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Fig. 2. Localization of SOX7 RNA. In situ hybridization of longitudinally bisected blastula stage embryos reveals the animal hemisphere concentration of SOX3 (A) RNA, while SOX17 RNA is localized to the vegetal region (B). While staining for SOX7 RNA was consistently quite weak, it was clearly restricted to the vegetal hemisphere (C). Embryos at stage 8 were dissected into animal caps (AC) and vegetal masses (VM) (indicated by white line in part A) and then analyzed for SOX7 or SOX11 mRNA by RT-PCR (D) either immediately (st8) or after they had been cultured until control embryos reached stage 16 (st16). Primers for ornithine decarboxylase (ODC) were used as a control for cDNA quality; the absence of reverse transcriptase (-RT) was used to control for the presence of contaminating DNA. At both time points, SOX7 mRNA was restricted to the vegetal mass; under similar conditions, SOX11 mRNA was restricted to the animal hemisphere. (E) A second set of SOX7-specific primers (Table 1 “SOX7 coding”) was used to confirm the vegetal localization of SOX7 RNA. Animal caps and vegetal masses were prepared at stage 8 and analyzed either immediately (“1”), after 2 h (“2”), or when control embryos had reached stage 27 (“3”); these primers amplified a fragment of the expected size (1kb) that was again restricted to the vegetal mass. |
