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Fig. 1. xMAK expression is dynamically regulated during Xenopus development. (A) The analysis of xMAK expression at different developmental stages. Total RNA from embryos isolated at different developmental stages was used for RT-PCR. FGFR served as a loading control. RT-, no reverse transcriptase. (B-M) Spatial distribution of xMAK RNA revealed by whole-mount in situ hybridization of albino embryos at indicated stages. (B-E,J,K) xMAK antisense probe. (F,G,I,L,M) xMAK sense probe. (D) Anterior view; e, eye; MHB, midbrain-hindbrain boundary. (H) En2 and Krox20 probes, anterior view. En2 is expressed as a bright band at the MHB, located anterior to Krox20, which marks rhombomeres 3 and 5. (B,F) Animal pole view; (C,G) lateral view; (E,I) dorsal view, anterior is towards the left. (J-M) Lateral view, anterior is towards the left. (K) e, eye; ov, otic vesicle; ba, branchial arches; tb, tailbud. (N,O) A cross-section of a stage 17 neurula embryo after in situ hybridization with xMAK antisense probe. Staining is observed in the deep (sensorial) layer of epidermal ectoderm (magnified view shown in O) and in somitogenic mesoderm.
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Fig. 7. MAK functions in regional brain patterning, but does not affect organizer markers. (A-O) MAK regulates midbrain-hindbrain boundary. Dorsal-animal region of four-cell embryos was injected with 50 ng of MOs or 2 ng of MAK RNAs, as indicated, together with nβgal RNA as a lineage tracer. At stage 20, Otx2, En2 and Gbx2 were assessed by whole-mount in situ hybridization. MAK MO inhibited Otx2 and En2 (D,E), but upregulated Gbx2 (F). MAK RNA expanded and posteriorly shifted Otx2 and En2 on the injected side (J,K), and inhibited Gbx2 (L). Red arrows indicate altered expression; black arrows indicate marker expression on the uninjected side. (P) MAK depletion or overexpression does not affect organizer marker expression. Two to four-cell embryos were injected twice in the dorsal margin with MAK MO or COMO (100 ng), β-catenin MO (30 ng) or indicated RNA (4 ng). RT-PCR was carried out with total RNA from stage 10 embryos. DNA fragments were separated in 6% SDS-polyacrylamide gel. EF-1α is a loading control. Uninj, uninjected embryos. RT-, no reverse transcriptase.
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hunk (hormonally up-regulated Neu-associated kinase) gene expression in Xenopus laevis , bissected, neural stage aembryo, assayed via in situ hybridization, NF stage 17, dorsal up.
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hunk (hormonally up-regulated Neu-associated kinase) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 23, lateral view, anterior left, dorsal up.
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Fig. 2. MAK (hunk) causes morphogenetic defects and head abnormalities. Four-cell stage embryos were injected into dorsal equatorial region (A, two injections) or animal pole region (B-D, four injections), with RNAs encoding wild-type MAK, MAK KD or GFP, as indicated, at 2 ng per injection. (A) Morphological analysis of MAK-expressing embryos. Embryos were fixed at stage 38 and scored for axis elongation and head defects. (B) The effect of MAK on morphogenetic movements. Animal caps were isolated from injected midblastula embryos and treated with 50 ng/ml of activin. MAK, but not MAK KD, inhibited animal cap elongation in response to activin. (C) Expression levels of Myc-tagged MAK and MAK-KD, revealed by Western blot analysis with anti-Myc antibodies in lysates of injected embryos at stage 10. (D) MAK does not inhibit mesoderm markers induced in animal caps by activin. Animal cap induction was as in B. The mesodermal markers MyoD, Chordin and Goosecoid were assessed in animal caps by RT-PCR, when sibling embryos reached stage 14. Whole embryo (WE) RNA is a positive control. Uninj., animal caps from uninjected embryos. RT-, no reverse transcriptase. EF-1α is a loading control.
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Fig. 3. MAK (hunk) is an activator of JNK. JNK activity was analyzed in embryonic lysates by in vitro phosphorylation of GST-Jun(1-135). Four-cell embryos were injected marginally four times with GFP, MAK or MAK-KD RNA (1.5 ng per injection), or δN-Fz8 RNA (0.5 ng per injection) as a positive control. Lysates were collected at stage 14 for JNK activity determination by western analysis with anti-phospho-Jun antibodies. Assays were carried out in duplicates, with 10 embryos in each experimental group. Anti-GST and anti-β-tubulin antibodies indicate loading.
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Fig. 4. MAK (hunk) interacts with Frizzled and Dishevelled. (A) Fz3 RNA recruits MAK to the cell membrane in animal pole cells. xMAK-GFP RNA was injected either with membrane-targeted dsRed RNA or with dsRed and Fz3 or Fz2 RNAs and GFP fluorescence was visualized in animal pole cells isolated from injected embryos at stage 10. xMAK-GFP is distributed in a punctate cytoplasmic pattern. Membrane-targeted dsRed RNA allows visualization of the cell membrane. Upon Fz3 RNA coexpression, xMAK-GFP is recruited to the cell membrane. The bottom right panel shows lack of effect of Fz2 RNA on xMAK-GFP localization (compare with top right panel). Bars on top left and top right panels are 5 μm. (B) MAK co-immunoprecipitates with Dsh. Lysates of embryos injected with Myc-MAK RNA (2 ng) and/or with HA-Dsh RNA (4 ng) into each cell of four-cell embryos were collected at stage 10 and immunoprecipitated with anti-HA antibodies. Western analysis with anti-Myc antibodies reveals a complex of Dsh and MAK. (C) In vitro phosphorylation of Dsh by MAK. Autoradiography (upper panel) reveals phosphorylation of in vitro translated Myc-Dsh that was co-immunoprecipitated with Myc-MAK or Myc-MAK-KD from in vitro translated lysates. Lower panel shows protein levels.
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Fig. 5. Axis elongation and eye defects in MAK-depleted embryos. (A) xMAK MO specifically inhibits in vitro translation of xMAK RNA. Autoradiography of in vitro translated 35S-methionine-labeled proteins is shown. No suppression is observed for mMAK RNA, which lacks morpholino target sequence. (B) MAK MO inhibits xMAK translation in vivo. Western analysis with anti-Flag antibodies shows that MAK MO, but not COMO, effectively downregulated levels of Cterminally tagged xMAK in injected embryos. Loading is controlled with anti-β-tubulin. (C) MAK MO or COMO was injected into two dorsal blastomeres of four-cell embryos. At stage 38, MAK-depleted embryos had shortened axes and eye deficiencies (see also Table 2). (D-H) Eye defects caused by MAK MO injection can be partially rescued by mouse MAK RNA in stage 38 embryos. Eight-cell embryos were injected into one animal dorsal blastomere with nβgal RNA as a lineage tracer, together with MAK MO (D,E), COMO (F) or MAK MO and mouse MAK RNA (G). (D,E) Both sides of the same injected embryo. Red staining (arrowheads) reflects lineage tracing. (H) The average eye index was calculated for each group of embryos at stage 38 as follows. 0, no visible eye; 1, severely disrupted retina with little pigmentation; 2, small or partially pigmented eye; 3, wild-type eye.
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Fig. 8. Depletion of β-catenin restores brain defects caused by MAK (hunk) MO. Embryos were injected with MAK MO (50 ng) and COMO (8 ng, A,C) or β-catenin MO (8 ng, B,D) into one dorsal animal blastomere at the four- to eight-cell stage. Whole-mount in situ hybridization of stage 19 embryos demonstrates that the effects of MAK MO on Otx2 and Gbx2 expression were partially reversed byβ -catenin MO (see also Table 2). Arrowheads in A and C indicate altered marker expression in MAK-depleted embryos. Nuclear βgal RNA served as a lineage tracer.
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