Mikryukov A , Moss T .

	Figure 2. Analyses of xTNIK and xMINK knockdown phenotypes.A) Posterior views of stage 13 and B) anterior views of stage 19 knockdown embryos. The indicated Morpholinos were injected into the two dorsal or ventral blastomeres of 4 cell embryos together with fluorescein dextran as lineage marker. See Figure S2A and B for additional examples and for dorsal views at stage 19. C) Stage 12.5 knockdown embryos before and after rescue by re-introduction of xMINK were also subjected to in situ hybridization for Xbra mRNA. Extension of the developing notochord is evident in the control embryos and after rescue. D) Similarly stage 10.5 and 14 xMINK knockdown embryos were also hybridized to reveal chordin mRNA. E) Hybridization at stage 11 reveals that knockdown of either xTNIK or xMINK locally suppresses the onset of Xbra expression. Where appropriate, ventral and dorsal sites of knockdown are indicated, otherwise knockdown was targeted to the dorsal blastomeres.
	Figure 3. xTNIK and xMINK interact dependent on the CNH domain of xMINK.A) Domain structure of xMINK and xTNIK their N-terminal Kinase domain and C-terminal CNH domain deletion mutants, respectively ΔN-TNIK (ΔNT aa301-1385), ΔN-MINK (ΔNM aa300-1270), TNIK-ΔC (TΔC aa2-1061) and MINK-ΔC (MΔC aa2-946). The inactivating point mutation K54 to R is indicated. B) Interaction between epitope tagged xMINK and xTNIK and their respective deletion mutants was determined by co-immunoprecipitation from co-transfected HEK293T cells. Immunoprecipitations were performed using the Flag epitope of the xTNIK constructs. C) xTNIK and xMINK co-localize in animal pole blastomeres. N-terminal YFP and RFP fusions of the kinases were expressed in animal pole cells, the animal caps excised at stage 9 and observed by confocal microscopy without fixation. D) and E) Phenotypic effects of xTNIK and xMINK mutants. The two dorsal blastomeres of four cell embryos were injected with the indicated RNAs and embryos allowed to develop to stage 39–40. See Figure S3D and E for the range of phenotypes observed. F) Examples of Keller explants from control and experimental embryos expressing the N-terminally deleted xTNIK (ΔNT) and xMINK (ΔNM) mutants.
	Figure 4. xTNIK and xMINK function downstream of Xdsh in Convergent Extension (CE).A) Dorsal view of stage 18 embryos expressing xTNIK and xMINK or the corresponding dominant negative N-terminal deletion mutants ΔNT and ΔNM in comparison with Xdsh. B), C) and D) Rescue of CE in embryos expressing ectopic Xdsh or the Xdsh mutant D2 by co-expression of the catalytically active C-terminally deleted xMINK mutant MΔC, but not the inactive M(K54R) ΔC or the N-terminally deleted mutant ΔNM. Embryos are shown at the equivalent of stage 18 in B) and stage 39–40 in C) and D) (see Figure S4B and C for the range of phenotypes). In A to D the dorsal blastomeres of four cell embryos were injected with the indicated amounts of the RNAs. E) N-terminal RFP fusions of xMINK and its deletion mutants were co-expressed with GFP-Xdsh or with a combination of GFP-Xdsh and Xfz7. Injections were made at the four cell stage into the animal poles of all four blastomeres, animal caps were excised at stage 9 and observed by confocal microscopy without fixation. See Figure S4D for the equivalent analyses of xTNIK and its dominant negative mutant.
	Figure 5. Secondary axis induction requires xTNIK and is inhibited by catalytically active xMINK.A) Secondary axis was induced by the ectopic expression of ß-catenin, and xTNIK, xMINK and their corresponding mutants were then tested for their ability to suppress the axis. Embryos were scored for complete axis duplication (I), partial duplication (II) and no secondary axis (WT). A selection of representative images is shown. B) Summary of embryo scoring for all constructs tested in the ß-catenin secondary axis assay. C) Effects of Morpholino knockdown of xTNIK or xMINK on the ability of Xdsh or a combination of Xwnt11 and Xfz7 to induce secondary axes. Scoring was as in A and B. In each case, embryos were injected at the four cell stage in the vegetal segment of one ventral blastomere.
	Figure 6. Proteolytic cleavage of xTNIK and xMINK in vivo regulates their subcellular localization.A) and B) Western analyses of endogenous and exogenous xTNIK and xMINK. The in vivo cleavage products of the endogenous proteins (1.5 embryo equivalents loaded per gel track) and proteins expressed from the respective injected mRNAs (4 ng/embryo, 0.5 embryo equivalents loaded per gel track) were detected using specific antibodies αT2/αT3 and αM5/αM7 raised to different regions of the Central domain. The N- and C-terminal epitope tags were also used to map the cleavage sites, cleavage products being sized by comparison with the epitope tagged truncation and deletion mutants of both xTNIK and xMINK run in parallel on the gels (data not shown). The accompanying diagrams indicate the epitopes recognized by specific antibodies and the xTNIK and xMINK-derived polypeptides Tf2 to 4 and Mf2 to 4 identified in vivo. C) The Kinase (KT aa2-312, KM aa2-313), Central (ΔNTΔC aa301-1061, ΔNMΔC aa300-946) and CNH (TC aa1062-1385, MC aa947-1270) domains of xTNIK (T) and xMINK (M) were fused to GFP, expressed in animal caps and viewed by confocal microscopy. D) The N- and C-terminally tagged xTNIK or xMINK were co-expressed in animal caps, detected using anti-Flag, -HA and -Myc antibodies and observed by confocal immunoflourescence microscopy. “n” indicates blastomere nuclei. The lower panels show enlargements of the indicated regions. E) Expression of the C-terminal deletion mutant of xMINK (myc-MΔC), that was shown to be able to rescue CE, displaces the CNH domain polypeptide (haM-CNH) from blastomere nuclei. The proteins were detected using anti-HA and anti-Myc antibodies and observed by confocal immunoflourescence microscopy.
	Figure 7. The isolated Kinase and CNH domains modulate signalling via both PCP and Canonical Wnt pathways.A) Four cell embryos were injected in the two dorsal blastomeres with the indicated RNAs and allowed to develop to stage 39–40. B) Secondary axis was induced by the ectopic expression of ß-catenin, and the isolated Kinase and CNH domains of xMINK and xTNIK were tested for their ability to suppress the axis. Embryos were scored for complete axis duplication (I), partial duplication (II) and no secondary axis (WT). In A and B a representative selection of images is shown, see Figure S6 for more information.
	Figure 8. Summary of xTNIK and xMINK products in the Canonical and Non-canonical Wnt pathways.The predominant subcellular localization (cytosolic or nuclear) of each polypeptide identified is also indicated. The level of action of xTNIK, xMINK and their proteolytic products act within each pathway, that is down stream of ß-catenin and Dishevelled, is indicated. The possible targets (ß-catenin/TCF and JNK/AP-2) are, however, purely based on the published literature.
	Figure 1. Organization and expression of the xTNIK and xMINK kinases.A) Diagrammatic representation of the domain structure of xTNIK and xMINK. The N-terminal Kinase and C-terminal CNH domains are indicated as well as the variably spliced regions within the Central domain of xTNIK. Expression constructs of xTNIK used throughout the manuscript were derived from a cDNA containing all four variable regions. B) In situ wholemount hybridization for xTNIK mRNA. “dbl” dorsal blastopore lip. C) Phenotypic effects of xTNIK and xMINK knockdown. Morpholinos against xTNIK (MoT#1 and -#2) and xMINK (MoM) mRNAs or control Morpholinos (Ctrl Mo) were injected singly and in combinations into the two dorsal blastomeres of four cell embryos and embryos allowed to develop until stage 39–40. Morpholino amounts injected per embryo are indicated as are the fractions of embryos showing the indicated phenotypes. D) Rescue of knockdown phenotypes. Morpholinos MoT and MoM were injected alone or coinjected with the indicated amounts of mRNA encoding the full-length xTNIK or xMINK.

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