Steiner AB , Engleka MJ , Lu Q , Piwarzyk EC , Yaklichkin S , Lefebvre JL , Walters JW , Pineda-Salgado L , Labosky PA , Kessler DS .

Paper published

	Fig. 1. Ectopic axis induction by FoxD3. (A) Control. At the four-cell stage a single ventral blastomere was injected with FoxD3 RNA (100 or 300 pg). Ectopic anterior axial structures, including ectopic eyes, were induced at the high dose (B) and ectopic tails were induced at the low dose (C). Embryos were analyzed at stage 35 by serial-section immunocytochemistry to detect muscle (12/101) (D), notochord (Tor70) (E) and neural tube (4d) (F) (transverse sections, dorsal up; arrowheads indicate stained tissues). Embryos were also analyzed at the early gastrula stage (stage 10.25) by whole-mount in situ hybridization for the expression of Goosecoid (G,H). FoxD3 induced ectopic Goosecoid expression (H) (vegetal views, dorsal up; arrowheads indicate dorsal blastopore lip and arrows indicate region of ectopic gene expression).
	Fig. 5. Mesodermal gene expression is dependent on FoxD3 function. (A-F) Control. At the four-cell stage, each blastomere was injected in the marginal zone with 500 pg of VP16-FoxD3 RNA (G-L), 25 ng of FoxD3MO (M-R), or 25 ng of mismatch MO (S-X). At the early gastrula stage (stage 10.25), embryos were analyzed by in situ hybridization for the expression of the indicated genes. The results shown are representative of three independent experiments (n=12-18 embryos per sample in each experiment). Vegetal views are shown for Brachyury, Chordin, Xwnt8, Mixer and Opl, animal views are shown for Dlx3, and dorsal is up for all panels.
	Fig. 7. FoxD3 is necessary and sufficient for Nodal expression. At the early gastrula stage, Xnr1 (A) and Xnr2 (B) are expressed in two distinct domains: strong expression in the dorsal marginal zone and punctate expression throughout the vegetal pole. In the experiment shown, vegetal expression is more apparent for Xnr2. For FoxD3 gain-of-function, 200 pg of FoxD3 RNA was injected into the marginal region of two blastomeres at the four-cell stage and the expression of Xnr1 (C) and Xnr2 (D) was examined by in situ hybridization at the early gastrula stage (stage 10.25). Ectopic expression of Xnr1 and Xnr2 is indicated with brackets. For FoxD3 loss-of-function, 0.5 ng of VP16-FoxD3 (E,F) or 25 ng of FoxD3MO (G,H) was injected into each blastomere at the four-cell stage and the expression of Xnr1 and Xnr2 was examined. As a negative control, 25 ng of mismatch MO (I,J) was injected. The results shown are representative of three independent experiments (n=20-25 embryos per sample in each experiment). Vegetal views with dorsal side up are shown. (K) At the one-cell stage, the animal pole was injected with FoxD3 RNA (300 pg) and animal explants prepared at the blastula stage (stage 9) were analyzed by RT-PCR at the early gastrula stage (stage 10.25) for the expression of Brachyury (Xbra), Xnr1, Xnr2, Xnr4 and Derriere (Der). PCR controls are as described in Fig. 2. (L) Lysates of FoxD3- or Xnr1-expressing animal explants were examined for the presence of phospho-Smad2 protein by western blotting with a phospho-specific anti-Smad2 antibody. Stripped blots were analyzed for total Smad2/3 proteins as a loading control.
	Fig. 6. Mesoderm induction by FoxD3 is non-cell-autonomous and dependent on the Nodal pathway. (A) At the one-cell stage the animal pole was injected with 100 pg of FoxD3 RNA and animal explants prepared at the early blastula (stage 7) were cultured intact or dissociated into individual cells in the absence of calcium (Disso.). The expression of Brachyury (Xbra), and MyoD was examined in uninjected (Control) and injected explants by RT-PCR at the gastrula stage (stage 11). (B) At the 32-cell stage a single animal pole blastomere was injected with 100 pg of FoxD3 RNA and explants prepared and fixed at the early gastrula stage (stage 10.5) were sequentially examined for Brachyury (Xbra) expression by in situ hybridization and FoxD3 protein expression by immunocytochemistry. To assess the dependence of FoxD3 function on Smad2 and Nodal, FoxD3 (100 pg) was injected alone, or in combination with 1 ng of the Smad2-interaction domain of Fast1 (SID) (C) or 1 ng of a truncated form of Cerberus (CerS) (D). Animal explants prepared at the midblastula stage (stage 9) were collected for RT-PCR analysis of Brachyury (Xbra) at the gastrula stage (stage 11) and Muscle Actin (M. Actin) at the tailbud stage (stage 25). Xnr1 (50 pg) was used as a positive control for the inhibitory activity of SID and CerS. PCR controls are as described in Fig. 2.
	Fig. S1. FoxD3 expression in the Spemann Organizer. Whole-mount in-situ hybridization analysis of FoxD3 at gastrula stages. Vegetal views (dorsal up) of early gastrula (stage 10.25) (A) and mid-gastrula (stage 11.5) (B) embryos are shown. FoxD3 mRNA is expressed in the Spemann Organizer domain of the dorsal marginal zone that converges to the midline and extends along the AP axis during gastrulation. Whole-mount immunocytochemistry analysis of the FoxD3 protein in the gastrula using an affinity-purified anti-Xenopus FoxD3 polyclonal antibody. High-magnification views of the dorsal marginal zone (C) and the ventral marginal zone (D) of a cleared mid-gastrula embryo (stage 11) are shown (animal up). FoxD3 protein is detected in the nuclei of cells of the Spemann Organizer domain, but not in ventral cells. (E) Onset of endogenous FoxD3 expression at the gastrula stage as shown by RT-PCR analysis of Xenopus embryos at the indicated stages. Numerical stage, as defined by Nieuwkoop and Faber (Nieuwkoop and Faber, 1967), is indicated in parentheses. EF1α is a control for RNA recovery and loading. Intact embryos (Embryo) served as a positive control and an identical reaction without reverse transcriptase controlled for PCR contamination (Embryo-RT).
	Fig. S4. FoxD3 rescue of axis formation in embryos injected with VP16-FoxD3 or FoxD3MO. The inhibition of axis formation by VP16-FoxD3 and FoxD3MO is predicted to result from a specific block of endogenous FoxD3 function. To determine the specificity of FoxD3 inhibition, FoxD3 was co-injected with VP16-FoxD3 or FoxD3MO in an attempt to rescue axis formation. At the four-cell stage, both dorsal blastomeres were injected with VP16-FoxD3 or FoxD3MO alone, or in combination with FoxD3 RNA, and axis formation was assessed at the tadpole stage. Whereas the majority of VP16-FoxD3-injected embryos had severe axial defects, only a minority displayed defects with FoxD3 co-injection (C,D). Similarly, the axial defects caused by FoxD3MO were rescued by FoxD3 RNA lacking the antisense target sequence (FoxD3-utr), but not by FoxD3 RNA containing the target sequence (FoxD3+utr) (E,F,H). As controls, injection of both dorsal blastomeres with FoxD3 RNA alone (B) or mismatch MO (G) did not perturb axis formation, by comparison with control (A). See Table 1 for quantification.