Fig. 6

lpp genetically interacts with scrib and trilobite for the regulation of C&E during zebrafish gastrulation. (A) GST fusion proteins, as indicated, and GST alone were expressed in E. coli, purified and analyzed by SDS-PAGE and GelCode Blue Stain Reagent staining. All proteins were expressed well. Protein markers are as indicated. (B) All four PDZ domains of wild-type zebrafish Scrib (AA 700–1517) were synthesized in vitro and ³⁵S-labeled. Labeled proteins were incubated with immobilized GST, GST-Lpp-LT_WT or GST-Lpp-LT_L557A and allowed to interact overnight at 4 °C. Bound proteins were eluted in sample buffer, separated by SDS-PAGE and visualized by autoradiography. The amount of synthesized protein loaded as a reference on the gel corresponds to 10% of the input used in each binding experiment. (C) Suboptimal doses (s.o.) of Lpp-MO/ATG (1 ng) or Scrib-MO (1 ng) were injected separately or were co-injected in the one- to two-cell stage; the phenotypes were scored at the five-somite stage and compared to embryos injected with optimal doses (o.) of either of the morpholinos (Lpp-MO, 5 ng; Scrib-MO, 5 ng). Phenotypes scored were: normal, C&E defects, and defects other than C&E defects (“other defects”). (D) Suboptimal doses (s.o.) of Lpp-MO/ATG (1 ng) or Tri-MO (0.25 ng) were injected separately or were co-injected in the one- to two-cell stage. The phenotypes were scored at 11 hpf and compared to embryos injected with optimal doses (o.) of either of the morpholinos (Lpp-MO, 5 ng; Tri-MO, 4 ng). Phenotypes scored were: normal or mild C&E defects (C&E+/-), moderate (C&E+) or strong (C&E++) C&E defects, and defects other than C&E defects (“other defects”). Pictures in panels C and D show for each condition embryos representative for the phenotype of the largest group(s). Abbreviations: NI, uninjected. n, total number of embryos per condition from two independent experiments.