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Fig. 3 Superficially located cells are specified to express cyt1 by late blastula stages. A: Schematic outline of experiment. Animal caps were dissected from late blastula (sphere stage, 4 hours postfertilization [hpf]) stage embryos and used intact or subdivided into explants enriched for external and internal cells (see Experimental Procedures section) before culture for various periods of time. All explants were cultured in aggregates of 10. At the end of the culture period, explants were harvested and processed for in situ hybridization or reverse transcriptase-polymerase chain reaction (RT-PCR) as described in the methods section. B: Whole-mount in situ hybridization analysis of cyt1 expression. Aggregates of whole animal cap explants (a-d) or deep cell explants (e, f) were cultured for 1 hr (until 5 hpf, equivalent to early gastrula; a), 4 hr (until 8 hpf, equivalent to mid-gastrula; b, e), 9 hr (until 13 hpf, equivalent to early somitogenesis, c), or 18 hr (22 hpf, equivalent to end of somitogenesis, d, f) followed by in situ hybridization for cyt1 expression. Explants cultured for 18 hr in d showed some variability in staining and ~50% were completely covered by cyt1 staining cells. The remaining 50% showed various degrees of covering by cyt1-positive cells, but all showed some staining. None of the internal explants cultured for 4 hr in e showed staining. Although all internal explants cultured for 18 hr in f stained, the intensity of staining was highly variable, as seen in f. C: RT-PCR analysis of cyt1 expression in explants. Aggregates of whole animal cap explants (lane 1), superficial cell explants (lane 2), or deep cell explants (lane 3) were cultured for 4 hr (until 8 hpf, equivalent to mid-gastrula), harvested, and analyzed by RT-PCR for cyt1 expression. Expression of α-tubulin (tub) was used as a loading control.