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Fibroblast growth factor receptor-1 phosphorylation requirement for cardiomyocyte differentiation in murine embryonic stem cells

Fibroblast growth factor receptor-1 (Fgfr1) gene knockout impairs cardiac and haematopoietic development in murine embryonic stem cells (mESC). In FGFR1, tyrosine residues Y653 and Y654 are responsible for its tyrosine kinase (TK) activity whereas phosphorylated Y463 and Y766 represent docking sites for intracellular substrates. Aim of this study was the characterization of FGFR1 signalling requirements necessary for cardiomyocyte differentiation in mESC. To this purpose, fgfr1−/− mESC were infected with lentiviral vectors harbouring human wild-type hFGFR1 or the Y653/654F, Y463F and Y766F hFGFR1 mutants. The resulting embryonic stem (ES) cell lines were differentiated as embryoid bodies (EBs) and beating foci formation was evaluated. In order to appraise the presence of cells belonging to cardiovascular and haematopoietic lineages, specific markers were analysed by quantitative PCR, whole mount in situ hybridization and immunofluorescence. Transduction with TK+ hFGFR1 or the TK+ Y766F-hFGFR1 mutant rescued cardiomyocyte beating foci formation in fgfr1−/− EBs whereas the TK− Y653/654F-hFGFR1 mutant and the TK+ Y463F-hFGFR1 mutant were both ineffective. Analysis of the expression of early and late cardiac markers in differentiating EBs confirmed these observations. At variance with cardiomyocyte differentiation, all the transduced TK+ FGFR1 forms were able to rescue haematopoietic differentiation in EBs originated by infected fgfr1−/− mESC, only the TK− Y653/654F-hFGFR1 mutant being ineffective. In keeping with these observations, treatment with different signalling pathway inhibitors indicates that protein kinase C and ERK activation are essential for cardiomyocyte but not for haematopoietic differentiation in EBs generated by fgfr1+/−∼ mESC. In conclusion, our results suggest that, although FGFR1 kinase activity is necessary for both cardiac and haematopoietic lineage maturation in mESC, phosphorylation of Y463 in the intracellular domain of the receptor is a specific requirement for cardiomyocyte differentiation.

Most of these recent developmental findings involved the use of animal models paralleled by in vitro differentiation studies on murine embryonic stem cells (mESC). mESC originate from the inner cell mass of the blastocyst and are capable of cell renewal and differentiation after in vitro aggregation into three-dimensional structures termed embryoid bodies (EBs) [12,13]. Cardiac development in mESC differentiation cultures is well established and is easily detected by the appearance of areas of contracting cells that display characteristics of mature cardiomyocytes [14]. All of the cardiac cell types have been generated from differentiating EBs, and gene expression analyses suggest that their development in culture recapitulates cardiogenesis in the early embryo [15,16]. Indeed, CP cells originated from both FHF and SHF, characterized by the common expression of nkx2.5 and by the selective expression of tbx5 and fgf10/isl1, have been isolated from differentiating ESC populations [2,3].
Fibroblast growth factor receptors (FGFRs) belong the subclass IV of membrane-spanning tyrosine kinase (TK) receptors [17]. Four fgfr genes have been identified: FGFR1-4 proteins share common structural features and interact with the members of the FGF family comprising of at least 23 polypeptides [18]. The FGF/FGFR system has been implicated in a variety of physiological and pathological conditions, including embryonic development, tissue growth and remodelling, inflammation, tumour growth and vascularization [19,20]. Following ligand binding and receptor dimerization, a number of tyrosine autophosphorylation sites have been identified in human FGFR1 (hFGFR1): Y653/654 are critical for TK activity [21], Y463 is involved in endothelial cell proliferation by binding to Src homology (SH)2/SH3 domain-containing adaptor protein Crk [22] and phosphorylated Y766 has been shown to bind phospholipase C-␥ (PLC-␥) in L6 myoblasts, Shb in endothelial cells and Grb14 in MDA-MB-231 human breast cancer cells [23][24][25]. Also, FGFR1 activation leads to FRS2 phosphorylation [26] followed by Grb2 and Shp2 interactions [27]. FRS2, Crk and Shb binding to FGFR1 affect the classical Ras/Raf-1/MEK/ERK/Jun proliferation pathway activated by TK receptors, while PLC␥1 activates protein kinase C (PKC) [28], whose role in cardiomyocyte differentiation has been demonstrated [29]. FGF/FGFR signalling plays important functions in mesoderm formation and development [30]. Accordingly, fgfr1 Ϫ/mice die during gastrulation, displaying defective mesoderm patterning with reduction in the amount of paraxial mesoderm and lack of somite formation [31,32]. Studies on chimeric embryos using FGFR1-deficient mESC revealed an early defect in the mesodermal and endodermal cell movement through the primitive streak, followed by deficiencies in contributing to anterior mesoderm, including heart tissue [33,34]. The pivotal contribution of FGF signalling in heart formation has been demonstrated in different animal models: in C. intestinalis, FGF signalling delineates the cardiac progenitor field [35]; in Drosophila, mesoderm spreading depends upon the expression of heartless, homologous to vertebrate fgfr1, and the heartless mutant is characterized by the absence of the heart [36,37]; in chicken, FGF signalling activated by FGF8 contributes to the heart-inducing properties of the endoderm [38]; in zebrafish, induction and differentiation of the heart requires FGF8 [39]; in mice, Fgf8 neo/Ϫ mutants show complex cardiac defects [40]. FGFR1 has been implicated in cardiac development also during murine EB differentiation. Indeed, analysis of the in vitro differentiation process of fgfr1 Ϫ/-mESC following EB formation led us to determine a non-redundant role for FGFR1 in cardiomyocyte development [41]. Moreover, the FGFR1 TK inhibitor SU 5402 [42], the MEK1/2 inhibitor U0126 [43] and the classical/novel PKC inhibitor GF109203X [44]

ES cell culture
Murine ESC [32,41] were adapted to grow without feeder cells and maintained in Dulbecco's Modified Eagle's Medium supplemented with 20% foetal bovine serum (Hyclone, South Logan, UT, USA), 0.1 mmol/l nonessential amino acids, 1 mmol/l sodium pyruvate, 0.1 mmol/l ␤-mercaptoethanol, 2 mmol/l L-glutamine and 1000 U/ml LIF (ESGRO, Millipore, Milan, Italy). At T0 of differentiation, exponentially growing mESC were resuspended in LIF-deprived EB medium and cultured in 30 l hanging drops (400 cells) for 2 days to allow cell aggregation. Then, aggregates were transferred onto 0.5% agarose-coated dishes and grown for 5 days in LIF-deprived EB medium. At day 7, EBs were transferred into 24-well tissue culture plates and allowed to adhere. Aggregates were monitored for the appearance of spontaneously contracting foci during the following days.

Vector production and transduction
Human FGFR1, Y653/654F-hFGFR1, Y463F-hFGFR1 and Y766F-hFGFR1 cDNAs [47] were independently cloned in the transfer vector pRRL-SIN-PPT-hPGK-GFP-WPRE by replacing green fluorescent protein (GFP) gene [48]. Viral particles were produced, purified by ultracentrifugation and used to infect murine fgfr1 Ϫ/-mESC, following a standard protocol [48,49]. As a control experiment, fgfr1 ϩ/and fgfr1 Ϫ/-mESC were infected with lentiviruses harbouring GFP under the ubiquitous phosphoglycerate kinase promoter [48]. In both cell types, GFP expression was observed in more than 90% of the undifferentiated cells and was maintained throughout the whole experimental differentiation protocol (data not shown). Moreover, GFP transduction did not affect beating foci formation in fgfr1 ϩ/-EBs (data not shown). 125 I-FGF2 binding assay was described previously [45]. Briefly, human recombinant FGF2 was labelled with 125 I (37 GBq/ml; GE Healthcare Life Science, Milan, Italy) using Iodogen (Pierce, Rockford, IL, USA) at a specific radioactivity equal to 1800 cpm/fmol. mESC were seeded in 24-well dishes at the density of 250,000 per cm 2 in complete medium. After 24 hrs, a standard binding assay was performed using 3 ng/ml 125 I-FGF2 in the absence or in the presence of an excess of unlabelled FGF2. At the end of the incubation, cells were washed with 2 M NaCl, 20 mM Hepes, pH 7.2. Then, radioactivity bound to high-affinity sites was stripped with 20 mM sodium acetate, pH 4, and measured in a gamma-counter (Wallac, Waltham, MA, USA).

Binding assay and immunoblot analysis
For immunoblot analysis mESC were seeded at 200,000 cells/cm 2 and starved in 1% FCS overnight. Then, cells were stimulated with FGF2 (100 ng/ml) for 15 min. and directly lysed in reducing sample buffer. Samples were run on 10% SDS-PAGE and transferred to Immobilon-P (Millipore, Milan, Italy). ERK1/2 phosphorylation was evaluated by membrane incubation with anti-phospho-ERK1/2 antibody (sc-7383, Santa Cruz Biotechnology Inc.), while uniform loading was judged by incubation of the same membrane with anti-ERK2 antibody (sc-1647, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA).

Whole mount in situ hybridization (WISH)
Total RNA from fgfr1 ϩ/Ϫ EBs at day 10 of differentiation was reverse transcribed to cDNA and used as template for PCR reactions using the following essential for cardiomyocyte differentiation [41]. The human FGFR1 gene product shares 98% amino acid sequence identity with the murine counterpart. Most of the differences between the two molecules are located in the first Ig-like loop of the extracellular domain, a region dispensable for the specificity of FGFs-FGFR1 interaction [50,51]. The aminoacidic identity of murine and human FGFR1 raises to 100% in the intracellular domain that comprises all the tyrosine autophosphorylation sites [21,51] (Fig. 1A). Moreover, infected cells show an increase in high-affinity 125 I-FGF2 binding when compared with control cells (Fig. 1B), indicating that both TK ϩ and TK Ϫ -transduced receptors are exposed on the cell surface. It must be pointed out that a certain amount of high-affinity 125 I-FGF2 binding was observed also in fgfr1 Ϫ/-mESC, probably due to the ability of FGF2 to bind other FGFRs [52]. Finally, to assess the signalling capacity of the transduced receptors, undifferentiated ES cell populations were stimulated with exogenous recombinant FGF2 and ERK1/2 phosphorylation was evaluated by Western blotting. As shown in Fig. 1C (Fig. 2B). We had previously demonstrated that the expression of the cardiogenic mesodermal marker mef2c is not affected in differentiating fgfr1 knockout mESC [41]. Since Mef2c cooperates with Nkx2.5 and other cardiac transcription factors in inducing downstream effectors [53], we hypothesized that FGFR1 may mediate cardiomyocyte differentiation by activating Nkx2.5 in Mef2c ϩ cardiogenic mesodermal cells. The present data confirm the nonredundant role of FGFR1 in nkx2.5 up-regulation and cardiomyocyte differentiation. However, in all our studies, the expression analysis of cardiac markers in differentiating mESC has been performed on the total RNA extracted from the whole EB. Considering that the expression of these markers is not always restricted to a single cell population, only the isolation of the different cardiomyocyte progenitor populations (reviewed in [5]) will allow the precise  identification of the step(s) in the cardiomyocyte differentiation pathway characterized by a non-redundant role for FGFR1. For instance, the FHF and SHF markers Tbx5 and Isl1, whose expression is not limited to heart tissue [54,55], show similar up-regulation kinetics in fgfr1 ϩ/Ϫ , fgfr1 Ϫ/-, and the infected v-wt-hFGFR1 and v-Y653/654F-hFGFR1 mESC populations (data not shown).
To further characterize differentiating transduced EBs, the expression of structural markers belonging to different lineages of mesodermal origin was evaluated by qPCR and whole mount in situ hybridization (WISH). FGFR1 is implicated in cardiac and haematopoietic development but is dispensable for endothelial cell differentiation in murine EBs [41,49]. Accordingly, fgfr1 ϩ/Ϫ and TK ϩ v-wt-hFGFR1 mESC, but not fgfr1 Ϫ/and TK Ϫ v-Y653/654F-hFGFR1 cells, express the cardiac ventricular myosin light chain (myl2) and the primitive haematopoietic marker CD41 (itga2b) at day 10 of differentiation ( Fig. 2C and D). All the cell lines express instead the endothelial markers vcam1 (Fig. 2C) and vascular endothelial cadherin (cdh5) (Fig. 2D)

Role of Y463 and Y766 FGFR1 residues in cardiomyocyte differentiation
Seven autophosphorylation sites have been identified in the cytoplasmic domain of hFGFR1 [21]. Among them, Y463 and Y766 represent docking sites for intracellular molecules. To assess the relative contribution of these residues in transducing signal(s) for cardiomyocyte development, we infected fgfr1 Ϫ/-mESC with lentiviral particles harbouring the cDNA for the TK ϩ hFGFR1 mutants Y463F or Y766F. Again, the efficient transduction of the receptor mutants was confirmed by RT-PCR (data not shown) and 125 I-FGF2 binding assay (Fig. 3A)  capacity was judged by FGF2-induced ERK1/2 phosphorylation (data not shown). Next, v-Y463F-hFGFR1 and v-Y766F-hFGFR1 mESC underwent the standard differentiation protocol and the appearance of beating cardiomyocytes was monitored. At day 10 of differentiation, v-Y463F-hFGFR1 EBs showed a dramatic impairment in generating beating foci when compared to v-Y766F-hFGFR1 EBs whose ability was only marginally reduced in respect to v-wt-hFGFR1 EBs (Fig. 3B). In agreement with the microscopic observations, analysis of Y463F-hFGFR1 EBs showed a strong reduction of the cardiac gene transcripts nkx2.5 and myl2 (Fig. 3C and E) and of cardiac structural protein troponin T (Fig. 3F). As expected, no down-regulation was seen in the levels of expression of the endothelial markers vcam1 and cdh5 (Fig. 3D and E).
Because of the involvement of FGFR1 in ES haematopoietic development (see above), v-Y463F-hFGFR1 and v-Y766F-hFGFR1 EBs were also evaluated for the expression of haematopoietic markers (Fig. 3D). In contrast with what observed for cardiac markers, the transcript levels of the primitive integrin itga2b, of the pan tyrosine phosphatase CD45 (ptprc), and of the embryonic beta-like globin gene hbb-bh1 were similar in v-Y463F-hFGFR1 and v-Y766F-hFGFR1 EBs and comparable to those detected in v-wt-hFGFR1 EBs. Thus, our data strongly suggest that Y463 residue in FGFR1 is specifically involved in cardiomyocyte differentiation of mESC. Moreover, this residue, together with residue Y766, is dispensable for FGFR1-mediated haematopoietic development.
We have previously observed that beating cardiomyocyte differentiation in fgfr1 ϩ/Ϫ EBs can be hampered by ERK1/2 and PKC inhibitors [44]. To get further insights about the signalling cascade that leads to cardiomyocyte differentiation in mESC and to assess the possibility to dissociate cardiac and haematopoietic differentiation at pharmacological level, as suggested by distinct tyrosine autophosphorylation requirements in FGFR1, differentiating fgfr1 ϩ/Ϫ EBs were treated with different signalling inhibitors and assessed for the expression of lineage-specific markers (Fig. 4). In keeping with previous observations [41], beating foci formation and the expression of the structural cardiac marker myl2 were strongly reduced by 10 M ERK1/2 inhibitor U0126 [43] or by 2 M PKC inhibitor GF109203X [44], whereas the expression of the haematopoietic marker itga2b and of the endothelial marker vcam1 were not affected. Moreover, MEK inhibitor PD98059 [56], PLC-␥ inhibitor U73122 [57], or p38 inhibitor SB203580 [58] (Fig. 4) Phosphorylation of Y463 in FGFR1 leads to the Crk-mediated activation of the Ras/Raf-1/MEK/ERK/Jun pathway in endothelial cells [22], whereas Y766 phosphorylation activates PLC␥ in myoblasts and triggers the hydrolysis of phosphatidylinositols that, in turn, stimulate PKC [62]. The ability of the Y766-hFGFR1 mutant to restore cardiomyocyte differentiation in transduced fgfr1 Ϫ/-mESC and the lack of inhibitory effect of the PLC-␥ inhibitor U73122 on the appearance of beating foci in fgfr1 ϩ/Ϫ EBs rule out a role for PLC-␥ in the PKC-dependent pathway involved in cardiomyocyte formation [29]. On the other hand, the ability of the ERK1/2 inhibitor U0126, but not of the MEK inhibitor PD98059, to hamper beating foci formation in fgfr1 ϩ/Ϫ EBs as well as in fgfr1 Ϫ/-EBs treated with the PKC activator phorbol miristate acetate [41] suggests that ERK1/2 activation required for cardiomyocyte differentiation is triggered by a PKC-dependent pathway rather than by the classical Ras/Raf-1/MEK pathway that FGFR1 dimerization activates in NIH3T3 fibroblasts [63]. Indeed, ERK2 can be activated by several PKC isoforms in Swiss 3T3 fibroblasts [29], including the novel PKC ␦ and ε isoforms involved in cardiomyocyte differentiation [64]. Clearly, the possibility exists that PKC and ERK pathways may play complementary roles at different times and/or in different cell types during EB differentiation.
At present, the signalling cascade triggered by the autophosphorylation of Y463 in FGFR1 and its cross-talk with PKC-and ERK-mediated signalling during cardiomyocyte differentiation of mESC remains to be elucidated. Indeed, Western blot analysis of total EB protein extract at day 9 of differentiation does not show any difference in CrkII and Shp2 phosphorylation levels among fgfr1 Ϫ/-, v-wt-hFGFR1, v-Y463F-hFGFR1 and v-Y653/654F-hFGFR1 EBs, thus indicating that the overall activation of these signalling molecules in differentiating EBs is not restricted to FGFR1 activity (data not shown). Also, FRS2 appears to be phosphorylated in both v-Y463F-hFGFR1 and v-wt-hFGFR1 EBs but not in TK Ϫ v-Y653/654F-hFGFR1 and fgfr1 Ϫ/-EBs, indicating that its activation depends on the TK activity of FGFR1 but not on Y463 phosphorylation (data not shown). Again, only the isolation of the different cardiomyocyte progenitor populations from these EBs will allow the identification of the FGFR1-dependent signalling pathway(s) involved in cardiomyocyte differentiation in mESC. Nevertheless, our data demonstrate for the first time a specific non-redundant role for residue Y463 of FGFR1 in cardiomyocyte differentiation.