Hwang YS , Luo T , Xu Y , Sargent TD .

Z01 HD001006-19 NICHD NIH HHS , Z01 HD001006-19 Intramural NIH HHS

	Figure 1. Spatial and temporal expression of MyoX in Xenopus. A: Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. MyoX mRNA was detected at all embryonic stages. B-H: Whole-mount in situ hybridization (ISH); stages are as follows: St. 16 neurula (B,C), St. 19 neurula (D), St. 21 tail bud (E), St. 24 tail bud (F), St. 33 tadpole (G), and St. 38 tadpole (H). Panel (C) is a transverse section of the embryo in panel (B). Early MyoX expression is most abundant in cranial neural crest (CNC), trunk neural crest (TNC), paraxial mesoderm (PM) and the anterior neural plate border (NPB). D-G: Later, strong expression is visible in placodes. In the tadpole, MyoX signal is elevated in cranial nerve V sim X, termini of nerve IX, X (arrowheads) and three termini of nerve VII (arrows, also see Supp. Fig. S3), and in epibranchial placodes (asterisks, G). aB, anterior branchial arch; Br, branchial arch; F, forebrain; H, hindbrain; Hy, hyoidal arch; IX, glossopharyngeal nerve; L, lens placode; M, midbrain; Md, mandibular arch; No, notochord; So, somites; pAD, anterodorsal placode; pAV, anteroventral placode; pB, posterior branchial arch; pM, middle lateral line placode; pOI, olfactory placode; pP, posterior lateral line placode; pPr, profundal placode; pV, trigeminal placode; V, trigeminal nerve; Vmd, mandibular branch of trigeminal nerve; VII, facial nerve; vOt, otic vesicle; Xb, branchial branch - cranial nerve X; Xl, lateral branch - cranial nerve X; Xv, visceral branch - cranial nerve X.
	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.
	Supplemental Fig. S3. MyoX expression on cranial nerve V and VII, somite, and brain. A: Expression of MyoX at St. 31 in cranial nerve V and VII. F: Cranial nerve VII starts branching at St. 32 (purple arrow). I,L: The ascending (red arrow) and descending (blue arrow) branches of VII become prominent at St. 35. The ascending branch approaches the anteriororsal aspect of the cement gland and reaches there at St. 38 MyoX expression demarcates nerve VII at this stage. D,G: Somites express MyoX throughout tail bud stages. M: MyoX is expressed in all brain regions. For anatomy of cranial nerves and brain see (Honore and Hemmati-Brivanlou, 1996; Nagata et al., 2003; Huang et al., 2007). F, forebrain; M, midbrain; H, hindbrain.

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