTA), and centrifuged at 13,000 rpm for 3 min. Concentration of proteins from the resulting “low speed supernatant” were estimated by the method of Bradford [5], using bovine serum albumin (BSA) as standard. Approximately 150 pg of protein from these samples were separated on 13% polyacrylamide gels according to Laemmli [31], and transferred onto 0.2 pm nitrocellulose (Schleicher & Schuell, Dassel, Germany) as described [30], the homogeneity of the protein levels being further controlled by Ponceau red staining. The blots were saturated with casein (2.5%) and probed with a rabbit polyclonal anti-stathmin antiserum directed against a synthetic peptide (peptide I [30]). Bound antibodies were detected with [‘251]-protein A (Amersham, UK) and autoradiography. Quantification of immunoreactive bands on autoradiograms was performed with an image analysis system (Samba, Grenoble, France), with increasing concentrations of a control sample as an internal reference, and with non saturated autoradiographic exposures providing values in the linear range of the reference curve. RNA isolation and analysis. Total RNA was isolated from the various cell lines using either the guanidinium isothiocyanate/cesium chloride method [ I l l or the single step method [13]. Northern blots were prepared according to [ 5 8 ] , with 30 pg of glyoxalated RNA, resolved on 1.5% agarose gel and transfered to Hybond N membrane (Amersham, UK). The stathmin probe was the 1-809 EcoRl -Drat fragment of the rat cDNA clone pS62b [17]. The mouse cytokeratin Endo A probe was the 2.3 kb EcoRl insert from clone p34R14 corresponding to the pseudogene 22 [60]. The Glyceraldehyde Phosphate Dehydrogenase (GAPDH) probe was a 0.6 kb insert corresponding to the 5’ end of a mouse cDNA [45]. The SCGlO probe was the whole plasmid containing the SCG 10-6 cDNA [54]. Probes were

labeled using ["PI dCTP in an oligonucleotide-primed reaction [20] (Amersham kit). Hybridizations were carried out at 42" C in the presence Of 50% formamide, and final washes were performed at 65" C in 0.2 x standard salina citrate (SSC), 0.1% sodium dodecyl sulfate (SDS) for the heterologous probe, or at 68" C in 0.1 x SSC, 0.1% SDS for the homologous probes. For reprobing, the RNA blots were washed at 75" C in 0.1 x SSC, 0.1% SDS, ioyo formamide.

The neural-specific, growth-associated protein SCGlO [54] being a member of the Same gene family with a high nucleotide sequence homology with stathmin [48], we also examined the expression of the mRNAs for SCG1O. As shown On Fig. 2, the two 1.9 and l.o kb SCGlO transcripts are clearly distinct from the stathmin

Results I

High expression of stathmin in embryonal curcinomu cells

I

I

I

- 2.7 kb

STATHMIN

The expression of stathmin at the protein level was assessed by immunoblotting with a stathmin-specific antiserum. Two dimensional gels confirmed that the 19 kDa immunoreactive band in EC cells corresponds to the unphosphorylated and phosphorylated states of the previously described isoforms of stathmin [3]. Mouse brain was used as a reference, since stathmin is most abundant in this tissue where it reaches a maximum around birth. The intensity of the 19 kDa stathmin immunoreactive band of various EC cell lines (1009, 1003 and F9) was intermediate between the signals obtained with the neonatal and the adult mouse brain tissues (Fig. l), and higher than in other cell lines such as the pheochromocytoma derived PC12 cells (not shown). Stathmin is thus highly expressed in embryonal carcinoma cells. The major 1.1 kb stathmin mRNA transcript was detected by Northern blot analysis in both the 1003 and F9 EC cell lines (Fig. 2). Comparison with neonatal mouse forebrain and with whole 12 day mouse embryo shows that stathmin is highly expressed also at the mRNA level in the multipotential 1003 and F9 EC cells.

NNB AdB 6okD-

1009

1003 F9

0 1 - 1.1 kb

SCGlO

- 1.9 kb 8 - 1.0 kb

Fig. 2. Stathmin and SCGlO mRNA levels in mouse neonatal brain (NNB), 12 days old embryo (12 dE), and EC cell lines. The Northern blot (20 pg of total RNA/sample) was successively hybridized with stathmin, SCGlO and glyceraldehyde phosphate dehydrogenase (GAPDH) probes. The stdthmin probe mostly hybridized with a 1.1 kb band, although a weak signal could be detected at 2.7 kb. The SCGlO probe specifically hybridized with two bands at 1.0 and 1.9 kb (a residual stathmin signal at 1.1 kb (*) is due to incomplete dehybridization). The hybridization with the GAPDH probe is presented as a control

c % 100

-

19 kD -

-

&

c

-a -

---

-

A

-

-

Fig. 1. Stathmin expression in mouse brain and embryonal carcinoma (EC) cell lines. Immunoblots of low speed supernatant extracts (140 pg protein) from neonatal ( N N B ) and adult (AdB) mouse forebrain and from the mouse embryonal carcinoma cell lines 1009, 1003, and F9 are shown (top). The apparent molecular weight of stathmin (19 kDa) and of a high molecular weight stathmin-related protein (60 kDa) are indicated. The 60 kDa immunoreactive band, previously described in neonatal brain [30]was not detected in any of the cell lines. The Ponceau red protein staining of the blot prior to antibody treatment is presented at the bottom

1

0 9 8 - lo g [RA]

7

0 1

3

5 days

7

9

Fig. 3. Dose response and kinetics of retinoic acid-induced decrease of stathmin in FY cells. Left: F9 cells were treated for 7 days with the indicated concentrations of retinoic acid ( R A ) .Right: cells were treated with retinoic acid (lo-' M ) alone (filled marks, three independent experiments) or together with dibutyryl-CAMP (dh-CAMP, M ) from day 2 (open marks, two independent experiments). Values on both curves were obtained by densitometry of the stathmin immunoreactive 19 kDa band of immunoblots, the value obtained with untreated cells being defined as 100%

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F9 c

1

2

3

5

TDM

7

STATHMIN

$&

- 1.1 kb

I

l

l

STATHMIN I - 0

1

I

o

I 0

-I

I

END0 A

Fig. 4. Expression of stathmin and EndoA mRNAs in differentiating F9 cells and in TDM cells. F9 cells were treated for 1 to 7 days with RA and dibutyryl-CAMP as described in Fig. 3. Twenty micrograms total RNA from these cells and from trophectodermal TDM cells were analysed by Northern blot. Comparison of the stathmin 1.1 kb mRNA signal with the GAPDH 1.3 kb signal indicates that stathmin mRNA levels are clearly decreased in F9 cells after 3 days of differentiation, while the cytokeratin EndoA 1.6 kb message becomes detectable

Fig. 5. Stathmin expression in immortalized undifferentiated (F9K4b2 and 1003pK4) or differentiated (1C11, H7, C1, and 11B) derivatives from F9 and 1003 cell lines. The cell lines and their properties are described in Methods. Top: stathmin immunoreactivc 19 kDa bands on immunoblots of low speed supernatants (140 pg protein) from the indicated cell lines; Bottom: Northern blot analysis of stathmin expression in the same cells. Approximately 20 pg of total mRNA were analyzed for stathmin mRNA in each case. Thc autoradiogram of control hybridization with a GAPDH cDNA probe is also presented

1.1 kb one. As expected, SCGlO mRNAs are highly expressed in neonatal brain, and also detectable in the whole 12 day embryo. However, the expression of SCGlO might not be strictly restricted to the nervous system, since its transcripts could also be detected, although at much lower levels, in the multipotential 1003 and F9 EC cell lines.

Figure4 shows that a decrease of stathmin mRNA parallels, at least in part, the decrease of the protein. However, whereas stathmin was significantly reduced as soon as after one day, the relative decrease of stathmin mRNA was clearly detected at 3 days, at the very time where the mRNA of the classical endodermal differentiation marker Endo A started to be expressed.

Decrease of stathmin expression during retinoic acid and dibutyryl-CAMP-induced endodermal differentiation of F9 cells

Stathmin expression is highly reduced in diflerentiated or commited cell lines of the extraembryonic and embryonic lineages derived from teratocarcinoma

When F9 cells were treated for 5 days with retinoic acid (RA), the expression of stathmin decreased, as indicated by the quantification of the corresponding Western blots (Fig. 3, left panel). This effect of RA was detected with concentrations as low as lo-’’ A4 and reached a maximum at around l o - @M , a range where retinoic acid induced the endodermal differentiation of F9 cells. With the maximally effective concentration of RA (lo-’ M), the decrease of stathmin expression was already detected after one day and reached a plateau at approximately 40% of the initial value after 4 days of treatment (Fig. 3, right panel). Addition of l o p 3 M dibutyryl-CAMP to the culture medium after two days of RA treatment induced a further decrease to less than 10% of the initial stathmin level. Interestingly, dibutyrylCAMP treatment without RA, a condition where the endodermal differentiation of F9 cells does not take place, did not induce any change in stathmin levels (not shown).

During the development of the mouse embryo, cells of the extraembryonic tissues differentiate first, formation of the trophectoderm being followed by that of the extraembryonic visceral and parietal endoderms. The teratocarcinoma derived cell lines TDM [41] and H7 [28] closely resemble cells of the trophectoderm and of the extraembryonic endoderm of the early embryo, respectively. Both cell lines display lower stathmin mRNA levels than the multipotent, undifferentiated F9 cells (Figs. 4 and 5). In the trophectodermal TDM cells (which correspond to the first differentiated extraembryonic derivatives appearing in the embryo and which characteristically express the Endo A cytokeratin) the level of stathmin mRNA is comparable to that present in the RA and dibutyryl-CAMP treated F9 cells, whose differentiation into parietal endoderm-like cells is also characterized by the expression of the Endo A mRNA (Fig. 4). Cell lines corresponding to early derivatives of the

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50 pm Fig. 6. lmmunocytochemicdl detection of stathmin in cultured mouse blastocysts. 3.5 day mouse blastocysts were maintained in culture for 3 days and indirect immunofluorescence (a, c, e) was performed as described in Methods. (a) anti-stathmin antiserum C (dilution 1 :200) specifically labeling the inner cell mass, (c) pre-

immune serum (1 :200) and (e) anti-Endo A cytokeratin TROMA-I monoclonal antibodies used as a negative control staining the peripheral trophectodermal cells. The corresponding phase contrast views are shown on the right (b, d, f), and the inner cell mass is indicated by an asterisk

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main embryonic lineages were derived from EC cell lines transformed with the immortalizing vector pK4. As shown on Fig. 5, the parental EC cell lines F9K4b2 and 1003pK4 expressed similar stathmin levels to that of their untrdnsformed counterparts F9 and 1003. On the other hand, stathmin expression was clearly lower, both at the protein and mRNA levels, in the C1 and 11B differentiation commited derivatives of 1003pK4 and F9K4b2. Due to the expression of the SV40 oncogenes, these cells still proliferate: the decrease in stathmin expression is thus associated with the differentiation commitment of the cells. Interestingly, stathmin levels were particularly low in the H7, C1 and 11B cell lines, which correspond to endodermal or mesodermal precursor clones, by comparison to the 1C11 clone which is a neuroectodermal precursor. Stathmin is expressed in the multipotential cells of’ the mouse blastocyst

To evaluate the relevance of the observations with cultured cell lines to the biological events actually taking place during normal embryonic development, we examined the distribution of stathmin in mouse embryos at the blastocyst stage. Immunocytochemical detection of stathmin was performed on embryos removed from the uterus at day 3.5 of gestation and kept in culture for three days to allow the attachment of the cells, the inner cell mass still forming characteristic cell clusters whereas the multinuclear trophectodermal cells spread out. With specific antistathmin antisera, stathmin appeared highly concentrated in the multipotential cells of the inner cell mass and barely detectable in the surrounding trophectodermal cells (Fig. 6a, b). The specificity of the intense fluorescence of the inner cell mass was further assessed with a corresponding preimmune serum (Fig. 6c, d). Furthermore, as expected, anti-Endo A cytokeratin antibodies strongly stained only the trophectodermal cells, leaving the clump of inner cell mass cells negative (Fig. 6e, f). Discussion The expression of stathmin was shown previously to be regulated during the late embryonic and the postnatal stages of development [30, 471. We report here that stathmin is highly abundant in embryonal carcinoma cells as well as in the multipotential cells of the blastocyst, and that its expression is down-regulated with the formation of the trophectoderm, i.e. the first differentiation of the mouse embryo. The high levels of stathmin detected in various embryonal carcinoma cell lines both at the protein and mRNA levels are remarkable, since no other cell line with similar levels of stathmin has been identified so far, with the possible exception of activated leukemic cell lines [6, 431. The high levels of stathmin detected by immunocytochemical methods in normal embryonic counterparts of EC cells, i.e. cells of the inner cell mass

of the blastocyst, confirm the physiological significance of the high expression of stathmin in the undifferentiated, multipotent cells that will give rise to the various layers of the embryo. Accordingly, preliminary results show that stathmin is present in significant amounts in cultured ES cells (Doye V. and Cohen-Tanoudji M., unpublished observations) and that it is biochemically detectable at the morula stage and increases in the blastula ~191. The high expression of stathmin is indeed characteristic of the multipotential EC cells, since it is dramatically decreased both in the immediate differentiated derivatives of EC cells and in the EC-derived differentiated cell lines, as well as in the embryo during its early development. F9 cells provide a good model for the differentiation events of early development. RA, a steroid hormone most likely acting as a differentiation signal during embryogenesis in vivo [7], induces the differentiation of the multipotential F9 cells into primitive endoderm-like cells, and further into parietal endoderm in the presence of dibutyryl-CAMP [57], thus reproducing in vitro early steps of mouse embryogenesis. Interestingly, the extent of the decrease of stathmin expression paralleled the degree of differentiation of F9 cells, dibutyryl-CAMP also potentiating the action of RA. Similarly, the low expression of stathmin mRNA and protein in the EC-derived cell lines of the extraembryonic (TDM, H7) and embryonic (11B, C1) lineages correlates well with a down-regulation related to the early differentiation steps of multipotential cells. That this correlation, first evidenced with the EC model system, is physiologically relevant, is demonstrated by the very low levels of stathmin detected by immunofluorescence in the first differentiated derivatives of the multipotential cells of the embryo, i.e. the trophectodermal cells of the blastula. Interestingly, the relatively high level of stathmin in the neuroectodermal precursor clone 1 C11 [lo], might be related to the high abundance of stathmin in differentiated neuronal structures [ 12, 301. Several possible mechanisms may underly the decrease of stathmin levels observed with early differentiation. The apparently more pronounced decrease of the protein than of its mRNA in F9 cells during the first two days of RA treatment indicates that RA may regulate the translation or the stability of the protein itself. In F9 cells treated with RA and which become thus responsive to dibutyryl-CAMP [%I, addition of dibutyryl-CAMP on day 2 greatly amplified the RA-induced decrease of stathmin at both the protein and mRNA levels. The effects on stathmin mRNA, mostly observed from day 3, may occur, as previously described for other down-regulated mRNAs in EC cells, through a decrease of either the transcription rate [26, 34,611 or the stability of the mRNA [16, 34, 371. Further analysis of stathmin transcription and translation, as well as of the stability of the protein and of its mRNA in both RA and RA+ dibutyryl-CAMP treated F9 cells will be necessary to provide new insights on the molecular mechanisms through which these two effectors cooperate during early development. In various biological systems, regulation of stathmin

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phosphorylation and/or expression has been related, in addition to the regulation of cell functions and differentiation [18], to the regulation of the proliferation of cells (reviewed in [50]). Furthermore, F9 cells induced to differentiate with RA and dibutyryl-CAMP display concomitantly a reduced level of DNA synthesis and growth rate [35], and they loose their tumorigenicity [56]. Altered patterns of expression of some protooncogenes [36], and in particular an early decrease of c-myc mRNA levels [16], have also been specifically associated with growth arrest during the differentiation of F9 cells, whereas the decreased expression of REX-1 gene was shown to be independent from the reduction in cell growth rate [25]. We show here that the expression of stathmin is also reduced in the committed or differentiated cell lines derived from multipotential EC cells maintained in a permanent state of proliferation by the expression of the transfected SV40 T antigen. The reduced level of stathmin expression in the differentiated derivatives of multipotential cells observed with F9 cells and in the embryo is therefore most likely not due to the decreased proliferation but rather to the differentiation process and/or to its regulation. Few markers of the multipotential cells of the early embryo or of the corresponding EC cell line models have been characterized so far. They include cell surface or structural molecules [46, 49, 531, several DNA binding proteins [14, 16, 25, 26, 32, 33, 37, 40, 421, and heat shock or heat shock-like proteins [2, 211. The high expression of stathmin in the multipotential cells of the embryo and its rapid down-regulation with their first differentiation stages makes it a good candidate for a new such marker. Stathmin expression reaches a peak at late stages of embryogenesis and at the neonatal stage, to decrease towards adulthood. Its high expression also in multipotential cells of the embryo further stresses its involvement in developmental regulations. Further characterization of its regulation will provide new clues on molecular mechanisms involved in early stages of embryogenesis and for the understanding of the precise function of stathmin. Acknowledgments. We wish to thank Dr. M. Fardeau for his constant support, Dr. P. Duprey for many stimulating discussions and the gift of the Endo A probe, and Dr. H. Chneiweiss for critical reading of the manuscript. We also gratefully acknowledge the help of M. Maury for the isolation of mouse blastocysts, and the generous gifts of the SCGlO probe from Dr. D.J. Anderson, and of ES cells from Dr. CohenTannoudji. This work was supported by the “Institut National de la Sante et de la Recherche Mkdicale”, the “Centre National de la Recherche Scientifique”, the “ Association Frangaise contre les Myopathies”, the “Association pour la Recherche contre le Cancer ”, the “Ligue frangaise de lutte contre le cancer” and the “Ministtre de la Recherche et de la Technologie”.

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