Barnett C , Yazgan O , Kuo HC , Malakar S , Thomas T , Fitzgerald A , Harbour W , Henry JJ , Krebs JE .

R01 EY009844 NEI NIH HHS , EY016029 NEI NIH HHS , EY09844 NEI NIH HHS , P20RR016466 NCRR NIH HHS , R15 EY016027 NEI NIH HHS , P20 GM103395 NIGMS NIH HHS , P20 RR016466 NCRR NIH HHS

	Fig. 2 – WSTF function is required for neural tissue formation in vivo. A: Two-cell stage embryos (dorsal view, anterior at top) were injected with carboxyfluorescein-labeled WSTF MO unilaterally into a single blastomere; fluorescence indicates the injected side. B-C: NCAM expression analyzed by whole mount in situ hybridization at late neurulation (stage 21; B: dorsal view, anterior at top; C: anterior view, dorsal at top). NCAM is normally expressed in the eyes and neural ectoderm. NCAM signal is diminished in the anterior on the WSTF MO-injected side. Asterisk denotes region of greatest loss of NCAM signal. C: Anterior view of embryo in (B), highlighting anterior reduction of NCAM expression in the eye rudiments and neural ectoderm on the injected side. Asterisk denotes loss of NCAM signal. D-E: NCAM expression analyzed by whole mount in situ hybridization at late tailbud stage (stage 35; side views, anterior to left). D: One-cell stage embryos were injected with WSTF-inverse MO (INV) and display normal NCAM expression in neural ectoderm, eye (e) and branchial arches (ba). E: One-cell stage embryos injected with WSTF MO (MO) display reduced NCAM expression at late tailbud stage. Bracket marked by asterisk indicates loss of NCAM staining in branchial arches in WSTF-MO injected embryos (E). Scale bars: 0.5 mm (A–C) and 1 mm (D and E).
	Fig. 3 – SHH, BMP4 and MRF4 are misexpressed in WSTF knockdown embryos. A: BMP4 expression in the neural plate analyzed by whole mount in situ hybridization at late neurulation (stage 17; dorsal view, anterior at top) in embryos injected with WSTF morpholino at the two-cell stage. BMP4 expression is expanded beyond its normal domain on the WSTF knockdown side (left) compared to the uninjected control side (right). B: Whole mount in situ hybridization displaying normal expression pattern of BMP4 compared to global WSTF knockdown embryos at late tailbud stage (stage 40; side view, anterior to left). One-cell stage embryos were injected with either WSTF MO (bottom) or WSTF-inverse MO (INV, top). Inverse MO globally injected embryos display normal BMP4 expression in the hindbrain, developing heart, and faintly in the otic vesicle. Global WSTF knockdowns display decreased BMP4 anterior expression as well as aberrant expression in dorsal tissues. C: Whole mount in situ hybridization displaying normal expression of pattern SHH compared to global WSTF knockdowns at late tailbud stage (stage 36–37; side view, anterior to left). One-cell stage embryos were injected with either WSTF MO (bottom) or WSTF-inverse MO (top). Globally injected inverse MO embryos display SHH expression in the head and notochord. Global WSTF knockdown embryos display decreased SHH expression throughout. D: Global WSTF knockdown embryo (right) displaying cyclopia/holoprosencephaly compared to globally injected inverse MO embryo (left) at stage 45 (dorsal views of heads, anterior at top). Arrows indicate location of eye(s). One-cell stage embryos were injected with either WSTF MO or WSTF-inverse MO. E: Real time RTPCR results for BMP4 and SHH graphed as a percentage of normal expression. BMP4 (stage 37) is significantly over-expressed and SHH expression (stage 15) is significantly reduced in WSTF knockdown embryos injected at the one-cell stage. Data are the average of 3–4 independent experiments and standard errors are shown. F: MRF4 expression in a stage 15 embryo (dorsal view, anterior at top) injected with WSTF MO at the two-cell stage. The injected side (left) displays a significant decrease in normal MRF4 expression in the developing somites. Scale bars: 0.5 mm (A and F) and 1 mm (B–D).
	Fig. 4 – PAX2, EPHA4, and SOX2 expression are reduced in WSTF knockdown embryos. A: PAX2 expression in the midbrain hindbrain boundary (MHB), the otic vesicle, optic stalk and the pronephros analyzed by whole mount in situ hybridization in control embryo at tailbud stage (Stage 33; lateral view, anterior at left) B: Tailbud stage embryo injected with WSTF morpholino at the one-cell stage. PAX2 expression is reduced in all expression domains. C: Side-by-side dorsal view of control embryo (left) with WSTF knockdown embryo (right) at tailbud stage, showing reduced expression of PAX2 in the MHB and the otic vesicle of WSTF knockdown embryo compared to control (Stage 33; dorsal view, anterior at top). D: EPHA4 expression in the forebrain, hindbrain, rhombomeres 3 and 5 and in the pronephros analyzed by whole mount in situ hybridization in control embryo at tailbud stage (Stage 33; lateral view, anterior at left) E: Tailbud stage embryo injected with WSTF morpholino at the one-cell stage. EPHA4 expression is reduced in all expression domains. F: Side-by-side dorsal view of control embryo (left) with WSTF knockdown embryo (right) at tailbud stage showing reduced expression of EPHA4 in the forebrain, hindbrain and rhombomeres 3 and 5 of WSTF knockdown embryo compared to control (stage 33; dorsal view, anterior at top). G-J: Tailbud stage embryos injected with WSTF morpholino at the two-cell stage. G: SOX2 expression in the brain, eye, otic vesicle, lateral line placodes and branchial arches analyzed by whole mount in situ hybridization on the uninjected side at tailbud stage (Stage 32; lateral view, right side of embryo). H: SOX2 expression is reduced in all expression domains on the injected side (left side of embryo). I and J: Dorsal views of two embryos injected at the two-cell stage (WSTF MO injected into the left side in both cases). Embryo in I is at stage 28, embryo in J is stage 32. Both show reduced expression of SOX2 in all expression domains on the injected side. STD: Standard control morpholino; MO: WSTF morpholino; os: optic stalk; br: brain; pd: pronephric duct; hb: hindbrain; pn: pronephros, MHB: mid-hindbrain boundary; ov: otic vesicle; r3, r5: rhombomeres 3 and 5; op: olfactory placode; cp: cranial placodes [otic and lateral line]; ba: branchial arches; e: eye. Asterisks indicate areas of reduced expression relative to controls. Scale bars: 0.5 mm (A, B, D, E, G, H), 0.1 mm (C, F, I, J).
	Fig. 5 – WSTF knockdown embryos display proper neural crest induction, but fail to maintain normal neural crest in later development. A–H: Two-cell stage embryos were injected with WSTF MO unilaterally into a single blastomere and analyzed by whole mount in situ hybridization at neural groove stage (stage 15) to display expression pattern of neural crest genes SNAIL and SLUG. Both genes are expressed at the neural crest boundary. WSTF knockdown has no effect on the stage 15 expression of neural crest genes SNAIL (A) and SLUG (E) (dorsal views, anterior at top). Identically treated embryos analyzed by whole mount in situ hybridization at tailbud stage (stage 30; side views of heads, anterior at top) display perturbed expression of neural crest genes SNAIL (B and C) and SLUG (F and G) on the WSTF-knockdown side (C and G). 60-lm sections through the branchial arches of unilateral WSTF knockdown embryos (anterior at top) display diminished expression of SNAIL (D) and SLUG (H) on the injected side, particularly in the internal structures (light blue staining). Arches are labeled in B–D and F–H. Asterisks indicate the relative position of missing/fused arch number 3 on the sides derived from the WSTF MOinjected blastomeres. Dashed lines in B and F indicate the plane of sectioning of D and H, respectively. Scale bars: 1 mm (A, E), 0.5 mm (B, C, F, G) and 0.1 mm (D, H).
	Fig. 6 – WSTF knockdown embryos display increased apoptosis of specific stages of development. A: TUNEL assay with a representative control embryo at tailbud stage showing no detectable levels of apoptosis (Stage 33; lateral view, anterior at left). B: A representative tailbud stage embryo injected with WSTF morpholino at the one-cell stage displaying a spatial increase in apoptosis in the hindbrain, olfactory placode and lateral anterior tissues. C: Side-by-side dorsal view of control embryo (left) with WSTF knockdown embryo (right) at tailbud stage, showing an increase in apoptosis in the hindbrain of the WSTF knockdown embryo compared to control (Stage 33; dorsal view, anterior at top). D: Transverse section of WSTF knockdown embryo (at a plane indicated by the dashed line in B) displays punctate staining in the peripheral tissues, lateral and ventral from the hindbrain, beneath the overlying epidermal ectoderm (Stage 33; sectional view, anterior at top). STD: standard control morpholino; MO: WSTF morpholino; nt: neural tube; nc: notochord. Scale bars: 0.5 mm (A and B), 0.1 mm (C and D).
	Supplementary Figure 2.

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