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Fig. 7. Opposite effects of PAR1 and aPKC on gene expression in separated
ectoderm cell layers. (A) Experimental scheme for the layer
separation assay. Two-cell embryos were injected with T560A or aPKC-CAAX mRNA.
Animal pole explants were dissected from injected or uninjected embryos at
stage 9, superficial (S) and inner (I) cell layers were separated based on
their different abilities to dissociate in a calcium- and magnesium-free
buffer and allowed to reaggregate. Cell aggregates were cultured until sibling
embryos reached stage 14 or stage 19. RNA was prepared from aggregate lysates
and analyzed by semi-quantitative RT-PCR. (B) A schematic section of
the frog embryo at stage 14 shows superficial and inner layers of non-neural
ectoderm with distinct sets of molecular markers. SLM, superficial layer
markers; ILM, inner layer markers. (C) Stage 14 aggregates: T560A RNA
upregulates the inner layer markers 
-tubulin and inca B and
downregulates the superficial layer markers ESR6e and grhl3
in both inner and outer layer explants. aPKC-CAAX has a complementary effect.
Stage 19 aggregates: T560A RNA upregulates the inner layer markers XDelta-1
and inca B and downregulates XK70 in the superficial layer.
aPKC-CAAX downregulates XDelta-1 and inca B in the superficial layer
and upregulates XK70, ESR6e and grhl3 in inner cell
aggregates. ODC is a control for loading. Uninj, no RNA injection; WE, whole
embryo; -RT, no reverse transcriptase control. The analysis of two
representative sets of cDNAs from several independent experiments is
shown.
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