Zelda Coordinates Early Gene Networks Figure 4. Zld potentiates Dl morphogentic activity. Wild-type (wt; A, C, E, G, I, K) and zld2 (B, D, F, H, J, L) embryos in nc 11–14 (e, early; m, mid; l, late) were hybridized with RNA probes synthesized against cDNA (A–D) or intronic DNA sequences (see nuclear dots in E–H and I–L insets) for genes indicated on the left. All embryos are oriented anterior to the left and dorsal up, except for the ventral views in E and F (top). (A–H) Nomarksi images. Target-gene transcripts are detectable at nc 11–12 in wild-type embryos, but are delayed by 1–2 nc in zld2. By nc 14, twi appears normal (B, bottom), while rho, brk and sog are all restricted to a narrow lateral domain (arrows in D, F, H). (I–L) Confocal FISH images of nc 14 embryos. DAPI stained nuclei expressing rho or sog are shown in yellow. Note the sporadic expression of rho and sog with irregular boundaries in zld2. (M) Box plot representing the fraction of active nuclei within the expression domains in the wt vs zld2 embryos (n = 10–15). Expression domains were variable among zld2 embryos (data not shown), and showed a significant decrease in the percentage of nuclei (on average 30%) with nascent transcripts (p = 1.02E-11 for rho and p = 1.82E-11 for sog). The expansion of brk dorsally in late nc 14 (D, bottom) is likely due to the absence of dpp in zld2. (N–P) IGB views of the genomic regions indicated. About 30 kb of the sog region is not shown (hash marks). Zld-bound regions coincide with known enhancers, including the shadow enhancer of sog (open red box) [35]. See brk primary and shadow enhancers [35] in Figure S1F. doi:10.1371/journal.pgen.1002339.g004 PLoS Genetics | www.plosgenetics.org 6 October 2011 | Volume 7 | Issue 10 | e1002339