Wu MY , Ramel MC , Howell M , Hill CS .

Cancer Research UK

	Figure 1. SNW1 was identified in a functional screen for neural crest fate in X. laevis. (A) A diagram of a stage 14 embryo depicting the classical model, which proposes that different levels of BMP signaling are required for ectodermal patterning and neural crest formation. (B) An overview of the functional screen. (C) The screen identified proteins whose overexpression modulated levels of Slug expression. 200 pg of mRNA expressing the X. tropicalis c-Myc, Dkk-1, Noggin2, or SNW1 was injected at the one-cell stage, with 250 pg of GFP mRNA as tracer. Control embryos received the GFP mRNA only. Embryos were fixed at stage 14 and analyzed for Slug expression by WISH. The number of embryos out of the total analyzed that showed the presented staining pattern is given. (D) X. laevis embryos were analyzed for SNW1 expression by WISH at the indicated stages. (E) Whole X. laevis embryo extracts were prepared from either wild-type (WT) embryos or embryos injected with 20 ng of a translation-blocking MO, MoSNW1a, at the one-cell stage, and analyzed by Western blotting using antibodies against SNW1 and Tubulin, the latter as a loading control. The developmental stages are indicated. (F) Zebrafish embryos were analyzed for SNW1 expression at the indicated times. Two gastrulation stages (the shield stage and 75% epiboly) and three segmentation stages (11, 12, and 13 hpf) are shown. Lateral views are shown, and for the later stages, also a dorsal-anterior view. (G) Whole zebrafish embryo extracts were prepared from wild-type embryos at the indicated times post-fertilization and analyzed by Western blotting using antibodies against SNW1 and MCM6, the latter as a loading control. The developmental stages are indicated. E, unfertilized egg. doi:10.1371/journal.pbio.1000593.g001
	Figure 2. SNW1 knockdown inhibits neural crest induction. (A) Injection of MoSNW1a (Figure S3) results in the loss of neural crest markers Snail, Slug, and Sox9 on the injected side at stage 14. MoSNW1a (20 ng) was co-injected with Fdx as a tracer in one cell of a two-cell Xenopus embryo. The tracer was detected using an anti-fluorescein antibody. Control embryos were uninjected. (B) Two-cell Xenopus embryos were injected in one cell with either 20 ng of control MO (MoControl) or 20 ng of MoSNW1a. WISH was performed at stage 28 using a probe against Twist. (C) Xenopus embryos were injected with control MO or MoSNW1a as in (B). At stage 42 the facial cartilage was stained with alcian blue and then dissected. The injected side is indicated with an arrow. (D) Zebrafish embryos were injected with 15 ng of either a control MO or MoZFSNW1 and fixed at 13 hpf for WISH using a probe against the neural crest marker Sox10, or at 12 hpf for WISH with probes against FoxD3 and the adaxial and paraxial mesoderm marker MyoD. A group of embryos is shown, as well as a representative single embryo. The neural crest cells are marked by grey arrowheads, and the MyoD-postive cells by an asterisk (righthand panels). For the MyoD staining, only the adaxial mesoderm is visible, and this is not affected by SNW1 depletion. In all cases the number of embryos out of the total analyzed that showed the presented staining pattern or phenotype is given. doi:10.1371/journal.pbio.1000593.g002
	Figure 3. Dorsally expressed SNW1 is required for neural crest specification, and this is independent of mesoderm. (A) Left panels. Wild-type (WT) zebrafish embryos at the indicated times post-fertilization were doubly stained for the neural crest marker Sox10 (dark blue) and SNW1 (grey-purple), which is localized in the neural plate (see Figure 1F). Right panels. Wild-type stage 14 Xenopus embryos were stained for either SNW1, the neural crest marker Slug or the neural plate marker Sox3. The orange lines mark the edges of the neural crest staining to enable direct comparison of expression domains. (B) One dorsal or one ventral blastomere of four-cell Xenopus embryos was injected with Fdx with or without 10 ng of MoSNW1a, as indicated. At stage 14 the embryos were stained for Slug and fluorescein. (C) Xenopus embryos were either uninjected (control), injected with 20 ng of MoSNW1a at the one-cell stage, injected with 250 pg of CerS mRNA into all four blastomeres at the four-cell stage, or injected with MoSNW1a at the one-cell stage followed by CerS mRNA at the four-cell stage. WISH was carried out for the mesoderm markers Xbra and Gsc at stage 12, or the neural plate marker Sox3 and the neural crest marker Slug at stage 14. OE, overexpression. In all cases the number of embryos out of the total analyzed that showed the presented staining pattern is given. doi:10.1371/journal.pbio.1000593.g003
	Figure 4. SNW1 is essential for specification of the border between neural and non-neural ectoderm. (A) Xenopus embryos were either uninjected (control) or injected with 20 ng of MoSNW1a at the one-cell stage. WISH was carried out for the dorsal mesoderm marker Goosecoid (Gsc), the dorsal midline markers Chordin (Chd), Xnot, and Noggin (Nog), the paraxial mesoderm markers Wnt8 and MyoD, the neural plate marker Sox3, the anterior neural marker Otx2, the anterior neural and neural plate border marker Zic3, the epidermal marker Epidermal keratin (EpiKer), and the neural plate border markers BMP4, Sox9, Snail, Slug, FoxD3, and Msx1. Arrows mark the expression of Xnot, Noggin, and Wnt8 at the anterior neural border. For EpiKer, a lateral view is shown in addition to the dorsal view. In all panels anterior is to the top. (B) Lateral and dorsal views of the Sox3 WISH are shown. Note that the sharp border of the neural plate is not formed when SNW1 is depleted. (C) Zebrafish embryos were injected with 15 ng of either a control MO (MoControl) or MoZFSNW1 and fixed at 11 hpf for double in situ hybridizations using probes against Sox10 (purple) and the neural plate marker N-cadherin (N-cad; turquoise). Lateral and anterior views of the same embryo are shown. (D) Xenopus embryos were either uninjected (control) or injected with 20 ng of MoSNW1a at the one-cell stage. Double in situ hybridizations were carried out at stage 14 for Slug (dark blue) and Sox2 (turquoise), or EpiKer (dark blue) and Sox2 (turquoise). The red line indicates the border between the neural plate and the epidermis. In all cases the number of embryos out of the total analyzed that showed the presented staining pattern is given. doi:10.1371/journal.pbio.1000593.g004
	Figure 5. SNW1 regulates BMP activity in Xenopus and zebrafish embryos and is essential for BMP activity at the border between neural and non-neural tissue. (A) One-cell Xenopus embryos were either uninjected (control) or injected with 20 ng of MoSNW1a or 500 pg of X. tropicalis (Xt) SNW1. Embryos were harvested at the stages indicated, and whole embryo extracts were analyzed by Western blotting using antibodies against p-Smad1 and Tubulin, the latter as a loading control. Quantification of p-Smad1 levels relative to Tubulin is shown below the blots. (B) Xenopus embryos were either uninjected (control) or injected with 20 ng of MoSNW1a or 500 pg of X. tropicalis SNW1 mRNA at the one-cell stage, and bisected at stage 14 into dorsal (D) and ventral (V) halves. Whole cell extracts were analyzed by Western blotting using antibodies against SNW1, p-Smad1, Smad1, and Tubulin, the last as a loading control. WE, whole embryo. (C) Zebrafish embryos were either uninjected (U) or injected with increasing amounts (5, 10, 15, or 20 ng) of the splice-blocking MO MoZFSNW1. Embryos were harvested at 8, 10, or 12 hpf. Whole embryo extracts were analyzed by Western blotting using antibodies against SNW1, p-Smad1, and MCM6, the last as a loading control. (D) Knockdown of SNW1 strongly reduces ventral p-Smad1 levels at stage 13, but has no effect on the p-Smad1 ventral/dorsal gradient at stage 10. Embryos that were either uninjected (control) or injected with 20 ng of MoSNW1a at the one-cell stage were fixed at either stage 10 or stage 13, sagittally bisected along the midline, and immunostained with an antibody against p-Smad1. For these panels and subsequent p-Smad1 immunostaining, the specific p-Smad1 staining is nuclear and punctate. A, anterior; D, dorsal; P, posterior; V, ventral. (E) Transgenic BRE-mRFP embryos were injected with 15 ng of control MO or MoZFSNW1. They were fixed at 11, 12, and 13 hpf, when WISH was performed for mRFP, which indicates domains of BMP activity. In each case three different views of the same embryo are shown. In control embryos, mRFP is expressed in the tailbud (see dorsal-posterior view) and in a horseshoe-shaped domain at the dorsal anterior at 11 hpf (red arrowheads), which becomes increasingly sharpened at 12 and 13 hpf. In SNW1 morphants, the mRFP in this dorsal-anterior domain is reduced or absent. However, mRFP expression, and hence BMP activity, in the posterior is only slightly reduced or unchanged. In (D) and (E) the number of embryos out of the total analyzed that showed the presented staining pattern is given. In (E), the MO starts to knockdown SNW1 only from 8 hpf, and thus its effects become apparent only after 10 hpf. This explains why only a small percentage of embryos at 11 hpf shows the presented phenotype. By 13 hpf, when the MO effectively knocks down SNW1, 66% of the embryos show the phenotype. doi:10.1371/journal.pbio.1000593.g005
	Figure 6. In post-gastrula zebrafish and Xenopus embryos the neural crest domain overlaps with the dorsal-anterior domain of BMP activity. (A) At early segmentation stages of zebrafish development the domains of expression of SNW1 and Sox10 partially overlap with the anterior �horseshoe� domain of BMP activity. Transgenic BRE-mRFP zebrafish embryos were stained for SNW1 (grey-purple) and Sox10 (dark blue), Sox10 (dark blue) and mRFP (grey-purple), mRFP (dark blue) and SNW1 (turquoise), mRFP (dark blue) and Hgg1 (maroon), or SNW1 (dark blue) and Hgg1 (maroon) at 11 hpf and 13 hpf. mRFP staining indicates regions of BMP activity. (B) A diagram representing the staining patterns of the four markers is shown. At 11 hpf, domains of expression of SNW1, Sox10, and mRFP partially overlap at the lateral edges of the dorsal-anterior horseshoe domain. However at 13 hpf, it is clear that although the neural crest marker Sox10 is still adjacent to the domain of SNW1 expression in the neural plate, the horseshoe mRFP expression domain (red arrowheads in [A]) is separated from the Sox10 and SNW1 expression domains. (C) Wild-type stage 13 Xenopus embryos were bisected transversely through the neural crest region. The anterior half was immunostained with an antibody against p-Smad1, and the posterior half was used for WISH against Slug. The embryos were then imaged along the plane of bisection and overlayed. The yellow lines outline the boundaries of detected p-Smad1 staining at the neural plate border, which overlaps with the Slug-positive neural crest cells. doi:10.1371/journal.pbio.1000593.g006
	Figure 7. SNW1 does not function downstream of the receptors in the BMP pathway in tissue culture cells or in Xenopus animal cap explants. (A) Knockdown of SNW1 had no effect on the levels of p-Smad1/5 or p-Smad2 in response to ligand stimulation. SNW1 was depleted in MDA-MB-231 cells using an siRNA pool containing four individual duplexes that specifically target the human SNW1 mRNA. 72 h after siRNA transfection, cells were either left uninduced or induced for 1 h with 20 ng/ml BMP4 (R&D Systems) or 2 ng/ml TGF-β (PeproTech). Whole cell extracts were analyzed by Western blotting using antibodies against SNW1, p-Smad1/5, p-Smad2, and Smad2/3, the last serving as a loading control. In this gel system p-Smad1 and p-Smad5 are resolved into two bands. RiscFree (RF) siRNA is a non-targeting duplex used as a control. (B) siRNA-mediated knockdown of SNW1 in mammalian cells does not affect a BMP-responsive reporter activity. An MDA-MB-231 cell line stably expressing TK-Renilla and BRE-Luciferase was transfected with a non-targeting siRNA (NT), an siRNA pool against SNW1, the four individual siRNAs that make up the pool, or an siRNA against Smad4. 72 h after transfection, cells were induced with 200 ng/ml BMP7 (PeproTech). Samples were taken for Western blotting after 1 h and for Luciferase/Renilla assays after 8 h. The efficiency of knockdown is demonstrated in the Western blot shown. (C) SNW1 depletion affects BMP activity only in whole embryos and not in animal caps. Embryos were either uninjected or injected at the one-cell stage with 20 ng of MoSNW1a or 10 pg of a plasmid expressing Noggin. Animal caps were cut at stage 8.5 and cultured until sibling embryos had reached stage 14. Whole cell extracts from whole embryos or the animal caps were analyzed by Western blotting using antibodies against p-Smad1, Smad1, SNW1, and Tubulin, the last serving as a loading control. doi:10.1371/journal.pbio.1000593.g007
	Figure 8. Reduction of BMP signaling by expression of Noggin in the dorsal ectoderm mimics SNW1 depletion, and overexpression of BMP2b rescues the effect of SNW1 depletion. (A�C) Embryos were either uninjected, injected with 20 ng of MoSNW1a at the one-cell stage, or injected with 5 pg of a Noggin expression plasmid and 100 pg of GFP mRNA as a tracer in one dorsal A tier blastomere at the 16-cell stage. Samples were fixed at stage 13 for immunostaining for p-Smad1 ([A], pseudo-colored magenta). Samples were taken at stage 14 for Western blot analysis using antibodies against p-Smad1 and Tubulin, the latter serving as a loading control (B), and samples were fixed at stage 14 for WISH using probes against Slug and Sox2 and at stage 28 for phenotype analysis (C). In (A) the nuclei are visualized with DAPI (pseudo-colored cyan) and the Noggin-injected cells with GFP. The dorsal side (D) is marked. In (B) two irrelevant lanes have been removed as indicated by the gap. In the bottom right panel of (C), the GFP marks the region of the embryo expressing exogenous Noggin. (D) Xenopus embryos were either uninjected or injected at the one-cell stage with 20 ng of MoSNW1a. At the 16- to 32-cell stage both groups of embryos were injected in one of the A1/B1/A2/B2 blastomeres with either 8 pg of plasmid expressing zebrafish BMP2b or 16 pg of the same plasmid together with Fdx as a tracer, as indicated. Embryos were fixed at stage 14, and neural crest was detected by staining for Slug by WISH. The Fdx was visualized with an antibody against fluorescein. doi:10.1371/journal.pbio.1000593.g008
	Figure 9. A model of SNW1 function. A schematic diagram summarizing our findings in both Xenopus and zebrafish embryos. A dorsal-anterior view of a schematized embryo is shown. doi:10.1371/journal.pbio.1000593.g009
	Fig S3 Depletion of SNW1 results in a dorsalized phenoype, reduces the number of Snail-positive neural crest cells, disrupts migration of neural crest cells into the branchial arches at the tailbud stages, and results in loss of cranial cartilage. (A) MOs used to deplete SNW1 in Xenopus and zebrafish. The two translation-blocking MOs targeting X. laevis (Xl) SNW1 are shown and aligned with both the X. laevis and X. tropicalis (Xt) sequences for SNW1. MoSNW1a overlaps the ATG (highlighted in green), while MoSNW1b binds in the 5′ UTR. The MOs are 100% complementary to the X. laevis SNW1 sequence, whereas there are five non-consecutive mismatches against the X. tropicalis sequence for each MO. The sequence targeted by the zebrafish SNW1 splice-blocking MO is shown. The exon is shown in uppercase and the intron in lowercase. (B) In vitro translation using rabbit reticulocyte lysate of either X. laevis or X. tropicalis SNW1 in the absence or presence of MoSNW1a or MoSNW1b. Translation of X. laevis SNW1 is inhibited by both MOs, with MoSNW1a being the more efficient. Neither MO inhibits translation of X. tropicalis SNW1. SNW1 levels were detected by labeling with 35S-methionine and autoradiography. Equal amounts of translation reaction were loaded in each lane. (C) One-cell embryos were injected with 40 ng of control MO (C), 20 ng of MoSNW1a (Ma), or 40 ng of MoSNW1b (Mb). Whole embryo extracts were prepared at the stages indicated and analyzed by Western blotting for SNW1, and Tubulin as a loading control. Both MOs are effective in blocking zygotic SNW1 translation, as evidenced by a reduction in SNW1 protein levels only from stage 12 onwards, with Ma being more efficient than Mb, consistent with (B). (D) One-cell embryos were injected as in (C). Embryos were fixed at stage 28 and analyzed by WISH for Snail, which reveals that cranial neural crest induction is disrupted. (E) Injection of MoSNW1a in one cell of a two-cell embryo reduces cranial cartilage formation on the injected side (arrows). Two-cell Xenopus embryos were injected in one cell with either 20 ng of control MO or 20 ng of MoSNW1a, along with 250 pg of GFP mRNA as a tracer. In (D) and (E) the number of embryos out of the total analyzed that showed the presented phenotype is given.
	Fig S4. Overexpression of SNW1 can rescue the effects of SNW1 depletion in Xenopus and zebrafish. (A) The effects of SNW1 knockdown in X. laevis embryos can be rescued by overexpression of X. tropicalis SNW1. Embryos injected with 20 ng of control MO, 20 ng of MoSNW1a, 500 pg of X. tropicalis SNW1 mRNA, or both MoSNW1a and X. tropicalis SNW1 mRNA at the one-cell stage. Neural crest induction was assayed by WISH for Slug and Sox9 at stage 16. The phenotype was analyzed at stage 30. The number of embryos out of the total analyzed that showed the presented staining pattern/phenotype is given. (B) Zebrafish embryos were injected with 15 ng of either control MO or MoZFSNW1. They were photographed for phenotype analysis at 28 hpf. The morphant embryos present a dorsalized-like phenotype, but also display necrosis in the head, which is not rescued by p53 MO co-injection (data not shown) [78]. (C) Overexpression of X. laevis SNW1 partially rescues the effects of SNW1 knockdown in zebrafish. Embryos were either uninjected or injected with 7.5 ng of MoZFSNW1, 125 pg of X. laevis SNW1 mRNA, or both. Embryos were cultured until 40 hpf, when they were analyzed for phenotype. MO-injected embryos and embryos injected with both the MO and the rescue mRNA were scored for a dorsalized phenotype (looking only at the extent of posterior structures) as in [71] (righthand graph).
	Fig S5. SNW1 knockdown has no effect on mesoderm induction or gastrulation and does not affect Activin-dependent induction of mesodermal tissue in Xenopus animal caps. (A) One-cell Xenopus embryos were injected with 20 ng of MoSNW1a. Uninjected control and MoSNW1a-injected embryos were fixed at stage 12 for WISH using probes against Xbra, Goosecoid, and Sizzled. (B) Animal caps dissected from MoSNW1a-injected embryos elongate similarly in response to 20 ng/ml Activin (PeproTech) as caps cut from control embryos. Thus mesoderm induction is not affected by injection of MoSNW1a. (C) Zebrafish embryos were injected with 15 ng of either control MO or MoZFSNW1. The embryos were fixed at 12 hpf and stained for the mesoderm markers No tail a (axial and tail), Even-skipped-like 1 (paraxial and tail), and Cdx4 (posterior axial and tail as well as some ectoderm). In SNW1 morphants, the expression of the mesoderm markers is largely unchanged, but there is a slight reduction in their expression in the tail mesoderm. Otx2 is a marker for anterior neural ectoderm and is normal albeit slightly expanded in SNW1 morphants. Otx2 and Cdx4 expression also indicate that anterior/posterior patterning is preserved in the absence of SNW1. In all cases the number of embryos out of the total analyzed that showed the presented staining pattern is given.
	Fig S7. (Zfish). BMP2b is the BMP family member in zebrafish that accounts for the BMP activity at the epidermis/neural ectoderm border that is dependent on SNW1. (A) Comparison of BMP2b, BMP4, and BMP7 expression in zebrafish embryos at 11 hpf (2�3 somite stage) using WISH. BMP2b transcripts are enriched at the epidermis/neural ectoderm border (arrows). In contrast, BMP4 is enriched in the anterior prechordal plate and the tailbud, while BMP7 appears to be expressed in the endoderm. (B) Transgenic BRE-mRFP embryos were injected with 15 ng of control MO or MoZFSNW1. They were fixed at 11 hpf, when WISH was performed for mRFP. SNW1 knockdown results in the strong loss of BMP activity at the epidermis/neural ectoderm border, as seen with mRFP WISH in BRE-mRFP embryos. Notably, some of the posterior mRFP staining is preserved in the SNW1 morphant, which is likely because of BMP4 activity. BMP2b expression, however, appears unaltered in SNW1 morphants, suggesting that SNW1 does not induce BMP activity at the epidermis/neural ectoderm border through the transcriptional regulation of BMP2b. Note that the same batch of injected embryos was stained for mRFP or BMP2b.
	Fig S8. Localized BMP activity is detected at the neural plate border region in Xenopus embryos, and this is dependent on SNW1. Fdx with or without 20 ng of MoSNW1a was injected into one cell of two-cell embryos. They were fixed at stage 13, bisected transversely through the neural crest region, and immunostained with antibodies against p-Smad1 and fluorescein. Nuclei were stained with DAPI. The specific p-Smad1 staining is nuclear and punctate. White arrowheads indicate a domain of p-Smad1 staining in the neural crest region that is lost on the injected side when SNW1 is depleted with the MO.
	Fig S9. siRNA-mediated knockdown of SNW1 in mammalian cells does not affect TGF-β signaling. (A) siRNA-mediated knockdown of SNW1 in mammalian cells does not affect a TGF-β-responsive reporter activity. An MDA-MB-231 cell line stably expressing TK-Renilla and CAGA12-Luciferase was transfected with a non-targeting siRNA (NT), an siRNA pool against SNW1, the four separate siRNAs that make up the pool, or an siRNA against Smad4. 72 h after transfection, cells were induced with 2 ng/ml TGF-β as indicated. Samples were taken for Western blotting after 1 h and for Luciferase/Renilla assays after 8 h. Whereas knockdown of Smad4 diminishes the CAGA12-Luciferase reporter activity, SNW1 depletion does not significantly alter TGF-β-dependent transcription. The efficiency of knockdown is demonstrated in the Western blots shown. (B) SNW1 does not interact with Ski or Smad proteins at endogenous levels in cells. MDA-MB-231 cells were preincubated for 3 h with 50 �M MG132 (Sigma) to prevent Ski degradation in response to TGF-β, before being induced or not with 2 ng/ml TGF-β for 1 h. Cell were then lysed in 150 mM co-IP buffer and immunoprecipitated with the antibodies indicated. A �beads alone� IP served as a negative control (lanes 3 and 4), and 10% of the total lysate used per IP was reserved for input (lanes 1 and 2). The IPs were blotted with the antibodies indicated. Smad2/3 and p-Smad2 as well as Ski can be co-immunoprecipitated with Smad4, as expected [28], while SNW1 was not co-immunoprecipitated (lanes 5 and 6). Immunoprecipitated SNW1 did not co-immunoprecipitate Smad2/3, Smad4, or Ski (lanes 7 and 8). Only Smad2/3, p-Smad2, and Smad4 co-immunoprecipitated with Ski (lanes 9 and 10). The blot for Smad2/3 is a re-probe of the SNW1 blot, and thus the SNW1 signal can additionally be seen on the Smad2/3 blot.

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