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Files in this Data Supplement:
Fig. S1. Sox and Tcf target specificity. RNA encoding the indicated Sox or Tcf (200 pg) was injected into the animal region of two-cell embryos. Animal caps were isolated at stage 9 and assayed at stage 11-12 by real-time RT-PCR. The results are normalized and shown as a ratio to ODC. This experiments was repeated three times and a representative is shown. With the exception that Sox3 and Lef induced Gata6 and Gsc, none of the other HMG box factors reproducibly induced significant expression of Sox17 targets.
Fig. S2. Tcf3 does not regulate Sox17 target genes in vivo. Embryos were injected at the two-cell stage with antisense morpholino oligos to b-catenin (20 ng) or Tcf3 (20 ng), as previously described (Heasman et al., 2000; Houston et al., 2002). At gastrula stage, five embryos from each condition were pooled and assayed by real-time RT-PCR and relative gene expression was normalized and presented as a ratio to ODC. As a control, the expression of the ubiquitously expressed gene plakoglobin was unchanged. As expected, Hex was downregulated by b-catenin depletion, but upregulated by Tcf3 depletion. This is the predicted result observed from all Tcf3-regulated genes (Houston et al., 2002) and is consistent with de-repressing Tcf3 activity. By contrast, the Sox17 target genes Hnf1b, Edd and Foxa2 were not upregulated in Tcf3-depleted embryos but were repressed in b-catenin depleted embryos. This indicates that they are not Tcf3 targets but are b-catenin targets. Consistent with the results presented in the paper, Foxa1 was not significantly regulated by b-catenin levels.
Houston, D. W., Kofron, M., Resnik, E., Langland, R., Destree, O., Wylie, C. and Heasman, J. (2002). Repression of organizer genes in dorsal and ventral Xenopus cells mediated by maternal XTcf3. Development 129, 4015-4025.
Fig. S3. Direct Sox17b targets. This experiment is a repeat of that presented in Fig. 2B, but analyzed by real time RT-PCR to better show the quantitation of the induced transcription. Isolated blastula animal cap explants either injected with mRNA encoding GR:Sox17b (150 pg) or uninjected were each cultured in three conditions; 1´MBS, 1´MBS + 10-6 dexamethasone (DEX) or in 1´MBS with 10-6 dexamethasone + 10 mg/ml cycloheximide (CHX). Dexamethasone activates the GR:Sox17b fusion protein and cycloheximide blocks translation. Control animal caps were treated with 5 ng/ml human activin A either with or without 10 mg/ml cycloheximide. At gastrula (stage 11), the explants were assayed by RT-PCR.
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