Requirement for Ntl and Spt for expression of Nodal ligands and antagonists around Kupffer's Vesicle. A-F: Arrowhead: perinodal regions flanking Kupffer's Vesicle. Arrows: spaw expression in the LPM. s: somites stage. A,B: Wild-type sibs and ntl^b160 homozygotes from a ntl^b160 incross, respectively; 7-10s. Dorsal view of tailbud. Dig-spaw probe. C,D: Wild-type sib and ntl^b160 homozygotes; 16-19 s. Lateral view, tail to bottom left. Dig-spaw probe. E: Sixteen to 18 s. Dorsal view, tailbud to bottom right. Dig-spaw probe. Left: Injected with 12 ng Gene Tool's Negative Control Morpholino (NC-MO). Right: Injected with 8 ng spt MO2. F,G: Sixteen to 19 s. Dig-spaw probe. F: Dorsal view of tailbud. Left: Injected with 4 ng spt MO1. Right: Injected with 8 ng 5-mis-spt MO1. G: Lateral view, tail to left. Left: Injected with 4 ng spt MO1. Right: Injected with 8 ng 5-mis-spt MO1. H-J: Progeny of a ntl^b160 incross; 7-10 s. DNP-ntl (red) + Dig-cha (purple, arrowhead) probes. Dorsal view of tailbud. H: Wild-type sib. I,J: ntl^b160 homozygotes. K: Eleven to 12 s. Dorsal view. Dig-cha probe. Left: Injected with 4 ng ntl-1 MO (high dose ntl-1 MO). Right: Uninjected counterpart. L: Eight to 9 s. Dorsal view of tailbud. Dig-cha probe. Left: 8 ng NC-MO; Middle: 8 ng spt2 MO; Right: Uninjected. M: Eight to 9 s. Dig-cha probe. Left: 4 ng spt MO1. Right: 8 ng 5-mis-spt MO1. N,O: Eight to 11 s. Dig-cha probe. N: Injected with 8 ng spaw-1 MO. O: Injected with 8 ng NC-MO. P: Seventeen to 21 s. Dig-spaw probe. Dorsal view of tail. Arrowhead: perinodal spaw expression. Left: Injected with 1 ng lrdr1 MO. Arrow denotes reversed (right-side) spaw expression in the LPM. Right: Injected with 8 ng NC-MO. Q: Twelve to 15 s. Lateral view, head to top. Dig-krox20 (hindbrain) plus Dig-cha probe (arrowhead). Left: Injected with 1 ng lrdr1 MO. Right: Injected with 8 ng NC-MO.

Characterization of low-dose ntl morphant embryos. A,B: Seven to 12 s, dorsal view. From a ntl^b160 incross. Arrowhead: notochord. A: Wild-type sib. B: ntl^b160 homozygote. C,D: Sixteen to 19 s, lateral view. n+s: ntl + spaw probes. Arrowhead: ntl mRNA in the notochord. C: Injected with 0.3 ng ntl-1 MO. D: U: Uninjected. E,F: Sixteen to 19 s, lateral view. sonic hedgehog (shh; arrowhead) + spaw probes. E: Injected with 0.2 ng ntl-1 MO. F: U: Uninjected. G-L: Arrows: Site of pdi-p5 expression in posterior notochord. Arrowhead: site where cha is normally expressed. G,H: Ten to 14 s. Dorsal view, close-up of tail and tailbud. Head to upper right. Two embryos from a ntl^b160 incross. pdi-p5 probe. I-L: Dorsal view of tailbud/KV region, head to lower right. pdi-p5± cha probes. Asterisk: pdi-p5 staining in anterior (out of field of view in J). I-K: Nine to 12 s. L: Seven to 9 s. I: Injected with 0.1 ng ntl-1 MO. J: Injected with 4 ng of ntl-1 MO. K: Injected with 4 ng NC-MO. L: U: Uninjected. M,N: Sixteen to 19 s, dorsal views of the same embryos shown in Figure 2C and D, respectively. Head at lower left, ntl + spaw probes. Arrows: spaw mRNA in lateral plate. M: Injected with 0.3 ng ntl-1 MO. N: U: uninjected. O,P: Nine to 12 s. Brightfield view of tailbud/KV region, live embryos. KV± denotes presence or absence of a visible Kupffer's Vesicle. Neg. MO: scrambled antisense cha MO. O: Injected with 0.85 ng Neg. MO. P: Injected with 0.5 ng ntl-1 MO.

charon expression in low-dose ntl morphants. A,B: Nine to 12 s. Dig-cha + DNP-ntl probes. Arrow: Notochord. Arrowhead: Site where cha is normally expressed. Developed for cha staining (left images, shown in dorsal view with tailbud to upper right) then redeveloped to show ntl staining (right images, shown in lateral view with tailbud to lower right). A: U: uninjected embryo. In right image, the ntl signal in the tail region obscures prior cha staining. B: Injected with 0.1 ng ntl-1 MO.

The chaTBE fragment drives KV expression of a GFP reporter. A,D: Endogenous charon expression. Reproduced from Hashimoto et al. ([2004]) with permission of the publisher. A: Six s embryo, vegetal view. D: Fourteen s, lateral view. Head to left. B, B′, C: Various views of the same pchaTBEd2eGFP-injected embryo. B: Eight s Bright field + fluorescence. Vegetal view. Red arrowhead points to Kupffer′s Vesicle (KV). B′: An enlarged image of the KV region of B. Red asterisk marks KV center. Red arrowheads point to d2eGFP-positive cells. C: Same embryo at 15 s. Head to left. Bright field + fluorescence. Red arrowhead: d2eGFP expression like cha (5D). E/E′, F/F′: Two pchaTBEd2eGFP-injected embryos from a separate experiment from that shown in B,C. Lateral view with superimposed bright field plus fluorescence. Yellow and black arrows: GFP fluorescence. Red arrowhead: KV. E: Fourteen-somite s. E′: Same embryo as in E. Enlarged view of tailbud/KV region. F: Twelve-somite s. F′: Same embryo as in F. Enlarged view of tailbud/KV region. G, G′, G′: Tailbud/KV region of a pchaTBEd2eGFP-injected embryo at 12 somites, shown in lateral, tilted, and dorsal views, respectively. Two-color RNA in situ with Dig-gfp and DNP-charon probes. In this embryo, the gfp (BM Purple) signal overlaps with the left half of the endogenous charon mRNA domain (INT Red/BCIP).

Effects of the γ-secretase inhibitor DAPT on charon and perinodal southpaw expression. A-F: Arrowheads: tailbud/KV region. Dig-cha probe. +DAPT: treated with 100 μM DAPT dissolved in DMSO. +DMSO: treated with 1% DMSO. A: Two to 3 s DAPT-treated embryo with reduced charon expression relative to DMSO control. B: Four to 6 s. Dorsal view of Kupffer′s Vesicle (arrowhead). C,D: Examples of mid-somite stage embryos, treated with DMSO alone or with DAPT. E,F: Enlarged views of embryos from C,D sets. E: Example of DAPT-treated embryo with no charon expression. F: Example of DAPT-treated embryo with reduced charon expression. G,H: Seventeen to 18 s. Dig-spaw probe. G: Dorsal view, head to upper right. Arrows: spaw expression in the LPM. H: Dorsal view of tail. Arrowheads: perinodal spaw.