Fig. 6

Nrz Knockdown Leads to Increased Myosin Phosphorylation at the Margin and Premature Actin-Myosin Contractile Ring Formation

(A) F-actin staining of control embryos (40% and 75% epiboly), nrz morphants (nrzMO), and thapsigargin-treated (5 μM) embryos. In control embryos, the actin-myosin ring is present at 75% epiboly but absent at 40% epiboly (white arrows). In nrz morphants the actin-myosin ring is already present before migrating blastomeres reached the equator. White asterisks materialize the location of vegetal F-actin inside the yolk cell. Scale bar: 100 μm.

(B) Immunofluorescence analysis of the margin region of 50% epiboly stage embryos: staining of phospho-MLC of nrz morphants versus control embryos showing MLC phosphorylation at the margin in nrz morphants, which is correlated with an increase in the F-actin signal (phalloidin staining). Merged images are shown on the right. Treatment of nrz morphants with the MLCK inhibitor ML7 or the calmodulin antagonist W13 inhibits MLC phosphorylation and F-actin polymerization. Thapsigargin-treated embryos were used as positive controls. Scale bar, 20 μm. Representative data from at least three embryos (50% epiboly).

(C) Immunoblot analysis of the phospho-MLC (Ser-19) content in the blastoderm of control embryos, nrz morphants, and embryos treated with increasing amounts of thapsigargin.

(D) Effect of ROCK inhibitor Y26732 (50 μM) and MLCK inhibitor ML7 (20 μM) on the mortality of nrz morphants. Mortality of ML7-treated embryos 7 hpf results in blastomere detachment due to the lack of actin-myosin ring formation. Representative results from three independent experiments are shown. See also Figure S5.