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Fig. 2. FGFs regulate Mkp3 transcripts through ERK MAP kinase in the
dorsal sclerotome. (A-E) Whole-mount in situ hybridisation with Mkp3
following FGF4 bead grafts. (A) HH13 embryo, (B) transverse section through
the trunk of an HH15 embryo, showing ectopic transcripts throughout somite
tissue, (C-E) Mkp3 expression after FGF and pharmacological inhibitor
bead implants, indicated by asterisks. (C) SU5402 (n=3/4), (D)
PD184352 (n=8/8) and (E) LY294002 (n=8/10). (F) Western blot
probed with anti-phospho-ERK antibody and tubulin. Protein isolated from
untreated somites (-) or somites dissected after exposure to PD184352 beads
(+). (G) SU5402 bead implants resulted in reduced endogenous Mkp3
expression (n=7/7). Double arrowheads indicate the `twin-stripe'
expression of Mkp3. (H,I) PD184352 bead implants (H) resulted in
reduced endogenous Mkp3 expression (red arrowhead n=11/16),
whereas a bead soaked in LY294002 (I) did not have any effect (red arrowhead
n=19/19). (J,K) In situ hybridisation for Mkp3 after
injection of (J) 2 mM PD184352 into somites (n=15/15) or (K) DMSO
(n=10/10). (L,M) Whole-mount in situ hybridisation for
scleraxis after injection of (L) 2 mM PD184352 into somites
(n=12/13) or (M) DMSO (n=10/10). Brackets in J-M show
injected somites. Bead grafts and inhibitor injections were analyzed after 5
hours. (N-Q) FGF receptor and ERK MAP kinase activity is required for
Mkp3 expression in somites. Whole-mount in situ hybridisation for
chick Mkp3 after electroporation of a FGFR1c-EYFP fusion construct
(n=5/5) (N), of pCS2+ encoding a FREK:Fc fusion protein
(n=5/10) (O), or of pCS2+ encoding mouse Spry2
(n=10/15) (Q). (O) Expression of sFREK:Fc is detected using a probe
against the Fc domain (red). (P) Electroporation of EGFP expression vector
alone (n=11/11). GFP transcripts were detected by in situ
hybridisation (red). Brackets indicate electroporated somites.
