A Second Zebrafish brachyury Gene

(A) Neighbor joining tree of vertebrate Brachyury proteins, with Ciona intestinalis (Ci) serving as the outgroup. Bootstrap percentages are shown for 1000 trials. The other organisms are human (Hs), mouse (Mm), chicken (Gg), Xenopus tropicalis (Xt), Xenopus laevis (Xl), fugu (Tr), stickleback (Ga), medaka (Ol), and zebrafish (Dr).

(B–J) Expression of bra from 50% epiboly to 24 hpf.

(B and C) At 50% epiboly, expression is restricted to the dorsal side of the margin (B) as shown by costaining with gsc (arrowhead) at shield stage (C).

(D and E) At 60% epiboly, expression encompasses the entire margin and the forming axial mesoderm.

(F) By bud stage, expression is present throughout the notochord (N) and in the bud.

(G) There is a brief period of expression in the ventral mesoderm around the eight-somite stage (arrow).

(H–J) Between 14 somites and 24 hpf, expression is restricted to the posterior notochord and the mesodermal precursors of the tailbud, as well as cells ventral to the posterior notochord (arrowhead).

Embryos are shown from an (B and C) animal view with dorsal to the right, (D) vegetal view with dorsal to the right, (E) dorsal view with animal pole to the top, (F) dorsal view with anterior to the left, (G) posterior view with dorsal to the top, and (H–J) lateral view with anterior to the left.

Bra Knockdown Enhances the ntl Phenotype

(A) A schematic of Bra knockdown strategies. MO1 targets the bra splice acceptor site at the intron 1-exon 2 junction. Primers in the first and third exons were used to analyze the bra transcript in uninjected (UC) and MO1-injected embryos. The improperly spliced bra transcript results in a truncated protein that lacks the T box. MO2 targets the translation start site of bra.

(B–E) Expression of myoD at 24 hpf in an (B) uninjected +/+ or +/- sibling, (C) uninjected ntl mutant, (D) MO1-injected ntl mutant, and (E) MO2-injected ntl mutant.

(F–I) Expression of tbx6 in four-somite embryos.

(J–Q) Expression of ntl at 75% epiboly and bud. Arrows and arrowheads show diminished notochord expression.

Embryos are shown from a (B-E) lateral view with anterior to the left, (F-I) posterior view with dorsal to the top, (J-M) dorsal view with animal pole to the top, and (N-Q) dorsal view with anterior to the left.

A Non-Cell-Autonomous Requirement for ntl/bra

(A–D) Confocal images of WT host embryos transplanted with (A) WT cells, (B) MO2 morphant cells, (C) ntl^-/- cells, or (D) ntl^-/- + MO2 cells. White arrowheads indicate the trunk/tail boundary. The horizontally elongated cells are muscles of the tail somites.

(E–J′) WT donor cells labeled with fluorescein dextran (green) were transplanted into unlabeled ntl/bra morphant host embryos. Somites were visualized with the MF20 antibody (red). Host embryos with WT donor cells in the mesodermal precursor region (I–J′) exhibited a nonautonomous rescue of some posterior somites, which was not seen when WT donor cells contributed to nonmesodermal tissues (G–H′) or in donorless mutants (E and F). White arrows in (E), (G), and (I) point to the 11th somite, which is the last somite to form in the embryo pictured in (G). Somites posterior to the white arrow in (I) represent rescued somites.

All embryos are shown from a lateral view with anterior to the left, except for (F), (H), (H′), (J), and (J′), which are dorsal views of the same embryos shown in adjacent panels.

Wild-Type Cells Nonautonomously Rescue tbx6 Expression in Embryos Lacking Ntl/Bra

Cells from wild-type embryos injected with fluorescein dextran were transplanted into unlabeled host embryos coinjected with MO2 and the ntl MO. At the indicated stage, host embryos were separated into groups based on the presence or absence of donor cells in the prospective posterior mesoderm (the tailbud at the four-somite stage) and stained for tbx6 (A–B′) or wnt8 (C and D). Host embryos containing donor cells in the prospective posterior mesoderm show expanded tbx6 expression (B) and patches of wnt8 expression (arrows in [D]).

(B′) A merged image of tbx6 expression with fluorescence from donor cells shows that tbx6 expansion occurs in both an autonomous and nonautonomous manner.

Embryos in (A), (B), and (B′) are dorsal views of flat-mounted embryos with anterior to the left, while those in (C) and (D) are vegetal views with dorsal to the right.

Paraxial FGF Signaling Is Maintained in ntl/bra Embryos

(A–P) pea3 and (Q–T) fgf8 expression at the indicated stages in uninjected and MO2-injected wild-type siblings and ntl mutants. Embryos are shown from an (A–D) animal view with dorsal to the right, (E–H) vegetal view with dorsal to the right, and (I–T) posterior view with dorsal to the top. Arrow marks the posterior notochord.

Ntl/Bra Maintains wnt8 and wnt3a Expression

(A-P) wnt8 and (Q-X) wnt3a expression at the indicated stages in uninjected and MO2-injected wild-type siblings and ntl mutants. Embryos are shown from a (A–D) ventral view with animal pole to the top, (E-H) vegetal view with dorsal to the right, and (I-X) posterior view with dorsal to the top. Arrowheads mark wnt expression domains. What appears to be very light staining around the edge of the embryo in (H) is caused by an edge effect and is not specific.

Ntl Regulates wnt8 and wnt3a Expression without Intervening Protein Synthesis

(A-F) The expression of wnt8 and (G-J) wnt3a in control EtOH-treated or dex-treated embryos injected with ntl-GR mRNA.

(B) Continuous treatment with dex until shield stage increased wnt8 expression compared to (A) controls.

(C-F) Chx treatment at shield stage followed by dex treatment for 1 or 2 hr also increased wnt8 expression.

(G-J) Chx treatment at 75% epiboly followed by 2 hr of dex treatment increased wnt3a expression. Control embryos were treated with EtOH (A) or EtOH plus chx (C, E, G, and I).

(A)-(H) are ventral views, animal pole at top; (I) and (J) are vegetal views.

Ntl/Bra Functions in an Autoregulatory Loop with Canonical Wnt Signaling

(A–H) Twenty-four hours postfertilization, embryos stained for myoD RNA expression from (A–G) the HS::dkk1-GFP line or (H) the ntl mutant. Temperature shifts (HS) were performed on hemizygous embryos at the indicated stage and then embryos were sorted based on GFP expression.

(B, C, and G) GFP⁺ embryos shifted at 75% epiboly phenocopy ntl/bra loss-of-function embryos.

(A–E) Lateral views; (F–H) dorsal views at the posterior end of the myoD-expressing region.

(I–L) Posterior view of four-somite (4s) HS::dkk1-GFP outcross embryos temperature shifted at 75% have a loss of (J) ntl and (L) bra expression in the mesodermal precursors of GFP⁺ embryos (arrowheads).

(M–P) Lateral view of HS::ΔTCF-GFP outcross embryos temperature shifted at the eight-somite stage and fixed 30 min afterward have a loss of (N) ntl and (P) bra expression in GFP⁺ embryos.

(Q) A schematic of a somitogenesis-stage embryo shown from a dorsal view with anterior to the top, illustrating two different functions of ntl and bra during mesoderm development. In the notochord (red), ntl and bra are involved in a putative autoregulatory loop with fgfs and are upstream of wnt11 and flh, whereas in the paraxial mesodermal precursors (green), ntl and bra function in an autoregulatory loop with canonical Wnts to maintain precursors throughout somitogenesis. Canonical Wnts also regulate the presomitic mesoderm.

bra Expression is Initiated and Maintained in Mesodermal Precursors of ntl -/- Embryos
(A–F) bra expression at the indicated stages in wildtype siblings and ntl -/- embryos shows proper initiation of bra in the margin and continued expression in the mesodermal precursors of ntl -/- embryos (arrows). Expression of bra is lost in the notochord of ntl -/- embryos (arrowheads). Embryos are shown from a (A, B) dorsal view with the animal pole to the top, (C, D) vegetal view with dorsal to the right, and (E, F) posterior view with dorsal to the top.

Muscle Morphology of Transplanted Cells
High magnification images of embryos shown in Figure 3 reveal elongated myotube morphology of transplanted cells (red) in tail somites. Nuclei are stained with DAPI (blue). All embryos are shown from a lateral view with anterior to the left.

pea3 Expression is Maintained in HS::ΔTCF-GFP Embryos
HS::ΔTCF-GFP outcross embryos temperature shifted at 8-somites and fixed 30 minutes afterwards show strong expression of pea3 in the tailbud of both GFP- and GFP+ embryos (A, B, arrows). This indicates that tailbud FGF signaling is normal in these embryos and therefore a loss of FGF signaling cannot account for the loss of ntl and bra expression in HS::ΔTCF-GFP embryos (Figure 7N, P). Embryos are shown from a lateral view with anterior to the left.

bra mRNA Rescues wnt8 Expression in MO1 Injected ntl -/- Embryos
(A–D) bra-GR mRNA and MO1 were co-injected into embryos from a ntl heterozygous intercross. Injected embryos were treated with (A, B) EtOH or (C, D) dex, fixed at 70% epiboly, and stained for wnt8 expression. (D) In the presence of dex, bra-GR mRNA rescued wnt8 expression in embryoS lacking Ntl and Bra function (compare to B, 100% rescued, N=9).