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Fig. 2. Over-expression of PACSIN2 inhibits gastrulation but does not perturb mesoderm induction. (A) Embryos were injected with 1 ng of mRNA encoding GFP, Wt-PACSIN2 or the ∂CC in both blastomeres at the two-cell stage. The embryos were scored for gastrulation inhibition when control embryos reached stage 13. The average of 3 independent experiments is plotted on the graph. The error bars represent the standard deviation to the mean. The number of embryos analyzed are as follow GFP = 74; Wt = 143; ∂CC = 104. The expression of proteins was monitored by western blot using both the anti-myc mAb 9E10 to detect GFP and the PACSIN2 mAb 3D8 to detect Wt and ∂CC. The equivalent of 0.5 embryos was loaded in each lane. (B, C) Injected embryos were fixed at early gastrula (B) or late gastrula stage (C) and treated by whole mount in situ hybridization with the markers chordin and brachyury (Xbra). At stage 10.5, all embryos started gastrulation at the same time as indicated by the presence of the blastopore lip (black arrowhead). At stage 13, embryos over-expressing Wt-PACSIN2 fail to close their blastopore (white double arrow) and the notochord fails to elongate (n) even though chordin is expressed. This indicates that the mesoderm induction occurred but that the gastrulation movements did not. Embryos expressing the ∂CC mutant or GFP gastrulated properly. : P < 0.05. |
