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First published online 14 July 2004
doi: 10.1242/dev.01275

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FGF acts directly on the somitic tendon progenitors through the Ets transcription factors Pea3 and Erm to regulate scleraxis expression

Department of Genetics, Harvard Medical School, Boston, MA 02115, USA

View larger version (94K): [in a new window]   Fig. 1. FGF-dependent induction of Scx in the somite may be direct or indirect. Section in situ hybridization on alternate frontal sections comparing expression of Scx (A), Fgf8 (B), Fgfr1 (C) and Frek/Fgfr4 (D) in a HH stage 20 embryo. (C) Red arrow indicates slight upregulation of Fgfr1 in the Scx-expressing region. (E,F) Models for direct or indirect induction of Scx in the anterior and posterior dorsal sclerotome. Four somites shown in frontal view, with myotomes represented as ovals, sclerotomes as squares. Anterior is towards the left, posterior towards the right. (E) In a model for direct Scx induction, FGFs (orange) expressed in the center of the myotome signal directly to Fgfr1 (blue) in the sclerotome, thereby activating expression of Scx (purple) in the sclerotome. Dark blue represents high expression levels of Fgfr1 in the sclerotome, light blue indicates lower expression levels of Fgfr1 in the myotome. (F) In a model for indirect Scx induction, FGFs (orange) signal through Frek/Fgfr4 (green), localized to the anterior and posterior myotome, to activate expression of a secondary factor that then signals to the underlying anterior and posterior sclerotome to induce Scx expression (purple). Light green indicates low levels of Frek/Fgfr4 in the myotome, dark green indicates higher levels of Frek/Fgfr4 in the ventral anterior and posterior myotome. Yellow represents myotome, aqua indicates sclerotome.

View larger version (82K): [in a new window]   Fig. 2. Fgf8 can induce ectopic expression of Scx in the sclerotome in the absence of the myotome. (A-D) Results of overexpression of Fgf8 in the psm with RCAS-FGF8, followed by surgical removal of dermomyotomes. (A,C) Whole-mount in situ hybridization for Scx following manipulation. Region of dermomyotome removal indicated by red bracket. (B,D) Double whole-mount in situ hybridization for Fgf8 on embryos shown in A,C. Antibody staining for phosphorylated MAPK/ERK (E,F) and myosin heavy chain (MF20) (F) on frontal sections of HH stage 20 embryos. (E) Phosphorylated MAPK/ERK (red) is seen in dorsal sclerotome, myotome and dermomyotome. Blue arrow indicates expression in the sclerotome, purple arrow in the dermomyotome, yellow arrow in the dorsal root ganglia. (F) Overlay of phosphorylated MAPK/ERK (red) and MF20 (green).

View larger version (111K): [in a new window]   Fig. 3. Scx is co-expressed with several members of the Fgf8 synexpression group. (A-C,G-I) Whole-mount in situ hybridization on HH stage 20 embryos. (D-F,J-L) Section in situ hybridization of frontal sections of HH stage 20 embryos. Comparison of Scx expression (A,D) with that of Pea3 (B,E), Erm (C,F), Mkp3 (G,J), Sef (H,K) and Spry2 (I,L). Red arrows in D-F and J-L indicate expression in the anterior and posterior dermomyotome.

View larger version (66K): [in a new window]   Fig. 4. Dominant-negative version of Pea3 blocks induction of Scx. (A-C) Whole-mount in situ hybridization for Scx following infection with either RCAS-Pea3EnR (A), an FGF8 bead implant (B) or RCAS-Pea3EnR combined with an FGF8 bead implant (C, asterisk indicates bead). (D) Domain structure of mouse Pea3 and Pea3EnR. Pea3 contains an acidic transcriptional activation domain (green), an Ets DNA-binding domain (red), and four inhibitory domains (blue). Dominant-negative Pea3 constructed by fusing the Ets DNA-binding domain to Engrailed (yellow). AD, activation domain; ID, inhibitory domain; Ets, Ets DNA-binding domain; EnR, Engrailed repressor.

View larger version (100K): [in a new window]   Fig. 5. Overexpression of Pea3 results in ectopic Scx expression in the dermomyotome and dorsal sclerotome. (A) Whole-mount in situ hybridization for Scx following infection with RCAS-Pea3. (B,C) Section in situ hybridization for Scx on frontal sections of control (B) or RCAS-Pea3-infected embryos (C). (D) Detection of viral infection using 3C2 antibody on section shown in C. (C) Blue arrow indicates ectopic Scx in dorsal sclerotome. (E,H) Section in situ hybridization for Scx (E,F) or Pea3 (G,H) on frontal sections of control (E,G) or RCAS-FGF8-infected embryos (F,H). (I-P) Whole-mount in situ hybridization for Scx (I-L), Myf5 (M,N) or Fgf8 (O,P) on trunks cultured in either DMSO (control) (I,K,M,O) or 30 µM SU5402 (J,L,N,P). Trunks shown in J and L were infected with RCAS-Pea3 on their right sides. Dm, dermomyotome; M, myotome; Sc, sclerotome.

View larger version (88K): [in a new window]   Fig. 6. Pea3 and Erm are induced prior to Scx following implantation of an Fgf8 bead. Whole-mount in situ hybridization for Pea3 (A,D,G), Erm (B,E,H), or Scx (C,F,I) following implantation of an Fgf8-soaked bead for 12 (A-C), 4 (D-F) or 3 hours (G-I). Asterisk in F indicates location of bead.

View larger version (89K): [in a new window]   Fig. 7. Fgf signaling is required for expression of Pea3 and Erm in the somites. Whole-mount in situ hybridization for Pea3 (A,B) or Erm (C,D) on trunks cultured for 24 hours with either DMSO (control) (A,C) or 30 µM SU5402 (B,D). (E-G) Whole mount in situ hybridization for Fgf8 (E), Pea3 (F) or Scx (G) in HH stage 17 embryos; somite stages VII, XII and XVI are indicated. (H-J) Section in situ hybridization for Fgf8 (H), Pea3 (I) or Scx (J) on alternate frontal sections of somite stages XII and XIII in a stage 17 embryo. (J) Red and blue arrows, indicate dermomyotomal and sclerotomal Scx, respectively.

View larger version (57K): [in a new window]   Fig. 8. Model for FGF-dependent activation of Pea3 and Erm, and subsequent induction of Scx in the somite. (A) FGF signaling leads to expression of Ets transcription factors Pea3 and Erm and inhibitors Mkp3, Sef and Spry in the anterior and posterior sclerotome and dermomyotome. FGFs secreted by the myotome bind to and activate an FGFR (green arrow). Receptor activation results in series of phosphorylation events (red arrows), culminating in direct or indirect transcriptional activation (black arrow) of target genes such as Pea3, Erm, Mkp3, Sef and Spry. (B) Once Pea3 and Erm expression domains have been established, further FGF signaling triggers phosphorylation and subsequent activation of Pea3 and Erm, which, in turn, activate transcription of target genes resulting in Scx expression. (C) Schematic of four somites, frontal view: dermomyotomes are beige; myotomes are yellow; sclerotomes are aqua. FGFs expressed in center of myotome (orange) can diffuse to surrounding dermomyotome, myotome and dorsal sclerotome (arrows). FGF signaling here results in expression of Pea3 (red) and Erm (green), in a nested pattern, within anterior and posterior dermomyotome and dorsal sclerotome. Scx expression (purple) is induced when myotomal FGFs signal to the Pea3- and Erm-expressing dermomyotome and sclerotome.