meis3 and pbx4 are required to prevent insulin expression in the anterior endoderm. (A–F) Ectopic insulin-expressing cells arise in the anterior endoderm of meis3 and pbx4-deficient embryos. Wild type embryos (A, D), lzr mutant embryos (B, E) and embryos injected with the dominant negative Meis construct (ΔCPbx4; C, F) were assayed for insulin expression at 24 hpf (A–C) and 48 hpf (D–F). Ectopic insulin expression is indicated by arrows. Note that all insulin positive cells become shifted anteriorly by 48 hpf (see text for details). (G–I) Cells injected with sox32 mRNA at the 32-cell stage primarily populate the endoderm. Wild type (G) or GutGFP transgenic (H, I; (Field et al., 2003)) embryos were injected with sox32 mRNA and rhodamine dextran at the 32 cell stage. At 24 hpf, the rhodamine signal is observed primarily in a deep layer of the embryo adjacent to the yolk (panel G shows a confocal stack of ∼125 μm that extends about halfway through the embryo). This region corresponds to the endoderm, since the rhodamine signal coincides with the GFP-expressing endoderm (white arrows in panels H, I), but not the GFP-expressing hatching gland (white asterisks in panels H, I) of GutGFP embryos. White arrows point to endoderm. (J, K) mRNA and rhodamine dextran co-localize in the endoderm following co-injection at the 32-cell stage. Wild type embryos were injected with sox32 mRNA, GFP mRNA and rhodamine dextran at the 32-cell stage and monitored for GFP (J) and rhodamine (K) expression at 24 hpf. White arrows point to the endoderm. (L–N) MOs co-localize with the rhodamine lineage label in endoderm following co-injection at the 32-cell stage. Wild type embryos were injected with sox32 mRNA, rhodamine dextran and fluorescein-tagged MO at the 32-cell stage. Embryos were raised to 24 hpf and monitored for fluorescein (L) and rhodamine (M) expression. Panel N is an overlay of panels L and M. White arrows point to endoderm. (O, P) Targeting of a meis3 MO (tMO1) to the endoderm produces ectopic anterior insulin expression (arrow in panel P). All panels are lateral views with anterior to the left. s1–s4 denotes the position of somites 1–4 as determined using Nomarski optics.
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