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In the frog, Xenopus laevis, fibroblast growth factor (FGF) signaling is required for both mesoderm formation and the morphogenetic movements that drive the elongation of the notochord, a dorsal mesodermal derivative; the coordination of these distinct roles is mediated by the Xenopus Ctr1 (Xctr1) protein: maternal Xctr1 is required for mesodermal differentiation, while the subsequent loss of Xctr1 promotes morphogenesis. The signaling cascade activated by FGF in the presence of Ctr1 has been well characterized; however, the Xctr1-independent, FGF-responsive network remains poorly defined. We have identified Xenopus Marginal Coil (Xmc) as a gene whose expression is highly enriched following Xctr1 knockdown. Zygotic initiation of Xmc expression in vivo coincides with a decrease in maternal Xctr1 transcripts; moreover, Xmc loss-of-function inhibits Xctr1 knockdown-mediated elongation of FGF-treated animal cap explants, implicating Xmc as a key effector of Xctr1-independent gastrular morphogenesis.
Fig. 1. Microarray analysis of Xctr1 knockdown. Data shown are from a representative Affymatrix gene chip hybridization; signal strength was visualized using Affymetrix GeneChip Operating Software 1.4. Transcripts that register hybridization signal with both probes (âXctr1MO and âXctr1MMâ) are shown in red. Transcripts that register âabsentâ or âmarginalâ hybridization signal with one probe are shown in blue; transcripts that register âabsentâ or âmarginalâ hybridization signal with both probes are shown in yellow. Examples of unaffected (EF1-α) and down-regulated (Muscle actin) transcripts are highlighted (arrows); number = fold change.
Fig. 2. Confirmation of genes with elevated transcription following Xctr1 knockdown. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of gene expression in stage 22 animal cap explants. Injection protocol and/or culture conditions are shown at top. A total of 10 ng/ml basic fibroblast growth factor (bFGF) was added, as shown. A total of 250 ng of Xctr1 morpholino (Xctr1MO) and 250 ng of Xctr1 mismatch morpholino (Xctr1MM) were injected, as shown. The induction by HoxB9, a marker of lateral plate mesoderm and spinal cord at this stage, demonstrates that FGF is active in this experiment (Wright et al.,1990). The ââRTâ lane contains all reagents except reverse transcriptase and was used as a negative control. Ornithine decarboxylase (ODC) is used as a loading control (Bassez et al.,1990).
Fig. 3. Regulation of Xmc by Xctr1. A: Xmc expression is suppressed by Xctr1. Microarray analysis of gene expression in embryos injected with RNA encoding either Xctr1 or β-galactosidase. Left column: genes that are expressed more highly in fibroblast growth factor (FGF) treated caps taken from Xctr1MO injected embryos than in FGF treated caps taken from Xctr1MM injected embryos. Right column: Relative expression of genes from left column in animal caps from embryos injected with either Xctr1 or β-gal RNA. Up-regulated genes are shown in red; down-regulated genes are shown in green. Arrows point to two transcripts that are strongly down-regulated by Xctr1 (top: 38%, bottom: 28% of signal in β-galactosidase-injected control samples); both transcripts encode Xmc. BâE: Reverse transcriptase-polymerase chain reaction (RT-PCR) confirmation of microarray data presented in A. B: Xmc expression is elevated following Xctr1 knockdown in FGF-treated animal caps. C: Xmc expression is elevated following Xctr1 knockdown in animal caps cultured in saline. D: Xmc expression is suppressed following Xctr1 misexpression in animal caps. E: Xctr1 does not inhibit Xmc induction by either FGF or Activin protein. Ectopic Xctr1 inhibited Activin-mediated cap elongation in these assays (data not shown). A total of 10 ng/ml bFGF, 0.5 ng/ml Activin was added, as shown. A total of 250 ng of Xctr1 morpholino (Xctr1MO) and 250 ng of Xctr1 mismatch morpholino (Xctr1MM) were injected, as shown. Also, 1 ng of Xctr1 and 1 ng of β-galactosidase RNA was injected, as shown.
Fig. 4. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of Xmc expression during early Xenopus development. Xmc expression is zygotic, and coincides with diminishment of maternal Xctr1 transcripts.
Fig. 5. XmcMO inhibits translation of 3ⲠMyc-tagged Xmc RNA (Xmc-Myc) containing the XmcMO binding site. Xmc-Myc translation is not inhibited by co-injection of a five base pair mismatch Xmc morpholino (XmcMM), or a control scrambled morpholino (CMO). Western blot of protein lysates from stage 10 Xenopus embryos, injected as listed, using anti-Myc and anti-β-tubulin antibodies, as shown (Sigma).
Fig. 6. Effects of Xmc loss-of-function on explant morphogenesis. A: Injection of morpholinos against Xmc (XmcMO) inhibits Xctr1 knockdown-mediated morphogenesis in fibroblast growth factor (FGF) -treated animal cap explants at stage 22; this effect is rescued by co-expression of Xmc RNA (Xmc) that lacks the XmcMO binding site. BâE: Quantification of the requirement for Xmc on explant morphogenesis. Error bars represent the standard error of the mean. y-axis = ratio of cap length:cap width. B: Xctr1 knockdown increases elongation of FGF-treated animal caps. For FGF, N = 17; for FGF/Xctr1MO, N = 19, with N = number of caps assayed. C: Xmc knockdown inhibits Xctr1 knockdown-mediated elongation. For FGF/Xctr1MO, N = 19; for FGF/Xctr1MO/XmcMO, N = 18. D: XmcMM does not inhibit Xctr1 knockdown-mediated elongation. For FGF/Xctr1MO/XmcMO, N = 25; for FGF/Xctr1MO/XmcMM, N = 26. E: Suppression of Xctr1 knockdown-mediated elongation by XmcMO is rescued by co-expression of Xmc RNA lacking the XmcMO binding site (Xmc). For FGF/Xctr1MO/XmcMO, N = 58; for FGF/Xctr1MO/XmcMO/Xmc, N = 57. F: Xmc knockdown does not affect Activin-mediated elongation. For Activin, N = 8; for Activin/XmcMO, N = 9. *P < 0.005; **P < 0.01. A total of 10 ng/ml bFGF, and 0.5 ng/ml Activin was added, as shown. Also, 2 ng of Xmc RNA, 250 ng of Xctr1MO, 25 ng of XmcMO, and 25 ng of XmcMM were injected, as shown.
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