Fig. 3

Relationship between Cell Organization and Non-autonomous Induction of egr2 Gene Expression

(A–C) Embryos injected with CNE1:egr2b-Myc and fixed at various time points to detect endogenous egr2b transcripts. At 12 hpf (A), endogenous egr2b gene expression (magenta) is upregulated in cells overexpressing Egr2b-Myc (green nuclei) and non-autonomous upregulation of egr2b is also observed in adjacent cells (hollow arrowheads, Ai and Aii). DAPI staining (grayscale) is shown in (Ai′) and (Aii′). By 17 hpf (B), Egr2b-Myc-expressing cells become segregated to the borders of r3 and r5, and non-autonomous upregulation of egr2b is no longer observed in adjacent cells (Bi and Bii, solid arrowheads). Knockdown of ephA4 (see also Figure S1) in embryos injected with CNE1:egr2b-Myc (C) causes cells overexpressing Egr2b-Myc to remain dispersed throughout the neuroepithelium at 17 hpf and non-autonomous upregulation of egr2b is now observed (Ci and Cii, hollow arrowheads).

(D–H) Transient expression of egr2b by heat shock of Tg[hsp70:egr2b-Myc] embryos (hs-egr2b) at 11.3 hpf induces upregulation of endogenous egr2b in r2, r4, and r6 (E); compare with control embryo shown in (D). H2B-GFP labeled wild-type donor cells were transplanted into hs-egr2b embryos at 4 hpf, which were heat shocked at 11.3 hpf to induce widespread expression of egr2b (F). Wild-type donor cells in r2 upregulate endogenous egr2b non-autonomously when isolated or dispersed (G, H, hollow arrowheads) but not when clustered together, indicated by the region encircled by white dashed line in (H). White dashed lines in (E), (G), and (H) indicate presumptive rhombomere borders. Single slices from a confocal z stack are shown. Scale bars: 50 μm.