Somite hematopoiesis is unaffected in embryos lacking primitive hematopoietic cells. (A-C) Somite-derived blood cells existed in clo mutants. (A) Confocal microscopic observation of somite-derived blood cells in a segment of the ICM in Tg(foxc1b:EOS;gata1a:DsRed);clo mutant and sibling. (B and C) Flow cytometry revealed that the number of DsRed+ cells in clo mutants was significantly reduced (B) whereas the number of EOS+;DsRed+ blood cells was not changed (C). The cell number per embryo for each type was averaged from 10 embryos. **P < 0.01; ns, non-significance (P > 0.05). (D and F) The number of gata1a+ primitive hematopoietic cells was significantly reduced in Tg(gata1a:DsRed) cdx4 morphants as indicated by confocal observation (D) and flow cytometry (F). (D) The ratio of embryos with the representative pattern was indicated. (F) Circulating blood cells taken from a pool of five embryos at 30 hpf were sorted by flow cytometry and the number of DsRed+ blood cells per embryo was calculated. *P < 0.05. The average was based on three independent experiments. (E, G, and H) The generation of somite-derived Red-EOS+ blood cells was unaffected in cdx4 morphants. Tg(foxc1b:EOS;fli1a:gfp) (B), or Tg(foxc1b:EOS) (D and E) embryos were injected with cMO or cdx4-MO at the one-cell stage. Five pairs of nascent somites of the injected embryos were photoactivated at the 20s stage, followed by confocal observation of Red-EOS+ cells (indicated by arrows) in the ICM at the 28s stage (E) and in the heart at 36 hpf (H) and by sorting of Red-EOS+ blood cells (F and G). ns, statistically non-significance (P > 0.05).
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