Mitchell T , Jones EA , Weeks DL , Sheets MD .

HD43996 NICHD NIH HHS , HL062178 NHLBI NIH HHS , P50 HL062178-050003 NHLBI NIH HHS , R01 GM069944-01 NIGMS NIH HHS , R01 GM069944-02 NIGMS NIH HHS , R01 GM069944-03 NIGMS NIH HHS , R01 GM069944-04 NIGMS NIH HHS , R55 GM056277-01 NIGMS NIH HHS , P50 HL062178 NHLBI NIH HHS , R01 GM069944 NIGMS NIH HHS , R55 GM056277 NIGMS NIH HHS , G19888 Biotechnology and Biological Sciences Research Council , R01 HD043996 NICHD NIH HHS , BB_G19888 Biotechnology and Biological Sciences Research Council

	Figure 1. Chordin expression correlated with the presence of pronephros in ultraviolet (UV) -irradiated embryos. A-E: Fertilized eggs were irradiated with different amounts of UV light before cortical rotation. At stage 10.5, 1/3 of the embryos at each UV dose were analyzed for chordin or goosecoid expression by in situ hybridization. Chordin expression in stage 10.5: DAI5 untreated embryos (A), embryos with an average DAI of 3.0 (B), embryos with an average DAI of 0.5 (C). Goosecoid expression in stage 10.5: DAI5 untreated embryos (D), embryos with an average DAI of 3.3 (E). F-K: Stage 35 embryos representative of each DAI level (5-0). Sibling embryos from each level of UV treatment were scored at stage 35 using the DAI scale and analyzed for pronephros and somitic muscle formation using in situ hybridization for XSMP-30 (blue, black arrowhead) and immunocytochemistry with the 12/101 antibody (brown, red arrowhead).
	Figure 2. Secondary trunks resulting from ectopic chordin expression contained pronephros. One posterior blastomere of four- to eight-cell stage embryos was injected with chordin mRNA. A,B: Embryos analyzed form muscle and notochord using immunocytochemistry 12/101 and Tor70 antibodies. C,D: At stage 33/34, some embryos were analyzed for pronephros (blue, yellow arrowhead) using in situ hybridization to detect XSMP-30 expression and analyzed for somitic muscle (brown, red arrowhead) formation immunocytochemistry with the 12/101 antibody. E,F: Some embryos were analyzed at stage 38-40 for pronephric duct formation using the 4A6 antibodies. The black arrows indicate pronephric ducts in the secondary and primary axis. G: Injected embryos analyzed at stage 38 for pronephric tubule formation using the 3G8 antibodies. The black arrows indicate pronephric tubules in the secondary and primary axis. H: One posterior blastomere of four- to eight-cell stage embryos was injected with noggin mRNA. Embryos were analyzed at stage 38-40 for pronephric duct formation using the 4A6 antibodies. The black arrows indicate pronephric ducts in the secondary and primary axis.
	Figure 3. Depletion of chordin mRNA in Xenopus embryos caused defects in pronephros and muscle formation. A: Blot hybridization of RNA isolated from uninjected control embryos (lane 1), embryos injected with increasing amounts of the control N,N-diethyl-ethylenediamine (DEED) oligonucleotide (lanes 2-4), and embryos injected with increasing amounts of the chordin DEED-antisense oligonucleotide (lanes 5-7). The blot was hybridized with probes to detect chordin and goosecoid mRNA. B-E: Morphology of chordin mRNA-depleted embryos. B: Embryos injected with chordin-antisense DEED oligo. C: Embryo injected with control DEED oligonucleotide. D: Uninjected control embryo. E: Embryo injected with chordin mRNA + chordin-antisense DEED oligo. F-H: Analysis of pronephric duct formation in chordin-depleted embryos using immunocytochemistry with the 4A6 antibody. F: Injected with chordin-antisense DEED oligo. G: Injected with control DEED oligo. H: Uninjected control. I-K: Analysis of pronephric tubule formation in chordin-depleted embryos using immunocytochemistry with the 3G8 antibody. I: Injected with chordin-antisense DEED oligo. J: Injected with control DEED oligo. K: Uninjected control. L-N: Analysis of somitic muscle formation in chordin-depleted embryos using immunocytochemistry with the 12-101 antibody. L: Uninjected control. M: Injected with control DEED oligo. N: Injected with chordin-antisense DEED oligo.
	Figure 4. Ectopic chordin expression promoted anterior somite formation and ectopic myf-5 expression. A,B: A C4 blastomere of 32-cell stage embryos was injected with biotin-labeled mini-ruby dextran chordin mRNA, and embryos were analyzed at stage 32 to detect the injected cells (black, horseradish peroxidase/streptavidin) and somitic muscle (brown, immunocytochemistry with the 12/101antibody). The red arrow indicates the anterior-most dextran-labeled muscle and, therefore, the anterior-most somite contribution from C4 progeny chordin mRNA. C,D: A single C4 blastomere of 32-cell stage embryos was injected with green fluorescent protein (GFP) mRNA chordin mRNA. Gastrulae exhibiting GFP fluorescence opposite the blastopore lip were split into two groups: half was analyzed at stage 10.5 for myf5 expression by in situ hybridization; the other half was analyzed at stage 33 for ectopic trunk formation and by in situ hybridization for pronephros and somitic muscle using XSMP30 and muscle actin probes (Table 5). The red arrow indicates the blastopore lip. The blue arrowhead indicates the normal myf5 expression (C). The black arrowhead indicates the ectopic myf5 expression (D).
	Figure 5. Pronephros formation occurred in association with somitic muscle in explants expressing different amounts of chordin. A-E: Both posterior blastomeres of four-cell embryos were injected with different amounts of chordin mRNA (0-500 pg). A 90-degree wedge of posterior tissue was removed from injected embryos at stage 10+, cultured to stage 33/34, and analyzed for both pronephros using in situ hybridization for XSMP-30 (blue stain, black arrowheads) and somitic muscle (brown stain, red arrowhead) using immunocytochemistry. Some of the explants expressing chordin mRNA occasionally developed a cement gland, and these are marked with an by an asterisk (*). None of the control explants from uninjected embryos ever formed a cement gland. The dashed box shows a magnified view of a single explant from each panel. F: Chordin-expressing explants were analyzed for pronephros (XSMP-30) and muscle (muscle actin) specific gene expression using reverse transcriptase-polymerase chain reaction (RT-PCR).

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