Snail is an immediate early target gene of parathyroid hormone related peptide signaling in parietal endoderm formation.

In mouse development, parietal endoderm (PE) is formed from both primitive endoderm (PrE) and visceral endoderm (VE). This process can be mimicked in vitro by using F9 embryonal carcinoma cells (EC) cells, differentiated to PrE or VE cells, and treating these with Parathyroid Hormone related Peptide (PTHrP). By means of differential display RT-PCR, we identified Snail (Sna) as a gene upregulated during the differentiation from F9 PrE to PE. We show that Sna is an immediate early target gene of PTHrP action in the formation of F9 PE cells. Using RT-PCR, we detected Sna transcripts in pre-implantation mouse embryos from the zygote-stage onwards. Sna was strongly upregulated in parallel with type 1 PTH/PTHrP Receptor (PTH(rP)-R1) mRNA in mouse blastocysts plated in culture, concomitant with detection of the PE-marker Follistatin and appearance of PE cells. By radioactive in situ hybridization on sections of mouse embryos, we found Sna expression in the earliest PE cells at E5.5. Sna remained expressed until at least E7.5. At this stage, we also observed clear expression in endoderm cells delaminating from the epithelial sheet of VE cells in the marginal zone. We conclude that PTH(rP)-R1 and Sna are expressed in endodermal cells that change from an epithelial to a mesenchymal phenotype. Since Sna expression has been described at other sites where epithelio-mesenchymal transitions (EMT) occur, such as the primitive streak at gastrulation and in pre-migratory neural crest cells, we hypothesize that Sna is instrumental in the action of PTHrP inducing PE formation, which we propose to be the first EMT in mouse development.

Dynamic connexin43 expression and gap junctional communication during endoderm differentiation of F9 embryonal carcinoma cells.

Gap junctional communication permits the direct intercellular exchange of small molecules and ions. In vertebrates, gap junctions are formed by the conjunction of two connexons, each consisting of a hexamer of connexin proteins, and are either established or degraded depending on the nature of the tissue formed. Gap junction function has been implicated in both directing developmental cell fate decisions and in tissue homeostasis/metabolite exchange. In mouse development, formation of the extra embryonal parietal endoderm from visceral endoderm is the first epithelial-mesenchyme transition to occur. This transition can be mimicked in vitro, by F9 embryonal carcinoma (EC) cells treated with retinoic acid, to form (epithelial) primitive or visceral endoderm, and then with parathyroid hormone-related peptide (PTHrP) to induce the transition to (mesenchymal) parietal endoderm. Here, we demonstrate that connexin43 mRNA and protein expression levels, protein phosphorylation and subcellular localization are dynamically regulated during F9 EC cell differentiation. Dye injection showed that this complex regulation of connexin43 is correlated with functional gap junctional communication. Similar patterns of connexin43 expression, localization and communication were found in visceral and parietal endoderm isolated ex vivo from mouse embryos at day 8.5 of gestation. However, in F9 cells this tightly regulated gap junctional communication does not appear to be required for the differentiation process as such.

Interdependent action of RalGEF and Erk in Ras-induced primitive endoderm differentiation of F9 embryonal carcinoma cells.

Previous work by us and others has implicated a role for Ral guanine exchange factors (RalGEFs) in Ras-induced cell growth and oncogenic transformation. Here we show for the first time that RalGEFs are involved in Ras-induced differentiation as well. Expression of oncogenic Ras in F9 embryonal carcinoma (EC) cells is known to induce differentiation to a primitive endoderm (PrE)-like phenotype, but the downstream signal transduction mechanisms involved are unclear. We found that PrE differentiation is induced by the Ras effector domain mutants, RasV12G37 and RasV12E38, but not by RasV12C40. Accordingly, expression of constitutively active forms of RalGEF (Rlf-CAAX) or Rafl (Raf-CAAX) is sufficient to induce differentiation. Inhibition of RalGEF activity by expression of dominant negative Ral completely abolishes Rlf-CAAX- and RasV12G37-induced differentiation, while it reduces differentiation by RasV12 and Raf-CAAX. Finally, while Rlf-CAAX does not increase Erk activity, inhibition of MEK blocks both Ras- as well as Rlf-CAAX-induced differentiation, suggesting that RalGEFs induce PrE differentiation in a manner depending on basal MEK or Erk activity. Based on these results we conclude that Ras induces PrE differentiation of F9 EC cells via an interplay of Erk-and RalGEF-mediated pathways.

Identification of a retinoic acid-inducible element in the murine PTH/PTHrP (parathyroid hormone/parathyroid hormone-related peptide) receptor gene.

We have shown previously that the PTH/PTHrP (PTH-related peptide) receptor mRNA becomes expressed very early in murine embryogenesis, i.e. during the formation of extraembryonic endoderm. Retinoic Acid (RA) is a potent inducer of extraembryonic endoderm formation and PTH/PTHrP-receptor expression in embryonal carcinoma (EC) and embryonal stem (ES) cells. Using the P19 EC cell line, we have characterized promoter elements of the murine PTH/PTHrP-receptor gene that are involved in this RA-induced expression. The data show that RA-induced expression of the PTH/ PTHrP-receptor gene is mediated by the downstream P2 promoter. Analysis of promoter reporter constructs in transiently transfected P19 cells treated with RA identified an enhancer region between nucleotides -2714 and -2702 upstream of the P2 transcription start site that is involved in the RA effect. This region matches a consensus hormone response element consisting of a direct repeat with an interspacing of 1 bp (R-DR1). The R-DR1 efficiently binds retinoic acid receptor-alpha (RARalpha)-retinoid X receptor-alpha (RXRalpha) and chicken ovalbumin upstream promoter (COUP)-transcription factor I (TFI)-RXRalpha heterodimers and RXRalpha and COUP-TFI homodimers in a bandshift assay using extracts of transiently transfected COS-7 cells. RA differentiation of P19 EC cells strongly increases protein binding to the R-DR1 in a band-shift assay. This is caused by increased expression of RXR (alpha, beta, or gamma) and by the induction of expression of RARbeta and COUP TFI/TFII, which bind to the R-DR1 as shown by supershifting antibodies. The presence of RXR (alpha, beta, or gamma) in the complexes binding to the R-DR1 suggests that RXR homodimers are involved in RA-induced expression of the PTH/PTHrP-receptor gene. The importance of the R-DR1 for RA-induced expression of PTH/ PTHrP-receptor was shown by an inactivating mutation of the R-DR1, which severely impairs RA-induced expression of PTH/PTHrP-receptor promoter reporter constructs. Since this mutation does not completely abolish RA-induced expression of PTH/PTHrP-receptor promoter reporter constructs, sequences other than the R-DR1 might also be involved in the RA effect. Finally, we show that the RA-responsive promoter region is also able to induce expression of a reporter gene in extraembryonic endoderm of 7.5 day-old transgenic mouse embryos.

Parathyroid hormone-related peptide (PTHrP) induces parietal endoderm formation exclusively via the type I PTH/PTHrP receptor.

A number of studies suggest a role for PTHrP and the classical PTH/PTHrP receptor (type I) in one of the first differentiation processes in mouse embryogenesis, i.e. the formation of parietal endoderm (PE). We previously reported that although in type I receptor (-/-) embryos PE formation seemed normal, the embryos were smaller from at least day 9.5 p.c. and 60% had died before day 12.5 p.c. Here we show that the observed growth defect commences even earlier, at day 8.5 p.c. Using two novel antibodies, we show that the expression of the type I receptor protein at this stage is confined to extraembryonic endoderm only. In addition, we show that large amounts of PTHrP protein are present in the adjacent trophoblast giant cells, suggesting a paracrine interaction of PTHrP and the type I PTH/PTHrP receptor in PE formation. The involvement in PE differentiation of other recently described receptors for PTHrP would explain a possible redundancy for the type I receptor in PE formation. However, deletion of the type I PTH/PTHrP receptor in ES cells by homologous recombination completely prevents PTHrP-induced PE differentiation. Based upon these observations, we propose that PTHrP and the type I PTH/PTHrP receptor, although not required for the initial formation of PE, are required for its proper differentiation and/or functioning.

The Ras/Erk pathway induces primitive endoderm but prevents parietal endoderm differentiation of F9 embryonal carcinoma cells.

The formation of parietal endoderm (PE) is one of the first differentiation processes during mouse development and can be studied in vitro using F9 embryonal carcinoma (EC) cells. Treatment of F9 EC cells with retinoic acid (RA) induces differentiation toward primitive endoderm (PrE), while differentiation toward PE is induced by subsequent addition of parathyroid hormone (PTH) or PTH-related peptide (PTHrP). The signal transduction mechanisms involved in this two-step process are largely unclear. We show that the RA-induced differentiation toward PrE is accompanied by a sustained increase in Ras activity and that ectopic expression of oncogenic Ha-Ras is sufficient to induce PrE differentiation. Ras activity subsequently decreases upon PTH-induced differentiation toward PE. This is a necessary event, since expression of oncogenic Ha-Ras in PrE-like cells prevents PTH-induced PE differentiation. Expression of active PKA in PrE-like F9 cells mimics PTH-induced PE differentiation and is again prevented by oncogenic Ha-Ras. The effect of oncogenic Ras on both differentiation steps is abolished by the MEK inhibitor PD98059 and can be mimicked by constitutively active forms of Raf and MEK. In conclusion, our data suggest that activation of the Ras/Erk is sufficient to induce differentiation to PrE and to prevent subsequent differentiation toward PE. Activation of PKA down-regulates Ras activity, resulting in disappearance of this blockade and transmission of signal(s) triggering PE differentiation.

Signals governing extraembryonic endoderm formation in the mouse: involvement of the type 1 parathyroid hormone-related peptide (PTHrP) receptor, p21Ras and cell adhesion molecules.

The formation of parietal endoderm (PE) from primitive endoderm (PrE) immediately after implantation of the early mouse embryo can be seen as the earliest example of an epithelio-mesenchyme transition (EMT) in murine development. Since EMT and EMI (epithelium-mesenchyme interactions) are at the very heart of morphogenesis, identifying molecular mechanisms governing these processes is of utmost importance. An excellent in vitro model system to study PE formation, i.e. F9 embryonal carcinoma cells, is available to this end. In the present paper we review our own recent results and those of others using these cells, and present our current view on the molecular mechanisms involved in PE formation.

Growth factor signalling.

Signalling between cells in the developing vertebrate embryo is essential for normal embryonic development. In the mid 1970's, signal transduction research started at the Hubrecht Laboratory with special emphasis on analysis of the signalling mechanisms that direct cell proliferation and differentiation. The introduction of in vitro model systems contributed tremendously to the success of the signal transduction research at the Hubrecht Laboratory. Initially neuroblastoma cell lines, and later embryonal carcinoma and embryonal stem cells played an important role in identification of the molecular key players in developmental signalling. For instance, embryonal carcinoma cells were used to identify and characterise polypeptide growth factors. Growth factor signalling research was extended to analysis of growth factor receptor activation. Moreover, the second messenger systems that are linked to growth factor receptors were studied, as well as the nuclear responses to growth factor receptor activation. Finally, the role of growth factor signalling in differentiation was established using embryonal carcinoma cells. Here, we will review work that was characteristic for the growth factor receptor signalling research that was done at the Hubrecht Laboratory between 1980 and the early 1990's.

Identification of connexin43 as a functional target for Wnt signalling.

Wnt mediated signal transduction is considered to regulate activity of target genes. In Xenopus embryos, ectopic Wnt1 and Wnt8 expression induces gap-junctional communication. During murine brain formation, Wnt1 and the gap-junctional protein connexin43 (Cx43) are co-expressed at the mid/hindbrain border, while interference with Wnt1 or Cx43 expression during embryogenesis leads to severe brain defects in the mid/hindbrain region. In PC12 cells, Wnt1 expression leads to an apparent increase in cell-cell adhesion. We investigated the effects of Wnt1 overexpression on gap-junctional communication in PC12 cells. Wnt1 expressing clones displayed an increased electrical and chemical coupling. This coincides with an increased expression of Cx43 mRNA and protein, while other connexins, Cx26, Cx32, Cx37, Cx40 and Cx45, were not up-regulated. Also, induction of Wnt1 expression in a mammary epithelial cell line leads to an increase in gap-junctional communication and Cx43 protein expression. In transient transactivation assays in P19 EC cells we found that Wnt1 and Li+, an ion that mimics Wnt signalling, increased transcription from the rat Cx43 promoter, potentially via TCF/LEF binding elements, in a pathway separate from cAMP-induced Cx43 transactivation. The results demonstrate that Cx43 acts as a functional target of Wnt1 signalling, and Cx43 expression can be regulated by Wnt1 at the transcriptional level. Our data suggest that Wnt1-induced cell fate determination is likely to involve regulation of gap-junctional communication.

Expression of the parathyroid hormone-related peptide gene in retinoic acid-induced differentiation: involvement of ETS and Sp1.

Differentiation of P19 embryonal carcinoma (EC) and embryonal stem (ES)-5 cells with retinoic acid (RA) induces expression of PTH-related peptide (PTHrP) mRNA. In this study we have characterized a region between nucleotide (nt) -88 and -58 relative to the transcription start site in the murine PTHrP gene that was involved in this expression. Sequence analysis identified two partially overlapping binding sites for the Ets family of transcription factors and an inverted Sp1-binding site. Two major specific bands were detected in a bandshift assay using an oligonucleotide spanning nt -88 and -58 as a probe and nuclear extracts from both undifferentiated and RA-differentiated P19 EC cells. The lower complex consisted of Ets-binding proteins as demonstrated by competition with consensus Ets-binding sites, while the upper complex contained Sp1-binding activity as demonstrated by competition with consensus Sp1-binding sites. The observed bandshift patterns using nuclear extracts of undifferentiated or RA-differentiated P19 cells were indistinguishable, suggesting that the differentiation-mediated expression was not caused by the induction of expression of new transcription factors. Mutations in either of the Ets-binding sites or the Sp1-binding site completely abolished RA-induced expression of PTHrP promoter reporter constructs, indicating that the RA effect was dependent on the simultaneous action of both Ets- and Sp1-like activities. Furthermore, these mutations also abolished promoter activity in cells that constitutively expressed PTHrP mRNA, suggesting a central role for the Ets and Sp1 families of transcription factors in the expression regulation of the mouse PTHrP gene.

Parathyroid hormone activates mitogen-activated protein kinase via a cAMP-mediated pathway independent of Ras.

In a previous study, we demonstrated that parathyroid hormone (PTH) inhibits mitogen-activated protein (MAP) kinase activation in osteosarcoma cells via a protein kinase A-dependent pathway. Here, we show that PTH can induce a transient activation of MAP kinase as well. This was observed in both Chinese hamster ovary R15 cells stably expressing high levels of rat PTH/PTH-related peptide receptor and parietal yolk sac carcinoma cells expressing the receptor endogenously. PTH was a strong activator of adenylate cyclase and phospholipase C in Chinese hamster ovary R15 cells. PTH-induced MAP kinase activation did not depend on activation of Gi, phorbol ester-sensitive protein kinase C, elevated intracellular calcium levels, or release of Gbetagamma subunits. It could, however, be mimicked by addition of forskolin or 8-bromo-cAMP to these cells. Prolonged treatment with forskolin caused sustained protein kinase A activity, whereas MAP kinase activity returned to basal levels. Subsequent treatment with PTH or 8-bromo-cAMP did not result in MAP kinase activation, whereas phorbol ester- or insulin-induced MAP kinase activation was unaffected. Finally, expression of a dominant negative form of Ras (RasAsn-17), which completely blocked insulin-induced MAP kinase activation, did not affect activation by PTH or cAMP. In conclusion, PTH regulates MAP kinase activity in a cell type-specific fashion. The activation of MAP kinase by PTH is mediated by cAMP and independent of Ras.

PTH/PTHrP receptor in early development and Indian hedgehog-regulated bone growth.

The PTH/PTHrP receptor binds to two ligands with distinct functions: the calcium-regulating hormone, parathyroid hormone (PTH), and the paracrine factor, PTH-related protein (PTHrP). Each ligand, in turn, is likely to activate more than one receptor. The functions of the PTH/PTHrP receptor were investigated by deletion of the murine gene by homologous recombination. Most PTH/PTHrP receptor (-/-) mutant mice died in mid-gestation, a phenotype not observed in PTHrP (-/-) mice, perhaps because of the effects of maternal PTHrP. Mice that survived exhibited accelerated differentiation of chondrocytes in bone, and their bones, grown in explant culture, were resistant to the effects of PTHrP and Sonic hedgehog. These results suggest that the PTH/PTHrP receptor mediates the effects of Indian Hedgehog and PTHrP on chondrocyte differentiation.

Parathyroid hormone related peptide mRNA expression during murine postimplantation development: evidence for involvement in multiple differentiation processes.

In this study we describe the spatio-temporal expression of Parathyroid Hormone related Peptide (PTHrP) mRNA during murine postimplantation development from day 5.5 post coitum (pc) until day 12.5 pc. From day 5.5 pc and onwards PTHrP mRNA was detected in the trophoblast. In addition, at day 5.5 and 6.5 pc epithelial cells of the antimesometrial crypt and cells of the inner zone of the decidua directly adjacent to the implanted embryo expressed PTHrP mRNA. This supported a previous model in which parietal endoderm formation is regulated by a paracrine mechanism involving PTHrP expressing trophoblast cells and receptor expressing extra-embryonal endoderm cells. The first embryonal PTHrP mRNA expression was detected in the roof of the hindbrain at gestation day 10.5 pc. From day 11.5 pc and onwards PTHrP mRNA was detected in the otic vesicle, the semilateral channels, the roof of the hindbrain and later in the choroid plexus, in epithelial cells of the lung and heart ventricle, mesenchymal cells lining the nasal pit, the dermis of the snout and at all sites of endochondral bone formation. The widespread expression of PTHrP mRNA during embryogenesis in extra-embryonic and embryonic tissues suggests the involvement of the peptide in multiple growth and differentiation processes.

Parathyroid hormone inhibits mitogen-activated protein kinase activation in osteosarcoma cells via a protein kinase A-dependent pathway.

Osteoblast-like cells, such as UMR 106 osteosarcoma cells, are known to be growth stimulated by growth factors such as EGF. In contrast, factors such as PTH and prostaglandin E2 inhibit their growth. The exact signal transduction mechanisms by which these latter factors act remain to be elucidated. Here we show that simultaneous treatment of UMR 106 cells with EGF and PTH-(1-34) resulted in a level of DNA synthesis intermediate between the levels of treatment with epidermal growth factor (EGF) and PTH alone. This correlated with the interference of PTH-(1-34) early in an EGF receptor-linked signal transduction pathway, i.e. the EGF-induced activation of p42 mitogen-activated protein (MAP) kinase. This effect was also found for prostaglandin E2, and could be potentiated by the phosphodiesterase inhibitor isobutyl-methylxanthine and mimicked by forskolin and 8-bromo-cAMP. There was a strict correlation between the lowest concentration of PTH-(1-34) required to enhance protein kinase A (PKA) activity and that required to inhibit MAP kinase activation, whereas saturating amounts of PTH-(3-34), a PTH analog unable to elevate PKA activity, had no effect. Lysophosphatidic acid- and 12-O-tetracanoylphorbol-13-acetate-induced MAP kinase activation were also inhibited by PTH-(1-34) and forskolin in these cells. Similar effects were seen on basic fibroblast growth factor-mediated MAP kinase activation in ROS 17/2.8 cells, indicating that this mechanism is a general feature of PTH in osteosarcoma cells. The inhibition of this mitogenic pathway through activation of PKA might play an important role in PTH-induced changes in proliferation and differentiation of osteoblasts.

Epidermal growth factor (EGF) induces serine phosphorylation-dependent activation and calcium-dependent translocation of the cytosolic phospholipase A2.

Phospholipase A2 (PLA2) is a key enzyme in the release of arachidonic acid and subsequent production of eicosanoids, which play an important role in a variety of biological processes, including mitogenic signalling by epidermal growth factor (EGF). In a previous study [Spaargaren, M. et al. (1992) Biochem J. 287, 37-43] we identified the EGF-activated PLA2 as being similar to the recently cloned high-molecular-mass cytosolic phospholipase A2 (cPLA2). In the present study we demonstrate a rapid transient EGF-induced activation of this cPLA2 and an EGF-induced increase in phosphorylation of the cPLA2. The EGF-induced activation of cPLA2 is reversed upon phosphatase treatment showing phosphorylation-dependent activation of the cPLA2. No direct association of the cPLA2 to the EGF receptor was detected under conditions where such an association with phospholipase C-gamma was demonstrated. Phosphoamino acid analysis of this cPLA2 showed that EGF induced an increase in serine phosphorylation exclusively, no tyrosine phosphorylation being observed. EGF treatment of the cells resulted in a Ca(2+)-dependent translocation of the cPLA2 from the cytosol to the membrane fraction. This is due to an EGF-induced [Ca2+]i rise which is dependent on the influx of extracellular Ca2+ via voltage-independent Ca2+ channels. It is shown that the Ca(2+)-dependent association of cPLA2 to membranes does not require accessory membrane molecules.

Expression pattern of parathyroid hormone/parathyroid hormone related peptide receptor mRNA in mouse postimplantation embryos indicates involvement in multiple developmental processes.

In this paper we describe the cloning of the mouse Parathyroid Hormone/Parathyroid Hormone related Peptide Receptor (PTH/PTHrPR) cDNA and expression of its mRNA during mouse postimplantation development from day 5.5 until day 15.5 post coitum (p.c.). In support of a model from previous studies, in which parietal endoderm differentiation is regulated by the interaction of the PTH/PTHrPR and Parathyroid Hormone related Peptide (PTHrP), high levels of PTH/PTHrPR mRNA levels were detected in developing parietal endoderm from day 5.5 p.c. and onwards. In the embryo proper, PTH/PTHrPR mRNA expression was mainly detected at sites of epithelium/mesenchyme interactions, starting at day 9.5 p.c. in the epithelium of the intestine and later in the mesenchyme of the lung, the epithelium of meso- and metanephric tubuli, the dermis and at all sites where bone formation takes place. The complexity of the PTH/PTHrPR expression pattern suggests tight developmental regulation and indicates multiple roles in embryogenesis for the receptor and its ligands, not only in extraembryonic tissue but also in the formation of various organs.

The rat, mouse and human genes encoding the receptor for parathyroid hormone and parathyroid hormone-related peptide are highly homologous.

The organization of the PTH/PTHrP receptor gene is highly homologous in three mammalian species, rat, human and mouse. This gene extends over 22 kb and contains at least 15 exons and 14 introns. The most 5' exon we have identified (exon U) is followed by an approximately 1kb intron. The second exon (exon S) encodes the initiator methionine and the putative signal peptide and is followed by the largest intron of this gene (about 11 kb). The amino-terminal extracellular region is encoded by 4 exons (E1, E2, E3 and G); exon G contains all 4 potential glycosylation sites. Membrane-spanning domains 1-4 and portions of their connecting intracellular and extracellular loops are encoded by 4 exons (M1, M2, M3 and M4). The second extracellular loop and portions of 4th and 5th membrane-spanning domains are encoded by one exon, EL2. The 5th membrane-spanning domain and portion of the 3rd intracellular loop are encoded by one exon, M5. The 6th membrane-spanning domain, the 3rd extracellular loop and the proximal part of the 7th membrane-spanning domain are encoded by one single exon (M6/7); the remaining sequence of the 7th membrane-spanning domain is encoded by a short exon, M7. The carboxy-terminal tail of the receptor and the 3' untranslated region are encoded by one single exon, exon T. The 3' untranslated region does not contain the classical polyadenylation signal, AATAAA. Expression in COS-7 cells of a minigene constructed of a 5' rat cDNA fragment (1.3 Kb) ligated in-frame to a 3' genomic fragment at the NsiI site, which is located in exon M6/7 resulted in a transcript that was translated into a functional receptor; it bound PTH and showed PTH-stimulated accumulation of intracellular cAMP. Therefore, the PTH/PTHrP receptor gene contains alternative 3' sequences that allow cleavage and polyadenylation of its transcript.

Glycogen synthase kinase 3 phosphorylates Jun family members in vitro and negatively regulates their transactivating potential in intact cells.

Expression of immediate-early genes involving the 12-O-tetradecanoyl phorbol 13-acetate (TPA)-responsive element (TRE) is modulated by post-translational modification of pre-existing activator protein 1 (AP-1) constituents. One of the components of AP-1, c-Jun, has been shown to be phosphorylated by glycogen synthase kinase 3 (GSK-3) in vitro in a region proximal to the DNA-binding domain, resulting in decreased DNA binding. Here, we have used transient transfection to show that AP-1 activity is inhibitable by coexpression of GSK-3 in intact cells. Furthermore, we show that the c-Jun-related proteins JunD and JunB are subject to similar regulation by GSK-3 in intact cells. Comparison of tryptic phosphopeptide maps of the three Jun proteins incubated with GSK-3 in vitro with maps of the same proteins immunoprecipitated from 32P-labelled cells indicates similar sites of phosphorylation. Together, these data support the hypothesis that GSK-3 is an important regulator of AP-1 activity in vivo.

Parathyroid hormone-related peptide as an endogenous inducer of parietal endoderm differentiation.

Parathyroid hormone related peptide (PTHrP), first identified in tumors from patients with the syndrome of "Humoral Hypercalcemia of Malignancy," can replace parathyroid hormone (PTH) in activating the PTH-receptor in responsive cells. Although PTHrP expression is widespread in various adult and fetal tissues, its normal biological function is as yet unknown. We have examined the possible role of PTHrP and the PTH/PTHrP-receptor in early mouse embryo development. Using F9 embryonal carcinoma (EC) cells and ES-5 embryonic stem (ES) cells as in vitro models, we demonstrate that during the differentiation of these cells towards primitive and parietal endoderm-like phenotypes, PTH/PTHrP-receptor mRNA is induced. This phenomenon is correlated with the appearance of functional adenylate cyclase coupled PTH/PTHrP-receptors. These receptors are the mouse homologues of the recently cloned rat bone and opossum kidney PTH/PTHrP-receptors. Addition of exogenous PTH or PTHrP to RA-treated EC or ES cells is an efficient replacement for dBcAMP in inducing full parietal endoderm differentiation. Endogenous PTHrP is detectable at very low levels in undifferentiated EC and ES cells, and is upregulated in their primitive and parietal endoderm-like derivatives as assessed by immunofluorescence. Using confocal laser scanning microscopy on preimplantation mouse embryos, PTHrP is detected from the late morula stage onwards in developing trophectoderm cells, but not in inner cell mass cells. In blastocyst stages PTHrP is in addition found in the first endoderm derivatives of the inner cell mass. Together these results indicate that the PTH/PTHrP-receptor signalling system serves as a para- or autocrine mechanism for parietal endoderm differentiation in the early mouse embryo, thus constituting the earliest hormone receptor system involved in embryogenesis defined to date.