In the cdh1rk3 mutant, defective adhesion between EVL and DEL cells was suggested to lead to delayed epiboly [4].
In addition, a very dynamic change in cell shape was observed in DEL cells of control embryos, but not in those of cfl1 morphants (Supplementary Movie S2).
As for the RNA in-situ analysis, uniform Chp immuno-staining was also observed in the EVL, the deep layer (DEL) cells and in the YSL (Figure 2C).
Based on the nuclei position in confocal analysis, the DEL cells were depleted from the leading edge region at 8 hpf (Figure 3B) relative to YSL and EVL nuclei.
In our studies, Chp knock down leads to a delayed migration of the YSL and EVL but an absence of DEL cells is noted at the migrating front (Figure 3), although the leading edge actin is essentially normal.
In normal embryos, cells were counted in regions corresponding to endogenous chick ets-1 expression during (n = 9; n(mid cells) = 1479; n(sur cells) = 1904) and after delamination (n = 6; n(del cells) = 624; n(mid cells) = 659; n(sur cells) = 1037) of cranial NCCs.
In L858R and E746-A750 del cells, the loss of morpholino-[I]IPQA accumulation could be characterized by a rapid washout, followed by a plateau, with a slow but slightly decreased (L858R cells, Figure 3 e)) (E746-A750 del cells, Figure 3(f)) CMR over time.
In L858R and E746-A750 del cells, the cell-to-medium ratio (CMR) of morpholino-[I]IPQA was about 60 65 at 60 minutes, which was ~1.6-fold higher than that of wild-type EGFR-transfected H1299 cells (Table 1).
At 120 min, the washout studies showed the higher retention of morpholino-[I]IPQA in L858R (8.9 ± 0.2 CMR, Figure 3 e) red dot line) and E746-A750 del cells (12.6 ± 3.2 CMR, Figure 3(f) red dot line) than that in EGFR-transfected H1299 (5.7 ± 0.4 CMR, Figure 3(g) red dot line) and vector-transfected H1299 (6.6 ± 0.3 CMR, data not shown).
Cell morphological analyses in 786-O yielded very similar to results as in RCC10, with type 1 control, RC161/2QW and del 114 178 cell lines demonstrating a fibroblastic, disorganized phenotype and all other cells displaying an epithelial phenotype, with the exception of the R167Q mutant, which had an intermediate morphology.
