Ras-MAPK and Rock synergize to activate MRLC. (A-D) Suboptimal inhibition conditions did not disrupt activation of pMRLC in the leading edge of the pLLp. Suboptimal doses and heat shock conditions were determined empirically by titrating treatments to the highest level at which there was no effect on MRLC activation (0.5 μM PD0325901, 10 μM Rockout, 36°C heat shock of hsp70:dn-Ras transgenics). All treatments were performed for 2 hours, from 28 to 30 hpf. hsp70:dn-Ras embryos were collected and fixed 2 hours following heat shock at 30 hpf. (A2-D2) Rosette formation was not disrupted when Ras-MAPK or Rock activity was blocked suboptimally. Pink arrowheads indicate the leading-most position in pLLp where pMRLC was detected. (E-F2) Combined suboptimal Rockout treatment with suboptimal MAPKK inhibition or suboptimal induction of hsp70:dn-Ras also resulted in failure of activation of MRLC in the leading edge and of formation of leading rosettes. (G) Quantification of pMRLC staining in the different treatment conditions. Percentages were derived by counting the number of cells caudal to the distalmost pMRLC signal (i.e. the number of leading cells that lacked the signal) and normalizing to the total number of cells in the pLLp. Suboptimal treatments or heat shock conditions did not cause a significant change in the pLLp position where MRLC is activated, but combinatorial treatments caused a significant difference (one-way ANOVA, P<0.0001; post-hoc Tukey-Kramer, **P<0.005). Scale bar: 20 μm.
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