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Figure 3. MyoX knockdown inhibited cranial neural crest (CNC) migration and delays trigeminal nerve outgrowth. A: MyoX knockdown does not disrupt early induction of NC. Embryos were injected into one cell at the two-cell stage with 30-ng MO2 and 5 ng of fluorescently labeled control MO, cultured to stage 16/17, sorted using a fluorescent dissecting microscope to identify the injected side (on right in all cases shown, indicated by asterisks) and processed for whole-mount in situ hybridization with probes for mesoderm (myoD), neural plate (sox2), epidermis (keratin), and NC (snail2). No effect was observed. B: MyoX knockdown inhibits cranial NC cell migration. Embryos unilaterally injected at two-cell stage with Control MO (cMO) or MO2, along with nuclear-localized lacZ RNA for lineage tracing, followed by Red-Gal staining (Research Organics), then whole-mount in situ hybridization with Sox9 and Sox10 probes. St. 22; ventral migration of hyoid arch NC (black arrow head) and branchial arch NC (red arrow heads, dashed lines) was inhibited compared with the uninjected side (Sox9, 88%, n = 25; Sox10, 96%, n = 24.). The control MO had no effect. St. 24; migration into all three arches occurred but was still retarded on the MO2-injected side (Sox9, 83%, n = 30; Sox10, 79%, n = 29). Enlarged views of the relevant regions from the MO2-injected side are shown to the right. C: The trigeminal nerve was affected by loss of MyoX. Control MO or MO2 injected into one cell at the two-cell stage. By St. 32, the distal region of the trigeminal nerve (arrows on the uninjected sides - left) was absent on the MO2-injected side (88%, n = 25). The control MO (n = 26) had no effect. Percentages and standard deviation error bars indicated to right as bar graphs. |
