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FGFR4 signaling is a necessary step in limb muscle differentiation

Irène Marics*, Françoise Padilla*, Jean-François Guillemot, Martin Scaal and Christophe Marcelle{dagger}

Developmental Biology Institute of Marseille, Laboratoire de Génétique et de Physiologie du Développement (LGPD), University Aix-Marseille II, Campus de Luminy, Case 907, 13288 Marseille Cedex 09, France
* These authors contributed equally to this work



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  		Fig. 1. FGFR4, but not FGFR1 is expressed in embryonic limb skeletal muscles. E5 embryos were hybridized to a FGFR4-specific RNA probe (A,C,E), or to a FGFR1-specific probe (B,D,F). Whole-mount in situs indicate that FGFR4 is specifically expressed in the developing muscles (A), whereas FGFR1 is widely expressed in the entire limb (B). Sections confirm that FGFR4 is specifically expressed in the skeletal muscles (C), which are recognized after immunohistochemistry with an embryonic myosin heavy chain-specific monoclonal antibody (E). In contrast, FGFR1 is strongly expressed in the dermis (D) and poorly, if at all, in the muscles (F). ec, ectoderm; de, dermis; mu, skeletal muscles; me, mesenchyme.

 


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  		Fig. 2. S-FR4-Fc and S-FR1-Fc bind to FGF in vitro. Heparin acrylic beads were first soaked with FGF2 (2) or FGF8 (8) and then incubated with 300 µl of cell supernatant from DF-1 cells infected with the S-FR4-Fc or S-FR1-Fc constructs. Control beads (He) were incubated with 2% BSA prior to exposure to the cell supernatants. Proteins adsorbed on these beads were separated by SDS-Page and blotted onto a membrane; they were recognized using an anti-human Fc fragment antibody. Both S-FR4-Fc and S-FR1-Fc bind to FGF2- and FGF8-loaded beads, but not heparin beads.

 


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  		Fig. 3. S-FR4-Fc and S-FR1-Fc disrupt gastrulation in Xenopus embryos. At gastrula stage, the expression of the mesodermal marker Brachury (in blue), which is normally expressed as a ring around the blastopore (E), is downregulated in cells co-injected with ß-galactosidase and S-FR4-Fc (A) or S-FR1-Fc (C) RNA. ß-galactosidase expression is detected in red. Arrowheads in A,C indicate the region in which Brachyury is downregulated. Overexpression of S-FR4-Fc (B) or S-FR1-Fc (D) in the dorsal blastomeres of early Xenopus embryos produce gastrulation defects, which lead later in development (around stage 28) to grossly abnormal embryos, when compared with control embryos injected with ß-galactosidase RNA only (F). Note that high and low doses of injected RNA were comparable with those used by Amaya et al. (Amaya et al., 1991Go; Amaya et al., 1993Go).

 


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  		Fig. 4. Inhibition of FGFR4 signaling results in a block of limb bud myogenesis. Two-day-old embryos were injected in the prospective limb bud domain with cells infected with a secreted form of FGFR4. Four days later, infected embryos were processed for double in situ hybridization (A-E,G) or whole-mount immunohistochemistry with a monoclonal antibody directed against the embryonic form of the myosin heavy chain (MyHC, F). In a first round of in situ hybridization, a Fc-specific probe enabled us to determine which embryos had been efficiently infected (such an embryo is presented in A). These were then destained and a second in situ reaction was performed with probes specific for various stages of myogenic differentiation. Although none of the infected embryos displayed a variation in Pax3 expression (B), all muscle markers (Myf5, D; MyoD, E), the embryonic myosin heavy chain (F) and FGFR4 itself (C) were strongly downregulated. (G,H) To estimate the amplitude of the inhibition, embryos were separated in two parts after in situ hybridization, and then photographed (G); the stained dorsal muscle masses were delineated manually with Adobe Photoshop, and their surfaces were compared by pixel counting (H). In the case presented here, MyoD staining was decreased by 77%. When control embryos were counted in a similar manner, a difference of no more than 8% was observed between both limbs.

 


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  		Fig. 5. Overexpression of FGF8 in somites promotes myogenic differentiation and FGFR4 activation. (A-C) Protocol used for electroporation of somites in vivo. FGF8 plasmid cDNA was injected into the somitocoel of the newly formed somites of (Hamburger-Hamilton) stage 15 chick embryos (A). These somites correspond to the prospective interlimb level (somites 22-27). By placing the positive electrode on the right-hand side of the embryo, we electroporated the lateral side of somites (B,C). Three days later, embryos were analyzed by in situ hybridization for MyoD (D) and FGFR4 (E) expression. Massive overexpression was observed with both probes (green arrowheads) along the entire mediolateral axis of somites (i.e. prospective epaxial and hypaxial domains). Red arrowheads in E indicate an overgrowth that, in some embryos, developed into a partial ectopic limb. HL, hindlimb; FL, forelimb.

 


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  		Fig. 6. Local inhibition of FGFR4 signaling does not modify muscle progenitor proliferation. S-FR4-Fc-expressing cells were injected into the limb buds of E6 embryos. These cells serve as a source of protein, which locally perturbs the signaling of endogenous FGFR4. After overnight incubation, the embryos were exposed to BrdU for 1 hour and analyzed for FGFR4 expression. The choice of the probe enabled us to examine the expression of endogenous FGFR4 and the position of the injected cell pellet (P). Two independent experiments are shown. (A,B,E) The first embryo; (C,D,F) the second embryo. (A,C) General views of the injected limbs. (B,D) Close up of the views presented in A,C. Broken lines in B,D represent the outline of the muscle bundles as they are observed in the contralateral, uninjected side. In B,D, a muscle bundle crosses the cell pellet. We observed that the muscles immediately adjacent to the injected cells display a strong downregulation of FGFR4 expression (red arrowheads), indicative of an efficient inhibition of muscle differentiation. (E,F) Sections of the embryos presented in B,D. Around the cell pellet, light, but clearly visible, blue staining (red arrowhead) enabled us to identify the position of affected muscle progenitors, which we delineated with a broken line. At 10-20 cell diameters away from the injected cells, FGFR4 expression was normal (dark blue, green arrowhead). BrdU counting was carried out by arbitrarily choosing a similar surface in the affected and unaffected regions and comparing the number of BrdU-positive nuclei in each. This was done in four embryos and in at least two adjacent sections each. No differences in BrdU-positive cells were observed between the two regions, demonstrating that an arrest of cell division cannot explain the disappearance of myogenic markers.

 


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  		Fig. 7. Muscle progenitors inhibited by S-FR4-Fc retain their ability to respond to FGF stimulation. S-FR4-Fc cells are injected in the segmental plate of two-day-old embryos. Two or 4 days later, heparin beads coated with FGF2 or FGF8 were implanted into the developing limb of embryos. In a first set of experiments (A,B), the beads were grafted in E4 embryos and re-incubated for an additional 2 days. (B) The contralateral, non-infected, non-implanted forelimb of the embryo shown in A. In a second set of experiments (C,D), the beads were implanted in E6 embryos and re-incubated for only 8 hours. We observed 2 days (A) or 8 hours (C) after implantation of the beads a rescue of MyoD expression immediately around implanted beads. No rescue was observed with control heparin beads (D).

 

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