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First published online 16 February 2005
doi: 10.1242/dev.01699

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Feedback interactions between MKP3 and ERK MAP kinase control scleraxis expression and the specification of rib progenitors in the developing chick somite

¹ School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, UK
² CRUK Molecular Pharmacology Unit, Biomedical Research Centre, Level 5, Ninewells Hospital, Dundee DD1 9SY, UK

View larger version (126K): [in a new window]   Fig. 1. Expression of Mkp3 during chick somite development correlates with the expression of scleraxis in somites and with the expression of Fgf4 and Fgf8 in myotomes. (A-C,G-L) Whole-mount in situ hybridisation of HH15, HH19, HH20 and HH23 chick embryos. (A,J) Mkp3. (J) Hind-limb buds were removed to show expression in posterior somites. (B,K) scleraxis, blue asterisk indicates the axial level of expression in somites, red arrow indicates scleraxis expression in presegmented mesoderm; (C,L) spry2; (G) Fgf4; (H) Fgf8; and (I) FGFR4/FREK. (D-F) Frontal sections through HH24 somites stained with MF20 antibody to visualise myosin heavy chain protein in the myotome (green) together with (D) Mkp3 transcripts (dark purple) in the dorsal sclerotome along the anterior and posterior somite edges. (E) MF20 and scleraxis transcripts (red). (F) MF20 and spry2. Brackets in J,G indicate the triangular shaped expression in posterior somites. ba, branchial arches; dm, dermomyotome; is, isthmus; lb, limb bud; my, myotome; ol, olfactory epithelium; op, optic vesicle; ot, otic vesicle; s, somite; sc, sclerotome; tb, tail bud.

View larger version (90K): [in a new window]   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.

View larger version (87K): [in a new window]   Fig. 3. The expression of Mkp3 over time correlates with differential levels of phosphorylated ERK in response to FGF bead implants. (A-C) Whole-mount in situ hybridisation of Mkp3 in chick embryos with FGF bead grafts, harvested after a 1-hour (A), 5-hour (B) or 24-hour exposure (C). (D) Western blot of untreated somites (-) or somites exposed to an FGF4 bead (+), harvested at the times indicated and probed with anti-phospho-ERK and -tubulin antibodies. (E) scleraxis was induced by an FGF4 bead after 5 hours. (F,G) Sections demonstrating the distribution of ectopic transcripts after bead grafting, (F) Mkp3 and (G) scleraxis. Red asterisks in C,E indicate the bead.

View larger version (54K): [in a new window]   Fig. 4. MKP3 regulates its own expression by regulating ERK MAP kinase. (A) Western blot of protein extracted from untreated somites (lane 1), somites electroporated with EGFP vector (lane 2) or electroporated with hMKP3-GFP vector (lane 3); 15 µg was loaded in each lane. Asterisks on the right identify the EGFP protein (lane 2) and the hMKP3-GFP fusion protein (lane 3). -tubulin served as a loading control. (B) PCR performed on plasmid DNA with specific primers. Human MKP3-GFP plasmid was detected with hMKP3-GFP primers but not with cMkp3 primers (lane 1). Chick Mkp3 plasmid was detected with cMkp3 primers but not with hMKP3-GFP primers (lane 2). (C) RT-PCR on cDNA obtained from somites expressing EGFP vector (lane 1) or hMKP3-GFP fusion protein (lane 2). (B,C) Templates used indicated at the top, primers used indicated on the left. (D) Immunohistochemistry on a frontal section using MF20 antibody detecting myosin heavy chain (green) and dpERK anti-body detecting active phosphorylated ERK MAP kinase (red). White arrowheads indicate rostrocaudal boundaries between somites. Dorsal root ganglia indicated by a blue arrow; dorsal sclerotome region indicated by a yellow arrow. (E) Section of somites electroporated with a GFP vector (in red) and hybridised with a probe detecting MyoD (dark blue). Arrows indicate extent of tissue electroporated, including dermomyotome, myotome and dorsal sclerotome. (F-I) Manipulating dpERK levels in somites results in loss of scleraxis transcripts, the following vectors were electroporated: (F,G) hMKP3-GFP, (H,I) hMKP3KIM-GFP. Single and double in situ hybridisation of electroporated embryos with the probes indicated on each panel. (F) Mkp3, bracket indicates detection of high levels of human transcripts, which crossreact; (G) scleraxis and hMKP3-GFP detected using a GFP probe (n=19/22); (H) scleraxis and hMKP3KIM-GFP detected using a GFP probe (n=8/8); (I) GFP fluorescence.

View larger version (91K): [in a new window]   Fig. 6. Manipulating dpERK levels leads to loss of scleraxis expression and rib defects. (A,D,F) Whole-mount in situ hybridisation of (A,D) scleraxis (dark blue) and (F) scleraxis with GFP (red). (A) hMKP3-GFP electroporation and (D) caMEK1 electroporation resulted in loss of scleraxis transcripts (n=19/22 and n=10/14). (F) GFP electroporation had no effect (n=16/16). (B,C,E,G) Alcian Blue staining of cartilage. Loss of dpERK resulted in loss of the distal part of rib one (B, n=8/12) or rib seven (C, n=9/16), depending on the somites targeted. (E) High levels of dpERK caused loss of the distal part of rib one (n=9/15). (G) GFP alone had no effect on rib development (n=12/12). (H) Infection of thoracic somites with RCAS-sFREK:Fc, at day 10 of development, multiple ribs failed to form (n=2/16). (I) In situ hybridisation detecting RCAS gag transcripts 48 hours after infection (red). (J) Transverse section through RCAS infected somite demonstrates presence of transcripts predominantly in the dorsal somite. Asterisks indicate missing ribs (B,C,E,H) compared with rib present in control (G). dm, dermomyotome; my, myotome; sc, sclerotome.

View larger version (98K): [in a new window]   Fig. 5. High levels of dpERK activity in somites results in loss of MyoD and Mkp3. (A) In situ hybridisation for Mkp3 after electroporation of a plasmid encoding caMEK1, a constitutively active form of MKK1; arrow indicates ectopic Mkp3 transcripts in the neural tube, bracket indicates the loss of Mkp3 transcripts in targeted somites (n=30/35). (B) Western blot detecting dpERK in somites electroporated with caMEK1 plasmid (+) compared with control somites (-); 7.5 µg of protein was loaded in each lane, compared with twice that amount in Fig. 3D and Fig. 4A. (D-G) In situ hybridisation for MyoD. (D) hMKP3-GFP electroporation has no effect on MyoD expression (n=16/16). INT/BCIP was used to detect GFP probe (red), it was washed out using methanol to visualise MyoD (E). (F) caMEK1 electroporation caused complete loss of MyoD transcripts (blue, indicated by bracket; n=10/15) but Pax3 (C) was not affected (n=16/16). (G) FGF8 beads (asterisk) resulted in loss of MyoD in adjacent somites (n=7/11). (H) caMEK1 electroporation caused a significant loss of Fgf4 transcripts (blue, indicated by bracket; n=13/18).