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First published online 8 November 2006
doi: 10.1242/dev.02663

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

Aaron B. Steiner, Mark J. Engleka, Qun Lu, Eileen C. Piwarzyk, Sergey Yaklichkin, Julie L. Lefebvre, James W. Walters, Liliam Pineda-Salgado, Patricia A. Labosky^* and Daniel S. Kessler

Department of Cell and Developmental Biology, University of Pennsylvania School of Medicine, 1110 BRB 2/3, 421 Curie Boulevard, Philadelphia, PA 19104, USA.

View larger version (71K): [in this window] [in a new window]   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).

View larger version (66K): [in this window] [in a new window]   Fig. 2. Mesoderm induction by FoxD3. At the one-cell stage, embryos were injected in the animal pole with 200 pg of Xenopus FoxD3 (xFoxD3) or mouse FoxD3 (mFoxD3), explants were prepared at the late blastula stage (stage 9), and explants were analyzed for morphogenesis, tissue differentiation and gene expression. At the tailbud stage (stage 25), convergent extension movements were observed in response to xFoxD3 and mFoxD3 (A-C), and differentiated somitic muscle was detected in the FoxD3-expressing explants (D-F) using a muscle-specific antibody (12/101). (G-I) Explants stained with 12/101 were sectioned and counterstained (H&E) to show the presence of somitic muscle (sm), notochord (nc) and neural tube (nt) in FoxD3-induced explants. (J) Gene expression in explants was examined by RT-PCR for Brachyury (Xbra), Goosecoid (Gsc) and Xwnt8 at the midgastrula stage (stage 11), and for Muscle Actin (M. Actin) and NCAM at the tailbud stage (stage 25). EF1 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).

View larger version (65K): [in this window] [in a new window]   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.

View larger version (70K): [in this window] [in a new window]   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 5'UTR and the complete antisense target sequence (FoxD3+utr) was inhibited by FoxD3MO, but not misMO. Translation of FoxD3 lacking the 5'UTR (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 5'UTR (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 tenfold 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.

View larger version (104K): [in this window] [in a new window]   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.

View larger version (70K): [in this window] [in a new window]   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.

View larger version (90K): [in this window] [in a new window]   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.

View larger version (81K): [in this window] [in a new window]   Fig. 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.