Young JJ , Kjolby RAS , Wu G , Wong D , Hsu SW , Harland RM .

R01 DC011901 NIDCD NIH HHS , R01 GM042341 NIGMS NIH HHS , R01 GM049346 NIGMS NIH HHS

	Fig. 1. Knockdown of Chordin and Follistatin results in a loss of dorsal structures in a subset of embryos produced by heterozygous noggin mutant adults. (A-C) sox2 expression. (D–F) myoD expression. (G–I) cytokeratin expression (A,D,G) Uninjected control embryos. (B,C,E,F,H,I) Embryos injected with 20 ng Chordin morpholino and 20 ng Follistatin morpholino. Dorsal views with anterior towards the top. Though we failed to successfully genotype the stained embryos, the embryos in C, F, and I are predicted to be the noggin nulls.
	Fig. 2. Homozygous nogRMH3 mutant Xenopus tropicalis have severe lower jaw deformities. (A–C) WT stage 46 tadpoles. (D–F) Homozygous nogRMH3 stage 46 tadpoles. Anterior towards the left. (A,D) Lateral views. (B,E) dorsal views. (C,F) Ventral views. (G–H) Sagittal views of whole-mount skeletal stains of WT (G) and mutant (H) and 10 µm sagittal sections stained with alcian blue and nuclear fast red of WT (G′) and mutant (H′) stage 46 skulls. (I–J) Flat-mounted cartilage from WT (I) and mutant (J) tadpoles. (I′–J′) Schematic of skeletal elements in (I–J).
	Fig. 3. Inhibition of BMP signaling rescues the craniofacial defects observed in the tadpoles homozygous for the nogRMH3allele. (A–D) Untreated tadpoles at stage 45 (E–H) Tadpoles at stage 45 treated with 0.1% DMSO for 24 h at stage 28. (I–L) Stage 45 tadpoles treated with 10 μM LDN-193189 for 24 h at stage 28. Genotyping showed that the following representative embryos are wild-type (A–B,E–F,I–J), while (C–D,G–H,K–L) are homozygous nogRMH3 tadpoles. (A,C,E,G,I,K) Dorsal views. (B,D,F,H,J,L) lateral views. White arrowheads show protruding suprarostral plate in the mutants, red arrowheads show rescued structures in mutants following treatment. (M) Quantification of phenotypes observed following treatments indicated. Black bars show the number of tadpoles with a wildtype phenotype, open bars show the number with the mutant phenotype and gray bars indicate tadpoles with non-specific defects. ** indicates p<0.0001.
	Fig. 4. Expression patterns of BMP pathway target genes are affected by the homozygous nogRMH3 mutanion in a stage specific manner. (A–D) Expression of msx1 in the head of representative WT (A,C) and homozygous nogRMH3 (B,D) tadpoles. (E–H) Expression of msx2 in the head of representative WT (E,G) and homozygous nogRMH3 (F,H) tadpoles. (I–L) Expression of edn1 in the head of representative WT (I,K) and homozygous nogRMH3 (J,L) tadpoles. (M–P) Expression of dlx1 in the head of representative WT (M,O) and homozygous nogRMH3 (N,P) tadpoles. (A–B,E–F,I–J,M–N) Stage 33. (C–D,G–H,K–L,O–P) Stage 38. Anterior to the left and dorsal towards the top. (A,C,E,G,I,K,M,O) Arrows show domains of expression of indicated genes in WT tadpoles. (B,D,F,H,J,L,N,P) Arrowheads show altered expression domains in homozygous nogRMH3 mutants.
	Fig. 5. hand2 and bapx1 expression is altered in homozygous nogRMH3 mutant tadpoles. (A–D) Expression of hand2 in the head of representative WT (A,C) and homozygous nogRMH3 (B,D) tadpoles. (E–H) Expression of bapx1 in the head of representative WT (E,G) and homozygous nogRMH3 (F,H) tadpoles. (A–B,E–F) Stage 33. (C–D,G–H) Stage 39. Anterior to the left and dorsal towards the top. (M) Quantification of bapx1 expression domain shift as measured by the cement gland (CG) to otic vesicle (OV) length divided by the stained mandibular arch (MA) to eye (Ey) length. Black bars indicate wildtype embryos and open bars show mutant embryos. ** indicates p<0.05 (T-test). (A,C,E,G) Arrows show domains of expression of indicated genes in WT tadpoles. (B,D,F,H) Arrowheads show altered expression domains in homozygous nogRMH3 mutants.
	Supplementary Figure 1: Stage series of representative embryos produced by heterozygous noggin mutant adults. (A-J) Phenotypically wild-type embryos and tadpoles. (K-L) Wild-type tadpoles. Total number of tadpoles and percentage within each phenotypic class. (M-N) Abnormal tadpoles with dorsal “horn.” Total number of tadpoles and percentage within each phenotypic class. (A,B,D,F,I,K,M) Dorsal views. (C,E,G,H,J,L,M) Lateral views. Anterior to the left.
	Supplementary Figure 2: Phospho-Histone H3 and TUNEL staining in Wild type and homozygous nogRMH3 mutant tadpoles. (A-B) Maximum intensity projections of WT (A) and homozygous nogRMH3 mutant (B) tadpole sections stained for Phospho-Histone H3 at stage 46. (A’,B’) higher magnification views of boxes shown in A and B. (C-D) Maximum intensity projections of WT (C) and homozygous nogRMH3 mutant (D) tadpole sections stained for TUNEL at stage 46. (C’,D’) higher magnification views of boxes shown in A and B. All scale bars = 100μm. (E) Quantification of cells positive for either phospho-histone H3 (n=4/genotype) or TUNEL (n=7/genotype). **: p<0.05.
	Supplementary Figure 3: Survival curve of Wild type and nogRMH3 mutant tadpoles Survival curve of 100 WT (closed boxes) and 100 mutant tadpoles (open triangles).

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