Li J , Perfetto M , Materna C , Li R , Thi Tran H , Duncan MK , Wei S .

G0A1515N Fonds Wetenschappelijk Onderzoek (Research Foundation Flanders), G029413N Fonds Wetenschappelijk Onderzoek (Research Foundation Flanders), EY015279 U.S. Department of Health & Human Services | NIH | National Eye Institute (NEI), GM114105 U.S. Department of Health & Human Services | NIH | National Institute of General Medical Sciences (NIGMS), GM104316 U.S. Department of Health & Human Services | NIH | National Institute of General Medical Sciences (NIGMS), GM094793 U.S. Department of Health & Human Services | NIH | National Institute of General Medical Sciences (NIGMS), DE022813 U.S. Department of Health & Human Services | NIH | National Institute of Dental and Craniofacial Research (NIDCR), 1-FY10-399 March of Dimes Foundation (March of Dimes), R01 GM114105 National Institute of General Medical Sciences, R03 DE022813 National Institute of Dental and Craniofacial Research, R01 EY015279 National Eye Institute, S10 RR027273 National Center for Research Resources, P20 GM104316 National Institute of General Medical Sciences, R01 EY015279 NEI NIH HHS , R01 GM114105 NIGMS NIH HHS , P20 GM104316 NIGMS NIH HHS , S10 RR027273 NCRR NIH HHS , R03 DE022813 NIDCR NIH HHS

	Figure 1. The snai2:eGFP transgenic embryos show eGFP expression in the CNC lineage at various stages. Heterozygous snai2:eGFP embryos were imaged at the indicated stages. (A–C) Dorsal view of neurula-stage embryos showing eGFP expression in pre-migratory (A), early migrating (B) and extensively migrating (C) CNC, with anterior at the top. Green fluorescence and bright-field images are merged in (B) to show the relative positions of migrating CNC streams in the whole embryo. (D,D’) Side (D) and dorsal (D’) views (with anterior to the left) of the head of a stage ~35 tadpole, with eGFP expression in the developing brain (br), lens (ln), and CNC cells forming condensing mesenchyme in the pharyngeal arches (pa). (E-E”) Dorsal (E), ventral (E’) and side (E”) views (with anterior to the left) of a stage ~42 tadpole. eGFP expression is detectable in the developing olfactory epithelium (oe), trigeminal nerves (nV), and CNC cells in the pharyngeal arches that begin to differentiate into branchial cartilage (bc) and ceratohyal cartilage (cc). (F-F”) Dorsal (F), ventral (F’) and side (F”) views (with anterior to the left) of a stage ~46 tadpole. eGFP is seen in the more differentiated trigeminal nerves and head cartilage structures, including Meckel’s cartilage (mc). Inset in (F”) is a higher-magnification image showing eGFP expression in tail somites. (G,G’) Dorsal (G) and ventral (G’) views (with anterior at the top) of a stage ~48 tadpole, with eGFP visible in both the olfactory (nI) and oculomotor (nIII) nerves. White arrowhead in (G’) indicates strong autofluorescence in the liver, which was also seen in wild-type tadpoles. (H,H’) Dorsal (H) and ventral (H’) views (with anterior at the top) of a stage ~53 tadpole. eGFP labels the thymus (tm) and highly differentiated head cartilage structures. Red scale bar in H = 500 μm.
	Figure 2. The transcripts of snai2 are downregulated during CNC migration but upregulated again as CNC differentiates. In situ hybridization was performed for snai2 with wild-type X. tropicalis embryos at the indicated stages. The expression of snai2 in the CNC decreases from stage ~22 to ~31 (A–E), but elevates again in the CNC cells that form condensing mesenchyme in the pharyngeal arches (pa; F) and persists in the differentiating head cartilage structures (G,H). All embryos are shown with anterior to the left. (A–G) side view; (H) dorsal view. Insets in (G,H) are ventral view of head cartilage dissected from tadpoles after in situ hybridization, with anterior at the top. ln, lens; so, somites.
	Figure 3. Localization of eGFP protein in snai2:eGFP embryos. (A–C”) Co-localization of eGFP with endogenous Snai2 protein in snai2:eGFP embryos. Immunohistochemistry was carried out for eGFP (green) and Snai2 (red) simultaneously at the indicated stages in snai2:eGFP embryos and tadpoles. (A-A”) eGFP and Snai2 are co-localized in the CNC at the onset of migration. A control embryo processed with secondary antibodies only but not either primary antibody did not display any signal (insets in A and A’). Embryos are shown in dorsal view with anterior at the top. (B–C”) Dorsal (B-B”) and ventral (C-C”) views of the head of a stage ~46 tadpole showing co-localization of eGFP and Snai2 in the branchial cartilage (bc), brain (br), lens (ln), trigeminal nerve (nV), and olfactory epithelium (oe), with anterior at the top. (D–E”) Transverse sections of anterior head cartilage. Immunohistochemistry for eGFP (green) and DAPI labeling for nuclei (blue) were carried out for stage ~46 snai2:eGFP (D-D”) and wild-type (E-E”) tadpoles, and images were taken with a Zeiss Axiozoom.V16 epifluorescence microscope. Sections are shown with anterior at the bottom (tilted toward the right in D-D”). Expression of eGFP is detectable in Meckel’s (mk) and infrarostral (ir) cartilage in snai2:eGFP but not wild-type tadpoles. Scale bar = 100 μm.
	Figure 4. Phenotypes of ADAM13 knockdown displayed by snai2:eGFP embryos. Eight-cell stage heterozygous snai2:eGFP embryos were injected with 1.5 ng MO 13-3 to target ADAM13 in one dorsal-animal blastomere, and cultured to the indicated stages; a red fluorescent dye was co-injected as a lineage tracer. The injected side is denoted with a white asterisk, and structures that are present on the uninjected side but absent on the injected side are denoted with white arrowheads. Insets show red fluorescence images of the same embryos. (A,B) Dorsal view (with anterior at the top) of stage ~18 (A) and ~22 (B) embryos displaying reduced CNC domain on the injected side, as determined by eGFP expression. In (B) CNC migration is normal on the uninjected side but inhibited on the injected side. (C–F) Injected embryos that did not show apparent defects in CNC induction or migration were selected and cultured to stage ~46. (C,C’) are dorsal and ventral views (with anterior at the top), respectively, of the same tadpole. The olfactory epithelium (oe) is not detectable (C) but branchial cartilage (bc) and trigeminal nerve (nV) appear normal (C’) on the injected side of this embryo. (D,E) Embryos with under-differentiated head cartilage structures on the injected side, as compared with the uninjected side. (F) An embryo with severely defective trigeminal nerve and head cartilage structures on the injected side. (D–F) are ventral views with anterior at the top.
	Figure 5. Wnt signaling activity in the CNC lineage. Heterozygous transgenic Wnt reporter embryos were imaged at the indicated stages. Expression of eGFP is detectable in the pre-migratory (A,B), migrating (C) and differentiating (D–F’) CNC. (A,B) dorsal view with anterior at the top; green fluorescence and bright-field images are merged to show the relative positions of CNC in the whole embryo. (C–E) side view with anterior to the left (C) or right (D,E). (F,F’) dorsal and ventral views (with anterior at the top), respectively, of the same tadpole. br, brain; ln, lens; bc, branchial cartilage; nV, trigeminal nerve; oe, olfactory epithelium; pa, pharyngeal arches.
	Figure 6. Wnt signaling is required for the differentiation of CNC into head cartilage structures. Snai2:eGFP or Wnt reporter embryos were treated with XAV939 (B,B’, F,F’, 20 μM; J,J’, 5 μM), IWR1-endo (C,C’, G,G’, 40 μM; K,K’, 10 μM) or DMSO (vehicle control) from stage ~28 to ~35. Embryos were washed and cultured again to the indicated stages, and images were taken with a Zeiss Axiozoom.V16 epifluorescence microscope. A representative embryo from each treatment group is shown on the left, with upper and lower panels displaying ventral and dorsal views (with anterior at the top), respectively, of the same embryos, and statistics is shown in the graphs on the right. ***P < 0.001.
	Figure 7. Inhibition of Wnt signaling in the post-migratory CNC reduces snai2 and sox9 expression. A,B. Wild-type embryos were treated with XAV939 (5 μM), IWR1-endo (10 μM) or DMSO from stage ~28 to ~35, and processed for in situ hybridization for snai2 (A) or sox9 (B). (C,D) One blastomere of 2-cell stage embryos was injected with 50 pg of mRNA encoding EnR-LefΔN-GR755A. Embryos were treated with DEX or DMSO (as control) from stage ~28 to ~35, and processed for in situ hybridization for snai2 (C) or sox9 (D). A representative tadpole from each treatment group is shown in side view in the upper panels (in C,D both the uninjected and injected sides of the same embryos are displayed side by side for comparison), and statistics is shown in the graphs. White arrowheads point to reduced staining in the condensing mesenchyme in the pharyngeal arches. **P < 0.01; ***P < 0.001.

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