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First published online February 18, 2004
doi: 10.1242/10.1242/dev.01000

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A Raf/MEK/ERK signaling pathway is required for development of the sea urchin embryo micromere lineage through phosphorylation of the transcription factor Ets

View larger version (61K): [in a new window]   Fig. 1. ERK is activated transiently in the PMC precursors a few hours after zygotic expression of ets1. (A-E) In situ hybridization with an ets1 probe. (B-D) Starting at the early blastula stage up to the mesenchyme blastula stage, ets1 expression is restricted to the PMCs (arrow in D). (E) At the end of gastrulation, ets1 transcripts are also enriched in the SMCs (arrow). (F-N) Whole-mount immunolocalization of activated ERK using an anti-MAPK-P antibody. (A) Sixty-cell stage, (F) early blastula, (B,C) prehatching blastula, (G,H) hatching blastula, (I,J) early mesenchyme blastula, (D,K) mesenchyme blastula, (L,M) early gastrula (arrows in M indicate staining in the ectoderm adjacent to the PMC clusters) and (E,N,O) late gastrula. (O) Control embryo stained with the secondary antibody alone. All the embryos are viewed from the side with the animal pole at the top except when mentioned: av, animal view; vv, vegetal view.

View larger version (124K): [in a new window]   Fig. 2. U0126 treatment and injections of mRNA encoding a MAP Kinase Phosphatase (MKP3) or a dominant-negative Raf (dnRAF) block PMC ingression and differentiation. (A-D) Control embryos. (E-H) Embryos treated with 10 µM U0126, (I-L) embryos injected with MKP3 mRNA, (M-P) dnRaf injections, (Q-T) dnRas injections. Note the absence of PMCs in the embryos treated with UO126 (E,F), or injected with MKP3 RNA (I,J) or dnRaf RNA (M,N). (A,E,I,M,Q) Mesenchyme blastula, (B,F,J,N,R) early gastrula, (C,G,K,O,S) late gastrula and (D,H,L,P,T) pluteus stage. anv, anal view. In control embryos, the PMCs ingress into the blastocoel (A) and form a ring around the archenteron and two bilateral clusters during gastrulation (B).

View larger version (31K): [in a new window]   Fig. 3. Activation of MAP kinase is essential for PMC ingression and skeletogenesis. Embryos were incubated at the indicated times with 10 µM of the MEK inhibitor and scored at the early gastrula stage for the presence of micromeres, and at the pluteus stage (48-72 hours) for the presence of spicules. Pictures of representative embryos obtained after the treatments are shown on the right. When U0126 treatment was started before hatching and was continued up to the pluteus stage, PMC ingression and spicule formation was blocked (A,B). If the inhibitor was added after beginning of hatching, PMC ingressed into the blastocoel but did not differentiate further (C,D). When the inhibitor was added at the two-cell stage or prehatching blastula stage PMCs formation was prevented (E), but if the inhibitor was removed soon after the beginning of invagination of the archenteron, spicules nevertheless formed (F). Spicules did not form when the inhibitor was removed after the late gastrula stage (G,H). ERK activity is required first between the prehatching blastula stage and the swimming blastula stage for ingression of the PMCs (green), and then during gastrulation for differentiation of the PMCs into spicules (yellow).

View larger version (85K): [in a new window]   Fig. 4. Effects of inhibiting MAP Kinase function on the gene expression program of the PMCs. Embryos were placed into MFSW containing DMSO (control) or 10 µM U0126 starting at the two-cell stage. After fixation at the desired stage, in situ hybridization was then performed with the following probes ets1 (A-F), alx1 (G-L), skeT (M-R) and msp130 (S-X). Control embryos on the left, U0126-treated embryos on the right of the figure. These experiments were repeated several times with different batches of embryos. Representative embryos are shown. U0126 treatment reduces the level of alx1, skeT and msp130 transcripts. Expression of ets1 remains unaffected by the treatment. HB, hatching blastula; lB, late blastula; mB, mesenchyme blastula; eG, early gastrula; lG, late gastrula; vv, vegetal view; ov, oral view.

View larger version (80K): [in a new window]   Fig. 5. Effects of inhibiting MAP kinase signaling on specification of the SMCs. Embryos were treated as above and whole-mount in situ hybridization was then performed for Delta (A-F), CyIa (G-L), AA29 (M-R) and 23F (S-X). Control embryos on the left, U0126-treated embryos on the right of the figure. U0126 treatment decreases the level of expression of Delta at the early blastula (B5) but not at later stages. The mesodermal expression of CyIa is suppressed at the mesenchyme blastula stage, but unaffected at later stages. The oral ectoderm expression (arrow in G) is unaffected. The expression of AA29 and 23F is reduced but not suppressed by the treatment with U0126. Stages are the same as in Fig. 4.

View larger version (59K): [in a new window]   Fig. 6. Inhibition of MAP Kinase signaling does not disrupt specification of the endoderm territory and formation of the oral/aboral axis. Expression of 42C (A-F), brachyury (G-L) and goosecoid (M-R) genes after inhibition of the ERK function. Control embryos on the left, U0126-treated embryos on the right. U0126 treatment does not affect the expression of endoderm (42C) and oral ectoderm (goosecoid and brachyury) marker genes. mB, mesenchyme blastula; lG, late gastrula; vv, vegetal view; ov, oral view.

View larger version (60K): [in a new window]   Fig. 7. Structural features of the sea urchin Ets1 and Alx1 proteins. (A,B) The P. lividus Ets1 protein contains a conserved MAPK phosphorylation consensus site and a putative ERK docking site in the Pointed domain. Partial sequence alignments between two sea urchin Ets1 protein sequences and the human Ets1, Ets2 and the Drosophila Pointed proteins showing the conservation of the PXTP motif (A) and ERK docking site (B). (C) The N-terminal region of the P. lividus Alx1 protein contains a putative MAPK phosphorylation consensus site (PSTP). Comparison of three different sea urchin Alx1 sequences showing conservation of this site. (D-K) Effects of mutations in the putative MAPK phosphorylation site of Ets1 on formation of PMCs and effects of activated Ras mRNA injections. (A) Control mesenchyme blastula stage. Overexpression of wild-type ets1 (E), ets1 T107D (F) or ets1 VP16 (H) converts a large number of cells into mesenchymal cells. Treatment with U0126 can block the effects of overexpressing ets1 (I) but not ets1 T107D (J). Mutating the putative MAPK phosphorylation site of the sea urchin Ets1 (etsT107A) abolishes its ability to promote epithelial-mesenchymal transition (G). (K) Overexpression of CA-Ras does not cause the same phenotype as overexpression of ets1 but causes a global epithelial remodeling. (vv) vegetal view.

View larger version (58K): [in a new window]   Fig. 8. Activation of the MAP Kinase is downstream of the Wnt/ß-Catenin pathway but does not requires intercellular communication. Immunolocalizations using an anti MAPK-P antibody on control embryos (A,D), zinc-treated (B), dnTCF RNA-injected (animalized) (E) or lithium treated (vegetalized) (C,F) embryos. Phenotypes of a control pluteus (G), dnTCF RNA injected animalized embryo (H) and a lithium vegetalized embryo (I). (J) Time course of MAPK activation analysed by western blot analysis and effect of cell dissociation. 16, sixteen-cell stage; 60, 60-cell stage; veB, very early blastula; eB, early blastula; phB, prehatching blastula; hB, hatching blastula; sB, swimming blastula; mB, mesenchyme blastula; lG, late gastrula; Pl, pluteus; vv, vegetal view. (K-M) Activation of ERK in dissociated blastomeres analysed by immunostaining. Note that the cellular aggregates visible on the pictures were not present during the culture but formed during fixation and the immunostaining protocol.

View larger version (15K): [in a new window]   Fig. 9. Model for the role of the Raf/MEK/ERK signaling pathway in the gene network regulating mesendoderm formation. At the blastula stage, activation of the Raf/MEK/ERK signaling pathway by an unknown factor Y, allows phosphorylation of Ets1, which is crucially required for the maintenance of alx1 and Delta expression in the micromeres, and for the initiation of skeT/Tbr and other downstream genes in this lineage.

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