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Fig. 1. A role of aPKC in epidermal ectoderm development. Four- to eight-cell embryos were unilaterally coinjected with aPKC-CAAX or aPKC-N RNAs and lacZ RNA as a lineage tracer (light blue staining). Injected embryos were cultured until stages 14-16, fixed and subjected to whole mount in situ hybridization with anti-sense probes to α-tubulin (A-H) and XK70 (L-S). aPKC constructs employed are shown above panels B and C. (A-K) Changes in ciliated cell differentiation. aPKC-CAAX decreases (B,E), whereas aPKC-N increases (C,F) ciliated cell differentiation (arrows) on the injected side as compared with the uninjected side (A,D). The same embryo is shown in A and B. (D-F) Sections corresponding to embryos in A-C. (D) A single row of α-tubulin-positive cells in the deep layer of uninjected ectoderm. (F) α-Tubulin-positive cells are found in the superficial layer (arrows). (G) Quantification of the results showing mean numbers of α-tubulin-positive cells per section±s.d. Sections of at least three representative embryos per group were analyzed. (H) Frequency of embryos with altered numbers of ciliated cells (data pooled from several experiments). Embryos were scored positive if the number of ciliated cells per injected area was increased by at least 50%. The data are representative of three independent experiments. (I-K) Cilia differentiation assessed by immunostaining with antibodies to acetylated tubulin in stage 18 embryos. (I) Uninjected control embryos with regular pattern of superficially located ciliated cells, (J) aPKC-CAAX injection inhibits cilia formation, (K) aPKC-N injection results in multiple cells with many differentiated cilia. (L-S) aPKC promotes superficial cell fate. (L,O) Control XK70 staining. (M,P) aPKC-CAAX expands XK70 (arrow) on the injected side. (N,Q) aPKC-N downregulates XK70 (arrow). (O-Q) Sections corresponding to embryos in L-N. Superficial expression of XK70 (O) is extended into the deep cell layer in aPKC-CAAX RNA-injected embryos (P, arrowhead). (Q) Inhibition of XK70 by aPKC-N (arrowhead). In all panels, arrows indicate altered staining as a result of injection. (R,S) Quantification of the effects, shown as average numbers of XK70-expressing cells in affected embryos (R), and frequency of embryos with visible changes in XK70 expression (S). Numbers of embryos per group are given above the bars. The data are from three representative experiments. |
