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Files in this Data Supplement:
Fig. S1. Wnt/β-catenin-mediated inhibition of cardiogenesis in Xenopus is mediated by GATA4/6 function. Analysis of gene expression using quantitative RT-PCR for Nkx2.5 and the cardiomyogenic markers MLC2 and TnIc in whole embryos at stage 32. Embryos were injected at the four-cell stage into the marginal zone of both dorsal blastomeres with β-cateninGR mRNA (βGR, 60pg per embryo) alone or together with GATA4GR (G4GR) (A) or GATA6GR (G6GR) (B) mRNA (100 pg per embryo), as indicated, and were cultured from stage 20 in dexamethasone (where indicated) to activate the injected constructs. Embryos were collected at stage 32 of development and total RNA was extracted from 15 embryos each using the RNeasy RNA Extraction kit (Qiagen). Synthesis of cDNA from this RNA was carried out using QantiTect Reverse Transcription kit (Qiagen). The cDNA was then analysed for relative fluorescence to ODC using Dynamo hotstart SYBR green enzyme mix (finenzymes) and analysed on an Opticon II Qpcr machine. Relative expression of the described cardiac markers were normalised to the housekeeping gene ODC and measured in comparison with control unmanipulated embryos normalized to equal 1 relative expression unit. Activation of β-catenin reduces the expression of the cardiomyogenic-specific markers MLC2 and TnIc more strongly than Nkx2.5, which is not exclusively expressed in the cardiac mesoderm targeted by our injections, but that cardiogenic gene expression is rescued by concomitant activation of GATA4GR or GATA6GR (comepare with Fig. 1C). UI, uninjected control.
Fig. S2. GATA4 and GATA6 differentially induce cardiomyogenesis in animal cap explants. RT-PCR analysis of gene expression of control stages 24 (A) and 32 (B) in animal cap explants injected with GATA4GR or GATA6GR mRNA (G4GR, G6GR, 100 pg) as indicated. Dexamethasone was added at stages 8 or 22 where indicated. G4GR is consistently the stronger inducer of cardiogenic marker gene expression when activated at early stages, whereas G6GR the relatively stronger inducer when activated at later stages (see also Fig. 1, Fig. 4).
Fig. S3. Expression profile of XWnt11. (A) Whole-mount in situ hybridisation with a XWnt11 probe and (B) in situ hybridisation on serial sections of stage 10.5 embryos using the same probe. At that stage, expression is not next to the dorsal blastopore lip (arrowhead) but further animal to it overlapping with that of the previously described GATA expression. RNA in situ hybridisation on whole-mount and serial cross-sections were as previously described (Afouda et al., 2005).
Movie 1. Non-beating animal cap from uninjected explants at control stage 45. None of the explants shows rhythmic movements.
Movie 2. Beating animal cap induced by injection of GATA4 mRNA and activated at stage 8. The explant shows rhythmic beating movements and the movement involves the entire explant.
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