Steiner AB et al. (2006), FoxD3 regulation of Nodal in the Spemann organi...

XB-ART-34853

Development 2006 Dec 01;13324:4827-38. doi: 10.1242/dev.02663.

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FoxD3 regulation of Nodal in the Spemann organizer is essential for Xenopus dorsal mesoderm development.

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

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Induction and patterning of the mesodermal germ layer is a key early step of vertebrate embryogenesis. We report that FoxD3 function in the Xenopus gastrula is essential for dorsal mesodermal development and for Nodal expression in the Spemann organizer. In embryos and explants, FoxD3 induced mesodermal genes, convergent extension movements and differentiation of axial tissues. Engrailed-FoxD3, but not VP16-FoxD3, was identical to native FoxD3 in mesoderm-inducing activity, indicating that FoxD3 functions as a transcriptional repressor to induce mesoderm. Antagonism of FoxD3 with VP16-FoxD3 or morpholino-knockdown of FoxD3 protein resulted in a complete block to axis formation, a loss of mesodermal gene expression, and an absence of axial mesoderm, indicating that transcriptional repression by FoxD3 is required for mesodermal development. FoxD3 induced mesoderm in a non-cell-autonomous manner, indicating a role for secreted inducing factors in the response to FoxD3. Consistent with this mechanism, FoxD3 was necessary and sufficient for the expression of multiple Nodal-related genes, and inhibitors of Nodal signaling blocked mesoderm induction by FoxD3. Therefore, FoxD3 is required for Nodal expression in the Spemann organizer and this function is essential for dorsal mesoderm formation.

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Species referenced: Xenopus laevis
Genes referenced: acta4 actc1 actl6a cer1 chrd dlx3 eng foxd3 foxh1 gdf3 gsc mapk1 mixer myod1 ncam1 nodal nodal1 nodal2 smad2 tbxt vegt wnt8a zic1
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	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. EF1Î± 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.

	Fig 3. Functional analysis of FoxD3 fusion proteins. (A) Schematic of the structure of FoxD3 and the FoxD3 fusion proteins. FoxD3 contains a conserved winged helix (WH) DNA-binding domain (residues 92-192). The repressor fusion protein (Eng-FoxD3) contains the repressor domain of Drosophila Engrailed (residues 1-298) fused to the FoxD3 WH domain. The activator fusion protein (VP16-FoxD3) contains the activation domain of HSV VP16 (residues 410-490). Embryos were injected with FoxD3 (100 pg), Eng-FoxD3 (100 pg) or VP16-FoxD3 (250 pg) and animal explants were analyzed at the tailbud stage (stage 25) for morphogenesis (B-E) and by RT-PCR for the expression of Muscle Actin (M. Actin) and Collagen Type II (Col II) (F). Like FoxD3, Eng-FoxD3 induced convergent extension movements and mesodermal gene expression, whereas VP16-FoxD3 did not. (G) Co-expression of VP16-FoxD3 and FoxD3 blocked induction of mesodermal genes by FoxD3. PCR controls are as described in Fig. 2.
	Fig 4. FoxD3 function is required for axis formation. (A) The sequence of FoxD3 flanking the initiator methionine with the sequence of the morpholino antisense oligonucleotide (181-158) highlighted in yellow. (B) Western analysis of animal explants prepared from embryos injected with FoxD3 RNAs (2 ng) alone, or in combination with antisense (FoxD3MO) or mismatch (misMO) morpholino oligonucleotides (50 ng). Translation of a FoxD3 RNA containing the 5â€²UTR and the complete antisense target sequence (FoxD3+utr) was inhibited by FoxD3MO, but not misMO. Translation of FoxD3 lacking the 5â€²UTR (FoxD3-utr) was unaffected by either oligonucleotide. Equal protein loading was confirmed by blotting for the ubiquitous MAPK. (C) RT-PCR analysis of Muscle Actin (M. Actin) induction in animal explants injected with FoxD3 RNAs containing or lacking the 5â€²UTR (200 pg) and FoxD3MO or misMO (50 ng). PCR controls are as described in Fig. 2. (D) At the four-cell stage each blastomere was injected in the marginal zone with FoxD3MO or misMO (25 ng), and extracts prepared at the midgastrula stage (stage 11) were analyzed by western blotting for the accumulation of endogenous FoxD3 protein. A single major band, migrating at the same position as overexpressed Xenopus FoxD3, was detected in uninjected and misMO-injected samples, and was reduced âˆ¼tenfold in FoxD3MO-injected samples. The exposure of the western blot in panel D was approximately eight times longer than that shown in panel B. (E-M) At the four-cell stage each blastomere was injected in the marginal zone with 250 pg of VP16-FoxD3 RNA (H,I), 15 ng of FoxD3MO (J,K) or 15 ng of misMO (L,M). At the tailbud stage (stage 30), embryos were sectioned (transverse, dorsal up) to examine the formation of axial structures, including notochord (nc), somitic muscle (sm) and neural tube (nt) (G,I,K,M). (E) Quantification of the combined results of five independent experiments.
	Figure 8. FoxD3 acts upstream of Nodal in axis formation and mesoderm induction. (A) Control. At the four-cell stage, both dorsal blastomeres were injected with FoxD3MO (25 ng) alone (C), or in combination with 10 pg of Xnr1 RNA (D). At the dose used, injection of Xnr1 alone (B) did not perturb axis formation in most embryos. (E) Quantification of a representative experiment. (F) To assess the dependence of Xnr1 and VegT activity on FoxD3, the animal pole was injected at the one-cell stage embryo with VegT (500 pg) or Xnr1 (100 pg) alone, or in combination with FoxD3MO or mismatch MO (50 ng). Animal explants were analyzed by RT-PCR at the gastrula stage (stage 11) for the expression of Brachyury (Xbra), MyoD, Goosecoid (Gsc), Xnr1 and Xnr2. As controls, the oligonucleotides were injected alone or in combination with FoxD3 RNA (300 pg). PCR controls are as described in Fig. 2.

References [+] :

Agius, Endodermal Nodal-related signals and mesoderm induction in Xenopus. 2000, Pubmed, Xenbase