Fig. S6
Dorsal expansion by sqt UTR is not mediated via miRNAs. (A) Model showing localization of sqt RNA (green) in 1-cell and 4-cell embryos, and expansion of the dorsal gsc expression domain (purple arc) upon injection of mutant sqt RNA (magenta). Yellow asterisks indicate presumed negative regulators (for e.g. miRNAs) bound by the sqt UTR. The model proposes that if the sqt UTR binds miRNAs that negatively regulate dorsal determinants, overexpressed mutant sqt RNA could act as a sink that binds to the miRNAs, allowing a larger zone of dorsal, and sqt 32UTR with mutations in these target sites should not expand dorsal. (B) Schematic showing predicted target sites (http://www.mirbase.org and http://www.ebi.ac.uk/enright-srv/microcosm/htdocs/targets/v5) for miR-430 (magenta bar), miR-19a* (purple bar) and miR-152 (orange bar) in the sqt UTR (blue line). Mutations in miRNA target sites are indicated with asterisks. (C) Schematic showing the venus reporter (green box) fused to either globin UTR (v:glo), sqt UTR (v:sqt), or sqt UTR with the miRNA target site mutations illustrated in B (v:sqtmm430, v:sqtmm19a* and v:sqtmm152). (D-H) Expression of gsc at 30% epiboly (D-H) still expands upon injection of v:sqtmm430, v:sqtmm19a* and v:sqtmm152 (E-H), similar to injection of v:sqt. (I-M) Expression of Venus protein at 24 hours shows the efficacy of the miRNA target site mutations. Venus expression is barely detected in v:sqt-injected embryos (J), in comparison to v:glo (I), v:sqtmm430 (K), v:sqtmm19a* (L) and v:sqtmm152 (M) injected embryos. (N-P) MZdicer embryos injected with lacZ:sqt (O) and sqtSTOP:sqt (P) RNA show expanded dorsal gsc in comparison to MZdicer embryos injected with lacZ:glo RNA (N). Therefore, dorsal activity of sqt 32UTR is not mediated via the miRNAs tested or dicer. (Q,R) The angle of gsc expression in injected embryos at 30% epiboly. Each blue dot represents a single embryo. N indicates number of injected embryos and μ(°) is the mean angle of gsc. (D-H,N-P) Animal pole views with dorsal to the right; (I-M) lateral views, dorsal to the right, anterior to the top. Red arrowheads (D-H,N-P) mark the extent of gsc expression. Scale bars: 100 μm. |