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	Figure 1. EphA4 mutants, their tyrosine phosphorylation state, and their kinase activity. Xenopus embryos were left uninjected or injected with RNA encoding wild type (WT) or various mutant EphA4 receptors. (A) Schematic representations of the kinase dead (KD, K653R), juxtamembrane tyrosine (Y596/602F), SAM domain deletion (ΔSAM), Y928F, and Y928E mutants are depicted. Embryos were harvested at stage 9, whole embryo lysates were immunoprecipitated with anti-EphA4 antibody and analyzed by Western blot analysis with antiphosphotyrosine antibodies (B) or in vitro kinase assays (C) were performed. SP, signal peptide; GD, globular ligand binding domain; TM, transmembrane domain; JM, juxtamembrane region; SAM, sterile alpha motif; PDZ, postsynaptic density protein, discs large, zona occludens binding motif.
	Figure 2. Comparison of EphA4 WT and EphA4 Y928F mutant expressing embryos. Embryos were left uninjected or injected with RNA encoding WT or Y928F mRNA. (A) Phenotypic effect and whole mount in situ hybridization with Xpo probe (posterior marker) of embryos expressing WT or Y928F mutant protein. Arrows indicate normal or ectopic posterior expression of the Xpo marker. (B) Summary of the frequency and percentage of Xenopus embryos displaying ectopic posterior structures when injected with either WT or Y928F EphA4 (number of embryos displaying protrusions/number of embryos injected). (C) WT or mutant EphA4 receptor expression was examined by Western blot analysis of stage 9 embryonic lysates. (D) Embryos stained for β-galactosidase activity after injection with mutant EphA4 receptor RNA plus β-galactosidase RNA or β-galactosidase RNA alone as a lineage tracer. Insert is enlargement of boxed area.
	Figure 3. Phenotypes and whole mount in situ hybridization of posterior and neural crest markers in Y928F injected embryos. (A) Cell dissociation at the blastula stage and ectopic protrusion formation at the stage 37/38 tadpole are observed in Y928F expressing embryos and not in uninjected controls. The arrow indicates a representative posterior protrusion. (B) Albino embryos were left uninjected or injected with Y928F mRNA (1.25 ng mRNA was injected into one blastomere at the 2-cell stage). Embryos at stage 26/28 were fixed with MEMPFA. Whole mount in situ hybridizations were performed with posterior markers (Xwnt3a and Xhox-3) and neural crest markers (Xslug and Xap-2). Filled arrowheads indicate staining of the posterior protrusions. Double-open arrowheads indicate third and fourth branchial arches; a single-open arrowhead denotes cephalic slug expression.
	Figure 4. Tyrosine phosphorylation state and kinase activity for various EphA4 Y928F double mutants. (A) Xenopus embryos were left uninjected or injected with RNA encoding the various Y928F double mutants depicted. (B) Embryos were harvested at stage 9 and the tyrosine phosphorylation state of the various EphA4 receptors was examined by IP-Western blot analysis. (C) In vitro kinase assays were performed on EphA4 immunoprecipitates to determine kinase activity of the various Y928F double mutants. (D) A Flag tag was introduced next to the signal peptide in ephrin binding domain mutants to create ΔGD-Flag-Y928F and ΔGD-Flag-Y928FKD mutants (noted in A). Various EphA4 mutants were immunoprecipitated with anti-Flag antibody and probed with antiphosphotyrosine antibody. Anti-EphA4 blots show equivalent levels of receptor expression. Abbreviations as in Figure 1.
	Figure 5. Morphological analysis and percentage of ectopic protrusions produced by the various Y928F double mutants. (A) Xenopus embryos were left uninjected or injected with RNA encoding the indicated Y928F double mutants. Photographs were taken at stage 26/28. Arrows indicate ectopic protrusions. Inset area represents dorsal view, and open arrowhead marks reference protrusion. (B) Embryos injected with Y928F RNA or Y928FY596/602E RNA were cultured and photographed at stage 9. (C) The frequency of the posterior protrusion phenotype was scored. Note: Both the Y928FM972K (Y928F SAM dimerization mutant) and Y928FY596/602E (Y928F juxtamembrane-SH2 binding mutant) induce budding protrusions (A and C), but Y928FY596/602E mutant does not induce cell dissociation (B).
	Figure 6. Activated FGFR1 induces ectopic posterior protrusions in whole embryos. (A) Morphology of embryos left uninjected or injected with 50 pg of an activated FGFR1 (actFGFR1). (B) Whole mount in situ hybridization analysis of ectopic protrusions produced by actFGFR1. Stage 26/28 embryos injected with actFGFR1 were fixed and probed with posterior markers (Xpo, Xwnt3a, and Xhox-3), neural crest marker (Xap-2), and FGF-8. Arrows indicate staining of ectopic protrusions by the indicated probes.
	Figure 7. EphA4 Y928F induces expression of eFGF, FGF-8, FGFR1, and FGFR4a. (A and B) Embryos were left uninjected or injected with Y928F mRNA (1 ng/embryo for A; 0.125 ng or 0.5 ng for B) into both blastomeres at the 2-cell stage. Animal caps were excised at stage 8 and cultured until stage 26 (A) or stage 10.5/11 (B). Expression of eFGF and FGF-8 were analyzed by RT-PCR. EF-1α was used to normalize cDNAs as a control. PCR reactions were performed in the absence of reverse transcriptase [RT(-)] as a negative control. A reaction performed using stage 26 (A) or stage 11 (B) whole embryo [WE RT (+)] RNA served as a positive control. (C) Analysis of ectopic protrusions by whole mount in situ hybridization of stage 26/28 embryos. Embryos left uninjected or injected with Y928F mRNA (2.5 ng/embryo) were fixed and probed with DIG-labeled FGF-8 (top three embryos), FGFR1 (middle three embryos), or FGFR4a (bottom three embryos). Arrowheads indicate staining of ectopic protrusions by the indicated probes.
	Figure 8. Inhibition of FGF-8 expression using antisense morpholino oligonucleotides represses Y928F-induced ectopic protrusion formation in vivo. (A) FGF-8-AS blocks translation of FGF-8 expression in vitro. HA-tagged FGF-8a or FGF-8b mRNAs (1 ng) were in vitro–translated in the presence or absence of FGF-8-AS (14 ng) or a control antisense reagent with 4 point mutations [FGF-8-AS-4PM] (14 ng) as indicated. Western blot analysis of the reaction products with anti-HA antibody was used to examine inhibition of FGF-8a and FGF-8b translation in the presence or absence of the indicated antisense reagent. The top and bottom arrows indicate the bands corresponding to FGF-8b and FGF-8a, respectively. (B) Antisense FGF-8 (FGF-8-AS) and (FGF-8-AS-4PM) were injected into the indicated number of Xenopus embryos with or without Y928F and scored for ectopic protrusions at stage 26.

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