Perfetto M , Xu X , Lu C , Shi Y , Yousaf N , Li J , Yien YY , Wei S .

P20 GM104316 NIGMS NIH HHS , R01 DE029802 NIDCR NIH HHS, R01 GM114105 NIGMS NIH HHS , P01 HL032262 NHLBI NIH HHS , R35 GM133560 NIGMS NIH HHS , U54 GM104942 NIGMS NIH HHS

Xtr.Tg(snai2:eGFP){Swei} + ddx3x MO (Fig.1.A)
Xtr.Tg(snai2:eGFP){Swei} + ddx3x MO (Fig.6.F)
Xtr.Tg(WntREs:dEGFP){Vlemx} + ddx3x MO (Fig.5.B)
Xtr Wt + AKTi (Fig.4.A)
Xtr Wt + AKTi (Fig.4.B)
Xtr Wt + ddx3x (Fig.S.2)
Xtr Wt + ddx3x MO (Fig.1.B-C)
Xtr Wt + ddx3x MO (Fig.2.A)
Xtr Wt + ddx3x MO (Fig.2.B)
Xtr Wt + ddx3x MO (Fig.5.A)
Xtr Wt + ddx3x MO (Fig.5.C)
Xtr Wt + {dn}Hsa.AKT1 (Fig.4.C)

	Fig. 1. DDX3 is necessary for normal NC induction and craniofacial cartilage development. (A) One anterodorsal (D1) blastomere of eight-cell stage snai2:eGFP embryos was injected with the indicated MO (1.5 ng each). Embryos were cultured to stage ∼46 and imaged for eGFP expression. (B,C) Wild-type X. tropicalis embryos were injected in one blastomere at the two-cell stage with the indicated MO (6 ng each) and mRNA encoding wild-type human DDX3X or encoding the AAA mutant (200 pg each), cultured to stage ∼12.5, and processed for in situ hybridization for the indicated markers. Representative embryos are shown in ventral (A) or dorsal (B,C) view with anterior at the top; injected side is indicated with a red asterisk. CT, control; WT, wild type; n, number of embryos scored. ***P<0.001; NS, not significant (χ2 tests). Scale bars: 500 μm.
	Fig. 2. DDX3 stabilizes β-catenin and activates Wnt signaling. Wnt reporter (A) or wild-type (B) embryos were injected into one blastomere at the two-cell stage with the indicated MO (6†ng each) and mRNA (200†pg), cultured to stage ∼12.5 (A) or ∼12 (B), and imaged for eGFP expression (A) or processed for in situ hybridization for gbx2 (B). Representative embryos are shown in dorsal view with anterior at the top. n, number of embryos scored. **P<0.01; ***P<0.001; NS, not significant (χ2 tests). (C-G) HEK293T cells were treated with DMSO or 10†µM RK-33 for 24†h (C,F), or transfected with the indicated siRNA (200†nM, D,E,G). In F and G, a plasmid encoding wild-type β-catenin or the stabilized mutant (Stab; 1†µg per well for 6-well plates), as indicated, was also transfected for 48†h. Cell lysates of three biological replicates were processed for TOP/FOPFLASH assays (C,D,F,G), or western blot analyses using the indicated antibodies (E). aβ-cat, active (unphosphorylated) β-catenin. Injected side is indicated with a red asterisk. *P<0.05; **P<0.01; ***P<0.001; NS, not significant (unpaired t-tests). (C,D,F,G) Data are mean±s.e.m. Scale bars: 250 μm.
	Fig. 3. KD of DDX3 reduces AKT activity, AKT-mediated GSK3β phosphorylation and ectopically expressed Snai1. (A,D,E,G) HEK293T cells were transfected with the indicated siRNA (200 nM) for 48 h (A,D,G), or treated with DMSO or 10 µM RK-33 for 24 h (E). In G, a plasmid encoding HA-tagged Snai1 (1 µg per well for six-well plates) was also transfected. (B,F) Embryos were injected at the one-cell stage with the indicated MO (12 ng each; 50 pg mRNA encoding HA-tagged Snai1 was co-injected in F), and cultured to stage ∼12.5. Cell or embryo lysates were processed for western blot analyses using the indicated antibodies. (C) Summary of the results of three independent experiments shown in B. Density of the target protein was normalized against that of β-actin, and fold change (DDX3 MO versus control MO) is shown. *P<0.05; **P<0.01 (unpaired t-tests). Data are mean±s.e.m.
	Fig. 4. AKT activity is required for Wnt signaling and NC induction. (A,B) Wild-type (A) or Wnt reporter (B) embryos were treated with 20†µM AKTi or DMSO from stage ∼10 to ∼12.5, and processed for in situ hybridization for the indicated markers (A) or imaged for eGFP expression (B). (C) Wnt reporter embryos were injected in one blastomere at the two-cell stage with dnAKT mRNA (50†pg), cultured to stage ∼12.5 and imaged for eGFP expression. Representative embryos are shown in dorsal view with anterior at the top. Injected side is indicated with a red asterisk. n, number of embryos scored. ***P<0.001 (χ2 tests). Scale bars: 250 μm.
	Fig. 5. DDX3 induces the NC through AKT, β-catenin and Snai1. Wild-type (A,C) or Wnt reporter (B) embryos were injected in one blastomere at the two-cell stage with the indicated MO (6†ng each), mRNA (50†pg for caAKT and 100†pg for Snai1) and plasmid (10†pg for β-catenin), cultured to stage ∼12.5, and processed for in situ hybridization for the indicated markers (A,C) or imaged for eGFP expression (B). Representative embryos are shown in dorsal view with anterior at the top. Injected side is indicated with a red asterisk. n, number of embryos scored. *P<0.05; ***P<0.001 (χ2 tests). Scale bars: 250 μm.
	Fog. 6. RAC1 is a downstream effector of DDX3 in NC induction. (A,B) HEK293T cells were treated with RK-33 (A) or transfected with the indicated siRNA (B) as in Fig. 3. Cell lysates were processed for western blotting for RAC1. (C) Embryo lysates shown in Fig. 3B were reblotted for RAC1. Results of three independent experiments are summarized in D. Density of RAC1 was normalized against that of β-actin, and fold change (DDX3 MO versus control MO) is shown. *P<0.05 (unpaired t-test). (E) The same batches of injected embryos in C and D (three biological replicates) were processed for RT-qPCR using two pairs of primers for rac1 mRNA. Results were normalized against gapdh mRNA (internal control). *P<0.05; NS, not significant (unpaired t-tests). (D,E) Data are mean±s.e.m. (F) One anterodorsal (D1) blastomere of eight-cell stage snai2:eGFP embryos was injected with the indicated MO (1.5†ng each) and mRNA (100†pg). Embryos were cultured to stage ∼46 and imaged for eGFP expression. Representative embryos are shown in ventral view with anterior at the top. n, number of embryos scored. ***P<0.001 (χ2 tests). (G) A model for DDX3 function in regulating downstream signaling during NC induction. Scale bars: 500 μm.
	Figure S1. Developmental expression of ddx3 during X. tropicalis embryogenesis. In situ hybridization was carried out for ddx3 with X. tropicalis embryos at indicated stages. A-D. Dorsal view with animal pole (A) or anterior (B-D) at the top. A’. Vegetal view of the same embryo in A with dorsal at the top. B’. Transverse section of a stage ~12.5 embryo with dorsal at the top, showing high ddx3 expression in the ectoderm. E. Side view with anterior to the right and dorsal at the top. Control in situ hybridization with sense probe is shown in insets, and arrows in A and A’ indicate the dorsal blastopore lip.
	Figure S2. Ectopically expressed X. tropicalis DDX3 causes expansion of NPB and NC specifiers. Wild-type X. tropicalis embryos were injected in one blastomere at 2-cell stage with 10 pg plasmid encoding X. tropicalis DDX3, cultured to stage ~12.5, and processed for in situ hybridization for the indicated markers. Embryos are shown in dorsal view with anterior at the top, and injected side is denoted with a red asterisk (same below). The number and percentage of injected embryos displaying the indicated phenotypes are shown underneath.
	Figure S3. The AAA mutant of human DDX3X does not effectively restore the reduced Wnt activity caused by DDX3 KD in X. tropicalis embryos. A. Wnt reporter embryos were injected in one blastomere at 2-cell stage with the indicated MO (6 ng each) and mRNA (200 pg), cultured to stage ~15, and imaged for GFP expression. Embryos are shown in dorsal view with anterior at the top. ***, P<0.001; NS, not significant (t-tests). B. Western blot with an anti-HA antibody showing the expression of HA-tagged human DDX3X AAA mutant.
	Figure S4. A constitutively active mutant of AKT activates Wnt signaling independently of DDX3. HEK293T cells were transfected with the indicated siRNA (200 nM) and a plasmid encoding caAKT (1 μg per well for 6-well plates) for 48 hr. Cell lysates of 3 biological replicates were processed for TOP/FOPFLASH assays. **, P<0.01; NS, not significant (t-tests).
	Figure S5. KD of DDX3 does not induce apparent apoptosis prior to neurulation. Wild-type X. tropicalis embryos were injected in one blastomere at 2-cell stage with the indicated MO (6 ng each), cultured to stage ~12.5, and processed for TUNEL staining (dark blue). Co-injected b- galactosidase mRNA was expressed and developed using Red-Gal. Three representative embryos from each injection group are shown in dorsal view with anterior at the top.
	Figure S6. Predicted secondary structures of the X. tropicalis rac1 and human RAC1 5’- UTRs. Prediction was carried out using the Mfold algorithm (http://unafold.rna.albany.edu/?q=mfold/download-mfold).
	Figure S7. Overexpression of RAC1 in one anterodorsal blastomere at 8-cell stage does not cause apparent defects in X. tropicalis embryos. One anterodorsal (D1) blastomere of 8-cell stage snai2:eGFP embryos was injected with 100 pg rac1 mRNA and a red fluorescent dye (Alexa Fluor 555) as a lineage tracer. Embryos were cultured to stage ~46; bright field (A-D) and fluorescence (with green and red fields merged; A’-D’) images are shown with anterior at the top.

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