Marquez J , Aslam F , Khokha MK .

NIH HHS , T32 GM007223 NIGMS NIH HHS , R01 HD081379 NICHD NIH HHS , R01 HD102186 NICHD NIH HHS , T32 GM007205 NIGMS NIH HHS , UL1 TR001863 NCATS NIH HHS

             Wnt signaling pathway

	Figure 1 Loss of EMC9 or EMC10 is predicted to result in abnormal protein folding. (a) Ribbon diagram of endoplasmic reticulum membrane protein complex. Stars indicate positions of EMC9 and EMC10. (b) Schematic diagram of EMC9 and EMC10 protein with domain annotations and locations and effects of variants. Red triangle indicates the splice variant and the red octagon indicates truncating variant. All of these variants would be predicted to result in loss of function and depletion of the protein. (c) Model of resultant protein misfolding due to EMC dysfunction predicted to result from loss of function variants in EMC9 and EMC10. Xs indicate losses of functional EMC9 or EMC10 in the EMC. C, cytoplasmic domain; ER, endoplasmic reticulum; H, helical transmembrane domain; MPN, Mpr1, Pad1 N-terminal domain; TM, transmembrane.
	Figure 2 Depletion of emc9 or emc10 in Xenopus results in defective Neural Crest Development. (a) Schematic of injection after holoblastic cleavage at the 2-cell stage (b) Schematic of experimental set up to assess sox9 and sox10 expression via WISH (c) Representative images of WISH for sox10 and sox9 demonstrate decreased expression at stage 20 in tissues depleted of emc9 or emc10 (injected halves of embryos indicated by asterisks) (d) Quantitation of sox10 and sox9 expression in tissues depleted of emc9 or emc10 (n = 60 per condition over three replicates for sox10 n = 24 per condition over three replicates for sox9). Scale bar: 500 μm. Statistical tests carried out as chi-squared tests with Yates correction for frequency of phenotype and two-tailed t-tests for expression area ratios. **** indicates p < .0001. Bars indicate mean and SD of replicate values for frequency and SD of individual values for area of expression.
	Figure 3 Depletion of emc9 or emc10 in Xenopus results in Craniofacial Cartilage Abnormalities. (a) Schematic of injection at the 1-cell stage (b) Representative images and quantitation of Alcian Blue staining of craniofacial cartilage demonstrates abnormalities at stage 45 in tissues depleted of emc9 or emc10 (n = 60 per condition over three replicates). Scale bar: 250 μm. Statistical tests were carried out as chi-squared tests with Yates correction. **** indicates p < .0001. Bars indicate SD of replicate values.
	Figure 4 Depletion of emc9 or emc10 in Xenopus affects embryo motility and neuromuscular acetylcholine receptor patterning. (a) Sample traces and measurement of control (n = 30), emc9 depleted (n = 30), and emc10 depleted tadpole movement over 10 s after stimulation (different colors differentiate distinct tadpoles) over three replicates. (b) Labeling of tail neuromuscular acetylcholine receptor distribution reveals sparse signals in (n = 30) emc9 and (n = 30) emc10 depleted junctions.
	Figure 5 Depletion of emc9 or emc10 in Xenopus affects WNT signaling via depletion of transmembrane protein levels. (a) Immunoblotting for Fzd7 showed a decrease in Fzd7 in pooled (n = 30 per stage per condition) emc9 depleted and emc10 depleted tadpoles as compared with control tadpoles at stage 45. (b) Immunoblotting for β-catenin showed a marked decrease in nuclear β-catenin in pooled (n = 30 per stage per condition) emc9 depleted and emc10 depleted tadpoles as compared with control tadpoles at stage 45.
	Figure S1: CRISPR Editing Insertions and Deletions Lead to Efficient Disruption of emc9 and emc10. Example ICE analysis of insertion and deletion sizes along with their predicted effects in pools of 3–5 injected embryos.
	Figure S2: Multiple guide RNAs recapitulate NCC phenotype observed in CRISPR depletion of emc9 or emc10. (A) Table of additional guide RNAs and targeted exons and sequences (B) Representative images of WISH for sox10 and sox9 demonstrates decreased expression at stage 20 in tissues depleted of emc9 or emc10 (injected halves of embryos indicated by asterisks). (C) Quantitation of sox10 and sox9 expression in tissues depleted of emc9 or emc10. Bars indicate SD of replicate values
	Figure S3: TUNEL Staining for Cell Death. St 20 embryos injected at the one-cell stage. Representative images of embryos injected with Cas9 only (as a negative control), emc9 gRNA+Cas9, emc10 gRNA+Cas9, or nup85 gRNA+Cas9 (as a positive control).
	Figure S4: Subdivision of emc9 and emc10 depletion in Xenopus by Craniofacial Cartilage Regions. (A) Schematic of ventral delineation of craniofacial cartilage regions assessed. (B) Quantitation of Alcian Blue staining of craniofacial cartilage demonstrates abnormalities at stage 45 in tissues depleted of emc9 or emc10 (n = 60 per condition over 3 replicates) most commonly in most anterior cartilage regions. Statistical tests carried out as chi-squared tests with Yates correction. **** indicates p < 0.0001.** indicates p < 0.01. Bars indicate SD of replicate values
	Figure S5: Depletion of emc9 or emc10 in Xenopus results in narrowing of the cardiac outflow tract. Representative examples of stage 45 hearts and quantitation of proximal outflow tract diameter. Statistical tests carried out two-tailed t-tests. **** indicates p < 0.0001. OFT: outflow tract, V: ventricle
	Figure S1: CRISPR Editing Insertions and Deletions Lead to Efficient Disruption of emc9 and emc10. Example ICE analysis of insertion and deletion sizes along with their predicted effects in pools of 3–5 injected embryos.
	Figure S2: Multiple guide RNAs recapitulate NCC phenotype observed in CRISPR depletion of emc9 or emc10. (A) Table of additional guide RNAs and targeted exons and sequences (B) Representative images of WISH for sox10 and sox9 demonstrates decreased expression at stage 20 in tissues depleted of emc9 or emc10 (injected halves of embryos indicated by asterisks). (C) Quantitation of sox10 and sox9 expression in tissues depleted of emc9 or emc10. Bars indicate SD of replicate values
	Figure S3: TUNEL Staining for Cell Death. St 20 embryos injected at the one-cell stage. Representative images of embryos injected with Cas9 only (as a negative control), emc9 gRNA+Cas9, emc10 gRNA+Cas9, or nup85 gRNA+Cas9 (as a positive control).
	Figure S4: Subdivision of emc9 and emc10 depletion in Xenopus by Craniofacial Cartilage Regions. (A) Schematic of ventral delineation of craniofacial cartilage regions assessed. (B) Quantitation of Alcian Blue staining of craniofacial cartilage demonstrates abnormalities at stage 45 in tissues depleted of emc9 or emc10 (n = 60 per condition over 3 replicates) most commonly in most anterior cartilage regions. Statistical tests carried out as chi-squared tests with Yates correction. **** indicates p < 0.0001.** indicates p < 0.01. Bars indicate SD of replicate values.
	Figure S5: Depletion of emc9 or emc10 in Xenopus results in narrowing of the cardiac outflow tract. Representative examples of stage 45 hearts and quantitation of proximal outflow tract diameter. Statistical tests carried out two-tailed t-tests. **** indicates p < 0.0001. OFT: outflow tract, V: ventricle.

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