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Fig. 1. Expression pattern of NRH1a and the design of NRH1 antisense morpholino oligonucleotides (MOs). (A) Expression pattern of NRH1a. RNA was extracted at the indicated stages from animal pole (AP), marginal zone (MZ) and vegetal pole (VP) regions, and the expression of NRH1a and ornithine decarboxylase (ODC) was analysed by real-time RT-PCR and normalised to levels of total RNA, with maximum observed expression defined as 100. Note significant levels of NRH1a expression in animal and vegetal pole regions. The inset shows expression of NRH1a at early gastrula stage 10 analysed by in situ hybridisation. There are high levels of NRH1a expression in the marginal zone, as previously reported (Bromley et al., 2004). (B) Expression pattern of Xbra, analysed and normalised as described for (A). Note that expression of this gene in animal pole and vegetal pole regions is lower than that of NRH1a. (C) The design of antisense morpholino oligonucleotides directed against NRH1 gene products in X. laevis and X. tropicalis. The initiating ATGs of X. laevis NRH1a and NRH1b and of X. tropicalis NRH1 are shown in red, preceded by their 5′ untranslated sequences. The sequences targeted by the indicated antisense morpholino oligonucleotides are coloured blue or green. A control MO differs by four bases (starred) from Xl MOb1. (D) Efficacies of the antisense morpholino oligonucleotides. Embryos were injected with 40 ng of the indicated MOs at one-cell stage, and 500 pg RNA encoding HA-tagged versions of NRH1a or NRH1b was injected at the two-cell stage. Embryos were cultured until mid blastula stage 8, and extracts were subjected to western blotting using an anti-HA antibody. All four X. laevis MOs block the translation of their cognate RNA while the control MO has little or no effect. |
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Fig. 6. Gene expression in embryos lacking NRH1 function. (A–E) Xenopus embryos were injected at the one-cell stage with 80 ng of a control MO or of Xl MOa1 or Xl MOb1. They were then assayed by real-time RT-PCR at stages 10, 11, 13 or 16 for expression of Xbra (A), Chordin (B), Xwnt11 (C), Goosecoid (D) or NRH1a (E). All values were normalised to those of ornithine decarboxylase and are presented as arbitrary units. Note that levels of Xbra, Chordin and NRH1b are substantially reduced by inhibition of NRH1 activity. (F) Scatter graphs summarising the results of four independent experiments investigating gene expression levels in embryos in which NRH1 activity is inhibited. The results show the fold reduction in gene expression caused by NRH1 MOs (such that a value of 1 would indicate no difference between control embryos and embryos injected with Xl MO1a or Xl MOb1). The ratios were calculated at the stage at which each gene was expressed at its maximum level (for example, at stage 11 for Xbra and stage 13 for Chordin). (G) Xenopus embryos were injected at the two-cell stage with 40 ng control MO into one blastomere and 40 ng Xl MOb1 into the other. The control and experimental sides of the embryos were distinguished by co-injection of 4 ng Lissamine-labelled control MO or fluorescein-labelled control MO. Embryos were allowed to develop to early gastrula stage 10 when they were analysed by in situ hybridisation using a probe specific for Xbra. Note that levels of Xbra are lower in the descendants of the blastomere injected with Xl MOb1 (right). Nine experiments of this kind have been carried out, together with five where one side of the embryo was injected with Xl MOb1 and the other left uninjected and six where one side of the embryo was injected with control MO and the other left uninjected. Only in the latter group were levels of Xbra similar in the two halves of the embryo. (H) Western blot of extracts from embryos injected at the cell stage with 80 ng of a control MO or Xl MOb1, probed with an antibody specific for Xbra (upper panel) or GAPDH (lower panel). Note that levels of Xbra are greatly reduced by inhibition of NRH1 protein function. |
