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Fig. 1. Myo10 is expressed in neural crest tissues during Xenopus development. Myo10 expression is first observed in Xenopus embryos at late gastrula stages, at the borders of the forming neural plate, where the neural crest will arise. Expression subsequently increases in the cranial neural crest and continues as they migrate towards the branchial arches. By tailbud stages, the expression decreases, mainly remaining in the cranial ganglia. All embryos are oriented with anterior to the left.
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Fig. 2. Myo10-MO disrupted expression of neural crest genes. (A) Embryos were injected with control or Myo10-MO (10 ng) in one cell at the two-cell stage, thus labeling one side (labeled with red-gal staining, bottom half in dorsal view and right panel in lateral view, marked by ). The subsequent expression of neural or neural crest genes was examined at later stages. While the expression of neural marker Sox3 was unaffected, the levels of neural crest genes Snail2, Sox10 and Twist were altered in the half of the embryo that received Myo10-MO. Co-expression of a low dose (50–100 pg) Myo10 RNA efficiently rescued the defect. (B) Percentages of embryos with different levels of neural crest gene expression (combining Snail2, Sox10 and Twist) at neurula stages. While over 75% of control-MO injected embryos showed symmetric neural crest gene expression, Myo10-MOs reduced the neural crest gene expression severely in over 25% of embryos and altered the expression mildly in 45% of the embryos. Nearly 70% of the embryos were rescued to normal levels by coexpressing Myo10 RNA. (C) Percentage of embryos with defective neural crest migration indicated by the expression of Sox10 and Twist at tailbud stages. Myo10-MOs lead to shorter and disorganized CNC migration in about 70% of the embryos and co-injection of Myo10 RNA reduced that to 18%.
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Fig. 3. High-resolution images of Snail2 expression. Myo10-MO injected embryos stained with Snail2 (as in Fig. 2) were imaged either at higher magnification or after transverse section. Upper panels were dorsal views of embryos in whole mount, with anterior to the left; bottom panels were 10 μm sections through the embryos with dorsal to the top. marks the injected side. np, neural plate. Although the areas expressing transcript were similar in size on left and right sides, the level of expression was markedly lower on the Myo10-MO injected side of the embryo.
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Fig. 5. Myo10 is not required for neural crest induction in AC assay. Animal caps were dissected from untreated embryos as well as those exposed to Wnt3a plus noggin, with or without Myo10-MO. Caps were stained for neural crest markers Snail2 and Twist at st.13 and st.14. While control explants remained round and did not express any neural crest genes, explants receiving Wnt3a and noggin started to express Snail2 and Twist weakly at st.13 and strongly at st.14 and the shapes of the caps became irregular. Myo10-MO did not inhibit either the expression of neural crest genes in the explants or the shape changes of the explants.
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Fig. 4. Cell proliferation and cell death are not affected by Myo10-MO. Embryos were stained with antibody to phospho-histone H3 to mark proliferating cells or processed for TUNEL staining to detect cells undergoing apoptosis. (A) Whole embryos were cleared to visualize the internal staining. Embryos are shown in dorsal view, with anterior to the left. Injections were done in the bottom half (marked by âŽ). (B) Transverse sections were oriented with dorsal side up, injected side to the right (marked by âŽ). Closing neural plate and closed neural tube were marked as np or nt. (C) Numbers of proliferating or dying cells on the injected side versus uninjected side were compared. Sections of 10 embryos were counted for each group and no significant changes were observed (p = 0.50, 0.36 for cell proliferation and cell death).
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Fig. 6. Myo10 is required for cranial neural crest (CNC) explants to spread and segregate on FN matrix. (A) CNC explants were dissected from early neurula embryos receiving control or Myo10-MO and plated on FN. By 7 h, control-MO expressing explants spread extensively and segregated into three cell streams. However, Myo10-MO expressing explants failed to spread on FN, but dissociated into loose and rounded cells. The addition of a low dose Myo10 partially rescued the effect. Distinct cell streams were marked by numbers. (B) The surface areas of each explant at the beginning and the end of the experiment were compared and the ratios of explant spreading summarized. Myo10-MOs reduced the ratio of spreading from 2.85-fold to 1.36-fold, while coexpressing Myo10 mRNA restored it to 2.77-fold. (C) The number of segments formed for each explants was counted and compared. While control-MO expressing explants were able to segregate into an average of 2.6 pieces, Myo10-MO reduced this to 1.3. With the addition of a low dose Myo10, explants made 2.2 segments on average. For both the spreading and segregation, the disruption by Myo10-MOs and the rescue by Myo10 were significantly different.
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Fig. 7. Myo10 is required for CNC migration in vivo. (A) GFP labeled CNC explants were grafted into unlabeled host embryos and imaged at tailbud stages. Fluorescent images and merged images are shown side by side. While control-MO receiving grafts migrated laterally into mandibular arch, hyoid arch, and branchial arches, Myo10-MOs expressing grafts failed to migrate efficiently and the migration paths were often indistinguishable. Co-expression with Myo10 restored this to normal levels. (B) Confocal images of the grafts show that control cells interacted with each other and followed their routes of migration to segregate cleanly into three streams. In contrast, Myo10-MO expressing cells did not appear to interact with neighboring cells. (C) Grafts were placed into four categories based on their migration behavior. While over 80% of control grafts migrated and segregated normally, over 24% of the Myo10-MO expressing grafts failed to migrate at all. Another 60% migrated much shorter distances, half of which did not segregate. Co-expressing Myo10 rescued the migration phenotype, with nearly 70% of the grafts migrating normally. All embryos are shown in lateral view, with dorsal up, anterior left.
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Fig. 8. Myo10-MO inhibited the formation of cranial cartilage. Cartilages in late tadpoles were stained with alcian blue. Ventral view of the cranial cartilage with anterior to the top, and injected side marked by âŽ. Cartilage on the Myo10-MO injected side was often malformed or even completely missing. Neural crest derived cartilages are marked as: M, meckel's cartilage; CH, ceratohyal cartilage; CB, ceratobranchial cartilage.
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Fig. 9. Myo10-MO disrupted cell adhesion and polarization of CNC cells. (A) CNC cells were dissociated and plated on FN-matrix. While control-MO expressing cells spread on FN and extend membrane protrusions actively, most Myo10-MO expressing cells remain round. A low dose of Myo10 RNA together with Myo10-MO efficiently rescued the phenotype, resulting in normal cell morphology. Bottom panels are higher magnification views. Arrows point to the filopodial protrusions while arrowheads indicate lamellipodia. For those few Myo10-MO expressing cells that appeared polarized, only very short protrusions were observed. (B) Spreading cells were counted and their ratios among the entire population were calculated. Myo10-MO dropped the percentage of spreading cells from over 15% to around 6% while Myo10-MO plus Myo10 RNA resulted in 13% cell spreading, significantly different from Myo10-MO but not from controls. (C) Cell–cell adhesion was examined by dissociation–reaggregation experiments. The same number of explants were used for each experiment, and the number of cell clusters with diameters over 100 μm were counted at the end of aggregation assay. While an average of 72 large cell clusters were formed by control-MO cells, only ∼ 20 large cell clusters were formed by Myo10-MOs treated cells. Coexpression of Myo10 increased the number to 48 on average, significantly different from that of MO alone. (D) Behaviors of labeled cells in CNC explant were recorded by time-lapse movies. Frames with 6′ intervals were shown. While control cells migrated and extended membrane protrusions dynamically, Myo10-MO expressing cells remained round and failed to form protrusions. The addition of Myo10 RNA efficiently rescued the ability to form protrusions and to migrate.
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myo10.2 (myosin 10, gene 2) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 20, dorsal view, anterior left.
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myo10.2 (myosin 10, gene 2) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 23, lateral view, anterior left, dorsal up.
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myo10.2 (myosin 10, gene 2) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 26, lateral view, anterior left, dorsal up.
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