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Supplementary Fig. S1. Temporal and spatial gene expression patterns of VegT during oogenesis and embryogenesis of X. tropicalis. (A)
Temporal expression was analyzed by semi-quantitative RT-PCR. Primer pairs specific for X. tropicalis ODC were described previously (Haramoto et al., 2004). The primers designed for X. tropicalis mVegT are as follows: 5 ATGAGAAACTGCTGTCAGGAACAC-3and 5TGAAACCTGGGCTTGTAGCG- 3 (B-R) Spatial expression was analyzed by WISH. Roman figure shows stage of oogenesis. |
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Fig. 7. Eomesodermin is essential for expression of zygotic VegT. (A) Diagram of the exon-intron structure of Eomes and design of two Eomes splice-inhibiting antisense MOs. The X. tropicalis Eomes gene has 6 exons. The splicing sites targeted by the Eomes MO1 and Eomes MO2 (purple) were the boundary of intron1-exon2 and exon2-intron2, respectively. The light blue arrows indicate the PCR primers to confirm Eomes splicing. (B) Eomes MOs effectively inhibited the proper splicing of Eomes, and suppress expression of zVegT. For these experiments, 12 ng of Eomes MO1 or Eomes MO2 was injected into the marginal zone of both blastomeres at the two-cell stage in X. tropicalis embryos and then harvested at stage 10+. Eomes MO1 or Eomes MO2 effectively inhibited the splicing of Eomes pre-mRNA and the expression of zVegT transcripts. Sequence analysis confirmed that Eomes MO1 and Eomes MO2 caused marked premature termination of Eomes transcripts (arrowheads). Arrows show the band corresponding to normal Eomes transcripts. (C-H) VegT expression is eliminated in the Eomes MOs-injected region. Eomes MOs or control MO (6 ng) and β-gal mRNA (100 pg) were coinjected into the marginal zone of one blastomere at the two-cell stage, and were fixed at stage 10 for WISH. Red gal staining indicates the injected side. Lateral views are shown. |
