Mechanisms of Ageing and Development 108 (1999) 77 – 85 Evidence that FGF receptor signaling is necessary for endoderm-regulated development of precardiac mesoderm Xiaolei Zhu a, Joachim Sasse b, John Lough a,* a Department of Cell Biology, Neurobiology and Anatomy and Cardio6ascular Research Center, Medical College of Wisconsin, 8701 W. Watertown Plank Road, Milwaukee, WI 53226, USA b Research Department, Shriners Hospital for Children, Tampa, FL 33612, USA Received 4 October 1998; received in revised form 3 January 1999; accepted 7 January 1999 Abstract Endoderm cells in the heart forming region (HFR endoderm) of stage 6 chicken embryos are required to support the proliferation and terminal differentiation of precardiac mesoderm cells in vitro. The endoderm’s effect can be substituted by growth factors, including members of the fibroblast growth factor (FGF) family. However, direct implication of FGFs in this process requires evidence that inhibition of FGF signaling interferes with proliferation and/or terminal differentiation. This report examines the consequences of treating endoderm/ precardiac mesoderm co-explants with agents that inactivate FGF receptors. Using sodium chlorate, which prevents FGF ligand-receptor interaction, it was observed that the percentage of S-phase precardiac mesoderm cells was markedly reduced, suggesting that cell proliferation was inhibited. To more specifically affect FGF signaling, the explants were treated with an antibody that recognizes an extracellular domain of FGF receptor-1 (FGFR-1). This treatment similarly inhibited cell proliferation. Although both agents modestly delayed cardiac myocyte differentiation as indicated by the contractile function, expression of a-sarcomeric actin was not affected. These findings provide additional evidence that an intact FGF signaling pathway is required during heart development. © 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Anterior endoderm; Cardiogenesis; FGF receptor antibody; Precardiac mesoderm; Sodium chlorate * Corresponding author. Tel.: +1-414-4568459; fax: + 1-414-4566517. E-mail address: jlough@mcw.edu (J. Lough) 0047-6374/99/$ - see front matter © 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 0 4 7 - 6 3 7 4 ( 9 9 ) 0 0 0 0 3 - 2 78 X. Zhu et al. / Mechanisms of Ageing and De6elopment 108 (1999) 77–85 1. Introduction During chicken embryogenesis, cells in the anterior half of the primitive streak are specified to the cardiac lineage by Hamburger–Hamilton stage 3b (Garcia-Martinez and Schoenwolf, 1993). Precardiac cells then migrate laterally and anteriorly, arriving by stage 6 in the bilateral areas of the anterior lateral plate known as the heart forming region (HFR) (Rawles, 1943; Rosenquist and DeHaan, 1966). Stage 6 precardiac mesoderm resides in close proximity to the endoderm cells that are required for terminal cardiac differentiation in vitro. The precardiac mesoderm does not survive in isolated culture, whereas only a small complement of endoderm cells is sufficient to induce the differentiation of a synchronously contractile multicellular vesicle (Sugi and Lough, 1994). Recent findings have shown that the endoderm’s cardiogenic effect can be replaced by endoderm-derived growth factors. At least three members of the fibroblast growth factor (FGF) family—FGFs 1, 2 and 4—that are present in the endoderm at stage 6 can mimic the endoderm’s effect (Zhu et al., 1996). However, no evidence has been presented to indicate that an intact FGF signaling pathway is required for the process. Several FGF receptors, termed FGFRs 1–4 and FREK (Marcelle et al., 1994), which bind cognate ligands with high-affinity have been characterized. FGFR-1 is expressed in the stage 6 precardiac mesoderm (Sugi et al., 1995). FGF binding to these receptors is mediated by low-affinity heparan sulfate proteoglycan (HSPG) co-receptors, and, inhibition of HSPG sulfation abolishes the receptor activity (Rapraeger et al., 1991, 1994). This report examines the effect of blocking FGF receptors on endoderm-supported cardiogenesis. Two approaches were utilized. Firstly, sodium chlorate was employed to determine the effect of inhibiting sulfation of low-affinity (HSPG) FGF receptors. Secondly, to more specifically block the FGF receptor, antibody neutralization was employed. The results indicate that receptor-mediated FGF signaling is an important component of the mechanism by which HFR endoderm supports the growth, but not necessarily the differentiation, of the precardiac mesoderm. 2. Materials and methods 2.1. Materials Anti-bromodeoxyuridine monoclonal antibody was purchased from BectonDickinson. Anti-sarcomeric a-actin monoclonal antibody was from Sigma (mouse IgM; cat. no. A-2172). Anti-FGF receptor polyclonal antibody was affinity purified from an antiserum (R-131) (Casscells et al., 1992) raised against a synthetic peptide (RITGEEVEVRDR) corresponding to residues 79–90 of FGFR-1 (Pasquale and Singer, 1989). This sequence is within the first immunoglobulin domain of the receptor; a search of the GenBank database revealed that this sequence, which is shared by FGFR-1 across various species, has no homology with other eukaryotic X. Zhu et al. / Mechanisms of Ageing and De6elopment 108 (1999) 77–85 79 proteins. Controls were performed utilizing IgG purified from normal rabbit serum (Sigma cat. no. I5006). Secondary antisera, fluorescent isothiocyanate (FITC)-conjugated goat anti-mouse IgG and FITC-labeled goat anti-mouse IgM, were from Cappel Laboratories (Charlotte, NC). 2.2. Culture of precardiac mesoderm/HFR endoderm explants Contiguous layers of HFR endoderm and anterior lateral plate mesoderm, or mesoderm alone, coinciding as much as possible with the heart-forming region (HFR), were dissected from stage 6 embryos and cultured as described previously (Sugi and Lough, 1994). Neutralizing antibody or sodium chlorate was added 1 h after placing the explants in culture, upon attachment of the explant to the fibronectin substrate. 2.3. Immunostaining and Western blotting Bromodeoxyuridine incorporation assays were performed as previously described (Sugi et al., 1993); percentages of BrdU-positive nuclei were determined by evaluating 500 nuclei in random fields of each explant. Sarcomeric a-actin immunostaining (Sugi and Lough, 1994) and Western blotting (Sugi et al., 1995) were performed as previously described. 3. Results This laboratory utilizes an in vitro bioassay to study the effects of the heart forming region (HFR) endoderm on the development of precardiac mesoderm explanted from embryos at stage 6. When cultured alone, the precardiac mesoderm explants do not survive; however, the presence of adjacent endoderm causes cardiogenic development that is characterized by the formation of a rhythmically contractile multicellular vesicle within 24 h. Multilayering of the precardiac mesoderm, which is highlighted by cell proliferation and aggregation, occurs in a defined explant region where cells express cardiac markers including smooth and sarcomeric a-actin (Sugi and Lough, 1994). Cells at the explant periphery remain as a monolayer; the identity of these cells, which express only smooth muscle a-actin as they migrate peripherally, is unknown. For these experiments, determinations were confined to the cardiogenic region, as depicted by the presence of sarcomeric a-actin-positive cells shown by the arrow in Fig. 1. 3.1. Effect of sodium chlorate Endoderm/precardiac mesoderm explants were continuously treated with sodium chlorate to prevent ligand-binding to high-affinity FGF receptors. The controls were treated with sodium chloride, or with sodium chlorate plus sodium sulfate, the latter serving as a competitor to prevent inhibition of proteoglycan sulfation. After 80 X. Zhu et al. / Mechanisms of Ageing and De6elopment 108 (1999) 77–85 two days, 5%-bromodeoxyuridine was added for 2 h, followed by fixation. The effects on the proliferation and differentiation of the cells in the cardiogenic region were determined by immunostaining to detect BrdU incorporation and a-sarcomeric actin expression. Fig. 2 shows the effect of chlorate treatment on explant size (Fig. 2A, panels a, c and e) and cell proliferation (Fig. 2A, panels b, d, and f); data from the latter were quantified by determining the percentage of nuclei that incorporated BrdU (Fig. 2B). The central cardiogenic region in the explants shown in Fig. 2A (a, c, and e) is denoted by arrowheads; note that the explant treated with chlorate (c) was only half as large as the controls (a and e), which was a typical finding. As shown in Fig. 2B, concentrations of 10–20 mM sodium chlorate reduced the percentage of cells in the S-phase of the cell-cycle by approximately 50%. Because sodium sulfate prevented this effect, it was suggested that chlorate specifically inhibited proteoglycan sulfation, rather than causing non-specific toxicity. Despite the chlorate-mediated reductions in the percentage of S-phase cells and explant size shown in Fig. 2, this was not accompanied by an appreciable reduction of sarcomeric a-actin expression as determined by immunostaining (not shown). Fig. 1. Sarcomeric a-actin immunostaining delineates the differentiated region of the precardiac mesoderm cellular multilayer. Endoderm and precardiac mesoderm explanted from the anterior lateral plate of a stage 6 embryo were co-cultured in defined medium as described in Section 2. As shown in this confocal micrograph, sarcomeric a-actin differentiation was confined to the surface layers of cells in the multicellular part of the explant (arrow). This region was evaluated for all of the determinations described in this paper. The mesoderm cells at the bottom of the explant were not differentiated. Endoderm cells, which were present in these cultures, are not shown. The scale bar is 25 mm. X. Zhu et al. / Mechanisms of Ageing and De6elopment 108 (1999) 77–85 81 Fig. 2. Sodium chlorate inhibition of endoderm-induced precardiac mesoderm proliferation. (A) Photomicrographs: endoderm/mesoderm co-explants were cultured with NaCl (20 mM), NaClO (20 mM) or NaClO plus Na2SO4 (10 mM) for 2 days. BrdU (50 mmol) was provided for the last 2 h of the culture period, followed by anti-BrdU immunohistochemistry. Panels a, c and e show whole explants. The arrowheads indicate the central cardiogenic region; note that the explant treated with chlorate (c) is smaller than controls (a and e). The scale bar in e is 500 mm. Panels b, d and f are confocal photomicrographs showing BrdU-labeled cells in the cardiogenic region of the explant; the white scale bar in f is 25 mm. (B) To quantitate these effects approximately 500 PI-stained nuclei in random fields of the cardiogenic region of 9–12 experimental explants were evaluated for BrdU incorporation. The vertical bars indicate 9 1 S.D. of the mean. The asterisk indicates statistical significance at the 1% confidence level using Student’s t-test. These findings suggest that endoderm-supported precardiac mesoderm proliferation is dependent upon an intact FGF receptor apparatus. To substantiate this interpretation it was determined that the replacement of the endoderm cells with insulin (Sugi and Lough, 1995), which is not an endoderm product and whose receptor activity is not affected by sodium chlorate, was not inhibited by this treatment (not shown). 3.2. Effect of anti-FGFR-1 antibody Although previous investigations utilized sodium chlorate to study the role of heparan sulfate proteoglycans (HSPG) in FGF signaling, (Rapraeger et al., 1991, 1994), chlorate-mediated inhibition of sulfation is not specific to HSPG co-receptors. To more rigorously evaluate whether HFR endoderm-induced cardiogenesis is dependent on FGF signaling, a neutralizing antibody that recognizes an extracellular domain in the FGFR-1 was used. Western blotting (Fig. 3) demonstrated the specificity of this affinity-purified antibody, which recognized a single band among proteins from whole embryos or isolated hearts at various developmental stages. Migration of this band at 145 kDa was consistent with the size of the monomeric FGFR-1 receptor. These blots also showed that FGFR-1 was detected as early as stage 5 (Fig. 3B) and that FGFR-1 was strongly expressed in the definitive heart 82 X. Zhu et al. / Mechanisms of Ageing and De6elopment 108 (1999) 77–85 (Fig. 3D). Unfortunately it was not feasible to assess the relative amount of FGFR-1 in isolated precardiac mesoderm due to the paucity of these samples. Fig. 4 shows the effect of anti-FGFR-1 on BrdU labeling in endoderm-induced precardiac mesoderm cells. As shown in Fig. 4B, IgG concentrations greater than 5 mg/ml reduced the percentage of cycling cells by approximately 50%, compared with control IgG. Fig. 4A shows representative confocal micrographs that were evaluated to generate the data in Fig. 4B. Similar to the effects of sodium chlorate, onset of contractility was delayed in anti-FGFR-1-treated cultures, which was not accompanied by decreases in expression of sarcomeric a-actin (not shown). 4. Discussion This study examined the hypothesis that receptor-mediated FGF signaling is a necessary component of the mechanism by which HFR endoderm cells regulate precardiac mesoderm development. These experiments, which utilized an inhibitor of HSPG sulfation as well as a specific antibody to block the FGF receptor, revealed that while cardiac myocyte differentiation was only delayed, the percentage Fig. 3. Western blot showing expression of anti-FGFR-1 (R-131) during development. Exactly 100 mg protein from whole embryos (panel A) and isolated hearts (panel C) at each indicated stage were separated in each lane of a 7.5% acrylamide/SDS gel, blotted onto nitrocellulose and immuno-reacted with affinity-purified anti-FGFR-1 (panels B and D). Panel B shows that FGFR-1, migrating at 145 kDa, was expressed as early as stage 5 among whole embryonic proteins. Panel D shows that FGFR-1 was strongly expressed in the developing heart at stage 11. X. Zhu et al. / Mechanisms of Ageing and De6elopment 108 (1999) 77–85 83 Fig. 4. Affinity-purified anti-FGFR-1 inhibits endoderm-induced precardiac mesoderm proliferation. (A) BrdU immunohistochemistry: endoderm/mesoderm co-explants were cultured with 10 mg/ml antiFGFR-1 (panels c and d). Control explants were treated with an equivalent amount of purified IgG from normal rabbit serum (panels a and b). As described in the legend to Fig. 2, BrdU was provided and immunofluorescent confocal microscopy was utilised to assess proliferating cells in the multicellular region of these explants (panels a and c), relative to the total number of cells detected by PI staining (panels b and d). (B) Quantitation of BrdU immunohistochemistry. To quantitate inhibition of cell proliferation 10–12 explants in each experimental group were subjected to BrdU/PI immunohistochemistry, followed by determination of the percentage of BrdU-positive cells as described in Fig. 2. The vertical bars indicate 91 S.D. of the mean. The asterisks indicate statistical significance at the 1% confidence level using Student’s t-test. of cycling cells in the precardiac mesoderm was markedly reduced, indicating that cell proliferation was inhibited. That these inhibitors affected FGF receptor activity was suggested by the ability of sodium sulfate to prevent the inhibitory effect of sodium chlorate (Fig. 2) and by the high specificity of the anti-FGFR-1 antibody (Fig. 3). These data complement earlier findings that FGF ligands and receptors are, respectively, present in endoderm (Zhu et al., 1996) and precardiac mesoderm (Sugi and Lough, 1995) at stage 6, as well as observations that purified FGF can replace the cardiogenic effect of endoderm (Sugi and Lough, 1995; Zhu et al., 1996). At least two dozen proteins are secreted by HFR endoderm (Kokan-Moore et al., 1991). In addition to the FGF ligands these include activin-A (Kokan-Moore et al., 1991), bone morphogenetic protein-2 (BMP-2) (Lough et al., 1996; Schultheiss et al., 1997) and the vitamin A transport proteins (transthyretin and retinol binding protein) (Barron et al., 1998). Considering the apparent complexity of endoderm signaling it is somewhat surprising that the inhibition of one component—FGF signal transduction—may affect mesoderm proliferation. These in vitro experiments indicate that FGF signaling is required to support the proliferation of precardiac mesoderm cells prior to their incorporation into the definitive myocardium. Previous studies have indicated a requirement for FGF signaling during heart development. In Drosophila, an FGF receptor (DFR1/heartless) has been shown to be required for mesoderm cell migration and subsequent heart formation 84 X. Zhu et al. / Mechanisms of Ageing and De6elopment 108 (1999) 77–85 (Beiman et al., 1996; Gisselbrecht et al., 1996; Shishido et al., 1997). In avians, appearance of the definitive myocardium at stage 9+ is accompanied by abrupt expression of FGF in cardiac myocytes (Parlow et al., 1991) which may serve an autocrine function that is required for myocardial growth (Sugi et al., 1993). In an in vivo study, Mima et al. (1995) demonstrated that expression of a dominant-negative FGFR-1 inhibits myocyte proliferation in the developing myocardium at the later stages. Approaches to transiently mis-express components of the FGF pathway in precardiac tissues in vivo prior to stage 7 would be of great value in clarifying the role of FGF signaling in early heart development. Although the percentage of S-phase cells was markedly reduced with both inhibitors, decreases greater than 50% were never attained. While this limitation might reflect the inhibitors’ inability to penetrate the cellular multilayer, this was unlikely due to the small size of the chlorate ion; moreover, cycling cells were uniformly distributed throughout the multilayer. One explanation for the inability to obtain complete inhibition is that FGF signaling was maintained via pathways that are transduced by atypical FGF receptors, such as the cysteine-rich non-tyrosine kinase FGF receptor which may function independent of heparan sulfate proteoglycan (Zhou et al., 1997). In addition, cell proliferation in FGFR-inhibited explants may reflect the presence of other FGFR isoforms. Also, cell proliferation may also be regulated by growth factors such as those that activate the insulin-like growth factor-II receptor, which is expressed in precardiac mesoderm (McCormick et al., 1996). Finally it must be considered that the developing precardiac multilayer may contain cell types that are differentially sensitive to FGF signaling; unfortunately, the structure of the multilayer as an aggregate of small, exceedingly condensed cells precludes double immunostaining approaches that would be required to test this possibility. Unlike skeletal myocytes in which cellular proliferation and differentiation are mutually exclusive processes, proliferation and differentiation of cardiac myocytes occur concomitantly. It is therefore difficult to discern whether a specific growth factor affects proliferative or differentiative processes in developing cardiac myocytes. The consistent finding that FGFR inhibition reduced cell proliferation while only modestly delaying cardiac myocyte differentiation was unexpected. This result suggests that FGF is an endoderm-derived cardiac myocyte ‘proliferation’ factor, in contradistinction to ‘differentiation’ factors that may also be secreted by HFR endoderm. This is consistent with our findings that whereas FGF supports survival and proliferation of non-precardiac mesoderm, BMP-2 is necessary to induce cardiogenic differentiation (Lough et al., 1996). Acknowledgements This research was supported by NIH Grant HL 39829. 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