Young JJ, Kjolby RA, Kong NR, Monica SD, Harland RM.

GM42341 NIGMS NIH HHS , R01 GM042341 NIGMS NIH HHS , T32 GM007127 NIGMS NIH HHS

	Fig. 1. Expression screen for direct transcriptional targets of Wnt signaling in neural tissue. (A) X. tropicalis embryos stained for transcripts identified as Wnt targets by RNA-seq. Stage 10.5 embryos show dorsovegetal views with the dorsal lip toward the top. Stage 12 and 15 embryos show dorsal views with anterior toward the top. (B,C) qPCR on 15-25 animal caps treated as indicated on the x-axis. The y-axis shows expression relative to odc. n=4 experiments. (D) Luciferase reporter assays in HEK293 cells treated with or without mouse Fgf. n=3 experiments. Error bars indicate s.e.m. All means were compared by one-way ANOVA followed by Tukey post-hoc analyses (*P<0.05).
	Fig. 2. sall4 is expressed in the neurectoderm. (A) Stage 10 embryo stained for sall4 RNA; dorsovegetal view with the dorsal lip of the blastopore toward the top. (B-D) Dorsal views of neurula stage embryos with anterior toward the top. (E) Sagittal section of stage 10 embryo stained for sall4 expression; animal pole is to the top and dorsal to the right. (F-K) Transverse sections at anterior (F,H,J) or posterior (G,I,K) of embryos stained for sall4 as indicated in B-D. (E-K) 50 μm sections, with (F-K) dorsal uppermost. SNE, sensorial neurectoderm; No, notochord; S, somite; PM, paraxial mesoderm; PSM, presomitic mesoderm.
	Fig. 3. Loss of Sall4 results in a loss of neural differentiation. Whole-mount in situ hybridizations of (A,C,E,G) uninjected control (UC) embryos and (B,D,F,H) embryos injected bilaterally with 40 ng Sall4 MO (20 ng/blastomere at the 2-cell stage), showing expression of sox2 (A,B), n-tub (C,D), snai2 and nkx6.1 (E,F) and myoD (G,H). Dorsal views with anterior to the top. The number of embryos showing the illustrated expression pattern among the total examined is indicated top right.
	Fig. 4. Expression of hindbrain markers is altered in Sall4 morphants. Whole-mount in situ hybridization of (A,D,G,J) uninjected control embryos, (B,E,H,K) embryos injected with 20 ng Sall4 MO into the right animal-dorsal (A/D) blastomere, and (C,F,I,L) embryos injected with 250 pg sall4 RNA into the right A/D blastomere, showing expression of gbx2 (A-C), mafb (D-F), pax2 (G-I) and meis3 (J-L). Dorsal views with anterior to the top.
	Fig. 5. Sall4 knockdown results in a loss of spinal cord differentiation. (A-H) Whole-mount in situ hybridization of (A,C,E,G) uninjected control embryos and (B,D,F,G) embryos injected with 20 ng Sall4 MO into the right A/D blastomere. (A,B) Expression of otx2, krox20 and hoxb9. Arrows indicate the relative anterior-posterior (A-P) position of krox20 and the anterior limit of hoxb9. Dorsal views with anterior to the top. (C-H) Posterior views of hoxb9 (C,D), hoxc10 (E,F) and hoxd10 (G,H) expression. (I-L) Quantification of A-P patterning defects associated with Sall4 knockdown. (I) Distance between the anteriormost expression of otx2 and the first krox20 stripe in arbitrary units (AU). (J-L) Length of the hoxb9 (J), hoxc10 (K) and hoxd10 (L) expression domains (AU). Error bars indicate s.e.m. Means were compared between left and right sides by Student's t-test (*P<0.05, **P<0.01, ***P<0.001). Data were generated from analyzing all embryos shown in A-H.
	Fig. 6. Knockdown of Sall4 causes an increase in pou5f3 expression. (A-I) Whole-mount in situ hybridization of (A,D,G) uninjected control embryos, (B,E,H) embryos injected with 20 ng Sall4 MO into the right A/D blastomere, and (C,F,I) embryos injected bilaterally with 40 ng Sall4 MO (20 ng/blastomere at the 2-cell stage), showing expression of (A-C) pou5f3.2, (D-F) pou5f3.3 and (G-I) pou5f3.1. Dorsal views, anterior to the top. (J-L) qPCR for pou5f3.2 (J), pou5f3.3 (K) or pou5f3.1 (L) in uninjected embryos, embryos injected with 40 ng Sall4 MO (20 ng/blastomere at the 2-cell stage), and embryos injected with 40 ng Sall4 MO (20 ng/blastomere at the 2-cell stage) plus 500 pg X. tropicalis sall4 RNA (250 pg/animal-dorsal blastomere at the 4-cell stage). The expression is relative to odc. Error bars indicate s.e.m. Means compared with uninjected control by one-way ANOVA followed by Tukey post-hoc analyses (*P<0.05). n=4 experiments.
	Fig. 7. Overexpression of pou5f3 represses neural differentiation. Whole-mount in situ hybridization of (A,C,E,G) uninjected control embryos and (B,D,F,H) embryos injected with 150 pg pou5f3 RNA (50 pg each of pou5f3.2, pou5f3.3 and pou5f3.1 RNAs) into the right A/D blastomere. Red staining is β-galactosidase used as a tracer for RNA injection. (C′-F′) Higher magnification views of the boxed regions in C-F. (A-D) otx2, krox20 and hoxb9 expression in anterior (A,B) or dorsal (C,D) view. (E-H) hoxc10 (E,F) and sox2 (G,H) expression in dorsal view.
	Fig. 8. Loss of spinal cord in Sall4 morphants is due to an increase in pou5f3 expression. (A-L) Whole-mount in situ hybridization of (A-D) uninjected control embryos, (E-H) embryos injected with 40 ng Sall4 MO (20 ng/blastomere at the 2-cell stage), or (I-L) embryos injected with 40 ng Sall4 MO, 20 ng Pou5f3.2 MO, 10 ng Pou5f3.3a MO, 10 ng Pou5f3.3b MO, and 20 ng Pou5f3.1 MO. (N) Embryos injected with 20 ng Pou5f3.2 MO, 10 ng Pou5f3.3a MO, 10 ng Pou5f3.3b MO and 20 ng Pou5f3.1 MO. Expression is shown for (A,E,I) otx2, krox20 and hoxb9, (B,F,J) hoxc10, (C,G,K) hoxd10 and (D,H,L,N) sox2. Dorsal views with anterior to the top. (M) Quantification of posterior neural gene expression as measured by expression domain length in arbitrary units (AU). White, uninjected control embryos. Gray, embryos injected with 40 ng Sall4 MO (20 ng/blastomere at the 2-cell stage). Black, embryos injected with 40 ng Sall4 MO, 20 ng Pou5f3.2 MO, 10 ng Pou5f3.3a MO, 10 ng Pou5f3.3b MO and 20 ng Pou5f3.1 MO. Error bars indicate s.e.m. Means compared with uninjected control by one-way ANOVA followed by Tukey post-hoc analyses (***P<0.001). Data were generated from analyzing all embryos shown in A-C, E-G, and I-K.
	Fig. 9. Fgf and RA signaling fail to posteriorize Sall4 morphants. Whole-mount in situ hybridization of (A,E) uninjected control embryos, (B,F) embryos injected with 40 ng Sall4 MO (20 ng/blastomere at the 2-cell stage), (C,G) embryos injected with 50 pg fgf8a RNA into the right A/D blastomere, (D,H) embryos injected with 40 ng Sall4 MO (20 ng/blastomere at the 2-cell stage) and 50 pg fgf8a RNA into the right A/D blastomere, (I,K) embryos treated with 1 μM all-trans retinoic acid (ATRA) and (J,L) embryos injected with 40 ng Sall4 MO (20 ng/blastomere at the 2-cell stage) and treated with 1 μM ATRA. Expression is shown for (A-D,I,J) otx2, krox20 and hoxb9 or (E-H,K,L) sox2. Arrowheads indicate the posterior limit of otx2 expression. Dorsal views, anterior to the top.
	sall1 (spalt-like transcription factor 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 10.5, blastoporal view, dorsal up.
	sall1 (spalt-like transcription factor 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 12, posterior blastoporal view, dorsal up.
	sall1 (spalt-like transcription factor 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 15, dorsal view, anterior up.
	sall4 (spalt-like transcription factor 4) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 10.5, blastoporal view, dorsal up.
	sall4 (spalt-like transcription factor 4) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 12, blastoporal view, dorsal up.
	sall4 (spalt-like transcription factor 4) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 15, dorsal view, anterior up.
	sf3b4 (splicing factor 3b subunit 4) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 10.5
	sf3b4 (splicing factor 3b subunit 4) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 12, dorsal view, anterior up.
	sf3b4 (splicing factor 3b subunit 4) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 15, dorsal view, anterior up.
	hnrnpk (heterogeneous nuclear ribonucleoprotein K) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 10.5, blastoporal view, dorsal up.
	hnrnpk (heterogeneous nuclear ribonucleoprotein K) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 12, dorsal view, anterior up.
	hnrnpk (heterogeneous nuclear ribonucleoprotein K) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 15, posterior view, dorsal up.
	sfrs7 (serine/arginine-rich splicing factor 7) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 10.5, blastoporal view, dorsal up.
	srsf7 (serine/arginine-rich splicing factor 7) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 12, dorso-posterior view, anterior up.
	sfrs7 (serine/arginine-rich splicing factor 7) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 15, dorsal view, anterior up.
	Figure S1: TVGR activates canonical Wnt signaling. (A) Quantification of secondary axis induction by ventral vegetal injection of TVGR at the 4-cell stage with representative tadpoles from each class. (B) RT-PCR on 5 whole embryos or 25 animal caps treated with the indicated reagents. -RT: reaction done in the absence of Reverse Transcriptase, epi. ker: epidermal keratin (epidermis), mus. act.: muscle actin (mesoderm) (C) Animal caps treated with the indicated reagents.
	extracted from Figure S1: TVGR activates canonical Wnt signaling. A. Representative tadpoles phenotypes, from each class.
	Figure S2: Intron 1 of sall4 binds β-catenin but does not mediate a Wnt signal. (A) Using animal caps to screen for direct transcriptional targets of Wnt in neural tissue. (B) qPCR on 15-25 animal caps treated as indicated on the X-axis. The Y-axis shows expression relative to odc. meis3 and hoxb9 serve as controls for known direct and indirect targets of Wnt, respectively. (C) Quantification of dorsalization in uninjected embryos (open bars) and embryos injected animally with 500 pg FLAG- tagged β-catenin RNA (250 pg/blastomere) at the 2-cell stage (filled bars) as scored by the dorsoanterior index (DAI). Error bars: 1 SEM. Images show a representative uninjected (UC) embryo with a DAI of 7 (normal) and a representative embryo with a DAI of 6 (kinked axis). (D) Schematic of the genomic locus of sall4 in Xenopus laevis (Xenbase.org). Blue boxes indicate exons and yellow circles indicate the location of putative TCF/LEF binding sites. Black ovals show the locations of the zinc-finger domains. Numbers indicate the position of putative binding sites relative to the transcription start site (TSS). (E) Chromatin immunoprecipitation of FLAG-tagged β-catenin in late gastrulae/early neurulae. Open bars represent uninjected embryos and closed bars represent embryos injected with 500 pg FLAG- tagged β-catenin (250 pg/blastomere at the 2-cell stage). Error bars: 1 SEM per cent input for each measurement. (F) Luciferase reporter assays in HEK293 cells treated with or without BIO and/or mouse FGF. Error bars: 1 SEM. All means were compared by one-way ANOVA followed by Tukey post-hoc analyses (*: p<0.05).

	Figure S3: cdx2 is activated by canonical Wnt signaling and not affected by Sall4 knockdown (A) qPCR on 5 whole embryos or 15 to 25 animal caps treated according to the conditions indicated on the X-axis. The Y- axis shows expression relative to odc. Error bars: 1 SEM. (B-C) cdx2 expression at stage 18. Dorsal views with the anterior oriented towards the top. (B) Uninjected control embryo. (C) Embryo injected with 20 ng Sall4 MO in one animal-dorsal cell at the 4-cell stage.
	Figure S4: sall1 is activated by canonical Wnt signaling and expressed during early embryogenesis. (A) qPCR on 5 whole embryos or 15 to 25 animal caps treated according to the conditions indicated on the X-axis. The Y-axis shows expression relative to odc. Error bars: 1 SEM. (B-E) Whole-mount in situ hybridizations of sall1 in Xenopus laevis embryos. (B) Whole mount stage 10 embryo stained for sall1, dorso-vegetal view with the dorsal lip of the blastopore oriented towards the top. (B’) Sagittal section of stage 10.5 embryo stained for sall1 expression, animal pole is to the top and dorsal is to the right. (C-D) Dorsal views of indicated neurula stage embryos, anterior is oriented towards the top. (C’-C’’) Transverse sections of stage 12 embryos stained for sall1, (C’) anterior and (C’’) posterior. (D’-D’’) Transverse sections of stage 15 embryos stained for sall1, (D’) anterior and (D’’) posterior. (E’-E’’) Transverse sections of stage 18 embryos stained for sall1, (E’) anterior and (E’’) posterior. (B’, C’-E’’) 50 μM sections, (C’-E’’) dorsal oriented towards the top. No: notochord, S: somite, PSM: presomitic mesoderm. 

	Figure S5: Injected embryos express functional FLAG-tagged β-catenin. (A) Western blot for the FLAG epitope in injected embryos. Actin serves as the loading control. (B) Ventralization of embryos injected with β- catenin MO and co-injection with FLAG-tagged β-catenin RNA. F-βcat: FLAG-tagged β-catenin.
	Figure S7: Sall4 does not rescue Dkk1-induced anteriorization. (A-D) Anterior views of whole-mount in situ hybridazations for otx2 and krox20 on Xenopus laevis embryos. (A) Uninjected control (UC). (B) Embryo injected with 400pg dkk1 RNA. (C) Embryo injected with 400pg dkk1 and 500pg sall4 RNA. (D) Embryo injected with 500pg sall4 RNA.

	cdx2 (caudal type homeobox 2) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 18, dorsal view, anterior up.

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