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Abstract
The extracellular signal-regulated kinase (ERK) is a key transducer of the epidermal growth factor receptor (EGFR) and fibroblast growth factor receptor (FGFR) signaling pathways, and its function is required in multiple processes during animal development. The activity of ERK depends on the phosphorylation state of conserved threonine and tyrosine residues, and this state is regulated by different kinases and phosphatases. A family of phosphatases with specificity toward both threonine and tyrosine residues in ERK (dual-specificity phosphatases) play a conserved role in its dephosphorylation and consequent inactivation. Here, we characterize the function of the dual-specificity phosphatase MKP3 in Drosophila EGFR and Xenopus FGFR signaling. The function of MKP3 is required during Drosophila wing vein formation and Xenopus anteroposterior neural patterning. We find that the expression of the MKP3 gene is localized in places of high EGFR and FGFR signaling. Furthermore, this restricted expression depends on ERK function both in Drosophila and Xenopus, suggesting that MKP3 constitutes a conserved negative feedback loop on the activity of the Ras/ERK signaling pathway.
Figure 7. The fibroblast growth factor mitogen activated protein kinase (FGF/MAPK) pathway regulates MKP3 expression during Xenopus development. All panels show XMKP3 expression pattern except K,L and Q-T, which show Fgf8 and Xbra expression, respectively. M-T: Injection of different mRNAs along with 300 pg of LacZ mRNA in one blastomere at the two-cell stage. The injection side was determined by XGal staining. A,B: Vegetal (A) and lateral (B) views of early gastrula embryos (stage 10) show that XMKP3 is expressed in the mesodermal marginal zone (B) and in the prospective neural ectoderm (B, arrowhead). C,D: Vegetal (A) and lateral (B) views of late gastrula embryos (stage 12). XMKP3 is expressed in all the prospective neuroectoderm (C and arrowhead in D). E-I: Dorsal views of different neurula stage embryos show similar XMKP3 mRNA distribution. At these stages, XMKP3 expression in the neuroectoderm becomes more restricted, being localized to the posteriorneuroectoderm (F, red arrowhead), at the midbrain-hindbrain boundary (F, black arrowhead), and in two horseshoe-shaped bands in the anteriorneuroectoderm (G, arrowheads). J: Lateral view of tail bud stage 34 embryos. XMKP3 is strongly detected in the branchial arch region (red arrowhead) and in the tail tip (black arrow). Indeed, these two domains of expression were the only ones detected in a previous report (Mason et al., [1996]). In addition, at this stage MKP3 is also expressed at the midbrain-hindbrain boundary (black arrowhead) and in the somites (red arrow). K,L: Anterior (K) and dorsal (L) views of stage 14 embryos showing the expression pattern of Fgf8. Note that Fgf8 and MKP3 are expressed in similar territories (compare K,G and F,L). M-P: Dorsal views of neurula embryos showing MKP3 expression in embryos injected with different mRNAs. These embryos are at a similar stage to the control embryo shown in H. Red and black arrowheads point at the uninjected (internal control) and the injected sides, respectively. Interfering (M) or increasing (N) Ras activity down-regulates or ectopically activates, respectively, MKP3 expression. Similarly, interfering (O) or increasing (P) FGF signaling down-regulates or ectopically activates, respectively, MKP3 expression. The effectiveness of these injections was determined by monitoring Xbra expression. Q-T: Interfering with FGF/MAPK pathway represses Xbra expression at early gastrula (vegetal views, Q,S) while increasing FGF/MAPK activity promotes ectopic Xbra expression at neurula stages (dorsal views, R,T). Compare the uninjected (red arrowheads) and the injected (black arrowheads) sides. Insets in Q and R show Xbra expression in control embryos at early gastrula and neurula stages, respectively. XMKP3, Xenopus extracellular signal-regulated kinase phosphatase 3.
Figure 8. MKP3 participates in anteroposterior neural patterning. Injection of different mRNAs along with 300 pg of LacZ mRNA in one blastomere at the two-cell stage. The injection side was determined by XGal staining. A,C,E: Lateral views of tail bud embryos. B,D,F: Dorsal views of neurula embryos. A,B: Overexpression of MKP3 causes anteriorization of the embryos, as determined by the shortening of the trunk and the slightly enlarged heads (A, arrowheads) and the posterior shift of neural markers (B, compare red with black arrowheads in the injected and control sides, respectively). These markers are Otx2, expressed in the anterior-most of the embryos; krox20, expressed in rombomeres 3 and 5; and HoxB9, expressed in the posterior spinal cord. C,D: Overexpression of an mRNA encoding a MKP3 kinase-dead mutant form (MKP3-mut) causes with low efficiency, the impairment of heads structures (C, arrowheads) and reduction of anterior neural markers (D, arrow; compare with an uninjected embryo in F). E: Coinjection of MKP3 and MKP3-mut partially rescues the defects observed in MKP3-injected embryos. F: Wild-type (WT) neurulaembryo showing the expression of Otx2, krox20, and HoxB9. MKP, extracellular signal-regulated kinase phosphatases.
dusp6 (dual specificity phosphatase 6) gene expression in Xenopus laevis embryo via in situ hybridization, NF stage 34, lateral view, anteriorleft, dorsal up.
dusp6 (dual specificity phosphatase 6) gene expression in Xenopus laevis embryo via in situ hybridization, NF stage 22, dorsal view, anteriorleft.
dusp6 (dual specificity phosphatase 6) gene expression in Xenopus laevis embryo via in situ hybridization, NF stage 14, G: dorsal view; G anterior view, dorsal up.
dusp6 (dual specificity phosphatase 6) gene expression in Xenopus laevis embryo via in situ hybridization, NF stage 12, lateral view, blastoporeright, dorsal up.
