wnt8a gene structure and generation of an EGFP reporter PAC. A: Schematic diagram (not to scale) of the wnt8a locus within PAC clone 42J21. Line represents the PAC insert. Boxes indicate exons (e.g., e1). Start and stop codons are shown for both wnt8a coding sequences, although we targeted our insertion only into wnt8a.1. B: An outline of the homologous recombination procedure. Targeting DNA contains 50-bp ends homologous to exon 2 upstream of the translation start site and to the second intron (double arrows). Correct recombination results in an insertion of EGFP at the Wnt8a.1 translation start site. Blue boxes represent 50-bp repeated vector sequences that recombine during the second Red-recombination step that removes the chloramphenicol resistance gene (Cm^r). This second Red recombination is stimulated by I-SceI cleavage at its target sequence, indicated by the jagged line. The EGFP coding sequence is followed by the SV40 poly adenylation signal (pA), then the rest of the wnt8a.1 locus. C: Transient EGFP expression from the modified PAC. Lateral view, anterior to the left. Note fluorescence in posterior mesoderm (arrow) and the YSL surrounding the yolk (asterisk).

EGFP fluorescence profile of Tg(wnt8aPAC:EGFP) heterozygotes. A,B: Expression at bud stage, lateral view, anterior to upper left. Arrow indicates tailbud, asterisk indicates anterior neural plate. Note strong fluorescence limited to the posterior embryo. C,D: Expression at <18-somite stage, lateral view, anterior to left. Fluorescence is observed in somites with an increasing gradient toward the tailbud. E,F: Expression at 24 hpf, lateral view. Strong fluorescence continues in somitic mesoderm, with continuing fluorescence detectable around the yolk. Expression is also observed in the heart (G, outlined by arrows) and pronephros (H, arrow). I,J: Comparison of transgene expression in embryos derived from males or females. I: Progeny from a heterozygous male crossed to wild-type female. Fifty percent of embryos fluoresce, 50% do not (non-fluorescing embryos indicated with asterisks). Note easily scored expression in somites of trunk and tail and around yolk. J: In contrast, 100% of progeny from a heterozygous female crossed to a wild-type male fluoresce throughout all tissues.

Transcription profile of Tg(wnt8aPAC:EGFP) compared to wnt8a. A,C,E,G,I,K: In situ hybridization for EGFP transcripts. B,D,F,H,J,L: In situ hybridizations for wnt8a transcripts. A,B: Forty percent epiboly, animal pole view, dorsal right. Both the transgene and wnt8a are expressed in the ventrolateral margin and are excluded from the dorsal margin. Inset in A: Lateral view of EGFP expression at dome-30% epiboly to illustrate YSL expression (arrow). C,D: Shield stage, animal pole view, dorsal right. Note continued expression in the ventrolateral margin and exclusion from the organizer. E,F: Ninety percent epiboly, lateral view, dorsal right. Note expression of EGFP and wnt8a in the embryonic margin. EGFP transcripts are detected in a broader band at the margin, likely reflecting a longer transcript half-life. G,H: Late bud stage, posterior view. Note strong expression of EGFP and wnt8a in the tailbud (arrows) and prospective pronephros (arrowheads, also in inset). EGFP transcripts are also detected in the paraxial mesoderm and adaxial cells (asterisk). Insets: Lateral views, anterior up. I,J: Eighteen-somite stage, lateral view, anterior left. EGFP transcripts are detected strongly in the tailbud, similar to wnt8a. EGFP transcripts continue to be detected in paraxial mesoderm. K,L: 24 hpf, lateral view, anterior left. EGFP transcripts are detected only at the tip of the tail (K) similar to wnt8a (L). Insets: Higher magnification of the tail. wnt8a is expressed at barely detectable levels at the tip of the tail (arrows).

Tg(wnt8aPAC:EGFP) expression depends on Nodal. A–J: In situ hybridizations in control (A,C,E,G,I) or SB-431542 treated embryos (B,D,F,H,J). A–F: Shield stage, animal pole views, dorsal right. A,B: wnt8a levels in the ventrolateral margin are reduced when Nodal signaling is inhibited, and the dorsal clearing is expanded (arrows). C,D: Tg(wnt8aPAC:EGFP) expression responds in an identical way to wnt8a. E,F: In situ hybridization for ntl to confirm effectiveness of SB-431542 treatment. Note dorsal clearing of ntl (arrows), corresponding to the loss of dorsal mesoderm; this phenotype resembles the cyc;sqt double mutant (compare to fig. 6U in Dougan et al., 2003). G–J: Late bud stage, posterior views. G,H: wnt8a expression persists in the tailbud of SB-431542-treated embryos but is not detected in the prospective pronephros. Arrows indicate prospective pronephros expression domain. I,J: Tg(wnt8aPAC:EGFP) expression responds identically to wnt8a. Persistent transcripts are observed in the tail paraxial mesoderm (asterisks). Note that no EGFP transcripts are detected in the position of the prospective pronephros, lateral to the presomitic mesoderm domain (arrow). K–P: The EGFP⁺ lineage forms tail mesoderm in SB-431542-treated embryos. K,L: Control Tg(wnt8aPAC:EGFP) 24-hpf embryo. Note strong expression in trunk and tail mesoderm. M,N: 24-hpf SB-431542-treated Tg(wnt8aPAC:EGFP) embryos. Note that developing tail mesoderm expresses EGFP. O: EGFP transcripts in a control Tg(wnt8aPAC:EGFP) embryo. Note expression in tailbud (arrow) but not in trunk or tail somitic mesoderm. P: EGFP transcripts are also detected in the tailbud of SB-431542-treated Tg(wnt8aPAC:EGFP) embryos (arrow).

Tg(wnt8aPAC:EGFP) expression depends on Ntl/Bra. A–F: In situ hybridizations to detect EGFP transcripts in wild-type (A–C) or ntl;bra morphants (D–F). A,D: Expression at shield stage, lateral view, dorsal right. Note slightly reduced EGFP staining in ntl;bra morphant. B,E: Expression at 90% epiboly, lateral view, dorsal right, anterior up. Note significantly reduced expression in the morphant. Insets: In situ hybridization for wnt8a expression to confirm knockdown. C,F: Bud stage, posterior view, dorsal up. Note significant reduction in EGFP transcripts in the morphant, with slight expression remaining in the tailbud and prospective pronephros (arrows). Asterisks indicate presomitic mesoderm. G–J: EGFP expression at 24 hpf. Note reduced EGFP fluorescence in somitic mesoderm that forms in Tg(wnt8aPAC:EGFP) embryos injected with ntl and bra morpholinos (arrows in H,J), but pronephros expression is not significantly diminished (arrowhead in J). J, Inset: In situ hybridization for EGFP in 24-hpf ntl;bra morphant. Expression is not detected, similar to the behavior of wnt8a.

Tg(wnt8aPAC:EGFP) response to different SB-431542 doses. Top row: Bright field view. Bottom row: EGFP fluorescence. Dose applied is indicated above each column. Note that even at the highest dose of 400 μM, which produces severely abnormal embryos, a domain of EGFP+ mesoderm still forms (arrows).