DEV02663 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  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).

• Supplemental Figure 2 -

  Fig. S2. FoxD3 expression constructs. The FoxD3 constructs described in this study were generated by subcloning into pCS2 or pCS2NLS (Rupp et al., 1994). Using the published sequence of Xenopus FoxD3 (Xfd6/Xfkh6) (Dirksen and Jamrich, 1995; Scheucher et al., 1995), RT-PCR was performed to generate a cDNA clone (nucleotides 105-1308) containing the ORF flanked by 67 nucleotides of 5′UTR and 21 nucleotides of 3′UTR. This subclone, referred to as pCS2-FoxD3, pCS2-xFoxD3 or pCS2-FoxD3+utr in this study, was used to generate subsequent FoxD3 constructs. A FoxD3 subclone lacking the 5′UTR (nucleotides 172-1308) was generated by PCR amplification (pCS2-FoxD3-utr). The FoxD3 DNA-binding domain (residues 82-204) was subcloned downstream of a NLS for expression of the FoxD3 winged helix alone (pCS2-NLS-FoxD3WH). The FoxD3 fusion constructs (pCS2-Eng-FoxD3 and pCS2-VP16-FoxD3) were generated by subcloning of the FoxD3 DNA-binding domain (residues 82-201) into pCS2-Eng and pCS2-VP16 (Kessler, 1997). Site-directed mutagenesis of the DNA-binding domain in pCS2-FoxD3, pCS2-Eng-FoxD3 and pCS2-VP16-FoxD3 was carried out with the QuikChange mutagenesis kit (Stratagene) to substitute alanine for glutamine 140 and histidine 144 (N140A/H144A).

• Supplemental Figure 3 -

  Fig. S3. FoxD3 dorsalizes ventral marginal-zone explants. FoxD3 mRNA (total dose 300 pg) was injected into the marginal zone of both blastomeres at the two-cell stage, and, at the early gastrula stage (stage 10.25), the ventral marginal zone was explanted. Cultured explants were harvested at the tailbud stage (stage 25) and analyzed by RT-PCR for the expression of Muscle Actin (M. Actin) and the blood marker αT4-Globin. Whereas uninjected ventral marginal zone explants (Control) expressed αT4-Globin but not Muscle Actin, FoxD3 induced Muscle Actin and suppressed αT4-Globin, consistent with the dorsal mesoderm-inducing activity of FoxD3. 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).

• Supplemental Figure 4 -

  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.