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Fig. S3. Spatial and temporal aspects of gene regulation by Geminin. (A) We compared the activity of full-length (FL) Geminin to the Gemininδcoil form with respect to their ability to suppress mesodermal expression of Brachyury and Xpo and endodermal expression of Sox17. These experiments used 120 pg Gemininδcoil or 12 pg of full-length Geminin mRNA (injected into one blastomere of an eight-cell stage embryos), to avoid cytotoxicity associated with higher level overexpression of the full-length form. We also assessed the temporal aspect of the response by comparison of these mRNA-injected embryos (where Geminin is overexpressed throughout early development) with embryos injected with expression plasmids for the Geminin FL and δcoil variants, where expression is driven from the CMV promoter and restricted to the late blastula-gastrula stages. Embryos were co-injected with 120 pg of each expression plasmid plus 30 pg β-galactosidase lineage tracer mRNA, were raised to stage 11.5 and were Red-gal stained to detect the lineage tracer (oriented rightward in images) and then assayed by in situ hybridization for the markers shown. The β-galactosidase mRNA lineage tracer defines only general areas, not individual cells, when plasmid DNA is injected. As is seen for Gemininδcoil mRNA injection (Fig. 1), either FL or δcoil forms of Geminin expressed as either capped mRNAs or from expression plasmids can effectively suppress mesodermal and endodermal expression. (B) Embryos were co-injected with 120 pg Geminin mRNA or 1 ng MO and with 30 pg β-galactosidase mRNA lineage tracer as before, stained for X-gal (blue) or Red-gal (pink in Fzd7), and in situ hybridized as indicated (purple). Magnified regions of Geminin overexpressing or MO knockdown (Xpo) embryos are shown, with the embryo they derive from in the inset (or at right for Oct25/Fzd7). The repression by Geminin of Epiker, Vent2, Xpo, Brachyury and Endodermin is restricted to the Geminin-injected cells. For morpholino knockdown, ectopic Xpo expression is also confined to the MO-injected territory, although the β-galactosidase mRNA lineage tracer would not be expected to reflect MO distribution at a single cell level. In contrast to the results seen for Geminin repression of gene expression, ectopic/expanded Oct25 and Fzd7 expression in neurula stage embryos occurs broadly in both unlabeled cells and in cells labeled with the X-gal (Oct25) or Red-gal (Fzd7) lineage tracer. |
